Chromosome Research (2009) 17 (Suppl 1):S29–S243 DOI 10.1007/s10577-009-9043-0

Oral and poster abstracts 1.1-O Band of origin versus band designation: morphological evidence provides the clue N. Chia (1) (1) ACT Pathology In 1971 reports of fluorescence and giemsa banding of human metaphase chromosomes flooded the literature. Each chromosome could be identified by a distinct pattern and described in terms of band, landmark and region. A system of nomenclature that assigned a designation for arms, region and bands was adopted, and idiograms were produced as the reference tool used to demonstrate banding patterns and define band designation. Whilst recognising the difficulty in the interpretation of the origin of certain bands and the stage at which they first appeared relative to other bands, these idiograms were constructed to demonstrate the constant pattern of banding with the assumption that all chromosome regions contract equally and simultaneously. Improvement in technology and the resulting quality of cell preparations enables us to see bands more clearly. Morphological assessment of banding patterns by intra and inter-chromosomal comparison at different levels of resolution shows that there is a consistent order to the appearance of bands, which correlate with the level of chromatin condensation. Interpretation of the progressive changes to the pattern of banding provides evidence of the origin of a band, and is significant in the accurate assignment of band nomenclature. This investigation demonstrates that several chromosomes show bands with origins that contradict the current band designation. Chromosome 1p31, 1q22– q25.2 and Xp21 are examples of regions that show a characteristic pattern of band generation hierarchies that have incorrect band assignments in relation to band of origin.

An indepth understanding of banding patterns is needed to optimise the accurate correlation of band designation with gene sequences at the molecular level. A thorough investigation using criteria such as morphology, individual band measurements and DNA content is required to produce a band nomenclature that will correlate more accurately with the localisation of gene loci. 1.2-O Relevance of array-analysis and karyotyping in the diagnosis of idiopathic developmental delay: a retrospective study of 36,325 patients in the Netherlands R. Hochstenbach (1) E. van Binsbergen (1) J. Engelen (2) A. Nieuwint (3) A. Polstra (4) P. Poddighe (5) C. Ruivenkamp (6) B. Sikkema-Raddatz (7) D. Smeets (8) M. Poot (1) (1) University Medical Centre Utrecht (2) University Medical Centre Utrecht (3) Maastricht University Medical Centre (4) VU University Medical Centre (5) Academic Medical Centre (6) Erasmus Medical Centre (7) Leiden University Medical Centre (8) University Medical Centre Groningen (9) Radboud University Nijmegen Medical Centre (10) University Medical Centre Utrecht High-resolution, genome-wide, array-based segmental aneusomy profiling has emerged as a sensitive technique for detecting pathogenic genomic imbalances in patients with idiopathic mental retardation and/or congenital abnormalities (DD/MR). We investigated whether karyotyping, combined with FISH, still hold relevance for the clinical investigation of these patients. To this end, we reviewed the outcome of cytogenetic studies in all 36,325 referrals in the Netherlands during the period 1996–2005, a period before the advent of array-based genome investigation. We estimate that in a minimum of 0.78% of all

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referrals a balanced chromosomal rearrangement would have remained undetected by array-based investigation only. These include familial rearrangements (0.48% of all referrals), apparently balanced, de novo translocations and inversions (0.23% of all referrals), and de novo, balanced Robertsonian translocations (0.04% of all referrals). In addition, 69,XXX triploidy is undetected in 0.03% of all referrals. We conclude that karyotyping would offer a very limited contribution to the detection of pathogenic aberrations in DD/MR patients with a normal array result. We propose that, because of its high diagnostic yield, high-resolution array-based genome investigation should be the first investigation performed in cases of DD/MR, detecting >99% of all pathogenic abnormalities. Based on a review of the literature, most of these are deletions of a single chromosomal segment. We discuss how confirmation studies and carrier detection by FISH can be performed most efficiently, using commercially available DNA-probes in as many instances as possible. 1.3-O Revision of breakpoints using molecular methods in a series of structural chromosome aberrations with previously determined breakpoints using banding M. Riegel (1) A. Baumer (1) R. Baldinger (1) A. Schinzel (1) (1) University of Zürich The aim of this project is to precisely define breakpoints of known chromosome aberrations. To better know and understand the genetic basis of the phenotypic alterations in rare chromosome aberrations, it is important to define the aneuploid segments for karyotype-phenotype correlation. We re-investigated cases examined by GTG banding during the preFISH period with the new molecular cytogenetic methods. The detection of deletions and duplications was determined using a combination of aCGH, FISH and MLPA methods. Some cases stemmed from a collaborative study with eastern European countries. Approximately 150 patients have been examined so far. The results of the studies proved to be very useful and, not rarely, surprising. There was almost no case in which the breakpoints suspected from former cytoge-

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netic investigations were fully confirmed. This does not or only to a minor extent point to poor earlier work, but rather to the impossibility of confidently determining some specific breakpoints. In some cases, the revision even revealed an aberration of which the aneuploid segments did not overlap with the originally determined segments. The discrepancy was particularly large in instances of duplication-deletions, rings and interstitial duplications or deletions, and in a few rare cases, the initially suspected unbalanced aberration was in fact not unbalanced. There was a clear tendency to localize breakpoints in light G bands. With the cytogenetic workup of cases in the context of this project we were able to clarify and better determine many aberrations. As the discrepancy frequently involved several megabases, genes previously thought to be within the deleted/duplicated segment were shown to be present in double copy and vice versa. From this work we have to conclude that many published chromosome aberrations that have been confidently classified will not have the breakpoints and thus the aneuploid segments as determined. This may lead to errors in karyotype-phenotype correlations. It seems necessary to re-investigate almost all structural chromosome aberrations if conclusions are to be drawn concerning the involvement of specific genes. 1.4-O X-chromosome disorders: Identification of underlying mechanisms P. Patsalis (1) G. Koumbaris (1) C. Hatzisevastou (2) A. Kurg (3) S. Kitsiou Tzeli (4) N. Scordis (5) Z. Kosmaidou (6) J. Vermeesch (7) I. Georgiou (8) N. Carter (9) (1) The Cyprus Institute of Neurology and Genetics (2) Ippokratio Thessaloniki General Hospital (3) University of Tartu (4) Agia Sophia Children’s Hospital (5) Makarios Hospital (6) Alexandra Hospital (7) University of Leuven (8) University of Ioannina (9) Wellcome Trust Sanger Institute Non-allelic homologous recombination (NAHR), non-homologous end joining (NHEJ), and recently Fork Stalling and Template Switching (FoSTeS) have been implicated as the main mechanisms for the creation of genomic disorders. In order to investigate the mechanisms responsible for the creation of X-

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chromosome disorders we analyzed 70 cases bearing cytogenetically visible X-chromosome abnormalities using whole genome tiling path BAC arrays and custom designed targeted ultra-high resolution oligoarrays. Using whole genome tiling path BAC CGH, we were able to accurately map the breakpoints of 35 cases bearing isochromosomes of the long arm of chromosome X at a resolution of 150 kb. Fifteen of these had breakpoints at the centromere and were considered monocentric. The remaining 20 were isodicentric and had breakpoints in proximal Xp in ChrX:51500000–58500000. This region of chromosome X is rich in segmental duplications and contains some of the largest and most homologous inverted repeats in the human genome. Based on the BACarray CGH findings, we designed custom oligo-arrays which cover this region in ultra-high resolution (44K and 385K oligos in 7 Mb region of interest) and feature enhanced coverage of segmental duplications. Screening the isodicentric cases with these ultra-high resolution arrays enabled us to identify previously undiscovered breakpoint complexity in 45% of the isodicentrics and demonstrate that they are formed by NAHR, facilitated by specific highly homologous inverted repeats. Twenty two percent of the isodicentrics were mapped within repetitive sequences and 33% have simple breakpoints that do not coincide with segmental duplications and are probably mediated by a nonhomologous recombination mechanism.

Numerical chromosomal abnormality was prevalent in 51% of the total. In numerical chromosomal abnormality the observed karyotype were pure line 45,X in 34.5% mosaic patterns such as 45,X/46,XX , 45,X/47,XXX , 45,X/46,XY and 45,X/46,Xi(Xq)in 17%. The other abnormalities were 46,Xi(Xq) and 46, Xi(Xp) in 12%, 46,XXdel p in5% and in 5% and 46, XX,t(X;3) t(X,3) in 2.5%. Male genotype was identified in 11 (29%) of the patients. Conclusion: The present study has emphasized that karyotyping is absolutely necessary in the evaluation of primary amenorrhea. This study also revealed the incidence of chromosomal abnormality in women with primary amenorrhea in southern of Iran is similar to that reported in the litreture. Correspondence author, Akbar Safaei, Department of Pathology , ShirazUniversity of Medical Sciences. Tel fax: 07112301784

1.1-P

1. 2. 3. 4.

Cytogenetic study in patients with primary amenorrhea in south of Iran: A. Safaei (1) M. Vasei (1) (1) Shiraz University of medical Sciences Objective: To estimate the incidence of the chromosomal abnormality referred for karyotyping in individual with primary amenorrhea in southern of Iran. Method: Chromosomal analyses have been carried out in 220 such cases that were referred from different parts of the south of Iran. The standard protocol for peripheral blood lymphocyte culture was followed for metaphase chromosome preparation and conventional analysis of G- banded chromosome. Result: The Frequency of the chromosomal abnormality was 18.8% in primary amenorrhea.

1.2-P Infertility and Cytogenetic Causes in men S. Soleimani (1) F. Mahjoobi (0) (1) The Blood Transfusion Organization research center Male infertility factor accounts for about half the cases of infertility in couples. Some of the chromosomal changes(aberrations) which have been found in infertilite men include: Balanced chromosomal translocation Chromosome inversion Marker chromosome Sex chromosome abnormality

Our investigation provides the circumstantial and direct evidence which confirms the importance of the sex chromosome in reproductive disorders. We have analyzed (10years study) 1021 blood samples from infertile men 664 of whom were oligospermic or azoospermic. Constitutional chromosome aberrations were diagnosed in 321 of these patients. We have observed 31.4% chromosomal abnormality in azoospermic men which is compatible with the data from literature. The following abnormal chromosome complements were found: 46,XX;47XXY;47,XYY;48,XXXY;45,X[10]/46, XY[134];46,XY[4]/47,XXY[82];

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We believe that many infertilities especially severe oligospermic and azoospermic cases raise the need for a cytogenetic analysis besides molecular techniques to reveal any cytogenetic abnormalities.

also several features in common. Herein presented are the clinical and molecular findings in a 1-year-old female with a karyotype 46,XX,der(4)t(4;13)(q34.1; q14.3)mat, whose phenotype exhibits the features of the two distinctive syndromes. The chromosome breakpoints and the size of segments involved in the rearrangement were determined by FISH applying chromosome 4 and 13 specific molecular banding (MCB), subtelomeric probes for 13qter and 4qter, and BACs mapped to 13q21.1 and 13q14.3, respectively. According to the labeling scheme for all 169 arraymapped MCB libraries (Weise et al., 2008) it was estimated that the duplication encompassed 73.95 Mb of 13q, and the deletion 4 14.6 Mb of chromosome. Reporting of further cases with very good clinical and molecular characterization will add to a more precise description of the deletion 4q syndrome and will pinpoint the critical region on chromosome 13 responsible for trisomy 13. Supported in parts by DAAD (D07/00070) and Evangelische Studienwerk e.V. Villigst.

1.3-P

1.4-P

Partial monosomy 4q and partial trisomy 13q: phenotype and molecular mapping of the breakpoints

Apparently identical subtelomere rearrangements revealed by FISH in two unrelated affected children with mental retardation and similar phenotypes

46,XX[11]/47,XXY[36];46,XY[6]/47,XYY [38];46,XY[10]/46,XX[26]/47,XXY[61]; 46,X,del(Y)(q11.23);46,X,inv(Y)(p11.2q11.22). We have found some patients with complex structural and aneupoloidy abnormalities: × 46,XX,inv(9)(p11q13)/47,XXY,inv(9)(p11q13) [4] × 47,XXY[93]/48,XXY+mar[4]/48,XXXY[2] × 47,XXY,inv(9)(p11q13) × 47,XXY,t(1;17)(p36.1;q21) × 46,X,del(q11.2)[98]/45,X[6] × 47,XXY,inv(9)(p11q13)/t(10;22)(q26.3;q13.1) × 46,X,idic(p11.32;q11.32)[27]/45,X[36]/46,XY [2]

J. Wagner (1) S. Dorner (2) F. Stipoljev (3) I. Skrlec (1) G. Lauc (1) A. Weise (4) K. Mrasek (4) T. Liehr (4) L. Brecevic (5) (1) School of Medicine (2) Clinical Hospital Osijek (3) General Hospital (4) FSU Jena (5) School of Medicine Phenotype in patients with segmental aneuploidy often vary in their clinical manifestation depending on the size of the chromosomal region involved. Deletions of chromosome regions 4q31, 4q32, and 4q33-4qter lead to a distinctive malformation syndrome (deletion 4q syndrome) of facial dysmorphism, cardiac and limb defects and developmental delay. More distal 4q deletions involving bands q34–q35 have been found in patients presenting with less characteristic features and less severe mental retardation. Partial trisomy 13q (13q14-qter) has also been shown to cause a characteristic phenotype associated with severe mental deficiency resembling that of trisomy 13. Although del4q and dup13q syndromes vary in their phenotypes, they have

Z. Vlckova (1) M. Simandlova (1) A. Puchmajerova (1) J. Camajova (1) P. Hedvicakova (1) J. Drabova (1) Z. Zmitkova (1) P. Goetz (1) D. Novotna (1) (1) University Hospital Motol We report on two patients with mental and motor retardation, growth retardation, similar congenital defects and similar facial dysmorphic features. The first one is an 8-year-old girl of Slavic origin, the second one is a 2.5-year-old boy of Vietnamese origin. More severe defects were diagnosed in the girl. Some of these were detected even before birth: IUGR, oligohydramnion, PCD of one kidney and the dilatation of brain cavities. After the birth (at 38. week of gestation, by C-section) muscular hypotonia, microcephaly with brachycephaly, atresia of ear canals and faint talipes were seen . Hydrocephalus, epilepsy, anaemia and latent hypothyroidism developed later. Some defects before the birth were also detected at the boy: dystopia of one kidney, cleft

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palate and septal defect of ventricles and also external ear anomalies. After the birth (at 30th week of gestation, by C-section) muscular hypotonia, equinovarus and umbilical hernia were found. Phenotypes of patients were not normal and do not correspond well with any clinical syndrome. Cytogenetic analysis was performed from G-banded chromosomes. The resolution level was 400–450 bands and did not bring any results. Both patients were recommended for investigation with subtelomere FISH panel. FISH was performed using Multiprobe Chromoprobe-T system (Cytocell) and revealed the nearly same chromosomal aberrations— duplication of 4qter together with a deletion of 18qter–t(4qter;18qter). Findings were confirmed by MLPA using SALSA MLPA kits PO36B, P019, and P070 (MRC-Holland). The size of segments was estimated using FISH whole chromosome painting probes for chromosome 4. However, the exact extent of aberrations was ascertained only using clinical arrayCGH at 1/2 Mb resolution level. More extensive aberration in the 8-year-girl corresponds with high probability with her more severe phenotype. It was interesting to note that the dysmorphic features and congenital defect were similar. Supported by grant of Health Ministry of Czech republic Nr 00064203. 1.5-P Ring chromosome 14 mosaicism: an unusual case associated to developmental delay and Epilepsy characterized by Genome array-CGH A. Nucaro (1) M. Falchi (2) T. Pisano (2) R. Rossino (4) A. Milia (5) F. Boscarelli (3) C. Montaldo (3) C. Cianchetti (2) D. Pruna (2) (1) Research National Council ( CNR) Ring chromosome 14 is a rare cytogenetic disorder, generally associated with developmental delay and seizures. Herein, we present a 10-year-old boy, born to young, healthy and non-consanguineous parents, with an unusual ring chromosome 14 mosaicism associated with mental retardation and epilepsy. At the age of five months he had seizures, confirmed by EEG as epilepsy. Seizures were resistant to common antiepileptic drugs. Cytogenetics investigation and FISH studies revealed an unusual chromosomal

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mosaicism 46,XY, r(14)(p13;q32)/ 46XY, dup r(14), ish (14wcp+). Out of 50 examined QFQ banded metaphases, 48 had a ring chromosome 14 and 2 had a duplicated ring chromosome 14 (4%).The parents showed a normal karyotype. Genome array-CGH studies revealed a microdeletion of about 150 kb at the site of ring formation at 14q32.33. To the best of our knowledge, this is the smallest deletion reported so far. Array- CGH also revealed a single BAC clone deletion in 14q22.1 and a single BAC clone deletion in 14q24.2. Previous linkage analysis has suggested a possible locus for susceptibility genes of epilepsy in this region. The objective of this study was to determine if there is a common minimal region of deletion on 14q32.33 in r(14) patients that could explain the phenotypic differences between this condition and the same deletion without the ring , and if the deletion in 14q22.1 and q14q24.2 could be associated with a susceptibility locus for epilepsy. 1.6-P A 7 Mb duplication 11q12.1q13.1 detected by array CGH in two mentally and developmentally retarded siblings U. Koehler (1) E. Holinski-Feder (1) H. Omran (2) K. Storm van’s Gravesande (2) (1) Medizinisch Genetisches Zentrum (2) Universität Freiburg We report two cases of an apparently de novo duplication 11q12.1q13.1 in two mentally retarded siblings. The 18 year old boy shows severe mental and developmental retardation with autistic signs, microcephaly, muscular hypotonia, and dysmorphic signs (brachycephalus, epicanthal fold, broad base to nose, high palate, oligodentia, auricular pits, kinky hair). In addition he has severe speech delay, strabism, myopathy, and joint laxity. His 24 years old sister exhibits a similiar but less severe pattern of features: mental and developmental retardation, and dysmorphic signs (small mid-face, low set ears, retrognathia, hypotelorism, epicanthal fold, broad base to nose, kinky hair). In addition she shows motor delay, scoliosis, and joint laxity. At birth both children were small for gestational age. Array CGH revealed a 7 Mb duplication of chromosomal segments homologuous to bands 11q12.1q13.1. Karyotyping has been per-

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formed subsequently as the initial reports were normal. However, GTG banding analysis demonstrates an interstitial duplication of bands 11q12.1 to 11q13.1 in both siblings. FISH analysis with probes for bands 11q12.1 and 11q13.1 respectively, revealed a direct duplication of this chromosomal segment. The healthy sister and both parents show normal karyotypes and normal FISH results. Thus, duplication is probably caused by gonadal mosaicism. The duplicated chromosomal region spans a high number of over a hundred genes, and a genotype-phenotype correlation is still ongoing. 1.7-P De novo unbalanced whole arm translocation resulting in 18p- syndrome I. Petkovic (1) I. Barisic (0) (1) Children’s Hospital Zagreb, University of Zagreb, Medical School (2) Children’s Hospital Zagreb, University of Zagreb, Medical School Whole arm translocations(WAT) are rare constitutional abnormalities. The aberration results from centromeric fission or juxtacentromeric breaks and reciprocal exchange of entire arms of two chromosomes. WAT involving chromosome 18 account for approximately 16% of all cases of monosomy 18p. To the best of our knowledge only three cases of t(15;18) leading to 18p monosomy have been reported so far. In this study we performed clinical, cytogenetic and FISH studies in oneadditional case of 18p monosomy due to an unbalanced WAT. Our patient is an 8-year-old girl. The child’s phenotype showed mental and somatic retardation, round and dysmorphic face, strabismus, skeletal anomalies, heart malformations and muscular hypotonia. Cytogenetic analyses were performed on slides obtained by peripheral blood culture. High-resolution G-banding method, RBG- and CBG- methods were used for chromosome identification. FISH method with whole chromosome 15 and 18 painting probes, D15Z1, SNRPN, PML, D18Z1 and D18S552 probes were used for precise characterization of the structural rearrangement. Analysis revealed monosomy 18p resulting from an unbalanced whole arm translocation between chromosome 15 and 18. The Aberrant chromosome presents two centromeric constrictions.

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CBG-banding did not show two separate centromeric regions, while FISH analysis confirmed the presence of centromeric regions from chromosome 15 and 18 on the derived chromosome. Breakpoints were attributed to 15p11.2 and 18p11 bands. Parental karyotypes were normal indicating de novo origin of the chromosome rearrangement. Mechanisms of formation of WAT are poorly understood. This study presents evidence that the unbalanced WAT between chromosomes 15 and 18 retained the centromeric region of both chromosomes involved in the rearrangement, and suggests that the translocation was not a result of recombination involving centromeric α-satellite DNA. Other factors like interphase chromosome domains, low-copy repeats or segmental duplications may be important in the etiology of unbalanced whole arm translocation 15;18 in our patient as well as in two other cases reported in literature. 1.8-P A case of congenital spina bifida in a newborn male with pseusodicentriric Y chromosome V. Antonenko (1) E. Shestopalova (2) N. Shilova (1) T. Zolotuhina (1) (1) Research Centre for Medical Genetics (2) Moscow Region Research Institution Pseudodicentric Y chromosome is a rare form of chromosomal abnormality. We present a newborn male with congenital spina bifida, hydrocephaly, ArnoldChiari anomaly, short neck, hyperplastic gums, hypoplastic penis and undescended testes. Cytogenetic analysis revealed two clones: one without Y chromosome and the other with an abnormal morphology of Y chromosome. C-banding revealed presence of two centromeres, only one of which was active. Presence of two copies of SRY gene in both arms of Y chromosome was revealed by FISH with LSI SRY DNA-probe. The karyotype of the child was: mos 46,X,psu idicY (q12)[21]/45,X[9]. Thus the patient had disomy for proximal and nullisomy for distal part of Y chromosome. The karyotypes of his parents were normal. Most cases of idic(Y)(q12) have been found in phenotypically normal men with anomalies of reproductive function. This is the first case of central nervous system abnormality in a patient with pseudodicentric Y chromosome.

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1.9-P Male Infertility due to 45,X[90]/46,X,nfY[10]: clinical and cytogenetic findings G. Kronberger (1) O. Rittinger (3) (1) Paracelsus Medical University Salzburg Rearrangements of the Y chromosome are rare causes of male infertility. Dicentric chromosomes derived from a Y following deletion of a segment of Yq are as a rule found in a mosaic form with a 45,X cell line, which arises as a consequence of mitotic instability of the derivative chromosome. Rearrangements of these dicentric Y are commonly observed. We report on a 34 year old male patient seeking advice for his infertility. He presented with an inconspicuous external habitus with normally developed external genitalia and normal blood levels of testosterone. Cytogenetic investigation revealed a predominant 45,X cell line and a second one with an additional small marker chromosome. Because of the inconclusive pattern of the small derivative chromosome, additional FISH investigations and PCR-based screening for Y-deletions were performed. FISH results were in accordance with an i(Yp) including a small amount of Yq (AZFa-b), the blood karyotype was 45,X[90]/46,X,nfY[10]. On the top of Xp some wcpY positive, but SRY and SHOX negative material was detected pointing to an additional premeiotic crossing over event. This observation is interesting with respect to the normal male appearance in spite of a quite small percentage of Y material in blood cells, suggesting different distributions in other tissues. Moreover, SRY was shown not to be involved in the X/Y recombination, as it would be expected for 45,X males without derivative Y chromosome. 1.10-P Interstitial de novo del(1)(q25.1q31.3): clinical presentation and molecular description with array-CGH E. Dimitriadou (1) K. Theodoropoulos (2) I. Lalou (1) M. Tzoufi (3) J. Vermeesch (4) J. Fryns (4) S. Kitsiou (5) M. Syrrou (1) (1) University of Ioannina (2) General Hospital of Ioannina “Hatzikosta” (3) University of Ioannina (4) University Hospital Leuven (5) University of Athens

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We report a 11-year-old male child with prenatal onset of growth retardation, growth hormone deficiency, developmental delay and several dysmorphic features including significant orthodontic problems, severe malocclusion, protrusion of upper jaw and narrow high-arched palate with triangular shaped palate, low hairline posteriorly, hypertelorism, long palpebral fissures, mild bilateral epicanthal folds , long eyelashes and reversion of lower eyelid, relatively prominent ears, small hands and digits with bilateral 5th finger clinodactyly and apparently persistent finger pads, small feet and toes with right 2nd– 3rd toe syndactyly and bilaterally increased gap between 1st and 2nd toes and bilateral contractures of the Achille’s tendon. He also has severe myopia, strabismus and mild astigmatism. Molecular karyotyping using a 1 Mb resolution BAC array demonstrated the presence of a 21 Mb sized deletion on the long arm of chromosome 1. The deletion flanking clones are RP11-552K17 and RP3433G19. The karyotype is arr cgh 1q25.1q31.3 (RP51045J21➔RP11-435N12)x1. The case is compared with similar cases from the literature of postnatally detected interstitial deletion on 1q. This is the first time that array-CGH analysis has been used for a more accurate assessment of the breakpoints in a patient carrying a deletion in the 1q25–q31 region. 1.11-P Submicroscopic subtelomeric 22q13 deletion: a recognizable phenotype? A. Jezela-Stanek (1) A. Gutkowska (1) M. Kugaudo (1) M. Gajdulewicz (1) M. Constantinou (2) K. Chrzanowska (1) M. Krajewska-Walasek (1) (1) The Children’s Memorial Health Institute (2) Medical University of Łódź 22q13 deletion syndrome is also known as PhelanMcDermid syndrome. Individuals with the disorder can suffer from a range of symptoms, with mild to very serious physical and behavioral characteristics, such as: general hypotonia, absent to delayed speech, normal to accelerated growth, global developmental delays with autistic behavior and minor dysmorphic features. The deletion occurs at the terminal region of chromosome 22 (from 22q13.3 to 22qter). While some clinical signs of del22q13 correlate with the size

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of the deletion, the main traits of the syndrome appear to be independent of the deletion size, and are related only to the presence of the SHANK3 gene (with a locus in 22q13.3; encodes for a structural protein of the postsynaptic density). The disorder may not be uncovered by routine karyotyping, therefore a specific FISH probe is recommended to confirm the diagnosis. The aim of this paper is to present 3 cases with deletion 22q13, emphasing the diagnostic approach and its clinical manifestation. All the cases were genetically evaluated because of noted developmental delay. In one—deletion was found in routine cytogenetic analysis and verified with the use of N85A3 probe [46,XX,del(22)(q13).ish del(22)(q13.3) (N85A3-)], while in two others—subtelomeric FISH screening allowed us to establish the diagnoses. In order to make precise the genotype-phenotype correlation, in one case further molecular tests have been performed (244k aCGH, Agilent), which revealed a deletion of 2,2 Mb. Knowledge of the pattern of the “22q13—phenotype” will help clinicians to diagnose this chromosomal abnormality in their patients and thus to counsel their parents accordingly. 1.12-P A novel mutation analysis of Tunisian Wilson disease patient ATP7B A. Mili (1) M. Gribaa (1) J. Bouguila (2) T. Ben Lazreg (1) I. Ben Charffedine (1) L. Adala (1) O. Mamai (1) M. Bost (3) A. Saad (1) (1) CHU Farhat HACHED. (2) CHU Farhat HACHED (3) Groupe Hospitalier Est Neurogénétique Moléculaire Wilson’s disease (WD), an autosomal recessive copper transport disorder, usually associated with symptoms involving the liver and central nervous system. The disease is caused by mutations in the ATP7B gene. Here we report a Tunisian consanguineous family with two brothers showing clinical and biological features of in favour of WD. STRs segregation using five microsatellite markers located around ATP7B gene showed a homozygote haplotype in our 2 patients. Mutation analysis of ATP7B gene find a homozygous substitution of an A by a G at position 3059 (3059AàG) leading to the replacement, in the

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protein, of the amino acid lysine by an arginine at position 1020 (K1020R). This causative new mutation has not been found among 200 tested controls. Key words: Copper, hepatitis, ATP7B gene, mutation analysis, Wilson’s disease 1.13-P Confusing diagnostics of subtelomeric 14q deletion syndrome. Genotype-phenotype correlation and review of the literature M. Krajewska-Walasek (1) A. Gutkowska (1) E. Ciara (1) A. Jezela-Stanek (1) K. Chrzanowska (1) (1) The Children’s Memorial Health Institute To date, the number of reported cases with a terminal 14q deletion is very small. Only 11 cases had a pure terminal deletion (14q3-14qter), while 6 were cryptic and were diagnosed by subtelomere screening. Major clinical features of the syndrome are mental retardation and dysmorphism, mainly blepharophimosis. However, the limited number of reported cases does not allow assigning specific features to specific regions of terminal 14q. Moreover, imprinting at 14q32 is an additional confounding factor in the establishing of genotype-phenotype correlations. We report a girl with mild developmental delay, hypotonia, microcephaly (which developed during the first year of life), facial dysmorphism (hypertelorism, blepharophimosis, epicanthic folds, iris coloboma, high forehead, short bulbous nose) and unilateral palmar crease. In contrast to the striking phenotype, her initial development was considered to be normal. Delays in motor milestones and intelligence became apparent from 6 months of age. Routine cytogenetic analysis (500-band stage) was normal and subsequently deletion of the 14q (14q?→14qter) was identified by subtelomeric FISH screening, and then verified by MLPA. Parental chromosome investigations for the 14qter deletion, including karyotyping and FISH, gave normal results. Unexpectedly, a balanced translocation involving chromosomes 4 and 12 was found in the patient’s mother [46,XX,t(4;12)(q21.3;q24.3).ish 14q(14qter x 2)] resulting in new implications for genetic counsel-

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ing in this family. In order to define the extent and the parental origin of the subtelomeric 14q deletion, a PCR-based analysis for microsatellite repeat polymorphisms was performed. We do believe that the features observed in patients with a terminal 14q deletion are specific enough to allow delineating of a clinically recognizable phenotype, especially in respect to a recognizable facial gestalt. Dysmorphologists should keep in mind this syndrome during evaluation of patients with blepharophimosis and mild mental retardation, hence differential diagnoses and parental origin of the deleted 14 chromosome are discussed. 1.14-P Incidence and distribution of Interchromosomal Effects in carriers of rearrangements E. Anton (1) J. Blanco (1) F. Vidal (1) (1) Universitat Autònoma de Barcelona

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frequencies of sex chromosome aneuploidies although in some cases, additional increase of autosome aneuploidies or diploidies were also observed. Discussion Evidences of an ICE have been detected in all kinds of carriers. Among them, rcp carriers appear to be the most prone to display such interferences followed by rob carriers and finally, by inv carriers. From these data, sex chromosome aneuploidies seem to be the most indicative parameter for ICE evaluation, as they have been detected in all the cases with positive results. However, we have not identified any common particular cytogenetic parameter among the rearrangements with ICE. This could be related to the limited size of the populations analysed, especially in inv carriers. Data from further studies would be helpful to shed more light on this aspect. Acknowledgments Project 2005SGR-00437 1.15-P

Introduction Carriers of rearrangements have an increased risk of producing gametes with numerical abnormalities. Such imbalances could result from meiotic interferences produced by the rearrangements in the segregation of other chromosome pairs, that is, an Interchromosomal Effect (ICE). Nevertheless, the occurrence of this phenomenon has been controversial. In our work, sperm FISH studies have been performed to obtain more data regarding the incidence and the distribution of ICE among carriers of Robertsonian (rob) and reciprocal (rcp) translocations and inversion (inv). Materials and methods Sperm samples from 10 rob, 14 rcp and 5 inv carriers were processed for FISH analysis as standardized in our laboratory. In all individuals, chromosomes X, Y and 18 were evaluated. Additionally, studies for chromosomes 21 and 22 were also performed in seven rob carriers and chromosomes 13 and 21 were analyzed in two rcp and four inv carriers. Results were statistically compared with internal control data. Results Significantly increased percentages of numerical abnormalities were detected in three rob, seven rcp and one inv carriers, representing 38% of total population analyzed. All of them presented altered

New euchromatic variant dup(11)(p15.3p15.1) transmitted through two generations S. Singer (1) M. Bonin (1) J. Prechtel (1) D. Pflumm (1) R. Ullmann (2) A. Tzschach (2) O. Riess (1) A. Dufke (1) (1) University of Tübingen (2) Max Planck Institute for Molecular Genetics A familial duplication of the chromosomal region 11p15.3-p15.1 due to an unbalanced insertion into the long arm of chromosome 14 was detected by chance at prenatal diagnosis for advanced maternal age. The duplication was also present in the father and in his brother, both clinically healthy. The derivative chromosome 14 was transmitted through the grandmother who is carrier of a balanced insertion ins(14;11) (q32.1;p15.3p15.1). A 7.1 Mb duplication of chromosomal region 11p15.3–p15.1 was identified by CGH on a submegabase-resolution whole genome tiling path BAC array and additionally verified by Affymetrix Human Mapping 100K SNP array. The region is located approximately 9 Mb centromeric to the Beckwith-Wiedemann syndrome critical region in 11p15.5. All three carriers present a very similar facial phenotype. Development of the meanwhile

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3 years old boy is normal. To the best of our knowledge, this is the first report of an unbalanced chromosome abnormality in this region that is not correlated with major clinical consequences.

to existing idiograms. Additional bands that were not previously documented but are constant and reproducible are described for the first time in the ISCN (2009).

1.16-P

1.17-P

ISCN (2009): A review of the idiograms

An unusual large 15q deletion due to an unbalanced translocation t(15;19)(q14;q13.4) resulting in Prader Willi syndrome

N. Chia (2) L. Shaffer (1) (1) Signature Genomics Laboratories (2) ACT Pathology The idiograms published in ISCN (2009) were constructed with the intention of providing a comprehensive set of idiograms that are representative of the chromosomes as observed using light microscopy. All levels of resolution show the differential shading thereby providing an improved correlation with the banding patterns observed by the analyst. A defined set of criteria was used in the selection of cells to ensure a consistent pattern of banding. By excluding external factors an accurate banding pattern could be achieved. Metaphase spreads with minimal overlapping and clumping of chromosomes and no interference of spreading by adjacent interphase nuclei were selected for pattern analysis. The slides used showed cells with a range of resolution which provided an internal comparison of coincident banding patterns at different levels of resolution. Interpretation of progressive changes to the banding patterns in relation to the level of chromatin condensation provided evidence of the origins of bands and the order of band sub-division. Intra and inter-chromosomal comparisons were used to determine a constant and reproducible banding pattern. Patterns of coincident band sub-division were recorded for each chromosome in the cell and in this manner a characteristic pattern of band generation hierarchies was produced. Inconsistencies of the comparative lengths of some chromosomes were observed in the ISCN (1985), ISCN (1995). To overcome this disparity, the length of each chromosome idiogram in the ISCN (2009) was calculated using the relative chromosome lengths (HAL) as a fraction of the length of chromosome 1 as published in the ISCN (1995). Improvements in the quality of cell preparations and the method employed in this study to ascertain banding patterns, has resulted in minor amendments

O. Rittinger (1) G. Kronberger (1) (1) Paracelsus Medical University Salzburg Background. Prader-Willi syndrome (PWS) is caused by lack of the paternally inherited copies or their expression of multiple genes in a 4Mb region on the 15q11–q13 region. In contrast to the predominantly interstitial deletion, translocations are rare causes of PWS involving apparently balanced translocations with breakpoints in SNRPN and unbalanced translocations involving the proximal 15q region and telomere ends of several other chromosomes. Case report. We report on a young PWS girl who was born at term to healthy young parents. Diminished fetal movements were noticed. Despite normal measurements for length and height she presented moderate hypotonia, particularly poor head posture, and poor sucking. Bottle feeding became possible not before the fifth week. No obvious dysmorphic features or malformations were found. Cytogenetic investigation showed an unbalanced reciprocal translocation t(15;19)(q14;q13.4) with loss of the derivative chromosome 15. The karyotypes of both parents were normal. PWS was confirmed by MS-PCR and FISH (SNRPN-). In addition, the subtelomeric region 19q was shown to be preserved making it unlikely a minor 19q duplication had an influence on the phenotype. Microsatellite genotyping indicated the deletion exceeding the usual distal breakpoints BP3-5 including D15S1007. A more refined breakpoint determination by means of array-CGH is under way. Conclusion. There are only a few reports on unusually large 15q deletions usually presenting with a more severe phenotype. In our patient to date no additional anomaly differing from the common PWS phenotype was found but the degree of mental disability can certainly not be anticipated. Therefore, a close followup of the development will be advisable.

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1.18-P Genetic investigations on 8 patients affected by a characteristic seizure disorder associated with ring 20 chromosome mosaicism D. Giardino (1) A. Vignoli (2) L. Ballarati (1) M. Recalcati (1) N. Camporeale (1) M. Marchi (1) P. Finelli (1) I. Giordano (3) M. Canevini (2) L. Larizza (4) (1) Istituto Auxologico Italiano (2) A.O. San Paolo (3) Spedali Civili Brescia (4) Università Studi Milano, A.O. San Paolo Chromosome 20 ring [r(20)] is a chromosomal disorder that has been associated with a rare syndrome characterized by a typical seizure phenotype consisting of complex partial seizures, a peculiar electroclinical pattern, cognitive impairment, behavioural problems and absence of a consistent pattern of dysmorphology. More than 60 cases have been reported till now: most are sporadic with de novo r(20) mosaicism and present with refractory epilepsy with periods of nonconvulsive status epilepticus (NCSE) and cognitive problems. The ratio of r(20) varies from 1 to 100% in lymphocytes, but the relationship of the percentage of r(20) cells with the clinical phenotype is controversial. The pathogenic mechanism underlying seizures disorder in r(20) syndrome remains to be elucidated. Only a few cases have been investigated with FISH techniques oriented at detecting subtelomeric 20 chromosome deletions associated with the ring formation and/or haploinsufficiency of two epilepsy genes, CHRNA4 and KCNQ2, located at 20qter. To the best of our knowledge, array-CGH technique aimed at discovering sequence copy number variations on r(20) and on the entire genome has not yet been applied to patients carrying r(20) chromosome. Here we describe 8 patients with r(20) chromosome mosaicism who have been subjected to extensive clinical, cytogenetic, FISH and array-CGH studies. The percentage of cells with r(20) varies from 8 to 71% in lymphocytes and from 0 to 81% in fibroblasts, with different concordance between the two lineages among patients. FISH investigations revealed the presence of both pan-telomeric and subtelomeric specific probes on r(20) on all probands. CHRNA4 and KCNQ2 gene sequences were found to

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be constantly maintained in the ring. Array-CGH analyses revealed a normal molecular karyotype in all of the probands except one, showing a 1,9 Mb deletion at 16q22.1 band. Chromosome 20 uniparental disomy as pathogenetic mechanism(s) underlying r(20) syndrome clinical features was also excluded in our series of patients. 1.19-P SNP-arrays analyses in 100 patients with normal karyotype and pathological phenotype V. Pecile (1) M. Rocca (2) P. D’Adamo (1) L. Esposito (3) P. Gasparini (1) (1) IRCCS Burlo Garofolo (2) University (3) CBM The underlying genetic defect remains difficult to diagnose in the majority of patients suffering from mental retardation. Recent developments in genomic microarray technology now allow for the genomewide detection of submicroscopic chromosomal alterations. SNP arrays currently represents the technique with the highest resolution power, since they allow identification of chromosomal abnormalities as for example variations in the number of copies (CNV) as duplications or deletions and uniparental disomy (UPD). To date, we have collected 100 samples from patients with normal karyotypes and some sampes from their unaffected family members. Enrolled patients presented moderate to severe MR, associated with at least one of the following clinical features: one major malformation and/or dysmorphism and/ or multiple minor anomalies. We have applied Infinium-2 genotyping assay with Human370CNVDuo or Quad BeadChips (Illumina Inc.) to detect CNVs and copy-neutral LOHs. SNP arrays consist of about 370,000 markers with an average spacing of 7 kb. The study-platform was validated using DNAs with 30 known aberrations and reference samples. Acquired data was analyzed with BeadStudio v3.1 (Illumina Inc.). To maintain all relevant information we developed an ad hoc database. We detected approximately 350 CNVs most of which have already been reported in the Database of Genomic Variants or recurrently present in our samples. We shall discus unique CNV and their associated phenotype. Patients with CNVs, who were not identified in the database or recurrent in our cases, were submitted to

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further investigation (FISH or SNP arrays on their parents). The results show that SNP arrays can be used to reliably detect and characterize subtelomeric and interstitial CNVs which are the cause of the pathological phenotype. 1.20-P Deletion 2p in two children with mental retardation identified with SNP arrays M. Rocca (1) P. Gasparini (2) A. Skabar (3) F. Faletra (1) L. Esposito (4) V. Pecile (2) (1) University of Trieste (2) IRCCS Burlo Garofolo (3) IRCCS Burlo Garofolo (4) CBM Deletions of chromosome bands 2p11.2 and 2p12 are rare and there are a few cases published in literature with deletions within these regions. These patients had developmental delay, mental retardation and variable additional problems in common. Here we report two cases with a deletion of chromosome bands 2p11.2–p12. The first patient was the second child of healthy and nonconsanguineous parents. She was a 7-year-old girl who presented speech delay, uncertain deambulation, blue-grey sclerae, facial dysmorphisms such as mild hypotelorism, strabism, broad nasal bridge, extroverse upper lip, downward slanted corners of mouth, big auricle. A brain MRI scan revealed no abnormalities. The second patient was the third child of healthy and nonconsanguineous parents. He was a 9-year-old boy who presented psychomotor delay, dyskinesia, epilepsy, craniofacial dysmorphisms such as greek helmet face, thin lips and arcuate palate. NMR revealed cysts in the atrium of right lateral ventricle and a mild frontal and bilateral temporal cortical atrophy. Chromosome analysis in both patients was normal. SNP array analysis, from whole blood sample, was carried out using the HumanCNV370-Quad platform (Illumina, San Diego, California) according to manufacturer’s protocol in order to detect chromosomal abnormalities not detectable using standard procedures. The analysis revealed a 9,2 Mb de novo deletion of chromosome 2p11.2–p12 in the first patient and a 2,6 Mb maternally inherited deletion of chromosome 2p11.2 in the second patient. We discuss these two cases, with different phenotype, who have a deletion of chromosome band 2p11.2 in common.

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1.21-P Characterization of 6p and 9q subtelomeric deletions: MLPA and FISH contribution M. Mota Freitas (1) J. Ribeiro (1) M. Martins (1) M. Barbosa (1) G. Soares (1) M. Fonseca e Silva (1) H. Correia (1) (1) Centro Genética Médica Jacinto Magalhães. INSA IP Mental retardation is a major social, educational, and health problem affecting 3% of the population. Subtelomeric chromosome aberrations are one of the major causes of mental retardation with or without multiple anomalies. Submicroscopic subtelomeric deletions of chromosome 9q, Chromosome 9q34.3 Deletion Syndrome (OMIM #610253), are mainly associated with severe mental retardation, hypotonia, microcephaly, hypertelorism, synophrys and heart defects. Deletions in 6p are rare events in the population and they may implicate two distinct phenotypes: i) 6p25 terminal deletions associated with hearing loss, anterior and structural eye anomalies, craniofacial defects, delayed bone maturation, heart and neuronal defects; and ii) 6p24–6p22 interstitial deletions that have been described with kidney abnormalities, short neck, structural eye anomalies and heart defects. We report on a 14 years old girl referred for subtelomeric studies, with normal karyotype, presenting with psychomotor retardation, microcephaly, dysmorphic features and cardiac abnormalities. MLPA investigation with two different panels of probes (P036 and P070, MRC-Holland) identified a 6p subtelomeric deletion (IRF4 gene in 6p25–p23) and a 9q subtelomeric deletion (EHMT1 and MRPL41 genes in 9q34.3), in both panels. FISH analysis for these regions showed normal hybridization for 6p subtelomeric region (6ptel48, AbbottVysis); a probe for locus D9S325 (Vysis-Abbott) was not conclusive for subtelomeric 9q, while the probe D9S2168 (Cytocell) showed normal hybridization. Molecular cytogenetics techniques are in progress to provide a better characterization of the delected regions. The subtelomeric regions of both parents were normal. To our knowledge, these two “de novo” subtelomeric deletions - 6p25–p23 and 9q34.3-have never

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occurred, simultaneously, in the same patient. The authors emphasize the importance of a good clinical characterization combined with the available molecular and cytogenetic technologies for a better definition of the breakpoints and, ultimately, the establishment of a correct genotype / phenotype correlation. 1.22-P A complex chromosomal rearrangement with 6 breakpoints involving chromosomes 2, 6 and 8 in an infertile female with normal phenothype R. Lasan Trcic (1) M. Kuspilic (0) L. Letica (0) K. Crkvenac Gornik (0) I. Tonkovic Gjurisevic (0) D. Muzinic (0) S. Huljev (0) D. Begovic (0) (1) University Hospital Centre Zagreb Complex chromosomal rearrangements (CCRs) may be described as are constitutional structural rearrangments involving three or more chromosomes, or having more than two breakpoints. They are rarely found in phenotipicaly normal individuals and are usualy detected in connection with reduced fertility. We report here on a clinically healthy, 35-year-old woman. She was referred for genetic counseling because of a history of three early abortions. GTG-banding results showed a CCR involving three chromosomes 2, 6 and 8. Molecular analysis of breakponts using FISH with region specific probes revealed a more complex character of the CCR. To determine apparently balanced translocation between chromosomes 2, 6 and 8 we performed array CGH, and results will be presented.

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(LD) and/or mental retardation (MR). Thirty per cent of these patients have no diagnosis. OBJECTIVE: We evaluated patients with DD history, taking account of their clinical aspects and laboratory results, who evolved to LD/MR. We identified subtelomeric rearrangements (SR) as etiology with Multiprobe T FISH®. METHODOLOGY: Fourteen patients were selected from the Genetic Outpatients Unity of Gaffrée and Guinle Universitary Hospital—UNIRIO, with DD history who evolved to DA/DM, with or without familial DD/MR history. Patient karyotypes were normal in G banding and in high resolution. Research in data base was done to find known syndromes. They were selected according to the De Vries et al criteria; we also excluded other pathologies like congenital infections and molecular X fragile syndrome in males. Ophthalmologic and auditive evaluations were performed too. Multiprobe T FISH® was used to SR screening. RESULTS: DD/MR familial history was present in 50% of patients, malformations, in 80%. Clinical score ranged from 4 to 7. Deletion of one 13q chromosome was found in one patient, with mosaicism, but specific probe do not confirm it. Soon, screening was negative in all patients. DISCUSSION: Negative result can be associated to the small number of the sample or to the presence of smaller rearrangements which were not sensible to this assay. CONCLUSION: Investigating idiopathic MR etiology is a challenge and a motivation to researchers who work in this area, where methodologies more sensible, like MLPA or CGH array, can be used to complement the investigation. 1.24-P

1.23-P Clinical-Laboratory Study in Patients with Developmental Delay who evolved with Mental Retardation: Multiprobe T FISH Assay S. Santos (1) D. Freire -Maia (2) (1) Federal University of Rio de Janeiro State UNIRIO (2) Medicine School of Federal University of São Paulo State INTRODUCTION: Developmental delay (DD) occurs in 5–10% of children under the age of five and, 1–3% of them develope learning disabilities

Subtelomeric rearrangements in 45 patients with idiopatic mental retardation and dysmorphic facial appearance Z. Yilmaz (1) S. Saygi (2) M. Derbent (3) O. Yüregir (1) I. Erol (2) (1) Baskent University (2) Baskent University (3) Baskent University Some submicroscopic subtelomeric rearrangements are associated with a specific phenotype. These chromosome abnormalities have been reported in 5–7% of children with moderate to severe mental retardation and in 0.5% of children with mild mental retardation. Thus,

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genetic testing for subtelomeric abnormalities among the patients with mental retardation has become an important diagnostic tool. We aimed to evaluate subtelomeric rearrangements in the patients with idiopatic mental retardation and dysmorphic facial appearances. We studied 45 unexplained mental retardation patients with facial dysmorphism. Standard cytogenetic analysis and subtelomeric region analysis with florescent in situ hybridization were performed on peripheral blood lymphocyte cultures of the patients. We did not detect any chromosome abnormalities or subtelomeric rearrangements in our group. Although, dysmorphic features are present in most of the patients with detected subtelomeric rearrangements, these phenotypic findings in mentally retarded patients may not be associated with subtelomeric abnormalities.

Results: In 19 patients fifteen subtelomeric chromosomal aberrations were recognized, which corresponds to a diagnostic yield of about 12,7%. Identified rearrangements were: del(1p36); der(1)t(1;3)(q44;p25) pat; der(1)t(1;22)(p36.1;q11.23)mat; del(4p); der(4)t (4;7)(pter−,pter+); del(5p); der(5)t(5;9)(pter−,pter+) mat; der(6)t(6;7)(q27;q36)mat; der(7)t(6;7)(q27;q36) mat; der(9)t(9;20)(pter−,pter+)pat; del(9qter); der(10)t (4;10)(pter−,pter+)mat; der(12)t(11;12)(pter−,qter−) pat; del(14qter); del(22q13). Moreover, in three cases variant del2q37 was identified. Conclusions:Since there is still limited knowledge of the phenotype of many subtelomeric aberrations, we believe that our study gives insight into the etiology of mental retardation by clinical delineation of subtelomeric aberrations.

1.25-P

1.26-P

Subtelomeric study in 149 Polish patients with mental retardation, dysmorphy and/or congenital anomalies of unexplained etiology—multiprobe FISH and/or MLPA now and what next ?

Isolation, lysis and amplification of single sperm cells for further assays: Preliminary results.

A. Gutkowska (1) A. Jezela-Stanek (1) . Marczak (1) M. Gajdulewicz (1) M. Kugaudo (1) M. Białecka (1) M. Juszczak (1) E. Ciara (1) K. Chrzanowska (1) M. Krajewska-Walasek (1) (1) The Children’s Memorial Health Institute Background:Subtelomeric chromosome aberrations are acknowledged as one of the significant causes of mental retardation (MR) with a prevalence of about 5.2%. Despite a large number of studies carried out to date, the phenotype and outcome of some subtelomeric rearrangements are still not recognized. Hence we report here the diagnostic yield of subtelomeric aberration in a series of 149 patients with an unexplained combination of mental retardation with dysmorphy and/or congenital anomalies, emphasizing the patient’s phenotype. Methods:Patients were evaluated for subtelomeric rearrangements using commercially available total subtelomeric FISH (TS). All had normal results of GTG-banded chromosomes at the 550-band level and were referred for TS based on clinical indications suggestive for chromosomal aberration. In some cases, for identification or confirmation of detected abnormalities, specific microdeletion probes and telomeric kits were used.

G. Daina (1) M. Rius (1) A. Obradors (1) A. GarciaPerió (1) B. Lledó (2) M. Oliver-Bonet (3) J. Benet (1) J. Navarro (1) (1) Universitat Autònoma de Barcelona (2) Instituto Bernabeu (3) Fundación Mateu Orfila de Investigación en Salud Islas Baleares To date there is an extensive knowledge about the whole genome amplification (WGA) of single cells (blastomeres, polar bodies, etc.) but the lack of information about this methodology on sperm cells is noteworthy. The aim of this study was to optimize the procedures to obtain a valid amplification product from a single sperm cell for further applications in molecular biology studies, as Comparative Genomic Hybridization (CGH), array-CGH, or mutation genotyping. The study was carried out in different samples of freshly obtained human spermatozoa of n=3 control individuals (46, XY). For the isolation, a micromanipulator was used to select single sperm cells prior to placing them into sterile PCR tubes. For the single sperm cell lysis and DNA decondensation, alkaline buffer (200 mM NaOH + 50 mM DTT) was used. A freezing process at −80°C and a lysis protocol at 65°C were performed before the whole genome amplification (WGA). A neutralizing solution; 900 mM Tris HCl, pH=8,3 was added next. The WGA of the

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single cell was carried out by MDA (Multiple Displacement Amplification) overnight, the WGA products underwent PCR for STR detection of both D7S2847 and D3S1537 in order to determine the success of amplification. The combination of the isolation protocol (getting optimal results using the micromanipulator), lysis and MDA, gave peaks of both STRs simultaneously with a mean value of 20.000 RFU (relative fluorescence units) in 66,7% of the single sperm cell amplified DNA. In conclusion, we found an optimal combination of spermatozoa isolation, single cell lysis, DNA decondensation and whole genome amplification protocol to obtain high amounts of amplification products from single sperm cells. Acknowledgements: FIS-ISCIII (PI 051395), FISISCIII (PI051834), FIS-ISCIII (PI081185), Grup de Suport a la Recerca. Generalitat de Catalunya (2005SGR00495) and Càtedra de Recerca Eugin funded this study. The first author has a predoctoral grant from the Fundació Crèdit Andorrà. 1.27-P A familial complex deletion/duplication rearrangement with variable phenotype in three members of a family. G. Velagaleti (1) T. Gliem (1) C. Hutchens (1) P. Gonzales (1) (1) Mayo Clinic Cytogenetically detectable imbalances of euchromatic regions are expected to result in an abnormal phenotype; however, there are several chromosomal regions that do not appear to cause abnormal phenotype despite an imbalance. Chromosome 5p is one such region, wherein imbalances of overlapping regions are known to cause variable phenotypic consequences ranging from severe to normal phenotype. In general, deletion of the 5p15.2 region is known to cause the ‘cri-du-chat” syndrome, while deletion of 5p14 is known to result in normal phenotype. Terminal deletions of 5p15 are also known to cause speech delay and microcephaly. Here we report on a family with 3 members observed to have a complex karyotype with deletion and duplication involving the 5p15 region with variable phenotype. A 5-year-old girl was referred for chromosome analysis because of microcephaly, developmental

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delay and neurological symptoms consistent with Rett-like syndrome without regression. Chromosome analysis showed a complex rearrangement with a deletion and a duplication of chromosome 5p. FISH analysis showed that the “cri-du-chat” critical region was not deleted while the 5p subtelomere probe was deleted. The karyotype was interpreted as 46,XX,dup (5)(p15.33p15.31),del(5)(p15.33).ish del(5)(pter−). Chromosome and FISH analyses of the family showed that the mother of the proband and a 14-year-old brother had identical complex rearrangements, while the father and a 9-year-old brother had normal karyotypes. The mother and the brother with the rearrangement do not have any neurological symptoms, although the mother was reported to have microcephaly also. Because of the phenotypic heterogeneity, array CGH studies were carried out on the mother and the brother to evaluate whether there were any other underlying genomic imbalances that might explain such heterogeneity. The array CGH results confirmed both the mother and brother to have similar genomic imbalances with an approximately 2.84 Mb terminal deletion and 4.6 Mb interstitial duplication. The duplicated region appears to be in a gene-poor region while the deleted region appears to harbor several genes. One such gene is the Iroquois homeobox 2 gene (IRX2) which is shown to have a role in central nervous system development processes. Given that microcephaly is one of the consistent features associated with 5p15 imbalances including 2 members of our family, the IRX2 gene may have a role. Further studies are in progress to illustrate the mechanism of formation of this rearrangement and the developmental stage at which such error occurred. 1.28-P Meiotic segregation, aneuploidy, and chromatin integrity in spermatozoa of translocation carriers M. Vozdova (1) E. Oracova (2) R. Rybar (1) R. Gaillyova (3) J. Rubes (1) (1) Veterinary Research Institute (2) Repromeda (3) Faculty Hospital Brno Constitutional Robertsonian and reciprocal translocations are the most frequent structural chromosomal aberrations in humans. They are not usually associated with any serious phenotypical manifestations. However,

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they represent an important factor affecting fertility and pregnancy outcomes due to the formation of unbalanced gametes in their carriers. Chromosomes involved in translocation form trivalents (in Robertsonian translocations) or quadrivalents (in reciprocal translocations) by pairing with their normal homologues during the first meiotic division. Gametes with partial or complete disomy and nulisomy of chromosomes involved in translocations arise as a consequence of meiotic segregation of a trivalent or a quadrivalent. Frequencies of all types of abnormal segregation of translocated chromosomes, as well as aneuploidy of other chromosomes, possibly resulting from an interchromosomal effect of translocations, were analysed in spermatozoa of 10 Robertsonian and 30 reciprocal translocation carriers by the fluorescence in situ hybridisation (FISH). The frequencies of abnormal sperm ranged from 4.2 to 23.5% in Robertsonian and from 40.1 to 70.3% in reciprocal translocation carriers. Some of the carriers showed increased frequencies of aneuploidy for chromosomes X, Y, 8, 18, and 21, and diploidy when compared with the results obtained in the control group with normal karyotypes. Sperm chromatin integrity was detected by Sperm Chromatin Structure Assay (SCSA). The level of the sperm chromatine damage expressed by DNA fragmentation index (DFI) and proportion of immature forms (HDS) was significantly higher in translocation carriers, in comparison with the control group. The sperm FISH analysis of meiotic segregation and aneuploidy helps to personalize the reproductive risk and choose the most effective assisted reproduction strategy for individual translocation carriers. Supported by the Grant Agency of the Czech Ministry of Health, grant No. NS 9842-4/2008

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The “ring syndrome” is characterized by an extreme growth failure together with an otherwise almost-normal appearance viz. No major malformation, no specific deletion syndrome, none or only a few unspecific minor anomalies. This syndrome is considered to occur independently of the chromosome involved. Clinical case We present a case of a ring chromosome 6 in a 18 months old male. He was born from healthy, unrelated parents. The birth weight was 2,590 g; lenght, 47 cm; and head circumference 33 cm. At the age of 2 months, he showed a microcephaly, growth retardation, pigmentary skin anomaly (only 3 spots) and signs of mild hypotrophy. At the age of 14 months, the patient was referred to the departments of dermatology and genetics because the spots had increased not only in size but also in number. Cytogenetic analysis of peripheral blood revealed an unstable ring chromosome 6 with chromosome breakpoints in 6p25 an 6q27. Further genetic characterization is in progress. At this moment, psychomotor development is satisfactory and the boy is attending a regular nursery school. Comments Prenatal and postnatal diagnosis of ring chromosomes are uncommon: in the literature, less than thirty cases of ring chromosome 6 have been reported. In the present case, the normal psychomotor development suggests that deletion of relevant genetic material is unlikely. The anomalies of our patient, especially the pigment skin anomalies are more probably related to ring instability. Microcephaly and growth retardation could be consequence both of ring instability and/or the loss of some telomeric material during ring chromosome formation. 1.30-P

1.29-P Ring Chromosome 6: Description of a new case Núria Palau¹, Elisabet Lloveras1, Sol Florensa2, Eulàlia Baselgas2, Miguel del Campo2,3, Marta Herrero1, Laura Barranco1, Anna Canellas1, Marta Costa1, Alberto Plaja1,3. 1 Departament de Citogenètica. General Lab. S.A. Laboratoris d’Anàlisis. Barcelona. Spain. 2 Departament de Pediatria del Institut Universitari Dexeus. Barcelona. Spain. 3 Unitat de Genètica. Hospital Materno- Infantil Vall d’Hebron. Barcelona. Spain.

Pericentric and paracentric inversions: a report of 35 cases L. Letica (1) R. Lasan (1) I. Tonkovic Durisevic (1) K. Crkvenac Gornik (1) D. Begovic (1) (1) University Hospital Centre Zagreb Pericentric and paracentric inversions are among the most frequent chromosomal rearrangements with a frequency of 1–2%. During the last 15 years we have detected 35 chromosome inversions excluding polymorphic inversions of chromosome 9. We have identified inversions for chromosome 1, 2, 3, 4, 5,

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7, 8, 9, 10, 12, 13, 14, 17, 21 and X chromosome. We found 24 (68,6%) cases of pericentric inverions and 11 (31,4%) cases of paracentric inversions. Twenty five cases were inherited and 2 cases were de novo. The most frequent inversion was inv(10)(p11.2q21) (17,1%). There were two cases with double inversions. Chromosomal analysis was performed using Gbanding according to standard procedure on peripheral lymphocytes. All patients were informed by a genetic counsellor. We have found that the presence of an inversion is rarely linked to an abnormal phenotype or abnormal progeny, nevertheless caution is recommended when counselling. 1.31-P Molecular and clinical characterization of patients with unbalanced cryptic chromosomal aberration L. Morozin Pohovski (1) I. Sansovic (1) I. Barisic (1) (1) Children’s University Hospital Zagreb We have studied a series of 50 unselected patients with idiopathic mental retardation and negative chromosomal analysis for subtelomeric aberrations by a Multiplex Ligation dependent Probe Amplification (MLPA) analysis. Two commercial kits from MRC Holland, SALSA MLPA kit P036C and SALSA MLPA kit P070 probe mixes were performed. Two rearrangements were detected by the P036 kit and confirmed by the P070 probes panel. The first was a 15-month-old girl with dysmorphic facial features, microcephaly and mild developmental delay. MLPA analysis revealed a common terminal deletion of the long arm of chromosome 22. Although the girl showed many clinical features of the 22q13.3 deletion syndrome, including normal growth, speech delay, and muscular hypotonia, she showed only mild delay of developmental milestones and had no behavioral characteristics consistent with autism spectrum disorders. The second patient was 6-year-old girl with dysmorphic features, microcephaly, prenatal and postnatal growth retardation and moderate developmental delay. Subtelomeric rearrangement consisting of a 12p deletion and 22q duplication was found. The chromosomal breakpoints and deletion size of this unique cryptic reciprocal translocation of paternal origin were

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subsequently characterized and evaluated by additional MLPA specific telomere probe mix and by quantitative fluorescent-polymerase chain reaction (QF-PCR). Our study confirms the clinical usefulness of the MLPA screening for subtelomeric chromosome aberrations in patients with idiopathic mental retardation. 1.32-P Ectrodactyly-Ectodermal Dysplasia-Clefting (EEC) Syndrome: Variable clinical presentations in a family E. Tug (1) S. Duzenli (1) H. Aydin (1) (1) Abant Izzet Baysal University Izzet Baysal Medical School Ectrodactyly-ectodermal dysplasia-clefting (EEC) syndrome is an autosomal dominant disorder involving abnormalities of the hands, feet, skin and teeth and clefts of the lip and palate. This rare syndrome is actually a multiple congenital anomaly syndrome leading to intra- and interfamilial differences in severity because of its variable penetrance and expressivity. Therefore, EEC syndrome is included within the broad spectrum of ectodermal dysplasias. Here we report two cases, a boy and his father with the EEC syndrome. The present patients illustrate the great phenotypic variability in the EEC syndrome. In our first case, aplasia cutis congenita (ACC) and dental caries were the major findings. Besides the urogenital findings such as cryptorchidism, hypospadias and nephrolitiasis, there also were findings like inguinal hernia and pectus carinatum. Furthermore, the patient had dismorphic findings such as complete transverse palmar crease, mildly upslanting palpebral fissures, prominent forehead, absent helical gyrus of the ears. The second case, which was the father of the first case, had no minor dysmorphic findings, but therefore had ACC at the same location as his son, and bilateral split hand (ectrodactyly), additionally. Metaphase chromosomes of both cases were analyzed by standard Gbanding technique. Their karyotypes were 46,XY. For, one single feature, including any of the three cardinal signs, is not enough for diagnosis, a meticulous examination of all family members is needed. This two cases present typical clinical symptoms of the EEC syndrome. After diagnosed two manifests in the father and one in the son, we can postulate that this complex is a multiple

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congenital anomaly syndrome with variable expressions. Thus, one can follow that the variability in penetrance and clinical expression of this autosomal dominant trait is changing through the two generations. 1.33-P Cytogenetic characterization, array-CGH delineation and phenotype genotype correlation of a de novo 9p partial duplication M. Maurin (1) A. Aboura (1) M. Gerard-Blanluet (2) E. Pipiras (3) A. Delahaye (3) L. Di Benedetto (1) B. Benzacken (3) A. Tabet (1) (1) AP-HP (2) AP-HP (3) AP-HP Few pure partial trisomies of short arm of chromosome 9 (9p) have been reported to date; most published 9p partial trisomies were inherited from a balanced carrier and thus involved imbalance of another chromosome. We report here on a 2 year-old girl carrying a de novo pure partial 9p duplication. Conventional and molecular cytogenetic studies, including BAC array-CGH, were performed, allowing us to make precise genotype-phenotype correlation in pure partial trisomy 9p. This child was the 3rd child of healthy unrelated parents. She was born after an uneventful 38.5 weeks pregnancy; birth weight was 3,660 kg, height 50 cm and head circumference 36 cm. Neonatal period was marked by important weight loss of this poorly awake baby, requiring hospitalization. Gastro oesophageal reflux was diagnosed. Development milestones were delayed with sitting alone at 12 months, and now at 24 months, walking on all fours. She performs no speech, only sounds, but understands verbal instructions. Socialization is good; she has no sleeping disorder but has started head banging. Cranio facial features are: high forehead, frontal bossing, deep set eyes, low inserted ears, slightly down slanted palpebral fissures, broad based nose with bulbous tip, well defined philtrum, down turned corners of the mouth. She also has transverse palmar crease and clinodactyly of the fifth finger. No visceral malformation was diagnosed. Conventional karyotype revealed a 46,XX,add(9) (p24). Parent’s karyotypes were normal. Molecular cytogenetic investigations using whole chromosome paint and loci specific FISH probes showed direct 9p

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terminal tandem duplication. Break points and size of the duplication were determined using a 4400 clones BAC array (Perkin Elmer): array-CGH showed 9p13pter duplication and confirmed that no other chromosomal pair is involved in this imbalance. Phenotype-genotype correlation and mechanism of this duplication will be discussed. 1.34-P Molecular karyotyping versus conventional karyotyping—or a combination of both? K. Hansson (1) A. Gijsbers (1) H. Dauwerse (1) E. Bijlsma (1) M. Breuning (1) Y. Hilhorst (1) E. Aanhane (1) M. Hoogenboom (1) E. den Ouden, Spruijt (1) C. Ruivenkamp (1) (1) LUMC With the advent of molecular karyotyping small copy number gains and losses not visible with conventional chromosome analysis can be detected. In the clinical diagnostic setting the use of high resolution genome wide array techniques as the first method of choice has therefore been shown to increase the diagnostic yield in patients with mental retardation with or without multiple congenital abnormalities. However, in some cases a combination of high resolution genome wide array analysis, conventional karyotyping and FISH analysis is warranted as deletions and duplications can be part of a more complex structural rearrangement not detectable with array analysis alone. To illustrate the necessity of using different methods we present three patients with interstitial deletions detected by 250K SNP-array analysis. In all cases, the deletions were demonstrated to be part of a more complex structural rearrangement visible only with conventional karyotyping. 1.35-P The 1q-syndrome: A case report and literature review A. Zagorac (1) N. Marcun Varda (1) N. Kokalj Vokac (1) (1) University Medical Centre Maribor (2) University Medical Centre Maribor (3) University Medical Centre Maribor

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Deletions of the long arm of chromosome 1 have been reported in numerous patients, with breakpoints occurring along the entire length of the q arm, causing clinical differences. Specific phenotypic findings, resembling a distinct syndrome, are present in cases with terminal deletions at 1q42 and 1q43. Monosomy 1q42-qter seems to be associated with characteristic manifestations. On these basis, molecular techniques are important in order to correctly define the deleted segment, and establish accurate genotype—phenotype correlations. We provide a clinical description of a girl with de novo terminal 1q43-qter deletion. The proband has been recognized as an infant with intrauterine growth retardation and generalized muscular hypotonia, microcephaly, epichanthal folds, telechantus, enophtalmus, short eye openings with lateral blepharophimosis, small, broad and flat nose, low-set ears, high-arched palate and short neck. Valgus deformity of feet was present with short right Achilles tendon. Magnetic resonance of the brain revealed agenesis of the corpus callosum, ventriculomegaly and enophtalmus. She has seizures. Cytogenetic analysis of peripheral blood showed a de novo terminal chromosome 1 long arm deletion, confirmed with Subtelomeric FISH (Multiprobe-TSystem, CytoCell). To define the extent of the deletion and the breakpoint location we performed 244k array-CGH (Agilent Technologies, USA) demonstrated a loss of 9.4 Mb genomic material and the breakpoint 1q43 (at 237.6 Mb). The patient’s features are compared with those of other patients with similar deletions, and variable phenotypic findings due to different deleted chromosomal segments are discussed. 1.36-P Polish Collection of Chromosomal Translocations A. Midro (1) B. Stasiewicz-Jarocka (0) B. Panasiuk (0) Polish group for Chromosome Translocation Evaluation* (0) (1) Medical University Despite the availability of prenatal cytogenetic services parents with balanced chromosome translocations should be informed about likelihood of chromosome imbalance leading to miscarriage, stillbirth or viable handicapped baby. This probability varies from

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translocation to translocation and empirical data are necessary to obtain risk figures. The degree of accuracy of the estimation of risk figures depends on the number of informative pregnancies conceived in carriers of reciprocal translocations (RCT) and on the precision of identification of breakpoints in the chromosomes involved. The aim of organizing a Polish Collection of Chromosome Translocations was to obtain empirical data of individual RCT carriers to improve genetic counseling and to collect clinical observations connected with chromosomal changes as available resource for locating the disease gene. Segregation analysis has been performed in 639 pedigrees according to Stengel-Rutkowski and Stene. The probability rates for unbalanced offspring for the group of 64 pedigrees (10%) was high, for the group of 45 pedigrees(7%) was medium, for the group of 370 pedigrees (58%) was low and for the group of 160 pedigrees (25%) no increased values were found. * Bocian E., Haus O.,Jakubowski L., Kałużewski B., Kostyk E., Krajewska-Walasek M., Lassota M., Latos-Bieleńska A., Limon J., Marcinkowska A., Pietrzyk J., Rączkiewicz B., Sąsiadek, SorbajSucharska G.,Zajączek S., Zaremba J. 1.37-P Cytogenetic and molecular analysis in five patients with dicentric Y chromosome and two males with Yp isochromosome V. Chernykh (1) L. Kurilo (1) H. Magomedova (1) N. Kuzina (1) O. Barkova (1) N. Shilova (1) T. Zolotukhina (1) O. Ryzhkova (1) A. Polyakov (1) (1) Research Centre for Medical Genetics of Russian Academy of Medical Sciences We have investigated five patients with a dicentric Y chromosome and two with an isochromosome of Yp. Chromosome analysis was performed on cultured peripheral blood lymphocytes in accordance with standard procedures. FISH analysis was performed on metaphase chromosomes and interphase nuclei using standard protocols with DXZ1, DYZ1, DYZ3 and LSI SRY probes. DNA was extracted from peripheral leukocytes by a standard method. Molecular analysis was performed using multiplex PCR amplifications of SRY, AMELX/AMELY, ZFY/ZFX, and up to 15 Yq specific STSs loci.

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Two of the three males with isodicentric Y were patients with male pseudohermaphrodism. In one patients a karyotype was ascertained initially as mos 45,X[13]/46,X,idicY(q11)[36]/47,XY,idicY(q11)[1]. In situ hybridization revealed mosaicism with the following clones: 45,X(37%), 46,XY (12%), 46,X, idicY(q11)(45%), 47,XY,idicY(q11)(6%). PCRs were positive for all analyzed loci, possibly because of a presence of 46,XY cells. In the other male with mos 45,X[23]/46,XY[17] karyotype FISH analysis demonstrated a presence of a pseudodicentric Yq; molecular analysis detected a deletion with a breakpoint between sY1197 and sY1192 loci. The third patient with idic Y was an infertile male with hypogonadism, short stature and mos 45,X[7]/46,X,idicY(q11)[23] karyotype. Molecular analysis showed Yq11.2 deletion with a breakpoint within the AZFb region between sY1302 and sY142 loci. FISH and molecular analyses demonstrated that the abnormal chromosomes were Yp isochromosomes in two males with markers (46X,mar; mos 46,XX/47,XX,+mar). Two SRY-positive phenotypic females with Turner syndrome had the following karyotypes: mos 45,X[25]/ 46,X,idic(Y)(q11.2)[75] and mos 45,X[85]/46,X,idic (Y)(p11.3)[15]. Deletion including the AZFb and AZFc regions was found in the first female patient. Two of the three patients with the Yp dicentrics showed proximal breakpoints that are typical for complete AZFb+c and AZFc deletions, respectively. Obtained data demonstrated that Y dicentrics commonly resulted from errors of the meiosis II during father’s spematotogenesis. However postzygotic origin of this structural Y chromosome rearrangement is possible in cases of dynamic mosaicism.

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male genitalia with hypoplastic testes descended in the scrotum and no sign of undervirilization. His weight was 74 kg, height—160 cm. Intelligence was normal. Hormonal examination demonstrated a hypergonadotropic hypogonadism. Semen analysis showed severe oligoasthenoteratozoospermia. Chromosome analysis was performed on cultured lymphocytes in accordance with standard procedures. FISH analysis was performed on metaphase chromosomes and interphase nuclei using standard protocols with DXZ1 and LSI SRY probes. DNA was extracted from peripheral leukocytes by a standard method. Molecular analysis was performed using multiplex PCR with SRY, AMELX/AMELY, ZFY/ZFX, and seven Yq STSs. X-chromosome inactivation (XCI) was determined in whole peripheral blood using by analysis of CAG repeat of the AR gene in the patient and his mother. Conventional cytogenetic examination showed 46, XX karyotype. FISH analysis has demonstrated a cryptic translocation of Yp11.3 material to the Xp22 and also revealed cryptic complex mosaicism with the presence of three SRY-positive cell lines: 45,X, SRY×1 (3.8%), 46,XX, SRY×1 (94.8%) and 47, XXX, SRY×2 (1.4%). PCRs were positive for SRY, AMELX, ZFX, ZFY and negative for all analyzed Yq loci. XCI analysis showed preferential inactivation of maternal X chromosome. Evidently the X-Y translocation in this patient is a result of abnormal X-Y interchange during paternal meiosis. The X chromosomal mosaicism is possibly due to the X-X non-disjunction during mitosis. However, non-disjunction between sister chromatids of the X derivate during paternal meiosis II is not excluded. 1.39-P

1.38-P Skewed X-inactivation in XX sex reversed patient with X chromosomal mosaicism E. Bliznetz (1) V. Chernykh (1) O. Ryzhkova (1) L. Kurilo (1) N. Shilova (1) T. Zolotukhina (1) A. Polyakov (1) (1) Research Centre for Medical Genetics of Russian Academy of Medical Sciences We report a 37-year-old 46,XX male with a complex X chromosomal mosaicism. The patient had fully mature

First case of a constitutional intrachromosomal triplication 1q43→q44: molecular- and cytogenetic investigations and characterization of the phenotype A. Roos (1) H. Tönnies (0) T. Goecke (0) T. Haaf (0) M. Baudis (0) S. Spengler (0) T. Eggermann (0) H. Schüler (0) (1) Institute of Human Genetics (2) Institute of Human Genetics (3) Institute of Human Genetics and Anthropology (4) Institute of Human Genetics (5) Institute of Molecularbiology

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Complex chromosome rearrangements and gene amplifications involving chromosome 1 sometimes creating weird chromosomes are well known phenomena of various malignant diseases. Whereas, the finding of a constitutional intrachromosomal triplication concerning an euchromatic segment of chromosome 1 has not been described before. We present the first case of a de novo intrachromosomal triplication of 1q43→q44 in a female patient with macrocephaly, facial dysmorphisms, tendency to infections, impaired hearing, and epilepsy. But she exhibited no inner malformations. This intrachromosomal aberration was detected by conventional cytogenetic analysis and further characterized by molecular-cytogenetic and moleculargenetic investigations, including FISH, CGH and SNP-array (500K) testing. Our investigations pointed out an inverted orientation of the middle segment, which is in line with the orientation of middle segments in triplications of other chromosomal regions described so far. A comparison of our clinical findings with the clinical picture of “pure” distal 1q duplications presents an almost identical phenotype. Surprisingly, the overall phenotype of our patient does not only resemble but also was milder than most of the cases with trisomy. Uptil now, intrachromosomal triplications have been described for nine different chromosomes or fourteen chromosomal regions. With this report, the number of chromosomes involved in this extremely rare type of aberration has now been raised to ten.

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cence in situ hybridization (FISH) using whole chromosome paint probe 18, the partial chromosome paint 20 (PCP RH17J3), RP11-15C15C (18q21.32–33) and YAC942B1 (20q12) clones were used to precisely define the translocation breakpoints. Based on GTG-banding and FISH analyses, the patient’s karyotype was interpreted as: 46,XX,t(18;20)(q21.3;q12)[20].isht(18;20) (YAC-942B1+;RP11-15C15+)[20]de novo. Aarraycomparative genomic hybridisation (a-CGH) examination using 44k and 244A (Agilent) oligonucleotide arrays did not detect a deletion or duplication, neither at the breakpoints or elsewhere in the genome. The abnormal phenotype observed in the patient could be explained by gene disruption, a position effect or could have occurred by coincidence. 1.41-P Neurofibromatosis 1 with r(17) in karyotype Oltová A.(1), Hořínová V.(3), Kuglík P.(2), Kadlecová J.(1), Vranová V.(1), Slámová I.(1), Šilhánová E. (4) (1) Department of Medical Genetics, University Hospital Brno, Czech Republic (2) Department of Genetics and Molecular Biology, Faculty of Science, Masaryk University, Brno, Czech Republic (3) Radioterapie Medical Center, Nový Jičín, Czech Republic (4) Department of Medical Genetics, University Hospital Ostrava, Czech Republic

1.40-P De novo balanced translocation t(18;20)(q21.3;q12) in a mentally retarded girl J. Vraneković (1) B. Brajenović - Milić (1) I. Babić - Božović (1) B. Peterlin (2) M. Kapović (1) M. Riegel (3) (1) school of medicine (2) Division of Medical Genetics,University Clinical Centre (3) Institute of Medical Genetics, University of Zurich We report a twenty-year-old girl with mental retardation, obesity, hirsutism and de novo balanced t(18;20)(q21.3;q12) translocation. Fluores-

The Neurofibromatosis 1 (NF1) is a severe autosomal dominant disorder with an incidence of about 1 in 3500 live births. We describe the history of a thirteenyear-old girl from healthy unrelated parents. She had a with short stature, degenerative stigmatization, café au lait spots distributed over the entire body except the face, and with distinct pubertal and mild psychomotoric retardation. Because NF 1 is caused by mutations in the NF1 gene, we tried to identify mutations in exons 6, 12b, 16, 28, 19, 30, 31 and 37. For more detailed results RNA based methodology was applied (analysis of cDNA NF1 gene) but no mutations were found. Cytogenetic examination revealed a ring chromosome 17, r(17), which is rare among chromosomal

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aberrations published in NF1. Using the fluorescence in situ hybridization method (FISH) we detected a subtelomeric deletion 17p. Currently we are analysing DNA of the NF1 gene using multiplex ligation-dependent probe amplification (MLPA) for the detection of deletions/duplications in the DNA of tested patient. The size of the deleted segment was assessed by array CGH. 1.42-P Errors at mitotic segregation early in oogenesis and at first meiotic division in IVF-oocytes from donor females: CGH analyses in MII oocytes and their 1PB. J. Navarro (1) A. Obradors (1) M. Rius (1) J. Cuzzi (2) A. Pujol (3) F. Marina (4) C. Màrquez (5) C. Gutiérrez-Mateo (1) J. Benet (1) (1) Universitat Autonoma de Barcelona (2) Universidade de São Paulo (3) Clinica Eugin (4) Instituto de Reproducción CEFER (5) Hospital Materno Infantil Valle d’Hebron Introduction The aim of this work is to analyze the frequencies and the mechanisms involved in the production of aneuploidy events in donor oocytes obtained just after follicular punction (“Fresh”) or matured in vitro (IVM). Methods A total of 61 oocytes, 28 “Fresh” and 33 IVM, obtained from 36 donors (mean donor age: 25.4 y.o.), were analyzed with Comparative Genomic Hybridization applied to the oocyte (MII) and its corresponding First Polar Body (1PB). Results A total of 18 doublet cells out of 61 (29.5%) were aneuploid. Two different mechanisms were involved in these aneuploidies (i.e., mitotic errors during oogenesis or first meiotic division errors). Considering that one of the oocytes contained both errors, 47.36% (9/ 19) of aneuploid events originated during the mitotic divisions errors, whereas 52.62% (10/19) were produced in first mitotic division early in oogenesis. Globally, 8 of the 36 donors (22.2%) produced aneuploid oocytes due to errors during the oogonial mitotic divisions, and the same rate (22.2%) originated at first meiotic divisions. Conclusion This unexpected high rate of donors containing mitotic errors has never been described

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before, although it had already been described in IVF patients. A significant prevalence in euploid fetus ovaries of a pool of aneuploid oogonia and the trigger effect of the hormonal stimulation of these aneuploid oogonia may explain this elevated incidence. Considering the importance of both meiotic and mitotic errors even in oocytes of young women, CGH-PGS, followed by confirmatory prenatal diagnosis, is highly recommended to avoid transferring potentially aneuploid embryos thus increasing their implantation rate. Acknowledgements This research study has been funded by the Ministerio de Sanidad y Consumo Fondo de Investigación Sanitaria Instituto de Salud Carlos III (FIS-ISCIII; PI 051395), the Grup de Suport a la Recerca de la Generalitat de Catalunya 2005SGR00495 and by the Càtedra de Recerca EUGIN. The first author has a predoctoral grant from the Generalitat de Catalunya (2005FI00108). 1.43-P Perinatal findings of proximal trisomy 13q and distal trisomy 20q due to a 3:1 segregation of maternal reciprocal translocation t(13;20)(q14;q13) N. Bouayed Abdelmoula (1) R. Louati (1) T. Rebai (1) (1) Medical University of Sfax Trisomy 13 is characterized by brain malformations (holoprosencephaly), facial dysmorphism (cleft lip/palate), postaxial polydactyly, visceral malformations and severe psychomotor retardation. Free trisomy 13 is found in around 75% of cases. In 20% of cases, trisomy 13 is associated with a Robertsonian translocation and in rare cases; the syndrome is caused by a reciprocal translocation. In such cases, clinical findings vary between partial proximal trisomy 13 (13pter—— 13q12 and 13q14) and partial distal trisomies 13 (13q22——13qter and 13q31 and 13q32––13qter). The influence of the second aneusomy must be also considered. We describe a male fetus who was found to have partial trisomy 13(pter-q13) and partial trisomy 20 (q13-qter); 47,XY,+der(13)t(13;20)(q14;q13). The chromosomal aberration was due to a 3:1 segregation with tertiary trisomy transmitted from a maternal reciprocal translocation. The fetus was a survivor of a twin pregnancy complicated by an early loss of a conceptus at 15 weeks’

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gestation (WG). Prenatal ultrasound at 20 WG showed bilateral cleft lip/palate, elevated cerebral ventricle size, high umbilical artery resistance indices and reduced abdominal circumference. The pregnancy was continued. At delivery (36 WG), a perinatal cytogenetic analysis showed an extra supernumerary marker chromosome whom chromosomal origin was ascertained after cytogenetic analysis of parents. The mother was in fact found to have a reciprocal translocation t(13;20)(q14;q13). Our observation further extends the clinical spectrum associated with proximal trisomy 13q and distal trisomy 20q. Reporting of such rare cases is important in order to enable this information to be used for genetic counselling in similar situations. 1.44-P Small rearrangements of chromosome Nr 7 unrecognised prenatally D. Novotna (1) M. Palanova (2) Z. Vlckova (3) M. Malikova (6) A. Puchmajerova (7) M. Koudova (8) J. Drabova (4) Z. Zmitkova (5) P. Hedvicakova (9) D. Chudoba (10) (1) University Hospital Motol Two cases of newborns with abnormal phenotypes are presented. There were abnormal facial features, severe hypotonia, perinatal problems, and failure to thrive in both children. Additionally small intestinal defects in the boy were supposed and abnormalities of limbs in the girl were observed. The boy’s karyotype was 46,XY,der(7)t(7;12) (p22.3;p13.31)mat, the girl’s karyotype was 46,XX, der(7)(pter→q36::p22→pter)de novo. The results were confirmed using subtelomeric FISH and MLPA and the extent of aberrations, including assessment of deleted—duplicated genes, was studied subsequently by arrayCGH. Both pregnancies were referred for amniocenteses, for ultrasound presence of two vessels in navelwort in the first case and because of positive screening in the second one. Neither karyotype was found to be abnormal, probably due to short chromosomes. The rearrangements are distinguishable in karyotypes with resolution 500 bands in the translocation and even more clearly in the inverted duplication—deletion case.

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Supported by grant of Health Ministry of Czech republic NR 00064203 and IGA grant NR/9457-3. 1.45-P Triplication within the 3q29 Del/Dup Syndrome Region in Two Cases C. Rudduck (1) A. Darmanian (1) L. St Heaps (1) M. Wilson (2) G. Peters (1) (1) Children’s Hospital, Westmead (2) Children’s Hospital, Westmead Both interstitial deletions and reciprocal microduplications of 3q29 have recently been described. The abnormalities are proposed to be mediated by nonallelic homologous recombination between low copy repeats in the region. There are basically two types of 3q29 deletion (or the reciprocal duplication) reported. These are (1): those with a 1.6 Mb common region only and (2) cases including the common region but of greater and variable size. The phenotypes are also varied with a few common features including mild to moderate mental retardation, developmental delay and microcephaly. Both de novo and inherited cases have been reported. We have utilised molecular cytogenetic analysis including CGH-array and FISH to further characterise two cases with an abnormal chromosome 3 at band 3q29. The cases were referred initially for cytogenetic analysis due to developmental delay and behavioural difficulties. Both these cases were found to have a triplication which included the common deletion/duplication region of 3q29. These amplified segments were of sizes 4.18 and 7.5 Mb respectively. These two cases, their molecular cytogenetic results, and clinical features will be described. 1.46-P De novo triple segmental aneuploidy of 1p, 1q and 4q in a girl with hypertrophic cardiomyopathy, muscle hypotonia and multiple congenital anomalies M. Chen (1) (1) Changhua Christian Hospital An 11-months-old girl with multiple congenital anomalies caused by a de novo triple segmental

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aneuploidy of 1p13-1p21 trisomy/1q32-1qter trisomy/ 4q35-1qter monosomy is presented. The patient showed severe hypertrophic cardiomyopathy, muscle hypotonia, facial dysmorphism, mental retardation and growth delay. Array-CGH confirmed no gene in the breakpoints of the rearranged regions, suggesting genomic imbalance is the major factor resulting in the phenotypic anomalies. Besides, by reviewing the literature, we found that most of the phenotypic defects correspond to a distal 1q trisomy syndrome, and suggested that the hypertrophic cardiomyopathy and muscle hypotonia may be associated with a cardiac gene TNNT2 in 1p32 and a FSH dystrophy critical region in 4q35, respectively. In short, patient with hypertrophic cardiomyopathy, muscle hypotonia and multiple segmental aneuploid has not been previously reported. 1.47-P A Down syndrome patient with tandem duplication of chromosome 21– reproductive counselling problems E. Braha (1) C. Rusu (1) V. Gorduza (1) M. Gramescu (1) M. Covic (1) (1) University of Medicine and Pharmacy Iasi Down syndrome is the most common chromosomal disorder due to trisomy for all or a large part of chromosome 21. A 5 years 8 months old girl, the third-born of a clinically normal and nonconsanguineous couple, was referred for chromosomal analysis with a clinical diagnosis of Down syndrome. The patient was born after an uncomplicated pregnancy. Chromosomal analysis and FISH test (Vysis probe) of the proband revealed a 21q tandem duplication. The karyotype of her parents was normal. De novo tandem duplication involving 21q chromosome is rare in Down syndrome in comparison to Robertsonian translocations. The anomaly in the proband could be the result of an intrachromosomal or interchromosomal exchange between long arms of chromosomes 21 in one of the parents. Other possibilities could be a para or a pericentric inversion or a translocation between two chromosomes 21 in a trisomy 21 zygote, at a very early stage of postzygotic development.

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We do not observe clear diferences between the clinical findings in 21q tandem duplication and free full trisomy 21. Because the anomaly in our patient has arisen de novo, we estimate that the risk of the parents of the child having another affected child is insignificant. However, there is an indication for prenatal diagnosis in the next pregnancy. 1.48-P Nine patients with a microdeletion 15q11.2 between breakpoints 1 and 2 of the Prader-Willi / Angelman critical region; clinically relevant or not? B. Sikkema-Raddatz (1) M. Doornbos (5) C. Ruivenkamp (3) T. Dijkhuizen (1) E. Bijlsma (3) A. Gijsbers (3) R. Hordijk (1) K. Kok (1) M. Breuning (3) C. van Ravenswaaij-Arts (1) (1) University Medical Centre Groningen (2) University Medical Centre Groningen/ Albert Schweitzer Hospital Dordrecht (3) Leiden University Medical Centre Behavioural differences have been described in patients with type I deletions (between breakpoints 1 and 3, (BP1-BP2)) or type II deletions (between breakpoints 2 and 3) of the 15q11.2 Prader-Willi/ Angelman region. The larger type I deletions appear to coincide with more severe behavioural problems (autism, ADHD, obsessive-compulsive disorder). The non-imprinted chromosomal segment between breakpoints 1 and 2 involves four highly conserved genes, TUBGCP5, NIPA1, NIPA2, and CYFIP1; the latter three are widely expressed in the central nervous system, while TUBGCP5 is expressed in the subthalamic nuclei. These genes might explain the more severe behavioural problems seen in type I deletions. We describe nine cases with a microdeletion at 15q11.2 between BP1-BP2, thus having an haploinsufficiency for TUBGCP5, NIPA1, NIPA2, and CYFIP1 without Prader-Willi/Angelman syndrome. The clinical significance of a pure BP1-BP2 microdeletion has been debated, however, our patients shared several clinical features, including delayed motor and speech development, dysmorphisms and behavioural problems (ADHD, autism, obsessivecompulsive behaviour). Although the deletion often

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appeared to be inherited from a normal or mildly affected parent, it was de novo in two cases and we did not find it in 350 healthy unrelated controls. Our results suggest a pathogenic nature for the BP1-BP2 microdeletion and, although there obviously is an incomplete penetrance, they support the existence of a novel microdeletion syndrome in 15q11.2.

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1.50-P Unusual Cases of Trisomy 9p and Tetrasomy 9p Detected Postnatally K. Adamová (1) P. Čapková (1) M. Holzerová (2) M. Jarošová (2) V. Curtisová (1) A. Šantavá (1) (1) University Hospital anf Palacky University (2) University Hospital anf Palacky University

1.49-P Structural sex chromosome abnormalities found in routine diagnostics A. Slunga-Tallberg (1) S. Kaukoranta (2) P. Lautala (3) J. Moilanen (2) M. Mäntymaa (4) M. Somer (6) M. Tuomi-Nikula (5) K. Uotila (7) N. HorelliKuitunen (1) (1) Medix Laboratories Ltd (2) Väestöliitto (3) Joint Authority for Päijät-Häme Social and health Care (4) Kymenlaakso Central Hospital (5) Felicitas Clinic (6) Väestöliitto (7) Kanta-Häme Central Hospital Structural abnormalities in either the X- or the Ychromosome in five healthy adults and in two children were detected using routine cytogenetic methods. The indications for the adults were infertility or primary amenorrea and for the children a mild vast developmental delay in a ten-year-old boy and a Turner fenotype in a 15-year-old girl. In three of the adult cases a translocation between the Y-chromosome and X chromosome or an autosome was detected, including the presence of the SRY-region on the autosome. In all male cases the AZFa, b and c regions had been deleted. A dicentric X-chromosome resulting in trisomy of both the short arm and a part of the long arm as well as a partial monosomy of the long arm was detected in a woman with primary amenorrea. In the ten-year-old boy a structurally abnormal Xchromosome with a cytogenetically balanced pericentric inversion was found. The 15-year old girl had an unbalanced translocation between chromosomes X and 4, which was inherited from her mother. All studies were done with standard cytogenetic methods, Gbanding and FISH and in 3 cases of a Y-chromosome microdeletion DNA-study was also performed. In conclusion, the standard chromosome study using Gband analysis combined with FISH is still a powerful tool to reveal potential genomic abnormalities.

Isochromosomes of autosomes are relatively rare chromosomal aberrations. Isochromosome formation results when one arm of a chromosome is lost and the remaining arm is duplicated. An isochromosome has morphologically identical genetic information in both arms. The clinical impact on the patient depends on the type of the chromosome and the length of the duplicated segment. Supernumerary isochromosomes may result in autosomal trisomy or much rarely in tetrasomy which has been described only in a limited number of chromosomes. We describe two cases of aneuploidy 9p both of which involved isochromosome 9p. The first case is a trisomy 9p (Rethore syndrom) in a form of isochromosome 9p detected in a child together with translocation of 9q to chromosome 11, confirmed by FISH and CGH. The second case describes a newborn with severe malformations. Postnatal examination revealed supernumerary chromosome 9, determined by FISH, as an isodicentric chromosome 9p. 1.51-P Molecular cytogenetic and clinical characterization of a maternally inherited 15q26.3-qter deletion m. avila (1) r. silveira-santos (1) s. serafim (1) a. sousa (1) b. marques (2) j. dupont (1) a. medeira (1) i. cordeiro (1) (1) Hospital de Santa Maria (2) Instituto Dr. Ricardo Jorge Subtelomeric deletions may account for about 2.5% of all mental retardation cases, with or without dysmorphic features. Some subtelomeric deletions are associated with a specific phenotype, allowing the identification of specific syndromes. Distal deletions of the terminal long arm of chromosome 15 are rare or seldom diagnosed. Only 12 cases of pure distal 15q26 deletions have been mentioned in the literature. Intrauterine growth retarda-

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tion (IUGR), post-natal growth failure, developmental delay, mild facial dysmorphism and minor abnormalities of the extremities, like clinodactyly and brachydactyly, were present in 8 documented cases. We report on a familial case of a 15qter (15q26.3) deletion, characterized clinically and at the molecular cytogenetic level, and compare it to similar published cases. The proband was a 10 year-old boy with a moderate developmental delay, short stature, minor facial anomalies and short hands with clinodactyly of the fingers. High-resolution karyotype was normal but subsequent FISH studies with subtelomeric probes revealed a maternally inherited 15qter (15q26.3) deletion. The child’s mother had a similar phenotype and moderate developmental delay. Both have abnormal fat distribution, finding not described before. No cardiac anomalies or other problems were identified. To the best of our knowledge, this is the most distal 15q deletion breakpoint reported to date and it could explain a relatively mild phenotype in these subjects. Further mapping of the deletion is still in progress.

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deletion in the short arm of chromosome 4 and a duplication in the long arm of chromosome 20. These results then were confirmed by multiplex FISH telomere. The karyotype was redefined as 46,XY, del(4) (p16).ish 4pter(GS10K2/T7x1)20qter(20QTEL14x3). Parents had a normal karyotype and results from FISH analysis using selective subtelomeric probes were also normal, suggesting that both deletion and duplication in the patient arose de novo. The additional clinical features, not typical of the WolfHisrschhorn Syndrome, could be related to dup(20q). Recently, new techniques have been used to study populations with developmental delay, with or without dysmorphology, and presenting a normal chromosome analysis by classic cytogenetics. We think that it is very important to carry out a retrospective study in patients with abnormal karyotype not clearly correlated to the clinical features. New techniques will allow to make a more accurate diagnosis. 1.53-P

1.52-P Patient with Wolf-Hirschhorn syndrome associated to a duplication 20q

Direct segregation analyses of large pedigrees together with sperm karyotype analyses. Presentation of two families of t(7;9)(q36.2;p21.2) and t(7;13)(q34; q13) carriers

Á. Pérez-Granero (1) N. Govea (0) M. Bernués (0) R. Alfaro (0) B. Sierra (0) E. Pestaña (0) J. Rosell (0) (1) Hospital Universitario Son Dureta (2) Hospital Universitario Son Dureta (3) Hospital Universitario Son Dureta (4) Hospital Universitario Son Dureta (5) Hospital Universitario Son Dureta (6) Hospital Universitario Son Dureta (7) Hospital Universitario Son Dureta

M. Kurpisz (1) B. Panasiuk (2) M. Debiec-Rychter (3) E. Wiland (1) E. Kostyk (4) A. Jakubiuk-Tomaszuk (2) B. Krzykwa (4) R. Lesniewicz (2) A. Midro (2) (1) Polish Academy of Science (2) Medical University (3) University of Leuven (4) Collegium Medicum Jagiellonian University

We report one patient with diagnosis of WolfHirschhorn Syndrome. He showed an apparent deletion in the short arm of chromosome 4. The karyotype was 46,XY,del(4)(p16) by conventional highresolution cytogenetic technology. Fluorescence in situ hybridization (FISH) analysis with the specific probe for Wolf-Hirschhorn Syndrome confirmed the deletion. In addition, the patient presented some features not related to this syndrome. We reviewed the case because of the pregnancy of the mother. We performed Multiplex Ligation-dependent Probe Amplification (MLPA) using SALSA P036E and SALSA P070 human telomere Kits. The patient showed evidence of a cryptic aberration. He had a

The segregation analysis of two relatively large pedigrees of T(7;9)(Q36.2;P21.2) and T(7;13)(Q34;Q13) carriers together with the sperm karyotype analyses using a tricolor fluorescence in situ hybridization (FISH) method were performed for genetic counseling. The families were ascertained because of probands with holoprosencephaly spectrum, dysmorphic features and congenital malformations. The chromosomal aberration detected in the proband of family 1 was a monosomy 7Q36.2-QTER associated with trisomy 9P21.2-PTER (conventional banding and multicolor FISH). By CGH-microarray analysis using the sanger 1 Mb clone set, breakpoints were localised to clones RP11-69O3/7Q36.2-36.3 (154.6 Mb) and PR11-27J8/9P21.1-21.2 (27.59 Mb). Unbalanced karyotypes with monosomy 7Q34→QTER

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and trisomy 13Q13→QTER were detected among stillborns/early death newborns in family 2. The individual probability assessment according to Stengel-Rutkowski and Stene for unbalanced progeny at birth after 2:2 disjunction and adjancent-1 segregation in family 1 WERE 30±14.5% (high) for the borns, 10±9.5% for miscarriages and 10±9.5% for stillbirth/early death. A very small probability rate (about 0.3%) of viable unbalanced progeny from 3:1 meiotic segregation was predicted for maternal carriers of family 2 and the probability rates for miscarriages and still birth/early death were 12.9±6.% (5/31) and 29±8.2% (9/31), respectively. Fish studies of spermatozoa for meiotic chromosomes segregation pattern in the father of family 1 revealed the relatively high level (48.8%) of different types of unbalanced spermatozoa with 20% products after 2:2 adjacent-1 segregation. The meiotic segregation pattern in the father of family 2 showed a rate of unbalanced spermatozoa of about 60%, with an unusual high rate (29.4%) of 3:1 segregants (i.e., 13.4% of tertiary segregation and 16% of interchange segregation). Adjacent-1 segregation followed with 23.5% and adjacent-2 with 7.2%. The high rate of unbalanced gametes in comparison to the low number of stillborn/ early death and miscarriages detected in family 2 suggests a strong selection against unbalanced karyotypes 1.54-P Multicolor FISH approaches reveal partial trisomy 3q27→qter in combination with partial monosomy 21q22.2→qter in a boy with neurodevelopmental delay and facial dysmorphism. C. Sarri (1) S. Douzgou (1) Y. Gyftodimou (1) A. Dinopoulos (2) E. Pandelia (1) K. Merou (1) N. Kosyakova (1) T. Liehr (3) A. Weise (3) M. Petersen (3) (1) Institute of Child Health, ‘Aghia Sophia’ Children’s Hospital, Athens, Greece (2) University of Athens, School of Medicine, ‘Attikon Hospital’, Athens, Greece (3) Institut fur Human Genetik and Anthropologie, Jena, Germany A 16-month-old boy was referred for genetic evaluation because of facial dysmorphism and neurodevelopmental delay, concerning, in particular, fine motor skills and oral motor control. He exhibited dolichocephaly, bitemporal narrowing, hypotelorism,

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broad nose with short columella, prominent philtrum and a capillary hemangioma of the right upper lip and oral mucosa. Conventional GTG-banding of lymphocytes revealed an abnormal chromosome 21. Parental examination with the application of subtelomeric probes showed that the abnormal chromosome 21 was the derivative result of a maternal balanced translocation between the distal part of the long arms of chromosomes 3 and 21. As a result the boy, who was re-evaluated by chromosome 21 specific subcen M-FISH, multicolor banding (MCB) for chromosome 3 and by subtelomeric probes for 3qter and 21qter, had partial trisomy 3q27 to 3qter in combination with partial monosomy 21q22.2 to 21qter. His final karyotype as follows: 46,XY,der(21)t(3;21)(q27;q22.2)mat. To the best of our knowledge, partial trisomy of the very distal part of the long arm of chromosome 3 is a rare event. We have found only one similar case in the literature. The genotype-phenotype correlation is discussed. Supported in parts by DAAD (D07/00070). 1.55-P Microduplication 22q11.2 in three generations K. Kuuse (1) T. Ilus (1) K. Varb (1) K. Männik (2) T. Reimand (1) (1) Tartu University Hospital, United Laboratories (2) Tartu University Microduplications in regions flanked by low-copy repeats are not rare, but have so far been underdiagnosed because of considerable variability in the clinical phenotype. 22q11 duplication syndrome phenotype ranges from mild to severe with considerable intrafamilial and interfamilial variability. We present here a case of familial microduplication 22q11 in grandmother, mother and daughter (diagnosed prenatally). The duplication was primarly diagnosed by FISH (DiGeorge/VCFS TUPLE1 Region Probe, Cytocell, UK) and further characterized in the mother and the daughter by using whole-genome Infinium Human370CNV SNP array (Illumina Inc.). The size of duplication in region 22q11.21 is ~2.5 Mb. The clinical problems are variable in the family: Grandmother (53 y)—mild facial dysmorphism, fertility problems, premature menopause, no seizures, no cardiac disturbances or psychiatric disorders.

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Mother (30 y)—facial dysmorphism, short stature, velopharyngeal insuffiency, anomalies of toes, mild learning disability, few seizure episodes without special findings in EEG, depression, arthropathy with contractures, no cardiac anomaly but some tahhycardial episodes, fertility problems, complicated pregnancy. Daughter (7 months)—prenatally (21wop): hypokinesis, limb contractures. Child was born at term with low birth weight and length, facial dysmorphism, severe contractures of upper and lower limbs, clinodactyly, heart US scan revealed FOA. FISH, microarray and clinical data will be presented and interfamilial phenotypic variability will be discussed. 1.56-P A complex low mosaic ring 18 chromosome in an adult patient with multiple congenital anomalies L. van der Veken (1) H. Douben (1) J. van de Brug (1) D. van Hassel (1) A. Hoogeboom (1) P. Poddighe (1) A. de Klein (1) (1) Erasmus Medical Center A 24 years old male patient was born with lower spinal anomalies, hypospadia, bifid scrotum, cryptorchism, anal atresia, kidney stones, urethra anomalies, radial dysplasia, and a hypoplastic thumb. Some of the anomalies overlap with the Vacterl association. Chromosome analysis of cultured peripheral blood lymphocytes revealed an additional ring chromosome in 13% of the metaphases. Both parents have a normal karyotype, demonstrating the de novo origin of this ring chromosome. FISH analysis using whole chromosome paints showed that the additional chromosomal material was derived from chromosome 18. A normal karyotype was found upon chromosome analysis of cultured fibroblasts (32 metaphases). To characterize the ring chromosome in more detail peripheral blood derived DNA was analyzed using Affymetrix 250K NSP1 SNP-arrays. In addition, Illumina 610QUAD arrays were used, since these arrays are better at detecting mosaic chromosomal imbalances. The array results indicated a 6 MB gain of the subcentromeric region of chromosome 18Q. Fish analysis using BACprobes from the subcentromeric region OF chromosome 18Q11 indicated the presence of 6–7 signals on

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the R(18) chromosome. In conclusion, the karyotype of the patient shows an additional ring 18 chromosome in 13% of the cells, and the ring chromosome represents a 6 Mb multiplication of the gene rich 18Q11 region. The phenotype of this patient, which is different from the phenotype of other patients with a ring chromosome 18, may be correlated to the unique structure and mosaic presence of the ring chromosome. This case illustrates the benefit of the application of array technology in combination with FISH techniques for the identification of marker chromosomes. 1.57-P Molecular characterization of a new case with a de novo non recurrent inv dup del 2q. Review of the mechanisms for the origin of this type of rearrangement. I. Lopez-Expósito (1) A. Vera (2) J. Bafalliu (3) M. Ballesta (4) G. Glover (5) C. Llopis (5) R. Moya (0) J. Suela (0) E. Guillén-Navarro (9) (1) Centro de Bioquimica y Genética Clinica (2) Centro de Bioquimica y Genética Clínica (3) Entro de Bioquimica y Genética Clinica (4) Servicio Pediatria (5) Bioquimica y Genética Clinica (6) Servicio Pediatria (7) Bioquimica y Genética Clinica (8) Nimgenetics (9) Servicio Pediatria Inverted duplication rearrangements associated with distal deletions are likely to be more frequent than initially thought using only classical cytogenetic methods. Several mechanisms have been proposed to explain their origin, including meiotic models in which a dicentric chromosome is formed by U-type exchange or by non-allelic homologous recombination (NAHR) mediated by LCRs. The main difference between these two models is the absence or presence of a single copy region between the duplication and deletion segment, which should indicate that the breakage is symmetric or asymmetric respectively. Another model is based on a pre-meiotic telomere loss followed by a breakage-fusionbridge (BFB) cycle to produce gametes with terminal deletions associated with interrupted inverted duplications. However, in only a few cases the exact breakpoints have been determined so the molecular basis of the mechanisms leading to inv dup del is still poorly clarified. We report on the clinical, cytogenetic, and molecular analysis of a new patient with a de novo inv dup

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del (2q). Molecular characterization using high resolution array-CGH defined a deletion of 2,6 Mb (2q37.3-2qter) followed by a duplication of 38,7 Mb (2q33.1–2q37.3). A single copy region with the size exceeding 12,9 Kb between the duplicated and deleted segments was not detected. FISH analysis demonstrated that the duplication was inverted and disclosed a polymorphic 2qter deletion in the father. Microsatellite analysis proved the maternal and intrachromosomal origin of the anomaly. Analysis of the breakpoints using the USCS genome browser did not reveal any segmental duplications that could have mediated the rearrangement through NAHR. The aberration might be the result of a U-type exchange or more likely, to be generated by a BFB cycle. Our patient showed features of the distal duplication as well as terminal deletion 2q syndrome.We compared the phenotype and clinical characteristics with other patients described in the literature. This study was supported by Fundación Séneca. Grant number: 05829/PPC/07"

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may be various defects in the development of organogenesis, such as the neural tube and the entire metanephros.Renal agenesis has been associated with some chromosomal abnormalities like trisomy, mosaicism or microdeletion, but in our case the cytogenetic analysis revealed normal karyotype (46, XY). Maternal allele of the polymorphic variant C677T of metylenetetrahydrofolate reductase (MTHFR) gene is said to be associated with NTDs in the offspring. After RT-PCR application for the MTHFR polymorphism, the genotype of the boy was found to be wild type while his mother was heterozygous for this allele. This result is supporting the above mentioned effect of the MTHFR C677T variant, to be one of the causes of NTD. The association between neural-tube defects and congenital renal anomalies probably reflects the fact that both systems are differentiating under the control of similar transcription factors and their genes. We suggest that individuals with neural tube defects associated with other anomalies such as especially congenital renal defects are not rare cases.

1.58-P 1.59-P Meningomyelocele and Renal Hypoplasia: A Rare Case Report H. Aydin (1) E. Tug (1) S. Duzenli (1) O. Erkal (3) A. Yoldas (2) M. Yirmibes Karaoguz (3) (1) Abant Izzet Baysal University Izzet Baysal Medical School (2) Abant Izzet Baysal University Izzet Baysal Medical School (3) Gazi University Faculty of Medicine Meningomyelocele (MMC) is the most common form of neural tube defects (NTDs) and may have other associated congenital defects including urinary tract. MMC prevalence is 2–4 in 1000 live births, but coexistence with renal anomalies is a very rare condition. We report a 9 year old boy with MMC at birth, who is the only patient in the family. When he was recruited in our clinics, his NTD was operated. In addition to this, right kidney hypoplasia, undiagnosed tumor at the left kidney, Arnold Chiari type II malformation, operated bilateral inguinal hernia, lower extremity plegia and urinary and faecal incontinence due to MMC were the other findings. As known, if the embryo is affected of unknown insults during the fourth week after fertilisation, there

Familial X;Y translocation with different phenotypic consequences. Contribution of FISH and CGH N. Bukvic (1) V. Delli Carri (2) A. Pansini (3) M. Di Cosola (3) C. Cesarano (2) L. Buonadonna (3) V. Bafunno (1) M. Chetta (1) G. D’Andrea (1) V. Longo (1) R. Santacroce (1) M. Sarno (1) F. Sessa (1) M. Gentile (3) M. Margaglione (1) (1) Sezione di Genetica Medica “Dipartimento di Scienze Biomediche, Universita degli Studi di Foggia, Italia” (2) OORR. Azienda Ospedaliero-Universitaria “Sezione di Biologia Molecolare e Citogenetica, Foggia, Italia” (3) P.O. “Di Venere”—Dipartimento Area Materno-Infantile “U.O.C. Genetica Medica, Bari, Italia.” X;Y translocations are relatively rare events in the human population and the majority of these aberrations, analyzed cytogenetically, have breakpoints at Xp22 and Yq11. All females with translocations t(X;Y)(p22;q11) are phenotypically normal except for short stature, while the males are either phenotipically normal or with various abnormalities. Terminal or interstitial Xp deletion (Xp22.2->Xpter) which lead to nullisomy of the deleted region and complete loss of the respective genes,

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have been recognized as reason for variable contiguous gene syndromes in males. The phenotype depends on the extent and the position of the deletion showing the variable association of apparently unrelated clinical manifestations such as ichthyosis (XLI), chondrodysplasia punctata (CDPX), hypogonadotropic hypogonadism with anosmia (Kallmann syndrome (KAL), ocular albinism (OA), short stature (SS) and mental retardation (MRX). In addition, some cases, have been reported either with symptoms of lack of attention and/or hyperactivity-impulsivity, both recognized as attention deficit hyperactivity disorder (ADHD). The extent of terminal Xp deletions is limited by the presence of male lethal genes in Xp22.2 at about 10–11 Mb from the telomere and in the most cases of viable reported male cases extended to the STS (~7.0 Mb) or to the KAL1 (~8.5 MB) locus, with respective manifestations. Herein, we present clinical, cytogenetic, FISH and array CGH study of an Italian family with an Xp;Yq translocation (mother, son and daughter). The chromosomal status is also discussed in the light of their phenotypic tracts. The final karyotypes of the patients were designated as: Patient 1: 46,Y,der(X),t(X;Y)(p22;q12).ish der(X) (Xptel-, Xcen+, Xqtel+)mat.arr cgh Xp22.31p22.33(RP11-60P14→RP13391G2) x 0; arr cgh Yq11.221qter (RP11-235I1→RP11-270H4) x 2 Patient 2: 46,X,der(X),t(X;Y)(p22;q12).ish der(X) (Xptel-, Xcen+, Xqtel+)mat.arr cgh Xp22.31p22.33(RP11-60P14→RP13391G2) x 1; arr cgh Yq11.221qter (RP11-235I1→RP11-270H4) x 1 1.60-P Cytogenetic evaluation of 1059 patients from Rio de Janeiro (RJ), Brazil

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chromosomal regions, which can not be identified by classical cytogenetic techniques. From 1996 to 2008, we checked 1059 patients for chromosomal abnormalities (517female/542male individuals) using GTG-banding and found 225 altered karyotypes (154F/71M). The majority of those abnormalities correlated with known syndromes. Out of those altered karyograms, 27.5% showed structural abnormalities. The aim of this poster is to report three cases, which need further molecular investigation for a precise diagnosis. One case was that of a 26-year-old woman, a variant of Turner syndrome, who was the seventh child of an old but nonconsanguineous couple. She had developmental delay, short stature, some Turner stigmata, hypoplasic and female genitalia, small breasts, genovalgus, no mental retardation, and no internal malformation. Her karyotype was 46,XX, add(X) (q21). The second case was that of a 3-year-old boy born from a young and nonconsanguineous couple, who presented hypotonia, pre- and postnatal growth retardation, developmental delay, seizures since five months old, microcephaly, facial dysmorphisms, excavatum pectus and small testis. His karyotype was 46,XY/47,XY, + marker de novo. His parents’ karyotypes were normal. He evolved with hyperactivity, seizures, unilateral cryptorchidia (R) and retinal scar, similar to that of toxoplasmosis, but with negative serology for this disease. In this case the clinical findings were suggestive of chromosomal abnormality, however the presence of a marker solely cannot explain the phenotypic alterations, since a marker can occur with or without abnormal phenotype. The third case was that of two brothers with Pierre Robin syndrome that had a pericentric inversion on chromosome 9, inherited from their mother. In conclusion classical cytogenetic techniques must be complemented by other molecular technique when structural alterations are found, as well as when the karyotype is normal but a chromosomal abnormality is suspected. 1.61-P

C. L. A. Paiva, E. M. M. Duarte, C. F. Rocha, L. de A. Agostinho, S R Middleton and S. R. dos Santos Laboratory of Cytogenetics, Department of Genetics and Molecular Biology, Universidade Federal do Estado do Rio de Janeiro (UNIRIO), RJ, Brazil. [email protected] GTG-banding analysis is the most used tool for identifying chromosomal alterations. Nowadays, molecular techniques can elucidate breakpoints and altered

A special case of Prader-Willi syndrome with 15q11–q13 deletion E. Gorduza (1) C. Rusu (1) E. Braha (1) M. Gramescu (1) C. Bujoran (2) I. Ivanov (3) M. Covic (1) (1) “Gr. T. Popa” University of Medicine and Pharmacy Iasi, Romania (2) “Sf. Maria” Children Hospital, Iasi, Romania (3) “Sf. Spiridon” Hospital Iasi, Romania

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Prader-Willi syndrome is a genetic disease that affects 1/10.000–1/15.000 newborns. The disease is produced by a lack of active genetic material in imprinted 15q11–q13 region of chromosome 15. This region called PWS/AS region may be lost by one of several genetic mechanisms which, in the majority of instances occurs through chance mutation. Usually, the disease is caused by a deletion of the paternal copies of the 15q11–q13 region. Other less common mechanisms include: uniparental disomy, sporadic mutations, chromosome translocations, and gene deletions. In majority of cases, the deletion is small (microdeletion) and the diagnosis is established by using FISH technique with probe for 15q11–q13 region. We present a Prader-Willi case with microscopic deletion in long arm of chromosome 15. The proband is the first child of a healthy, unconsanguinous, young couple. The pregnancy was normal and the delivery was by a natural way. The patient presented repeated febrile convulsions, starting at 6 weeks and a slight retardation of neurological achievements. The first genetic consultation was made at 16 years. The clinical features observed were: slight mental retardation (poor school results) generalized obesity (+2.04 SD), short stature (−2.60 SD) almond shape eyes, microstomia, thoracic kyphosis, small penis, small hands and feet. The clinical supposition of Prader-Willi syndrome was confirmed by cytogenetic analysis, that revealed a 46,XY,del(15)(q11–13). The karyotype was made also in both parents of proband, and the results were normal. The ophthalmological examination was normal, and the endocrinological examination revealed a slight hypogonadism. This case shows the importance of careful examination in all individuals with short stature, borderline mental retardation and hypogonadism. Also, our presentation reflects the importance of classical cytogenetic examination in all patients with Prader-Willi syndrome.

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Often, cytogenetic studies have an important role in the evaluation of couples with a poor obstetric history, and in roughly half of the cases male-related factors are involved. A male-specific infertility locus on chromosome 1 has been proposed recently and the regions concerned by breakpoints were 1q21, 1q32, 1q24, 1p22, and 1q12 in decreasing number and significance. The present study deals with the analysis of chromosome 1 cytogenetic rearrangements, in couples with reproductive failure that occur in the form of infertility, spontaneous abortions or chromosomally unbalanced children. We report five reciprocal translocations that involve chromosome 1. These translocations were detected, among a total of 16 reciprocal translocations (5/16: 31%) identified during our cytogenetic activity (2001 to 2009) that concern reproduction failure. Two translocations: t(1;4) (q31;q26) and t(1;2)(p35;q33) were identified respectively in a female and a male with recurrent spontaneous abortions. A third translocation t (1;18)(p21,q12) was identified in two males who have asthenotertospermia and failure of ICSI attempts. The last translocation was t(1;6)(p32; p32) which was detected in an extreme oligospermic man with 60% of multiflagellar large headed sperm. We suggest that chromosome 1 harbours a critical domain whose integrity is essential for male and female fertility. 1.63-P Heterozygous deletion of exon 8 in WFS1 gene in two Wolfram Syndrome probands with hearing loss

F. Rim (1) A. Ahlem (2) R. Tarek (1) B. Nouha (1) (1) Medical University of Sfax, Tunisia (2) 2. Pasteur Institute of Tunis, Tunisia

I. Sezgin (2) E. Altuntas (1) A. Bora (1) H. Kurtulgan (2) B. Koksal (2) S. Muderris (1) O. Ozdemir (2) (1) Cumhuriyet University Faculty of Medicine (2) Cumhuriyet University Faculty of Medicine (3) Cumhuriyet University Faculty of Medicine (4) Cumhuriyet University Faculty of Medicine (5) Cumhuriyet University Faculty of Medicine (6) Cumhuriyet University Faculty of Medicine (7) Cumhuriyet University Faculty of Medicine

Reproductive failure is a devastating health problem that affects many couples who are trying to conceive.

Objectives: Point mutations in the wolfram syndrome 1 gene (WFS1) are associated with the autosomal

1.62-P Report of chromosome 1 rearrangements in reproductive failure

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dominant and/or resessive mild type sensorineural hearing loss in first degree relatives. The purpose of this study was to enhance the molecular diagnosis of Wolfram Syndrome by using multiplex ligationdependent probe amplification technique (MLPA). Methods: Total genomic DNA was isolated from peripheral blood of affected probands and a control. Multiplex PCR was performed followed by MLPA analysis. Results: We identified heterozygous deletion in exon 8 of WFS1 gene (wolframin protein) in the father and in one of his daughters with low hearing loss and optic athropy. No mutation was detected in both GJB 2 (connexin 26) and GJB6 (connexin 30) gene regions in both probands and the control. Key words: Wolfram Syndrome, Heterozygous deletion, WFS1 gene—exon 8, hearing loss

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(Q35;Q14) in an infertile male with severe oligozoospermia (sperm count 2.2 mln/ml). Subsequently performed fluorescence in situ hybridization (FISH) with WCP5 and WCP13 (cytocell) revealed that material from chromosome 13q was translocated to chromosome 5q, and the small terminal part of 5q was reciprocally translocated to 13q. The breakpoints at 5q35 and 13q14 have not been reported before in infertile males. In conclusion, our study confirmed the elevated impact of chromosomal abnormalities in reproductive failure of males. The revealed new case of translocation with new breakevents might be helpful for the detection of the chromosomal regions, where is possible to localize new genes of male infertility. Supported by the project SF 0180096S08. 1.65-P

1.64-P Chromosomal study in male infertility: survey and a case with unusual reciprocal translocation t(5;13)(q35;q14) R. Mikelsaar (1) H. Margus (1) P. Korrovits (2) (1) Institute of General and Molecular Pathology (2) Centre of Andrology Chromosomal abnormalities disturb the reproductivity in males. The frequency of chromosomal abnormalities in infertile males varies from 2.2% to 15.2%, where 3.8% involve the sex chromosomes and 1.3% autosomes. The aim of this study was to find chromosomal abnormalities associated with male infertility. We have cytogenetically studied 239 infertile males. chromosome analyses were performed from peripheral blood lymphocyte cultures using GTG, c-banding and FISH methods. From the 239 infertile men major chromosome abnormalities were revealed in 24 patients (10%): autosome abnormalities in 8 (3.3%) cases, and sex chromosome abnormalities in 16 (6.7%) patients. The more frequent structural aberrations are Robertsonian translocations and sex chromosome abnormalities. Reciprocal translocations have been found in approximately 1% of infertile men and are more rare in oligozoospermic males than in azoospermics. Here we report the first case of a translocation T(5;13)

Association of semen parameter variation with genetic abnormalities of spermatozoa E. Shilnikova (1) I. Fedorova (2) O. Chiryaeva (2) L. Petrova (2) V. Dudkina (2) N. Sadik (2) M. Bogdanova (3) (1) St-Petersburg State University (2) Ott’s Institution of Obstetrics and Gynecology (3) Medical centre InAlMed Semen analysis provides essential information on male reproduction property. However, main parameters (concentration, motility and percentage of morphologically normal spermatozoa) vary considerably over several months. Most investigations concerning correlation of semen parameters and spermatozoa chromosome features were carried out on large groups of patients. Variations in semen parameter association with heteroploidy frequency in spermatozoa along with percentage of spermatozoa with degraded DNA of the same individual was a principal goal of our study. Semen samples from the patient with Robertsonian translocation 45,XY, der(13;14)(q10;q10) were collected twice a few months apart. In the first semen sample severe oligoasthenoteratozoospermia was diagnosed. Disomy frequency of autosomes 3, 7, 9, 10 and 18 (1,1–1,48%) was increased in comparison with sex chromosome disomy (0,35–0,52%) (p<0,0001). The percentage of

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DNA degraded spermatozoa estimated by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) was estimated as 1,22%. In the second sample all scored parameters demonstrated noticeable improvement: concentration and motility were normal, percentage of morphologically normal spermatozoa was 2% (teratozoospermia). Disomy frequency of autosomes 3, 7, 9, 10 and 18 varied from 0,39% to 0,84% and sex chromosomes— 0,23–0,39%. Disomy frequency of chromosome 10 and diploidy were lower than in first semen sample (0,45% vs. 1,48% and 0,88% vs. 2,47%, respectively p<0,0001). Also the percentage of DNA degraded spermatozoa was lower than in first sample (0,6%, p= 0,0091). Our data suggest association of semen parameters with the number of spermatozoa bearing chromosome abnormalities and degraded chromatine. The latter parameter should be considered for male infertility treatment strategy in reproductive technology clinics. 1.66-P Meiotic recombination and sperm aneuploidy in post-vasectomy obstructive azoospermic patients V. Peinado (1) N. Al-Asmar (1) M. Susiarjo (3) J. Gruhn (2) L. Rodrigo (1) M. Gil-Salom (1) J. Martinez- Jabaloyas (1) C. Rubio (1) T. Hassold (2) (1) Instituto Valenciano de Infertilidad -IVI-Valencia (2) Washington State University, Pullman (3) University of Pennsylvania School of Medicine

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Results: OA-1: 47 years old, 18 years MLH1 ± SD=50.2±3.9 OA-2: 60 years old, 24 years MLH1 ± SD=46.0±2.7 OA-3: 38 years old, 12 years MLH1 ± SD=48.1±4.0 OA-4: 53 years old, 18 years MLH1 ± SD=48.3±1.4

after vasectomy, mean after vasectomy, mean after vasectomy, mean after vasectomy, mean

Statistical comparisons: – – – – –

Mean MLH1: OA-1 vs. OA-2, p<0.001 No significant differences in the percentage of SCs with 0 and 1 exchanges among the four patients. % of SCs = 2 exchanges AO-1 (49.8) vs. AO-2 (60.6), p<0.01 % SCs with >2 exchanges AO-1(34.9) vs. AO-2 (21.2), p<0.001;AO-1 (34.9) vs. AO-3(29.8), p<0.05. No statistical differences in sperm aneuploidy rates.

Conclusions: Mean recombination levels were similar to previously published studies in post-vasectomized patients, but there was a significant decrease in MLH1 foci in OA2, the oldest patient and the one with the longest period of time post-vasectomy. No significant increase in aneuploidy rates were observed in the four patients compared to the control group of normozoospermic individuals. 1.67-P

Objectives: To study meiotic recombination in pachytene cells and aneuploidy in spermatozoa in testicular biopsies from 4 post-vasectomy obstructive azoospermic patients (OA). Material and methods: Meiotic recombination was assessed using immunocytogenetics with three primary monoclonal antibodies: anti-SCP3 (axial/lateral elements of the synaptonemal complex); anti-CREST (centromeric regions) and anti- MLH1 (mismatch repair protein MutL homolog1 that co-localizes to sites of meiotic cross-over). Aneuploidy rates for chromosomes 13, 18, 21, X, and Y were analyzed by fluorescence “in situ” hybridization (FISH) and compared with a control group of 5 normozoospermic fertile donors.

De novo partial trisomy of the subtelomeric region of 1q on 1pter causing dysmorphic features F. Duzcan (1) G. Cetin (1) T. Kalkan (1) K. Erdogan (1) S. Semiz (2) (1) Pamukkale University (2) Pamukkale University Partial trisomy of the subtelomeric region of 1q is rarely reported. Here we report a case with 46,XX, der(1)(qter➔q43::pter➔qter). The proband was a 10 years old daughter of first cousins once removed marriage. She attended our unit with motor and mental retardation and hirsutism suspected to be a case of de Lange syndrome. Physical examination

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revealed growth retardation (<3p), triangular face, prominent forehead, low frontal and posterior hair line, synophrys, anteverted nostrils, long philtrum, high arched palate, downward corners of mouth, pointed chin, prominent and simple ears, hirsutism on the back and dorsal parts of the arms and legs, long and thin fingers, bilateral long and overridded 2nd toes. Her bone age was 6 years at the age of 9, and cranial MRI showed agenesis of corpus callosum. Her hormonal status was normal. Cytogenetic analysis with GTG-banding showed additional material on the terminal region of 1p. FISH analysis with whole chromosome painting probe for chromosome 1 revealed no aberrant signals. Finally, subtelomeric analysis of the chromosome 1 showed partial trisomy of the subtelomeric region of 1q on 1pter. Subsequent aCGH analysis of the case for further delineation of the length of the duplicated region is planned. 1.68-P Partial duplication of 18q including Edwards Syndrome distal critical region in a patient with normal phenotype and oligoasthenospermia: case report. R. Quiroga (1) M. Roselló (1) S. Monfort (1) S. Oltra (1) I. Ferrer-Bolufer (1) F. Martinez (1) C. Orellana (1) (1) Hospital La Fe Introduction: Several individuals with partial trisomies of chromosome 18 have been reported. The patients display a range of severity from mild phenotype to classic Edwards Syndrome (ES). Different candidates for the critical region (CR) have been proposed. Here, we described an oligoasthenospermic young man with a derivative chromosome 18 and a normal phenotype. Clinical data: A 29 year patient with severe oligoasthenospermia was referred to the Genetics Unit for karyotyping. Personal and family history was unremarkable. No clinical signs was found at physical examination. Material and Methods: Standard karyotype, MLPA (subtelomeric screeningsalsa P036D), FISH (telomeric and centromeric probes for chromosome 18) and Array-CGH 44KAgilent were performed.

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Results: Standard karyotype: 46,XY,add(18p)[25]. Parent’s karyotype: normal. MLPA: deletion of 18p and duplication of 18q probes. FISH in peripheral blood metaphases and buccal mucosa interphase nuclei was in agreement with the results of MLPA, discarding mosaicism in these tissues. Array-CGH: Deletion of 0.25 Mb (18pter) and duplication of 17.4 Mb (18q22.1-qter bands) were observed. Discussion: Several authors have attemped to map the ES critical region. However, not all the karyotypes were performed with high-resolution banding techniques. Duplicated region in our patient involves distal ES region. Lack of clinical features, except for oligoasthenospermia, is remarkable. Partial duplications of 18q, involving the distal ES region have been associated to some clinical features of ES. Two recent reports described smaller duplications with no clinical signs: a healthy father with a 18q23 duplication (450 kb) transmitted to the fetus with congenital anomalies; and a patient with recombinant chromosome (dup18q/del18p), where 3 Mb duplicated segment corresponded to band 18q23. The latter was physically healthy, except for mild myopia and deficient social behavior. In conclusion, we described a large duplication on chromosome 18, that involves the distal CR with no clinical features. This duplicated region would not be critical for ES phenotype. 1.69-P Instability evaluation in ring chromosomes M. Melaragno (1) L. Kulikowski (3) C. Kim (2) R. Honjo (2) V. Meloni (1) A. Pacanaro (1) D. Christofolini (1) F. Bellucco (1) D. Brunoni (1) R. Guilherme (1) (1) Universidade Federal de São Paulo (2) Faculdade de Medicina da USP (3) Faculdade de Medicina da USP Ring chromosomes usually result from two breaks in both chromosome arms followed by rejoining of the ends or by fusion of a telomeric region to a terminal breakage end or to another telomeric sequence. Ring chromosomes are prone to instabil-

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ity, due to sister chromatid exchanges during mitosis that can result in ring loss or other secondary aberrations. Instability and mosaicism rate were investigated in nine patients with ring chromosomes 3, 4, 10 (two cases), 14, 15, 18 and 22 (two cases). We analyzed 200 metaphases with G-banding and 100 using FISH with centromeric probes. NOR staining was present in all acrocentric rings except one r(22). MLPA with subtelomeric probes in r(3) and r(10) revealed deletions 3p and 10q. FISH with a pantelomeric probe showed no hybridization in r(14) and r(15), but the r(14) hybridized to the RP1176N15 14q subtelomeric probe. In the patients with r(3), r(4), r(10), r(10), r(14), r(15), r(18), r(22) and r(22), the percentage of cells with one monocentric ring was 87.3%, 95.7%, 83.7%, 84.3%, 86.7%, 92.3%, 90.7%, 96.0%, and 95.0%, respectively; of cells with 45 chromosomes and ring loss it was 6.3%, 1.7%, 12.7%, 11.0%, 11.7%, 3.7%, 6.7%, 3.7% and 4.3%, respectively; and of cells with secondary aberrations it was 6.3%, 2.7%, 3.7%, 4.7%, 1.7%, 4.0%, 2.7%, 0.3% and 0.7%, respectively. These secondary aberrations were, in decreasing order, dicentric ring chromosomes, open rings, two or three monocentric or dicentric ring chromosomes in the same cell, and chromosome fragments. Our r(3), r(10), r(14), r(15) and r(18) can be considered instable, since secondary aberrations were observed in >5% of the cells. According to the literature, larger ring chromosomes are more instable than smaller ones, but our r(4) was stable, indicating that instability is not only correlated to ring size but also to genetic content. (Financial Support: FAPESP, Brazil).

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The frequency and pathogenesis of these additional disorders are still in big part unknown. The aim of this study was to analyze the association of alpha-1 antitrypsin (AAT) phenotypes and serum concentrations with these disorders in Klinefelter syndrome patients. We have analyzed alpha-1 antitrypsin in thirteen azoospermic patients with Klinefelter syndrome. All patients had the karyotype 47,XXY. Nine patients (69%) reported repeated pneumonias and chronic bronchitis in their medical history (10.5% in the general population). Three persons had kidney problems and one had infected chronic trophic leg ulcers. The AAT level ranged from 0.74 to 1.48 g/l (normal range 0.9 to 2.0 g/l) with the mean value of 1.25 g/l (normal 1.45 g/l). The most prevalent Pi phenotype was M1M1. Two phenotypes with AAT deficiency alleles were identified. One patient with AAT phenotype XZ and AAT level of 0.74 g/l reported several episodes of bronchitis and pneumonia. Another patient with AAT phenotype M10 and AAT level of 1.44 g/l had infected chronic trophic leg ulcers. One patient with AAT phenotype M1M1 had a borderline AAT serum level (1.09 g/l) and repeated bronchitis. In conclusion, the frequencies of these additional disorders in klinefelter syndrome are higher than in the general population. These patients frequently have lowered AAT level. AAT deficiency may be underdiagnosed among the klinefelter syndrome patients, especially among those who have pulmonary diseases or additional health problems. the treatment with aat could improve the life quality of klinefelter syndrome patients. Supported by the project sf 0180096s08. 1.71-P

1.70-P Association of Klinefelter syndrome with alpha-1 antitrypsin E. Vaidla (1) J. Lissitsina (1) H. Margus (1) K. Ausmees (2) M. Punab (2) P. Korrovits (2) R. Mikelsaar (1) (1) Tartu University (2) Tartu University Clinics Klinefelter syndrome is a well characterized abnormality of sex chromosomes. Apart from the main symptoms (infertility, small and firm testes, gynaecomastia) several other problems indifferent organ systems, especially lung diseases, may be present.

Prenatally diagnosed de novo inverted duplication of 8p11.2–p23 region with deletion of 8pter in a dysmorphic case G. Cetin (1) K. Erdogan (1) O. Ozdemir (2) T. Kalkan (1) B. Kaleli (3) I. Kilic (2) F. Duzcan (1) (1) Pamukkale University Medical School (2) Pamukkale University Medical School (3) Pamukkale University Medical School Common rearrangements of the chromosome 8p include inv dup of (p11.2p23) region. Here we report a postnatal case of 3 days old boy. The proband was

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the only child of healthy non-consanguineous parents and delivered at 39 weeks of gestation with a birth weight of 2150 g. The first pregnancy of the couple was aborted spontaneously at 8 weeks of gestation. He was first diagnosed prenatally by amniocentesis when fetal MRI revealed agenesis of corpus callosum and DandyWalker malformation. The GTG-banding of the amniocentesis revealed inv dup of 8p11.2–p23 region. FISH analysis with whole chromosome painting probe for chromosome 8 showed no painting defect. The family decided to continue the pregnancy. The proband was referred to our unit with multiple congenital anomalies after the birth. Physical examination revealed generalised hypotonia, arched eyebrows, periorbital fullness of subcutaneous tissues, long philtrum, hypoplasia of the antihelix, anteverted nares, depressed nasal root, bulbous nasal tip, micrognathia, widely spaced nipples, preaxial polydactly on the left hand, bilateral inguinal hernia, hyperpigmentation of the scrotum and hydrocele. Cranial MRI showed agenesis of the corpus callosum. ECG resulted in biventricular dominancy and ECHO scan for cardiac malformations evidenced single ventricule, transposition of great arteries, and PDA. Karyotyping from peripheral blood of the baby was consistent with prenatal karyotyping. Subtelomeric FISH analysis for chromosome 8 showed deletion of the subtelomeric region of 8p. Further, aCGH analysis is planned for determining the length of the rearrangement and the involved genes in the region. 1.72-P 2p16.1p16.1 microdeletion syndrome: narrowing the critical region. P. Prontera (1) D. Rogaia (1) L. Bernardini (2) M. Barbieri (1) A. Capalbo (2) M. Giuffrida (2) C. Ardisia (1) E. Donti (1) (1) Università di Perugia (2) CSS Hospital, IRCSS San Giovani Rotondo and CSS-Mendel Institute Microdeletion at 2p15–16.1 has been recently reported in three patients, delineating an emerging recurrent microdeletion syndrome associated with a recognizable phenotype. We describe a 9-years-old female with dismorphisms, development and mental retardation in which molecular-cytogenetic analysis disclosed a microdeletion within the 2p16.1 chromosome band.

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The proband was born at 40-weeks gestation because of diagnosis of intrauterine growth retardation (IUGR). At birth, weight was 2300 g (<10° centile), length 44 cm (<3° centile) and OFC 33,5 cm (10°–25° centile). Developmental milestones were delayed and during the first months of life she suffered from gastrointestinal reflux, feeding problems and inguinal hernia. A postnatal growth retardation and a moderate intellectual disability became evident later. At the time of our evaluation height was 120 cm (<3° centile), weight 20 Kg (<3° centile) and OFC 50 cm (3°–10° centile) and she showed flat occiput, short neck, high palate, large ears with wide concha, high nasal root, short philtrum, prognatism, upslanted and short palpebral fissures and strabismus. G-banding 400 bands resolution karyotype performed on peripheral blood lymphocytes revealed an apparently balanced translocation between a chromosome 1 and a chromosome 7 which already carried a paracentric inversion on the long arm [46, XX, der(7)inv(7)(q22q32)t(1;7)(q31;q36)]. All the rearrangements were confirmed by WCP1 and WCP7. Their de novo origin was assessed by analysis of the parents. A subsequent 75 Kb array-CGH investigation disclosed no segmental aneusomies on chromosomes 1 and 7 but revealed a deletion spanning about 3.5 Mb in the 2p16.1p16.1 region not detectable at cytogenetic level. FISH analysis with locus-specific probes demonstrated the de novo origin of the microdeletion. The deleted region, clinical outcome and medical history in this patient are similar to those reported in other published case, further suggesting that this genomic segment is prone to rearrangements and its hemizygosity gives rise to a clinically recognizable syndrome. Moreover, the deletion in our case is the smallest reported, narrowing the critical region, in particular, regarding cognitive function. 1.73-P Molecular characterisation of a ring chromosome 22 in a boy with severe language delay, hypertonia and autistic features H. Hanene (1) I. Ben Abdallah (1) S. Mougou (1) N. Kahloul (1) S. Mehri (1) H. Elghezal (1) A. Saad (1) (1) Farhat Hached University Teaching Hospital

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The common clinical features reported in ring chromosome 22 cases include mental retardation with severe language impairment, hypotonia, and autistic disorders. In many cases, dysmorphic facial features, microcephaly, syndactyly, and seizures or abnormal EEG have also been reported. However, phenotype genotype correlation was not clear because the variability of the deleted region in the ring chromosome and the presense or not of mosaicism with normal and/or monosomy 22 cells. We report, here, a new case of de novo homogenous ring chromosome 22 in a 7 year-old boy. He was born at term of uneventful pregnancy from healthy non consanguineous parents, with a birth weight of 2,7 kg. He was the first child of a family without any history of mental retardation and consisting of a 35 years old mother, a 39 years old father and a normal 3 years younger brother. He acquired sitting position at 10 months and walked at 27 months. On examination, he had a head circumference of 52 cm (normal), a body weight of 21 kg (normal) and a height of 125 cm (normal). He presented minimal cranio-facial dismorphic traits, toes and fingers did not show syndactyly. Severe language disability was noted and his speech was limited to few syllables. Also, he presented behavioral problems such as hypertonia, aggressive behavior, and autistic features. FISH analysis using specific locus probes was performed to identify the deleted region in ring chromosome 22. The distal non deleted signal in ring chromosome 22 was obtained with the BAC clone CTA-299D3 located in 22q13.2 and the size of telomeric deletion is estimated to 2.5 Mb. We conclude that in our case, the patient presented the clinical features of 22q13.3 deletion syndrome but not the other reported signs of ring 22 cases because the small terminal deletion associated with this case of ring chromosome 22. 1.74-P Deletion in 6q21 region detected by array-CGH in a patient with microphthalmia I. Carreira (1) E. Matoso (1) S. Ferreira (1) J. Melo (1) E. Siva (2) (1) Faculdade de Medicina Universidade de Coimbra (2) Hospitais da Universidade de Coimbra

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Microphthalmia, anophthalmia and coloboma (MAC) are rare structural abnormalities of eye development with a minimum live birth prevalence varying between 1,8– 2,0:10000, that may appear isolated or as part of complex dysmorphological syndromes. Cytogenetic approaches have already identified numerous chromosomal changes associated with MAC spectrum, but these do not account for the totality of MAC pathologies. Recently, the use of molecular cytogenetics techniques such array-CGH has provided a further step towards a better definition of candidate genomic regions. We report the case of a child with microphthalmia, in whom conventional cytogenetic studies revealed a translocation (13;15), maternal in origin. Although having the same translocation as the child, the mother has a normal phenotype, which made us suspect that other chromosomal alteration could be present in the young boy. In order to evaluate this, array-CGH using a whole genome BAC array was performed with the constitutional chip 4.0 (PerkinElmer), with an average resolution of 650 Kb. This array-CGH profile showed a 6q21 deletion, without alterations in chromosomes 13 and 15. A search for the genes localized in this region revealed the presence of 17 genes, including SNX3, a member of the sorting nexin family involved in intracellular trafficking. SNX3 has been considered a strong candidate gene for Microphthalmia syndromic 8, a disorder characterized by an association of phenotypes that include microphthalmia, microcephaly, ectrodactyly and prognathism (MMEP). This case illustrates how array-CGH can be a valuable tool for deciphering conventional cytogenetics unexplained cases, and lead to the identification of novel genes, or at least novel genomic regions, involved in a particular phenotype. 1.75-P Lesson from 5 years experience in male infertility management S. Kalantar (1) H. Fazli (1) H. Khodai (1) A. Ebrahimi (1) M. Seyed Hassani (1) M. Sheikha (1) (1) Research & Clinical Centre for Infertility, Yazd Medical Sciences University Introduction: Fifteen percent of couples do not achieved pregnancy after one year of sexual inter-

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course & at the end of their reproductive life, 2–7% of couples remain childless. Male factor infertility accounts for about half the cases of couple infertility. Up to 40% of MF the etiology remains unknown. In these cases of idiopathic MF the question arises whether these could be explained, at least in part, by genetic factors. Chromosomal abnormalities is one of genetic causes of infertility. Materials & Methods: Genetic abnormalities detected clinically in 625 infertile men were retrospectively reviewed. Metaphase spreads were made from phytohaemaglutinin-stimulated peripheral lymphocytes using standard cytogenetic techniques. The chromosomal status was analyzed using CytoVision Ultra ver.4.0 from Applied Imaging. Results: In this study, out of 625 infertile males, 53 men had chromosomal aberration including 35 numerical aberrations; 47,XXY, 46,XY/47,XXY, 46,XY/47,XY +mar, 48XXYY and 18 cases with structural aberration (partly variants and balanced aberrations); 46,XY,dup (4)(q31.1q32), and 46XY,qh+ (variant), 46,XY,del 13p (variant), 46,XY/47,XY,+8, del (8)(pter), 46,XY,t(7;14) (p10;q10) (balanced rearrangement). Conclusion: The potential of various sperm retrieval techniques to transmit genetic defects causing male infertility raises the need for a systematic genetic screening of these patients. An algorithm was designed to be used by andrology evaluation for referring to Genetic counseling during an infertility program. 1.76-P A case of terminal 14q deletion with de novo unbalanced t(Y;14)(q?;q32) translocation.

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our clinic because of facial dysmorphic features and mental retardation at the age of 4 years. His physical examination revealed the following dysmorphic features: low frontal hairline, asymmetric head, down-slanting palpebral fissures, epicanthal folds, a bulbous nose with a marked nasal bridge, synophyris, high arched palate, irregular teeth, broad and long philtrum, hirsutism on the back and dorsal sides of arms, bilateral Simian line and tapering fingers, unilateral scrotal hernia, pes planus and insecure gait. Magnetic resonance imaging (MRI) scan of the brain showed corpus callosum hypoplasia and blood and urine metabolic screen was normal. His first cytogenetic evaluation with conventional GTG banding showed additional material on the q arm of chromosome 14. Chromosome analysis of his parents showed normal constitutional 46,XX and 46,XY karyotypes. Fluorescence in situ hybridization (FISH) analysis with a whole chromosome painting panel was performed and showed that the additional material had originating from an unbalanced translocation between the terminal q arms of chromosome Y and 14. Subsequent, subtelomeric FISH analysis using a panel of subtelomeric probes revealed a deletion of 14q32 region. Finally, we detected a 4.1 Mb deletion arr.cgh 14q32.2q32.3 (102295758-106368585)x1 by array CGH experiments and the karyotype of the case was designated as 46,X,der(14)t(Y;14)(q?;q32). nuc ish 14q32 (D14S1420x1). arr.cgh 14q32.2q32.3 (102295758-106368585)x1. There are 42 genes in the deleted region and two of them are OMIM genes (AKT1 and AMN1). 1.77-P Case report. A newborn with double aneuploidy.

G. Bagci (1) G. Cetin (1) C. Semerci (1) G. Toruner (2) M. Cinbis (3) (1) Pamukkale University Medical School (2) UMDNJ-NJ Medical School (3) Pamukkale University Medical School Terminal deletions of chromosome 14q are rarely reported. Here we report a case with terminal 14q32 deletion associated with a derivative 14 chromosome from a de novo t(Y;14)(q?;q32) translocation. The proband was the first child of healthy non-consanguineous parents. He was referred to

I. Vool (1) K. Joost (1) T. Ilus (2) (1) Tallinn Children’s Hospital (2) Tartu University Hospital Mosaicism with double aneuploidyof two different chromosomes is very rare. In this case we found mosaicism Turner syndrome and male Down syndrome. Female patients with diploid cell lines may reveal variable features of Down syndrome and Turner syndrome, but males in addition may show ambiguous external genitalia.

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A blood sample from patient with cryptorchism and hypospadia was investigated. Cytogenetic findings with G-banding revealed mosaicism in peripheral blood: mos45,X[66]/47,XY,+21[34]. No normal cell lines was observed. Cytogenetic investigation from skin cells revealed the same proportion of both cell lines in fibroblasts: karyotype 45,X[21]/47,XY,+21[9]. Based on the cytogenetic findings Down-UllrichTurner (DUT-) syndrome was diagnosed. It is a rare syndrome with frequency 1: 2000000 newborn. The origin of DUT-syndrome may be explained by a gain of one chromosome and a loss of an other during early embryogenesis. However, chimaerism cannot be excluded. 1.78-P A novel NSD1 intragenic deletion in a patient with a Sotos/5q35 terminal deletion compound phenotype C. Castronovo (1) D. Rusconi (1) C. Gervasini (2) D. Milani (3) A. Cereda (3) L. Larizza (2) A. Selicorni (3) P. Finelli (1) (1) Istituto Auxologico Italiano (2) Università di Milano (3) Clinica Pediatrica De Marchi Pure constitutional deletions of the very distal 5q35 band have only been reported in a few cases. The pure 3.5 Mb subtelomeric deletion syndrome is characterized by muscular hypotonia in infancy, borderline intelligence, postnatal short stature, multiple minor anomalies and typical facial gestalt. On the contrary terminal deletions including the adiacent ~ 2 Mb NSD1-locus show a compound phenotype with overlap to Sotos syndrome, which is characterized by overgrowth, macrocephaly, developmental delay and distinctive facial features. Sotos syndrome results from mutations and deletions of the NSD1 gene, located at chromosome 5q35.3. A small percentage of NSD1 rearrangements (5–6%) is represented by exonic deletions/duplications; to date only 14 partial NSD1 deletions and 1 partial duplication have been reported in patients with typical facial gestalt in association with one out of three major criteria, namely overgrowth, macrocephaly, and developmental delay. All these rearrangements are de novo events and of unique size.

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We report on a 4 and half year old patient showing, at clinical evaluation, facial dysmorphism (frontal bossing, mild hypertelorism, deep set eyes, large nasal bridge, anteverted nostrils, malar hypoplasia, small ears with thick helix, thick phyltrum, macrostomia), normal growth and psychomotor delay. A high resolution CGH array analysis on the patient and his parents was carried out identifying a hemizygous de novo intragenic NSD1 deletion of 38 kb including part of intron 2 and exon 3. This novel exonic deletion affects part of NSD1 PWWP domain which is thought to be involved in proteinprotein interactions. The clinical features of our patient, not typical of Sotos syndrome, remind those underlying 5q35.3 deletion syndrome (including NSD1 deletion), being thus only partially explained by NSD1 intragenic deletion. We are testing the hypothesis that other mutations in 5q35 (point mutations and/or cryptic balanced microrearrangements) may dilute the Sotos features resulting in a combined phenotype. 1.79-P A new cryptic balanced rearrangement in 15q13 in autistic patients L. Larizza (1) M. Recalcati (2) C. Castronovo (2) M. Crippa (2) D. Rusconi (2) S. Russo (2) M. Masciadri (2) M. Della Monica (3) M. Bonati (4) P. Finelli (2) (1) Università degli Studi di Milano (2) Istituto Auxologico Italiano (3) A.O.R.N. G. Rummo (4) Istituto Auxologico Italiano Poster presentation Autism spectrum disorder (ASD) (MIM 290850) is a common and heterogeneous neurodevelopmental heritable disorder, characterized by language impairments, social and communicative deficits and restricted and repetitive behaviours. Linkage study and chromosomal rearrangements allowed autism (AD) to be associated with several susceptibility genomic regions, including 15q11– q13. Among chromosomal rearrangements reported in autistic patients, maternal duplications and small supernumerary inv dup(15q) markers, both involving 15q11–q13 region, are the most frequent, suggesting the presence in this region of dosage-

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sensitive genes that may contribute to the disorder onset. Given the relevance of 15q11–q13 in autism etiopathogenesis and overrepresentation in the region of segmental duplications, we looked for 15q11–q13 cryptic balanced microrearrangements in a cohort of 48 AD patients, diagnosed by quantitative ADI/ ADOS scales, as well as in 58 healthy individuals. Both groups underwent multicolour interphase FISH analysis by using a BAC probe panel specific for 15q11–q13 region. Nine out of 48 patients (18.8%) were found to carry a 15q13 1.5 Mb inversion either in heterozygous (8) or homozygous (1) condition. In 3 out of 9 inversion carriers it was possible to establish that the cryptic microrearrangement was inherited from one unaffected parent. Only 3 out of 58 healthy controls (5.2%) were carriers of the same heterozygous inversion. The difference between inversion frequency in AD patients and controls was statistically significant (P<0.01). The higher frequency of inversions in AD patients lends support to the hypothesis that this region may have a role in susceptibility to AD. In order to assess how the inversion may contribute to AD phenotype, we are currently testing whether or not this balanced rearrangement may slightly perturb the expression of genes flanking the inversion breakpoints by methylation and quantitative expression studies.

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she did not sit or walk and had no verbal contact though she showed visual and emotional contact. Epileptic seizures were recorded at 12, 18 and 19 months. The abnormalities were: low weight (7200 g) and delayed growth (73 cm); microcephaly (42 cm); thick and wide eyebrows, depressed nasal tip, short philtrum, micrognathia, high vaulted narrow palate, delayed teeth eruption; large, low-set, prominent and ‘simple’ ears with bilateral tags; clinodactyly of III-V toes with over-riding II toe; few small haemangiomas, nevi, and local patchy hypopigmentation; agenesis of corpus callosum, centrencepalic cyst, and white matter abnormality on MRI; horseshoe kidneys on ultrasound investigation. Both parents had normal karyotypes. The patient’s karyotype in cultured peripheral blood lymphocytes by conventional method was 46,XX,add(4)(p15). Duplication of 4p was established by mFISH analysis. However, FISH with TelVysion 4p DNA probe showed a subtelomeric deletion of 4p. Molecular-cytogenetic investigation revealed an interstitial duplication 4p with simultaneous subtelomeric deletion of 4p in this patient. High resolution chromosome analysis allowed to ascertain the patient’s karyotype as 46,XX,dup(4) (p16.2p13)dn. We suppose that patient’s phenotype mainly depends on subtelomeric deletion of 4p. 1.81-P Trilaminar germ approach: A girl of mosaic trisomy of chromosome 8 with hemangioma

1.80-P A case of dup 4p associated with a cryptic subtelomeric deletion 4p. T. Tsvetkova (1) G. Rudenskaya (0) T. Zolotukhina (0) N. Shilova (0) I. Mandron (0) (1) Research Centre for Medical Genetics of RAMS The patient, an only child of an Azebaijanian family, was examined at 20 months. The mother’s first pregnancy ended in an early spontaneous abortion. The girl was born of a second full-term pregnancy that threatened to abort at 13 weeks and showed fetal intrauterine growth retardation since 24 weeks. The birthweight was 2270 g, length 49 cm. After birth, the development was significantly delayed. At 20 months,

O. Cilingir (1) M. Ozdemir (1) B. Durak (1) M. Canturk (1) R. Emre (1) E. Satilmis (1) S. Artan (1) (1) Eskisehir Osmangazi University A 14 months old girl referred to medical genetic policlinic with cleft palate. She was a product of a term gestation pregnancy from a 31-year-old gravida 2, para 1, abortus 0 mother. Her birth length was 51 cm(50p), weight 3250gr(25–50p) and current length is 72 cm(10–25p), weight 6150gr (<3p), head circumference is 43.5 cm(3–10p). At her physical examination we found abnormalities consistent with mosaic trisomy 8 such as neuromotor growth retardation, dysmorphic craniofacial features, skeletal abnormalities and abnormal dermatoglyphics. In addition to these were abnormalities in cervical, thoracal, sacral regions; with

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variable sizes, correlated with cutaneous hemangioma, mild pink macular lesions were observed. Family history: uncle with epilepsy, first degree cousin marriage between her grandmother and grandfather. Chromosomal analysis was done on peripheral blood lymphocytes, skin biopsy and buccal mucosa smear cells. 47,XX,+8[31]/46,XX [24] chromosome constitution was found in cells from lymphocytes cultures and mosaicism ratio was found 56% for 47,+8 in cultured lymphocytes. Fluorescence in-situ hybridization using a chromosome 8-specific probe(D8Z1) showed evidence of mosaicism for trisomy 8 in the lymphocytes(48%), buccal mucosa smear cells(16%) and cultured fibroblasts(69%). According to our investigation, over 70 cases have been reported, and the estimated frequency for mosaic trisomy 8 syndrome is about 1:25,000 to 50,000 births. The clinical features of our case correlated well with literature but additionally we observed hemangiomas which has not been reported. The severity of the clinical features is dependent on the proportion of trisomic cells in endodermal, mesodermal and ectodermal tissues. So, we think it is necessary to examine the ratio of three germ line in order to directly determine phenotype genotype correlation. Our case is important for determination of the mosaicism ratio of three germ line tissues. The patient should be periodically investigated for clinical features, especially with respect to the degree of mental retardation, autism, infertility and malignancy. 1.82-P Double partial deletions of proximal 21q and distal 22q in an adult mentally retarded patient derived from a maternal translocation F. Behjati (1) S. Ghasemi Firouzabadi (1) K. Kahrizi (1) H. Yazdan (4) S. Arzhangi (1) S. Banihashemi (1) H. Najmabadi (1) (1) University of Social Welfare and Rehabilitation Sciences (2) University of Social Welfare and Rehabilitation Sciences (3) University of Social Welfare and Rehabilitation Sciences (4) Welfare (Behzisty) Organization (5) University of Social Welfare and Rehabilitation Sciences (6) University of Social Welfare and Rehabilitation Sciences (7) University of Social Welfare and Rehabilitation Sciences

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We describe an 18 year mentally retarded Iranian male patient with partial monosomy for proximal 21q and partial monosomy for distal 22q. The patient’s karyotype using conventional cytogenetics, is 45, XY,-21, der(22)t(21;22)(q21.1;q13.33)mat. The mother was a carrier of a balanced reciprocal translocation described as 46,XX,t(21;22)(q21.1; q13.33). The parents are consanguineous, with six children, three normal and three affected (one expired). The patient has severe mental retardation, with clinical features characteristic of partial deletions of both proximal 21q and distal 22q. They include self mutilation, limited speech, developmental delay, antimongoloid look, epicanthal folds, dolichocephaly, full brow, thick lips, wide nasal bridge, hypertelorism, bulbous nose, absence of urula, large low set ears, and dysplastic toenails. In our patient, as most other reported cases, the partial monosomy 21 has resulted from an unbalanced translocation leading to the deletion of centromeric segment of chromosome 21. 1.83-P Transposition of great arteries associated with 15q deletion M. Orera (2) P. Blanco (1) J. Arades (1) R. Fernandez (1) (1) HGU Gregorio Maranon (2) CIRCAGEN Congenital cardiac malformations are present in 0.8% of live births. Transposition of great arteries (TGA) is the most common cyanotic cardiac malformation and accounts for 5–7% of all cardiac malformations. We present the case of a 28 years old pregnant woman that presented in the 30 week with polyhydramnios. The prenatal ultrasound showed TGA and paucity of fetal movements and brachycephaly. We performed a diagnostic and therapeutic amniocentesis where we studied chromosomal aneuploidies via FISH ( Aneuvysion, Vysis), and obtained a result compatible with normal female. The delivery was at 39+3 weeks, with a weight of 2.040 gm and APGAR 4/8. During the immediate neonatal period we observed marked hypotony with arthrogryposis of hands and feet. A new blood sample was obtained for karyotyping and FISH analysis for the probes SNRPN/PML/CEP 15 (Vysis). The conventional

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karyotype showed a deletion spanning 15q11–q15 that was confirmed by the deletion of SNRPN locus in one chromosome 15. The final result was 46,XX del(15)(q11–q15). To our knowledge this is the first association reported of 15q deletion and TGA. So far there are no cardiogenesis genes reported in this area. This case may indicate the presence of such gene/s in this location 1.84-P Interchromosomal insertion between chromosomes 2 and 5 in an infertile man with azoospermia O. Ozer (1) F. Sahin (1) U. Gul (2) Z. Yilmaz (1) (1) Baskent University Faculty of Medicine (2) Baskent University Faculty of Medicine Chromosomal rearrangements can result in reproductive problems such as infertility or habitual abortion. Interchromosomal insertions are the most common form of unusual chromosomal rearrangements. Previous studies have estimated the prevalence of insertions to be 1 in 80000. No notable difference in fertility between male and female insertional translocation carriers have been reported. A theoretical total of 10 different 2:2 segregations and 16 different 3:1 segregations can result from an insertional translocation during gametogenesis. Among all chromosomal rearrangements, the average risk of having an abnormal child is the highest in insertional translocations; with an average of 32% for the male and 30% for the female carriers. The viability of the conceptus depends on the size of aneuploid chromosome segments. Haploid autosomal length (HAL) can be used to determine aneuploid states. Here we report a 36-year-old male who was referred to our department because of infertility due to azoospermia. His karyotype was 46,XY,ins(2;5)(q13;q13.1q32) and Y chromosome microdeletion screening was normal. The patient was seen in a genetic counseling session and his pedigree analysis did not reveal recurrent miscarriages in any of the family members. We thought that the presence of the insertion might inhibit spermatogenesis and might be responsible for azoospermia. The high rate of chromosomal abnormalities in infertile patients strongly suggests the need

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for routine cytogenetic analysis, detailed pedigree analysis and genetic counseling. 1.85-P Familial submicroscopic rearrangement resulting in partial trisomy 2p and partial monosomy 8p M. Moreno-Garcia (1) J. Sánchez el Pozo (2) J. CruzRojo (2) G. Pérez de Nanclares (3) M. Fernandez Navas (1) B. Azqueta (1) A. Perez Muñoz (1) E. Barriro Miranda (1) (1) Hospital 12 de Octubre (2) Hospital 12 de Octubre (3) Hospital de Cruces Subtelomeric aberrations are a significant cause of idiopathic mental retardation and congenital anomalies. We report a familial subtelomeric rearrangement in a family with two patients with severe mental retardation and additional congenital anomalies. The index patient is a 19 year old boy with severe mental retardation, dysmorphic features, facial anomalies, foot syndactyly, camptodactyly, joint contractures and genital anomalies. High-resolution G-banding analysis showed normal results. Multiprobe telomere fluorescent in situ hybridization revealed a derivative chromosome 8 resulting in partial trisomy 2p and partial monosomy 8p. The patient inherited the derivative chromosome 8 from his father, who carries a cryptic apparently balanced reciprocal translocation involving the terminal regions of 2p and 8p. His paternal cousin is a 9 year old girl with severe mental retardation, dysmorphic features, facial anomalies, microcephaly and joint contractures. FISH studies using subtelomere specific probes 2p and 8p revealed the same derivative chromosome. The patient inherited the derivative 8 chromosome from her mother, who carries the apparently balanced cryptic reciprocal translocation. 1.86-P Chromosomal changes in a boy with disharmonic development and allaly I. Aganovic-Musinovic (1) S. Ibrulj (1) M. MackicDjurovic (1) (1) Medical faculty Univerziti

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We report the case of the boy with disharmonic development and allaly and a chromosome 1q deletion as well as a derivate of a chromosome 2. Segment 1q (32q→qter) has been translocated on 2 qter. Boy has been diagnosed to have disharmonic development at the age of three. Wide range of examination and analyses have been done, including EEG of the brain and audiogram, that have been within normal range. Possibility of articulation is present but allaly is existing. At age of nine, he went through observation in school for children with special needs; disharmonic development of speech and motor capacities has been registered. He recognizes colors but cannot name them. During evaluation of complex movement imitation for age of 6–10, he accomplishes the result for age of 3–4 years. Intellectual abilities are lower compared to age. He has been categorized like a person with combined disabilities that include disharmonic development and slow speech development. Chromosomal analysis was performed from cultures of peripheral blood lymphocytes, using GTG bend method, and revealed translocation t(1q;2q), that is present in all mitotic cells. Karyotypes of parents using the same method showed that they have normal chromosomes, so we consider this translocation to have occurred de novo. 1.87-P A 22q11.2 microduplication syndrome family; concurrent additional duplication pattern in LZTR-1 gene region may be the cause of clinical variability

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syndromes share some overlapping clinical features ranging from mild to severe abnormalities. Here, a 4 year-old male proband in a consanguineous family with mental retardation/learning and speech disability, delayed psychomotor development, hypotonia, congenital heart diseases and mild facial dysmorphism findings was presented. Family history revealed that one of his uncles was carrier of balanced t (14; 22) (q10; q10). Chromosome analyses of all healthy family members, including mother, father, and siblings of the proband revealed no abnormality. In fluorescence in situ hybridization (FISH) analyses with TUPLE1 probe, two signals (one of them was much more intense) in metaphase chromosomes and three signals in interphase cells were observed in proband which was reflecting interstitial duplication of 22q11.2 region. FISH analyses of mother and the healthy sibling revealed the same interstitial duplication pattern, which indicated a familial inheritance. For detecting the duplicated area on 22q11 region, Multiplex Ligation-dependent Probe Amplification (MLPA) analyses were performed. While mother and healthy sibling carried the same duplication pattern, proband’s analysis revealed an extra duplicated area in LZTR-1 probe region beside the others. LZTR-1 gene is a member of the BTB-Kelch superfamily which has a role in the Golgi network. The duplication of this region only in the proband may reflect the possible role of this region in clinical variability and pathophysiology of 22q11.2 microduplication syndrome. At this point, a large number of cases are necessary to resolve genotypic/phenotypic correlations in 22q11.2 microduplication syndrome. 1.88-P

D. Torun (1) S. Kozan (1) E. Tepeli (1) A. Uludag (1) A. Yesilyurt (1) M. Bahce (1) S. Guran (2) (1) Gulhane Military Medical Faculty (2) Gulhane Military Medical Faculty The chromosome 22q11 region is susceptible to rearrangements that are associated with congenital anomaly disorders and malignant tumors due to misalignments of low copy repeats (LCRs). Different congenital anomaly syndromes (e.g. DiGeorge/velocardiofacial syndrome (DG/VCFS), der (22), cat-eye syndrome (CES) and 22q11.2 microduplication syndrome) are caused by decreased or increased gene dosage for the part of chromosome 22q11. These

Identification of a small supernumerary marker chromosome in a case of Down syndrome. A. Lungeanu (1) A. Arghir (1) M. Budisteanu (2) S. Chirieac (1) G. Cardos (1) (1) Victor Babes” National Institute of Pathology (2) 2. “Prof. Dr. Alex. Obregia”Clinical Hopsital of Psychiatry, Bucharest, Romania Small supernumerary chromosomes (sSMC) are structurally abnormal chromosomes present in 0.043% of newborn children and in 0.288% of mentally retarded cases (Leihr T., Weis A., 2007).

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Here we describe a Down syndrome case that possesses a small supernumerary chromosome apart from 21 free trisomy in all peripheral blood lymphocytes. The case, a seven months old girl with facial and limb abnormalities, sever psychomotor retardation, growth failures and muscular hypotonia was referred for cytogenetic investigation for confirmation of the diagnosis of Down syndrome. The child was the product of the twelfth pregnancy of non-consanguineous parents. At birth, the mother was 42 years old. All the other brothers are healthy. Peripheral blood chromosome analysis was performed with motorized Zeiss microscope, Metafer4 AutoCapt images, Ikaros Metasystems karyotype and Isis Metasystems for FISH analysis softwares. Classical cytogenetics using GTG banding revealed a hyperdiploid karyotype with 48,XX,+21, +mar. The marker was about the same size as chromosome 20. Other cytogenetic techniques like NOR and CBG failed to add new information about the chromosomal origin of sSMC. Uptil now, FISH using WC 15(R), WC 21(R), MD di George T-box1 (22q11)/22qter (Kreatech-Poseidon), CTA −154 H4 (22q11.21) and CTD −2293P9 (17p13.3)(R)/RP11 14P20 (18p11.32) (G) BAC probes, excluded the involvement of the short arms of chromosomes 15, 17, 18, 21 and 22 as origin of sSMC, but confirmed once more the free trisomy 21. In conclusion, the chromosomal origin of sSMC identified in the presented case proved to be quite difficult and more investigations like reverse FISH and array-CGH will be performed for a detailed marker structure elucidation and accurate genotype/ phenotype correlation. Acknowledgements: PN II 42-130/2008 and PN0933.0203 /2009 Projects. We thank Maria Cristea and Ioana Borcan for technical support and DEXTER COM srl for financial support of poster presentation. 1.89-P Subtelomeric duplication of 5p in a patient with psychomotor delay. M. Ermakova (1) E. Dadali (0) T. Zolotukhina (0) N. Shilova (0) T. Tsvetkova (0) T. Malinovskaya (0) (1) Research Centre for Medical Genetics of RAMS

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The boy was the third child of a 28-year-old mother and 28-year-old father of Caucasian origin. Birth weight was 3000 g, length 50 cm. Since birth he showed a degree of psychomotor delay and some facial dysmorphisms. On clinical examination at 16 month psychomotor delay was noted: he did not sit or walk without support,was unable to make verbal contact and had spastic quadriparesis. The dysmorphic features were: proportionate growth retardation, microcephaly (41 cm), high-arched palate, micrognathia, short philtrum, ptosis, ephicantus, strabismus, congenital anomaly of optic nerve. The MRT of brain and visceral ultrasound examinations were not done. There was no family history of developmental delay and the parents were not consanguineous. Proband’s parents had normal karyotypes. The patient’s karyotype in cultured peripheral blood lymphocytes by conventional method was 46,XY,add(5)(p15.1). FISH analysis with wcp 5 DNA-probe showed an absence of additional material from another chromosome. FISH with TelVysion 5p DNA-probe revealed 5p subtelomeric duplication. Both conventional cytogenetic and molecular-cytogenetic analyses allowed us to ascertain the patient’s karyotype as 46,XY,dup(5) (p15.1p15.33)dn. Our results support that a subtelomeric duplication as well as a subtelomeric deletion may result in the phenotypic abnormalities. 1.90-P Incidence of syndrome Down during the period 1998-2008 period M. Mackic-Djurovic (1) I. Aganovic-Musinovic (1) S. Ibrulj (1) (1) , Medical faculty Univerziti of Sarajevo In the Center for Genetics of theMedical faculty in Sarajevo 3248 karyotypes from peripheral blood lymphocytes and using GTG bend techique have been analyzed during last ten years. During that time we have registrated 216 persons with Down syndrome. The most frequent was complete free trisomy 21, but we have also registrated the mosaic type and translocation type of Down syndrome. In our report we will present different types and female/male ratio of Down syndrome as well as a table that presents the incidence of Down syndrome during that period.

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1.91-P

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zygotic X chromosomes loss (normal and derivate) in different cell lines.

XX male with Robertsonian translocation (13;14) 1.92-P V. Galkina (1) V. Chernykh (1) T. Tsvetkova (1) N. Kosyakova (1) L. Kurilo (1) L. Shileiko (1) N. Shilova (1) T. Zolotukhina (1) O. Ryzhkova (1) A. Polyakov (1) (1) Research Centre for Medical Genetics of Russian Academy of Medical Sciences We report about 26-year-old XX male with a Robertsonian translocation (13;14). The patient had male genitalia with hypoplastic testes descended in the scrotum and no sign of undervirilization. His weight was 62 kg, height—164 cm. Intelligence was normal. Hormonal examination revealed elevated LH and FSH levels and normal testosterone level. Semen analysis showed severe oligoasthenoteratozoospermia. Chromosome analysis was done on cultured lymphocytes in accordance with standard procedures. FISH analysis was performed on metaphase chromosomes and interphase nuclei using standard protocols with DXZ1 and LSI SRY probes. DNA was extracted from peripheral leukocytes by a standard method. Molecular analysis was performed using multiplex PCRs with SRY, AMELX/AMELY, ZFY/ZFX, and 20 Yq STSs. Conventional cytogenetic examination showed 45, XX,der(13;14)(q10;q10) karyotype. FISH analysis has demonstrated a cryptic translocation of Yp11 material onto the Xp22 and also revealed mosaicism with the presence of two XX cell lines: SRY×1 (92%) and SRY×2 (8%). PCR amplifications were positive for SRY, AMELX, AMELY, ZFX, ZFY loci and negative for all analyzed Yq STS markers. The origin of Robertsonian translocation is still unevaluated because of blood samples from proband’s parents were not available for examination. This case of XX male syndrome is unusual because of: 1) X chromosomal mosacism; 2) uncommon Yp breakpoint localized distal to AMELY; 3) combination with Robertsonian translocation. Evidently the X-Y translocation in patient is a result of abnormal X-Y interchange during paternal meiosis I. We suppose that revealed X chromosomal mosaicism is due to several mutations: nondisjunction between sister chromatids of SRY-carrying X derivate during meiosis II resulted in formation of 47, XXSRY+XSRY+ zygote with following selective post-

Rare chromosomal abnormalities associated with mental retardation and multiple malformations M. Budisteanu (1) A. Arghir (2) S. Chirieac (2) G. Cardos (2) C. Burloiu (1) I. Minciu (1) A. Lungeanu (2) (1) Clinical Hospital of Psychiatry (2) “Victor Babes” National Institute of Pathology The mental retardation (MR) is one of the most frequent conditions (3% in general population). 30–40% children with MR have a chromosomal abnormality. In this paper we present five cases of rare chromosomal abnormalities associated with MR and multiple malformations. These cases are part of a national research project, aimed at studying mental retardation in children. First case is a 2-year-old girl with MR, tetraparesis, dysmorphic features, heart congenital malformation; the karyotype with GTG banding showed a 4p deletion, and FISH and array-CGH confirmed a 4p16.2–p15.3 deletion. The second case is a 5-year-old boy with sever MR, dysmorphic features, cardiac, ears and genital malformations; the cytogenetic studies showed a 3p14.1 deletion. Another case is that of a 3-year-old boy with sever MR, dysmorphic features, hypotonia, hypoacusia, hypogamaglobulinemia, cerebral dysmielination; the karyotype with GTG banding and FISH revealed a 18q21-qter deletion. The fourth case is a 6-year-old girl with sever MR, dysmorphic features, behavioural problemes; the cytogenetic studies showed a partial trisomy 18. Another case is that of a 1-year-old girl with a phenotype suggesting Edwards syndrome (sever psychomotor retardation, dysmorphic features, failure to thrive, cerebral malformation, epileptic seizures, arthrogriposis); the cytogenetic investigations showed a duplication of 18q. All these case are important in understanding of different genetic mechanisms involved in occurrence of mental retardation. Acknowledgments: The authors thank Prof. Jean Michel Dupont and Mrs. Dominique Blancho for kindly providing BAC-FISH probes andarray-CGH experiments.

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Also, we thank Mrs. Mioara Cristea and Mrs. Ioana Borcam for technical assistence. Financial support: PN II 42-130 Project, CAPACITATI 29/2007 Project, Bilateral Collaboration Project Romania-France “Brancusi” 1.93-P 22q11.2-Distal Deletion In A Patient With Complex Congenital Heart Malformation C. Fagerberg (1) J. Graakjaer (1) U. Heinl (2) A. Rasmussen (1) C. Brandt (1) (1) Vejle Hospital (2) Kolding Hospital 22q11.2-distal deletion is a recently described recurrent genomic disorder distinct from DiGeorge Syndrome and Velocardiofacial Syndrome. The 22q11.2-distal deletion is localized just telomeric to the frequent 22q11.2 deletion, and is thought to be mediated by low copy repeats. A phenotype is emerging with premature birth, pre- and postnatal growth retardation, mild psychomotor retardation and facial dysmorphism as the most frequent clinical features. Mild congenital heart defects have been reported in a few patients. We present a patient with a de novo 22q11.2-distal deletion and a complex congenital heart malformation, thus expanding the phenotypic spectrum. 1.94-P Infertility and miscarriage in t(13;14) Robertsonian translocation carriers T. Bulakbasi Balci (1) Z. Yilmaz (1) B. Haydardedeoglu (2) F. Sahin (1) (1) Baskent University Faculty of Medicine (2) Baskent University Faculty of Medicine Robertsonian translocations are among the most common balanced structural rearrangements seen in the general population with a frequency of about 1 in 1000 in newborn surveys. The great majority of balanced Robertsonian translocations involve two different chromosomes; those involving the fusion of homologs are very rare. Heterologous translocations can be transmitted through many generations of phenotypically normal individuals. However, trans-

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locations involving non homologous chromosomes are occasionally associated with repeated spontaneous abortions and male infertility. It is thought that in adjacent segregants and in some infertile males the translocation may disrupt spermatogenesis. Here we report 9 patients with Robertsonian translocations, all with rob(13;14). Four of these patients were female; three of them had recurrent miscarriages and one had infertility. The remaining 5 were male patients with infertility due to spermatogenesis defects. They were all phenotypically normal and their karyotypes were 45,XX,rob(13;14)(q10;q10) or 45,XY,rob(13;14)(q10; q10). In two couples, one with a female carrier and one with a male carrier, preimplantation genetic diagnosis (PGD) was performed. The couple with the male carrier had a healthy baby boy with a normal karyotype. In the second couple with a female carrier we did not achieve a pregnancy. The Robertsonian translocation rob(13;14), although harmless, often causes infertility or miscarriages. Even though this abnormality is of autosomal origin, its gametogenetic effects may lead to different outcomes in males and females. These aspects outline the nature of genetic counseling and guide through further management in couples with Robertsonian translocations. 1.95-P Rare carrier of three chromosomal aberrations t(1;18)(q42.1;q23),t(4;8)(p14;q22.3),inv(10) (p11q21) in a male with reproductive malfunctions N. Huleyuk (1) E. Turak (2) H. Akopyan (1) (1) Institute of hereditary pathology of AMS Ukraine (2) Transcarpathian medical genetic consultation institution A family with a newborn girl having defects characteristic for Wolf-Hirschhorn syndrome, has applied for investigation to medical genetic consultation institution. It was found that this newborn girl was born from the fourth pregnancy. Two previous pregnancies have ended with spontaneous miscarriage during first trimester, the third pregnancy ended with the birth of a healthy boy. Analysis of girl’s karyotype has revealed aberrations in two chromosomes, resulting in 46,XX, del(4)(p14), inv (10)(p11q21) karyotype. A cytogenetic investigation of parents and girl’s sibling was suggested. Father’s karyotype revealed three aberrations: balanced trans-

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locations t(1;18)(q42.1;q23) and t(4;8)(p14;q22.3), and a pericentric inversion inv(10)(p11q21), resulting into 46,XY,t(1;18)(q42.1;q23),t(4;8)(p14;q22.3),inv(10) (p11q21) karyotype. In the phenotypically healthy boy, two chromosomal aberrations were found: t(1;18) (q42.1;q23) and inv(10)(p11q21), resulting in 46,XY,t (1;18)(q42.1;q23),inv(10)(p11q21) karyotype. 1.96-P FSHR deficiency in a patient with a familial translocation t(2;8) A. Kuechler (1) B. Albrecht (1) A. Köninger (2) B. Hauffa (3) J. Gromoll (4) B. Horsthemke (1) (1) Institut für Humangenetik (2) Klinik für Frauenheilkunde und Geburtshilfe (3) Klinik für Kinder- und Jugendmedizin (4) Centrum für Reproduktionsmedizin und Andrologie We report on a 17-year old female patient referred to us because of a primary amenorrhoea. Her body measurements and childhood development were normal. She did not show dysmorphic signs. Endocrinological investigations revealed a hypergonadotropic hypogonadism. Breast and pubic development corresponded to Tanner stage 3. Laparoscopy showed normal ovaries but subsequent histological investigations revealed a disturbed folliculogenesis. A cytogenetically balanced translocation t(2;8)(p16.3or21; p23.1) was detected by chromosome analysis. The translocation was also found in her healthy mother. Her father had a normal male karyotype. Because of the endocrinological and histological results, a FSHR deficiency was suspected. Sequence analysis of the FSHR gene, which is located in 2p16.3, revealed a non-synonymous point mutation in exon 10 (Pro587His) but no wildtype allele. The mutation affects the 6th transmembrane domain and most likely results in inactivation of the FSH receptor. The mutation was also found in the father, but not in the mother. Molecular-cytogenetic analyses with two BAC probes derived from the FSHR locus showed the translocation to be unbalanced with one deletion breakpoint within the FSHR gene. To further characterize the deletion size, an array analysis was performed (GeneChip Human Mapping 250K Sty Array, Affymetrix). This revealed a deletion of approximately 163 kb in 2p16.3 involving exons 9 and 10 of the FSHR gene. The

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deletion on the maternal allele demasked the mutation on the paternal allele. In summary, the coincidence of two rare changes in our patient caused the clinical symptoms consistent with an FSH resistance. 1.97-P Rare case of balanced translocation (7;21) (q32;q22) and additional derived chromosome 21 in girl born after in vitro fertilisation M. (1) (2) (3)

Sobol (1) V. Badiuk (2) M. Cygankova (3) National T.Shevchenko University of Kyiv; 2. National University of Kyiv-Mohyla Academy National Children’s Hospital

A 7-year-old girl (proband) was referred for cytogenetic analysis because of congenital abnormalities. Proband was born after 36 weeks gestation to a 30 yearsold woman. Pregnancy was obtained by using IVF techniques. Caesarean section was performed at 36 weeks gestation because of fetal hypoxia. The newborns weight was 2250 g. Intrinsic jaundice was observed. At age 4 months periodic convulsions appeared but had passed by age 7 months after drugs treatment. Head computed tomography showed brain pathology. Physical examination at age 7 years showed the following abnormalities: mental retardation, macrocephaly (head circumference of 53 cm), posteriorly rotated ear auricle, small teeth, spastic inferior paresis. The growth of theproband was within the normal range. Deficiencies of visual system were not observed. Abnormalities evoked the diagnosis atypical or mild Down syndrome. G-banding analysis on proband’s metaphase chromosomes from cultured peripheral blood lymphocytes revealed balanced translocation (7;21) and additional derived chromosome 21. It gave rise to parent’s karyotyping. Father had normal male karyotype. Mother carried balanced translocation: 46, XX,t(7;21)(q32;q22). The couple passed IVF procedures for two times but karyotype was not done. History: the woman had five pregnancies. First and second pregnancy were terminated at woman’s option. The third pregnancy was obtained by using IVF techniques but it resulted in spontaneous abortion. The fourth pregnancy resulted in the proband’s birth. In both cases own woman’s oocytes were used for IVF. The fifth pregnancy had naturally occurred, and a boy with normal male karyotype was born.

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Consequently proband’s karyotype is 47,XX,t(7;21) (q32;q22)mat,+der(21)t(7;21)(q32;q22), and the patient has partial trisomy 7q and 21. Such combination of chromosome abnormalities is quite rare especially among children born after IVF. We deal with a failure of principle of couple’s preparation for IVF that leaded to the birth of ill child. 1.98-P The Spectrum of Phenotypes in Females with Balanced X-Autosome Translocations

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TUBGCP5, CYFIP1, NIPA2and NIPA1 that might be causative for the girl’s phenotype. Our patients illustrate the spectrum of phenotypes in females with balanced X-autosome translocations. None of them exhibit Turner stigmata or known X-linked disorders. We discuss our findings with respect to the literature. Furthermore, array-CGH helps to detect relevant genes which may be important for interpretation of indistinct phenotype and allowing new insights into cytogenetic diagnosis. 1.99-P

I. Dietze-Armana (1) M. Schneider (1) G. Rettenberger (1) K. Mehnert (1) (1) genetikum (2) genetikum (3) genetikum (4) genetikum Females with balanced x-autosome translocations represent a clinically heterogeneous group depending on the x breakpoint position and replication behaviour of the x chromosomes. they may be fertile, have Gonadal dysgenesis, reveal multiple congenital anomalies and mental retardation or suffer from an x-linked disorder (Schinzel 2001). Here we report clinical, cytogenetic and molecular data of five cases with apparently balanced xautosome translocations. Case 1 t(X;2)(q22.1;q31.1) and case 2 t(X;15) (p22.1;q13) are apparently de novo balanced translocations ascertained at amniocentesis. At about 3 years both girls are phenotypically normal. However, long-term follow-up with respect to mental and physical development and fertility is required. Case 3 with t(X;7)(q22.1;q42) was present in a 34 years old woman with small uterus, delayed menarche at 17 years and irregular ovulation. An association between ovarian dysgenesis/ovarian failure and abnormalities of the X chromosome has been extensively reported in the literature. Case 4, 16G/2P, is a 30 year old phenotypically normal women with irregular menstruation and increased genetic risk for chromosomal imbalance in her offspring. Chromosome analysis revealed a mosaic karyotype with t(X;2)(p11.4;q42). Case 5, a 4 years old girl with profound speech delay and mental retardation revealed a t(X;22)(p22.1; q22.1). Array-CGH detected normal profiles for chromosomes X and 22, but a novel 300 kb deletion in 15q11.2 including four not imprinted genes

Clinical and molecular characterization of the 17q21.31 microdeletion syndrome in 11 French patients with mental retardation C. Dubourg (1) J. Andrieux (2) M. Doco-Fenzy (3) C. Missirian (4) C. Le Caignec (5) S. Jaillard (6) C. Schluth-Bolard (7) E. Landais (3) A. Moncla (4) D. Sanlaville (7) (1) CHU Pontchaillou (2) Hôpital Jeanne de Flandre (3) CHRU (4) Hôpital d’Enfants de la Timone (5) CHU (6) CHU Pontchaillou (7) Hospices Civils de Lyon Array comparative genomic hybridization has recently led to the characterization of novel microdeletion and microduplication syndromes like the 17q21.31 microdeletion syndrome. Here we report the clinical and molecular characterization of 11 French patients with mental retardation and with the 17q21.31 microdeletion syndrome. We also present here the genotyping for H1/H2 and parent-of-origin analysis. Several clinical features such as moderate mental retardation, childhood hypotonia, low birth weight and facial dysmorphisms (long face, tubular or pearshaped nose and bulbous nasal tip) are common. Other inconstant clinically features include epilepsy, heart defects and kidney/urologic anomalies. The described 17q21.31 critical region covers 424 kb and encompasses five reference genes (CRHR1, IMP5, MAPT, STH and KIAA1267). We report here a smaller ~ 200 kb deletion which encompasses only MAPT, STH and KIAA1267. This narrowing of the critical region point out the MAPT gene as candidate gene, especially since MAPT has been associated with several neurodegenerative disorders.

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All these deletions arise de novo. A 900 kb inversion polymorphism exists in the 17q21.31 region and chromosomes with the inverted segment in different orientations represent two distinct haplotypes, H1 and H2. These different orientations are likely to facilitate the generation of the microdeletion through an established mechanism of NAHR and the offspring of carriers of the H2 lineage are predisposed to deletion. In each trio tested, the parent-of-origin of the deleted chromosome 17 carries at least one H2 chromosome and, for informative cases, 2/3 out of deletions were of maternal origin and 1/3 of paternal origin. 1.100-P A de novo partial monosomy 10q in one of IVF twins A. Kuskucu (1) K. Yararbas (1) M. Celik (2) C. Aydogmus (2) H. Aldemir (2) (1) Dept. of Medical Genetics Bakirkoy Women’s & Children’s Health Education Research Hospital, Istanbul TURKEY (2) Dept. of Pediatrics Bakirkoy Women’s & Children’s Health Education Research Hospital, Istanbul TURKEY Deletions of long arm of chromosome 10 is a rare entity and the breakpoints show a heterogeneous pattern. Phenotype usually shows frontal bossing, broad nasal bridge, cleft palate, micrognathy and other facial features next to growth retardation, mental deficit, cardiac and urinary abnormalities. We present one of IVF twins with de novo partial monosomy 10q with dysmorphic features, tatralogy of Fallot, duodenal atresia and recurrent pneumonia. 1.101-P A Unique Complex Translocation in a Child With Dysmorphic Features S. Artan (1) B. Durak (1) M. Ozdemir (1) C. Yarar (2) M. Muslumanoglu (1) H. Aslan (1) E. Satilmis (1) (1) Eskisehir Osmangazi University (2) Eskisehir Osmangazi University Complex chromosome rearrangements are rare structural abnormalities that involve at least two chromosomes and more than two breakpoints and are often associated

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with developmental delay, mental retardation and congenital anomalies. We report de novo, apparently balanced translocation t(18;8;12) involving three chromosomes with four breakpoints in a 3-year old girl with profound motor and mental retardation, seizures, craniofacial dysmorphism, developmental delay. During prenatal period, cleft palate and IUGR were noted by obstetric ultrasonography. Diffuse cerebral atrophy was determined by cerebral MRI. PFO was detected by ECO. The physical examinations of the parents were normal. Conventional cytogenetic analysis revealed an apparently balanced 46,XX,t(18;8;12). Molecular cytogenetic analysis with whole chromosome, sub-telomeric and locus specific FISH probes showed breaks at 18q21.3, 18q22?, 8p22 and 12q13?. Further molecular analyses were performed to determine possibility of any genomic unbalance as a result of complex rearrangement. To our knowledge, a complex translocation including these chromosomes has not been reported. The patient has features similar to cases with partial deletion of chromosome 18q. However detailed molecular analysis has shownshow us the molecular pathology in the breakpoints for the clinical features of the case. 1.102-P A familial 18q23 deletion transmitted from mother to two daughters E. Margarit (1) C. Morales (1) A. Soler (1) N. Clusellas (1) A. Sánchez (1) (1) Hospital Clinic de Barcelona The deletion of the long arm of chromosome 18 constitutes one of the commonest deletion syndromes, occurring in 1/10000 live births. Clinical features are variable, usually related to the extension of the deleted region, and can include: mental retardation, short stature with growth hormone deficiency, delayed bone maturation, hypotonia, midfacial hypoplasia, microcephalia, hypertelorism, congenital aural atresia with hearing impairment and feet abnormalities. Here we report a family with an 18q23 deletion transmitted from the mother to two daughters, all three with short stature. The first daughter, aged 6, was referred for karyotyping because of short stature and developmental delay, with poor growth since birth, bone delayed maturation and GH hormone deficiency. The second child, aged 2, also had growth deficiency and developmental delay. Additional-

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ly, she had respiratory difficulties, hyperplasic thymus and laringomalacia. Hormonal studies revealed slightly altered levels of ACTH and cortisol hormones. The two children and their mother were found to have an 18q23 deletion visible at conventional cytogenetic analysis. Additional FISH (fluorescence in situ hybridisation) studies using subtelomeric and painting probes for chromosome 18 confirmed our cytogenetic observations and excluded rearrangements with other chromosomes. Although the mother has not been yet carefully examined by a clinician, she has low stature (149 cm) like the daughters. Additional analyses are in progress using molecular techniques that will allow delineating precisely the extension of the deletion in the three individuals. A more detailed and focused clinical examination will facilitate a good phenotype-genotype correlation and genetic counselling for the family members. Moreover, all these data, added to the information reported so far, will eventually facilitate the genetic counselling for new detected cases and also the search for candidate genes controlling the phenotypic traits of the syndrome. 1.103-P Inherited complex translocation involving three chromosomes V. Lima (1) S. Dória (1) C. Madureira (1) S. Pereira (1) P. Xavier (2) A. Barros (1) (1) Faculty of Medicine (2) Hospital de São João EPE Introduction: Complex Chromosomal Rearrangements (CCR) occurring in phenotypically normal persons are rare. Three or more chromosomes chromosomes are involved and a considerable variety of rearrangements is possible. CCR are usually considered to induce severe reproductive impairment by disturbing the meiotic process and producing unbalanced gametes responsible for high reproductive risk. Most of CCR are reported to be de novo. Material and Methods: The proband is a 35 years old woman referred for infertility. She is one of the four sibs from unrelated parents. The three older sibs are two females (41 and 47 years old) and one male (44 years old), all are married and with children. Chromosome analysis was performed from PHA-stimulated blood lymphocytes cultures, using GTL banding, according to standard techniques. FISH studies were carried out using whole chromosome painting and centromeric probes.

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Results: Cytogenetic analysis revealed a female karyotype with an apparent balanced reciprocal translocation involving three derivative chromosomes, 46,XX,t (4;9;11)(q21.3;q32;q21). The same CCR was present in the father and in the eldest sister. FISH studies with WCP4, WCP9 and WCP11 show one additional signal in the distal long arm of the derivatives chromosome 11, chromosome 4 and chromosome 9, respectively. Centromeric probes complemented these results. Discussion: We report the identification of a balanced paternally inherited CCR in a phenotypically normal woman with infertility. The same CCR was found in a healthy sister with one child. This rearrangement involves three chromosomes and three different breakpoints. CCR are at high risk of producing unbalanced gametes, leading to infertility and spontaneous abortion if the segregation results in an unbalanced zygote or to a live born child with multiple congenital malformations if the unbalanced zygote is viable. Our data show that offspring with normal karyotype or with the same balanced CCR as the parent can also occur. This means a good prognosis for genetic reproductive counseling. 1.104-P 46,XX,i(19) karyotype in a case of cerebral ventriculomegaly M. Pérez Sánchez (1) A. González Ramírez (2) A. Mora Guijosa (1) (1) Hospital Virgen de las Nieves. (2) Hospital Universitario San Cecilio Sometimes the finding of a chromosomal alteration can cause problems for offering correct genetic counselling because the chromosomal anomaly may be specific for a patient or for a family. We describe here a case of a 46, XX, i(19) karyotype in a new born female with cerebral ventriculomegaly without any other phenotypic anomaly. A foetal ventriculomegaly with no other anomalies was detected during a routine prenatal ultrasound examination at 20 weeks of gestation. An amniocentesis for prenatal diagnosis was performed. The karyotype of the foetus was 46,XX,der(19). The parents had a normal karyotype (46,XX and 46,XY). The patient decided to continue the pregnancy. At the next ultrasound examination the ventriculomegaly had not increased in size and no other anomalies were detected. Intrauterine

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growth retardation, with lacunar placenta and probably foetal malnutrition were detected at 29 weeks of gestation. It was decided to deliver the foetus. The newborn was an apparently normal premature female. Karyotyping was done to confirm the prenatal findings and the abnormal chromosome 19 was identified as an isochromosome of the long arm of chromosome 19, 46,XX,i(19). The patient was hospitalized in the postnatal period with systemic infection and respiratory distress. Medical examination at the age of 8 months, showed that the ventriculomegaly was still present but no other anomalies were detected. There was normal growth and spicomotor development. The patient’s development will be followed in the future to watch out for any problems that could be attributed to her genetic status. The possible effects of this genetic anomaly and the problems associated with a good genetic counselling will be discussed.

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(D14S1012, D14S1046, D14S1069) for SPTB gene localization and found out positive LOD-scores and determine the max LOD-score as 4.62. Conclusions: Linkage analysis is an effective, cheap and quick pre-screening method in genetically heterogeneous diseases. There is no way, by examining the phenotype, to find out the gene responsible for the genetically heterogeneous diseases like HS. After the prescreening, DNA sequence analysis will be the best method to determine the disease. 1.106-P Case report: paracentric inversion of chromosome 1 in a man with azoospermia M. Virdis (1) V. Licheri (1) F. Spina (1) R. Murru (1) L. Martorana (1) A. Azzena (1) S. Deidda (1) L. Balestrino (1) S. Orrù (1) C. Carcassi (1) (1) University of Cagliari

1.105-P Linkage analysis of a four generation herediter spherocytosis family with spectrin deficiency P. Tasdemir (1) N. Akarsu (2) S. Demirel (3) (1) selcuk university (2) Hacettepe University Medical Faculty (3) selcuk university Medical Faculty Background: Hereditary Spherocytosis (HS) is an inherited hemolytic anemia caused by the defects on membrane proteins and characterized by icterus, anemia and splenomegaly. Hereditary Spherocytosis clinically and genetically comprises a heterogeneous group of hemolytic anemias. Methods: We tried to find out the linkage to the gene which causes autosomal dominant HS in a large family by using LINKAGE analysis. Here we report a four generation Turkish family consisting of 86 individuals of whom 19 are affected. The linkage to the major genes in hereditary spherocytosis which is SPTA1, SPTB, ANK1 ve SLC4A1 was searched by using LINKAGE and MERLIN analysis. Results: We pointed out the linkage to the SPTB gene which takes an important role in the formation of autosomal dominant HS. The SPTB gene, synthesis β spectrin protein and maps on 14q12–q21 choromosomal region. We choose three microsatellite markers

The correlation between chromosomal rearrangements and impaired sperm is well-known and it is due to an interference with chromosome synapsis during meiosis. In rearrangement carriers, such a correlation may depend on the chromosomes involved in the rearrangement and on their breakpoints. Pachytene configurations of some structural rearrangements may more directly affect the progression of meiosis and the production of gametes. However, the female meiotic checkpoint process seems to be less efficient than the male analogous mechanism (LeMaireAdkins et al., 1997). Disturbances in the pairing process can (adversely) block the male meiotic progression. The commonest rearrangement among the infertile males was reported to be inv(1), observed in 6,7% of the examined individuals, with a strong prevalence of pericentric inversions. The highest number of reported breakpoints among the infertile males was found on 1q21 (Bache et al. 2004). We report on an 35 years-old infertile patient presenting with azoospermia. Ultrasound exam revealed normal deferent ducts, while the testicular biopsy showed germinal aplasia. He did not present any other relevant disease and had not a relevant familial history. The hormonal dosages (assays) were normal. The GTG banding karyotype revealed a paracentric inversion of chromosome 1: 46,XY,inv(1) (q21q32).

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The clinical history of the patient gives evidence of a primitive testicular defect affecting gametogenesis, without any other cause apart from the chromosomal rearrangement, that is thereby strongly suspected to be the responsible of the disturbance. This observation is coherent with data reported by Bache et al., which suggest that chromosome 1 harbours a critical domain whose integrity is essential for male fertility, with a special suspect for region 1q21. Consequently chromosome 1 region 1q21 should be object of further investigations to elucidate a possible correlation with male infertility. 1.107-P Clinical and cytogenetic finding in a patient with 45,X/47,XXX mosaicism. A case report

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thought the higher frequency of the monosomic cell line causes, however, a Turner Like phenotype. It is now important to confirm the absence of cell line 46,XX and to establish the correct percentage of the mosaicism in the other tissue because it is known that the degree of development and the presence of malformation are both influenced by the proportion and distribution of the haploid cell line. Other investigations are now in progress in order to better define the phenotypic picture: skin fibroblast karyotype, microsatellite analysis to determine the parental origin of the X chromosome involved in the nondisjunction event, cardiac ultrasonography to evaluate the presence of congenital heart defects, hormone assay to study functional activity of the hypothalamus-hypophysis axis. 1.108-P

F. Meloni (1) S. Deidda (1) R. Murru (1) L. Martorana (1) A. Azzena (1) V. Licheri (1) A. Sammarco (3) A. Milia (2) S. Orrù (1) C. Carcassi (1) (1) University of Cagliari (2) University of Cagliari (3) University of Cagliari Turner syndrome is a common chromosomal abnormality affecting 1:2000 live female births. The syndrome is characterized by growth retardation, ovarian dysgenesis, and phenotypic abnormalities. The rare 45,X/47,XXX mosaicism karyotypes affects 1,7% of patients with Turner syndrome and is associated with a milder phenotype than usual. We reported a case of 17 years old patient who underwent cytogenetic analysis because of the presence of primary amenorrhea. The clinical examination showed features of hypogonadism such as absence of secondary sexual characteristics and short stature. A laboratory evaluation demonstrated the presence of a subclinical hypothyroidism. A pelvic ultrasonography revealed unfunctional ovaries and thin uterus. Both familial and pathological history were negative. Peripheral blood karyotype revelead the presence of two different cell lines 45,X/47,XXX (86%,14% respectively). The origin of mosaicism was consistent with a mitotic nondisjunction event in a disomic zygote. The absence of a 46,XX cell line led us to hypothesize that this event has happened during the first post zygotic division, even if the two cell lines are not present in equal proportions. The presence of cell line 47,XXX mitigated the patient’s phenotype even

Atypical deletion in the Prader Willi region A. Nordgren (1) J. Lundin (1) H. Malmgren (1) M. Lehtihet (1) B. Anderlid (1) (1) Karolinska Institutet Prader-Willi (PWS) syndrome is characterized by obesity, cognitive impairment, hypotonia, hypogonadism, short stature and a distinctive behavioural phenotype including an obsessive–compulsive personality. As neonates the PWS patients have severe hypotonia and great feeding difficulties, but between 18–36 months of age they become hyperphagic and obese. Short stature is common as well as characteristic dysmorphic features including almond shaped eyes, strabismus, small hands and feet and scoliosis. Non-insulin-dependent diabetes mellitus often occurs. PWS is caused by abnormal parental specific imprinting in the PW critical region, and the majority of cases are resulting from the deletion of paternal copies of several imprinted genes, making PWS a contiguous gene syndrome. Two common classes of deletions of bands 15q11.2–q13 have previously been described in individuals with PWS; one between BP1 to BP3 including seven genes and the other between BP2 to BP3 including four genes. Deletion sizes vary between 4 and 8 Mb. Small deletions comprising only, or parts of, SNRPN have been reported as well as deletions and imprinting defects in mosaic state.

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We have performed a 244 kb Agilent-array in a 38-years old male with cognitive function and behaviour within the normal range, obesity, type 2 diabetes, hypogonadism and small hands and feet as part of an endocrinology evaluation. An 847-kb deletion including the SNRPN gene and one open reading frame, C15ORF2, was found. Further analyses revealed normal imprinting in the region, and the deletion was mosaic and present in 58% of leucocytes analysed. Microsatellite analysis could not detect parental origin since the patient was homozygous for one of the markers and the other marker in the region indicated bi-allelic inheritance. In addition, the results are difficult to interpret because of the mosaic state of the deletion. The phenotype of our patient is well concordant with P-W syndrome. However, his cognition is within normal limits and there was no neonatal hypotonia or feeding problems. The mild phenotype is most probably due to the mosaic deletion in combination with the normal imprinting pattern. 1.109-P Microduplication of 700 kb within Xq26.2q26.3 in a developmentally retarded 5-year-old boy L. Bernardini (1) P. Prontera (2) A. Capalbo (1) A. Mencarelli (2) M. Giuffrida (1) I. Manes (2) C. Gradassi (2) B. Dallapiccola (1) E. Donti (2) (1) CSS Mendel Institute (2) University of Perugia Interstitial microduplications within Xq26q27 chromosome bands are very rare and hemizygous males show a form of X-linked mental retardation with or without isolated growth hormone deficiency or hypopituitarism, probably due to an increased dosage of the SOX3 gene, which maps at Xq27.1. Here we describe a 5-years-old male patient showing global developmental and growth delay in whom molecular cytogenetic analysis disclosed a microduplication on Xq. The proband was born at 36-weeks gestation by caesarean section following a diagnosis of oligohydramnios and IUGR. Developmental milestones were delayed and height, weight and OFC steadily remain under the 3rd centile. Clinical history also includes convulsive episodes, dilatation of brain ventricles, hypoplastic corpus callosum, delayed bone age and GH deficiency.

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High-resolution G-banding karyotype performed on peripheral blood lymphocytes revealed a normal male karyotype, whilearray-CGH analysis with a resolution of 100 Kb (44K chip; Agilent Technologies) disclosed a duplication of about 700 Kb at Xq26.2q26.3. FISH analysis with locus-specific probes carried out on metaphases revealed a duplication both in the proband and his apparently healthy mother. The duplicated region encompasses 12 RefSeq genes, including PHF6 (OMIM 300414) but not SOX3. PHF6 encodes a protein with unknown function and loss of function (LOF) mutations affecting its coding regions are causative of Borjeson-Forssman-Lehmann syndrome (BFLS), a disorder characterized by mental retardation, epilepsy, hypogonadism, hypometabolism and obesity. In conclusion, our observation suggests that a) an increased dosage of gene/s located at Xq26.2q26.3, thus not including SOX3 locus, could be responsible for a form of X-linked mental retardation associated with GH deficiency; b) the clinical phenotype characterized by relevant weight loss (hypermetabolism?) could be due to gain of function mutations of PHF6 gene; c) this evidence, together with the hypometabolism and obesity associated with LOF mutations, suggests a role of PHF6 on regulation of basal metabolism. 1.110-P Do chromosomal polymorphisms have any impact on reproductive outcome? M. Gaytan Muñoz (1) F. Bronet Campos (1) E. Martinez (1) R. Herrer (1) M. Ariza (1) A. Liñan (1) J. Landeras (2) M. Martinez (2) J. Garcia-Velasco (1) (1) IVI Madrid (2) IVI Murcia Objective: Chromosomal alterations in males can affect spermatogenesis. These patients could transmit chromosomal imbalances to descendants and cause miscarriage and/or newborns with malformations. Patients under assisted reproduction treatment occasionally show chromosomal polymorphisms in their karyotype, and it is not clear whether these alterations do or do not have any effect on seminal parameters and/or in the success rate in assisted reproductive cycles. Design and method: We performed high resolution karyotypes on 109 males under assisted reproduction treatment from our clinic, carrying sex chromosomal

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polymorphisms (n=34, group 1), acrocentric polymorphism (n=36, group 2) or heterochromatin polymorphisms (n=39, group 3). As a control group we selected 199 males with similar seminal parameters. Partners of women over 39 years of age were excluded. We evaluated sperm concentration, motility and morphology, recovery of capacitated spermatozoa, fertilization rate, implantation failure and previous miscarriages. Statistical analysis was performed by analysis of variance (ANOVA) for those variables with normal distribution and Kruskal-Wallis test for the rest of variables (significance p<0.05). Results: Male carriers of chromosomal polymorphisms showed normal sperm concentration (>20 million per ml). All groups showed a decrease in sperm motility (33%, 34% and 38% for groups 1, 2 and 3 respectively) when compared with the normal values established by the World Health Organization (WHO), thus indicating astenozoospermia. We did not find statistical differences when fertilization rate, implantation failure and number of previous miscarriages were compared with the control group. Conclusions: In summary, sperm motility was the only parameter showing significant differences with respect to the WHO criteria, although these patients did not show a decrease in fertility rate. We did not find differences for the rest of parameters. 1.111-P

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retardation, hyperactivity, and obesity. She was the second child of non consanguineous parents who had no previous genetic history. The girl started to walk at 18mo, and to talk at 48mo. She presented subtle abnormal phenotypic features, such as downslanted palpebral fissures, small mouth with lifted upper lip, fifth fingers clinodactyly, and obesity. She was a lively, hyperactive and very talkative child. Psychological tests revealed IQ=66 (mild mental retardation). Neurological examination did not show focal CNS damage. Head MRI disclosed the enlargement of liquid spaces of the brain and subtle asymetry of lateral cerebral ventricles. GTG-banded karyotype analysis revealed presence of small, dicentric marker chromosome. Both parents presented normal karyotypes. For better identification of the marker’s origin FISH analysis with whole chromosome painting probe (wcp15) and Prader-Willi/ Angelman syndrome critical region probe (PWSASCRSNRPN) was performed. FISH showed two adjacent PWSASCR signals on the marker chromosome. The karyotype: 47,XX,+psu idic(15)(q11.2)(15pter➔ cen➔15q11.2::15q11.2➔cen➔15pter)[30]. ish(wcp15+)[10].ish(15q11.2x4)[10], and a cytogenetic diagnosis of 15pter-15q11.2 tetrasomy were then established. The SNRPN methylation pattern was normal, the microsatellite analysis showed the maternal origin of the marker. In contrast to the patients described by others, our patient did not show autistic or stereotypic behavior, nor did she have epilepsy. She was only mildly retarded, with subtle dysmorphic features.

Tetrasomy 15q syndrome—case report 1.112-P M. Drozniewska (1) T. Janiszewska (2) O. Haus (1) (1) Collegium Medicum in Bydgoszcz, UMK in Torun (2) J. Biziel University Hospital

Genotype-phenotype correlations in two large deletions 13q

Small supernumerary marker chromosomes (SSMC) are often associated with developmental delay and malformations. They can occur in 0,05% of postnatal cases. Among them, the most common is inv dup(15) [also known as idic(15)], that can represent up to 50% of all SSMC cases. The phenotype of tetrasomy 15q carriers may vary from non affected to severely retarded depending on the size and type of marker. SSMC(15) are generally of maternal origin. The presence of additional copies of 15q11–13(PWSASCR) strongly correlates with autistic behavior in children. We report on a 5-years old girl who was referred to the genetic counselling clinic by a neurologist because of mental

Tosca L1,2,3, Latour S4, Metay C5, Guerin N1, Toujani S1, Lebas A6, Senat MV7, Goossens M5, Tachdjian G1,2,3 and Brisset S1,2,8 1 AP-HP, Cytogénétique, Hôpital Antoine Béclère, Clamart, F-92140 2Univ Paris Sud, Le KremlinBicêtre, F-94276 3INSERM U935, Villejuif, F-94801 4 APHP, Pharmacie, Hôpital Antoine Béclère, Clamart, F-92140 5APHP, Plateforme de Génomique IMRB 955, Hôpital Henri Mondor, Créteil, F-94010 6AP-HP, Neuropédiatrie, Hôpital Bicêtre, Le Kremlin-Bicêtre, F-94275 7AP-HP, Gynécologie Obstétrique, Hôpital Antoine Béclère, Clamart, F-92140 8INSERM U782, Clamart, F-92140

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13q- syndrome is caused by structural 13q regions abnormality associated with varying phenotypes. Current clinical features include moderate-severe mental/growth retardation, craniofacial dysmorphy, abnormalities of the extremities and brain defects. We report on two independent deletions of the long arm of chromosome 13. The first case corresponded to a prenatal diagnosis for exencephaly and acrania detected during the first trimester ultrasound. The second case corresponded to a 3-year-old girl referred to our laboratory because of psychomotor/developmental delay, behavior and walking difficulties. She was tall (+3SD) and presented an anteverted basin. Trophoblast chromosome analysis in the first case identified a 46,XY,del(13q) karyotype. Fluorescent in situ hybridization (FISH) experiments showed deletion of 13q subtelomere probe and of LSI 13q14 locus. Using BACs probes, we identified a large and terminal deletion (75–80 Mb) del(13)(q13.3qter). In the second case, a 46, XX,del(13q) karyotype was found. FISH studies showed LSI 13q14 deletion. Use of BACs probes showed an interstitial deletion with proximal and distal breakpoints in 13q13.3–13q14.2 and 13q14.2–13q21.32 regions, respectively. Array comparative genomic hybridization (arrayCGH) on the DNA of the propositus is under current investigation in order to precisely define the breakpoints and to identify the deleted genes. Comparison of our cases with previous reports of 13q deletions underlines the importance of 13q22–34 region in Dandy-Walker malformation (encephalocoele and hypoplasia of the vermis cerebelli) and neural tube defects.

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families. Most phenotypes associated with 6q duplication included moderate mental delay and facial dysmorphisms (hypertelorism, epicanthic folds). Renal malformations were reported in two cases and obesity was described in one family with 6q21.q22 duplication. We report a six members family in which the mother and two (male and female) children had similar phenotypes with facial dysmorphism (sloping forehead, epicanthus), gross obesity (W>3SD) and mild mental delay. The boy suffered also from chronic renal failure and had dysplastic kidneys. The two other children had normal mental development and the father had normal weight. Classical cytogenetic analysis showed an interstitial duplication of a 6q segment in the first three patients. Array-CGH confirmed and sized the duplication to 6q16.1q21 region (16.80 Mb). Investigations in the other children showed the presence of a smaller duplication implicating only the 6q16.3q21 segment (7.05 Mb). In order to characterise the breakpoints of the rearrangements, mono-locus FISH and M-banding were realized and displayed, in the first three patients, two separate duplicated segments 6q16.1q16.3 and 6q16.3q21, and only one duplicated 6q16.3q21 segment in the two other children. The segments were inserted in the 6q25q27 region. In conclusions, we report the first case of an interstitial 6q16.1q21 duplication, transmitted totally or partially to the offspring, associated with obesity, facial dysmorphisms and mild psychomotor delay, and characterised using array-CGH. 1.114-P

1.113-P Familial 6q16.1q21 duplications investigated by array-CGH and M-Banding M. Doco-Fenzy (1) É. Landais (1) S. Brunet (1) V. Koubi (2) P. Kleinfinger (2) P. Souchon (3) M. Beri (4) P. Jonveaux (4) R. Garnotel (5) D. Gaillard (1) (1) CHRU-Reims (2) Pasteur Cerba (3) CHRU-Reims (4) CHRU-Nancy (5) CHRU-Reims Duplications of the chromosome 6 long arm were reported in more than 30 cases and concerned mostly distal 6qter regions. In only four cases interstitial duplications of the 6q13–q22 segment was reported. Familial interstitial 6q duplications were reported in five

Incomplete penetrance and variable expressivity in a series of 10 French patients with 15q13.3 recurrent microdeletion detected using array-CGH D. Sanlaville (1) A. Masurel-Paulet (2) J. Andrieux (3) C. Le Caignec (4) P. Callier (2) E. Flori (5) S. Jaillard (6) F. Mugneret (2) P. Jonveaux (7) L. Faivre (2) (1) Hospices Civils de Lyon (2) CHU de Dijon (3) CHU de Lille (4) CHU de Nantes (5) CHU de Strasbourg (6) CHU de Rennes (7) CHU de Nancy Since the large implementation of array-CGH in the diagnostic work-up of mental retardation, novel microdeletion syndromes have been described. In particular, the 15q13.3 microdeletion has been iden-

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tified in 0.2–0.3% of individuals with mental retardation and epilepsy, schizophrenia, autism and other neuropsychiatric features. The critical region between BP4 and BP5 contains at least seven genes, including CHRNA7, which is considered a good candidate gene for the epilepsy phenotype. We report a series of 10 patients (7 index cases and 3 affected parents) presenting a developmental delay and a 1.5 Mb 15q13.3 recurrent microdeletion ascertained through 11 French CGH-array platforms. All 7 index cases presented mild to moderate mental retardation with absent or non-specific dysmorphic features. Only one had seizures, 3 had an abnormal electroencephalogram and none had an autistic behaviour. One patient presented a highly different phenotype, including hydrocephaly, joint dislocations, congenital lymphoedema and notable dysmorphic features associated to mental retardation. Familial studies could be performed in 5/7 index patients. Interestingly, all 15q13.3 microdeletions were inherited (3 from the mother, 2 from the father). The affected parents had mild mental retardation with epilepsy in one. In particular, the microdeletion was found in 2 completely asymptomatic mothers. This study is in favour of incomplete penetrance and more variable clinical expressivity than previously published. Therefore, the 15q13.3 recurrent microdeletion might be only considered as a risk factor for mental retardation. The search for mutations on the second allele of the CHRNA7 gene is in progress in order to explain this incomplete penetrance. 1.115-P Spontaneuos pregnancy in a woman with an isochromosome of the long arm of the X chromosome M. Garcia-Barcina (1) B. Barreña (1) E. Sarasola (1) M. Onaindia (1) M. Tapia (1) J. Garcia (1) M. Angulo (1) M. Fiaño (1) (1) Osakidetza (Servicio Vasco de Salud) The isochromosome of the long arm of the X chromosome, i(Xq), is the most common structural aberration found in patients with Turner syndrome (TS). Here we report the case of a 10 years old female who had been investigated because of her short stature and was found to have a karyotype 46,X,i(Xq). Her family was informed about TS and its clinical features, including infertility.

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At the age of 30 she became pregnant she was referred to our hospital at 15 weeks of gestation for prenatal genetic counselling and amniocentesis because of her karyotype. The presence of the i(Xq) was confirmed by GTGbanded chromosome analysis of blood lymphocytes from the mother and cultured amniocytes from the fetus. The alpha fetoprotein level on amniotic fluid was within normal limits and the ultrasound examination, including detailed echocardiography, at 20 weeks of gestation showed a normal female fetus. The couple decided to continue the pregnancy. Metaphase arm-specific chromosome painting was peformed with XCAP X long (Texas Red) and XCAP X short (FITC) probes, revealing in both samples the presence of two red and two green signals, which is not consistent with the presence of an X isochromosome. Furthermore, a region without hybridization could be observed adjacent to the labelled short arm of the derivative chromosome. Using the C-banding technique this region was defined as heterochromatine. In order to identify the origin of this extra region, MLPA with the primer sets P036 and P070 (specific for the subtelomeric regions of the 22 autosomes and both sex chromosomes) was performed showing deletion of SHOX, an increase in VAMP7 (SYBL1) copy numbers and presence of UTY and DDX3Y. Now, the chromosome breakpoints of this X-Y translocation must be defined and the correlation with the patient’s phenotype must be done. Our case indicates the clinical importance of combining molecular and cytogenetic studies in a prenatal setting. 1.116-P A de novo inverted duplication of 2p with terminal deletion in a patient with the classical phenotype of trisomy 2p23-pter M. Chaabouni (1) R. Meddeb (1) I. Ouertani (1) F. Maazoul (1) L. Ben Jemaa (1) H. Chaabouni (1) (1) Charles Nicolle Hospital Inverted duplications with terminal deletions have been reported in an increasing number of chromosomes such as the chromosome 8, chromosome 4 and more recently chromosome 2. Here we describe the clinical, cytogenetic and molecular characterization of an inverted

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duplication of chromosome 2p in a 12-months-old girl. She has a history of intrauterine growth retardation and breathing distress. Physical examination revealed major hypotonia, dysmorphic features with prominent high forehead, hypertelorism, left epicanthus, wide nasal bridge, abnormal ears, high arched palate and congenital heart defect with ventricular septum defect. She presents with overlapping 2nd and 3rd toes. Her psychomotor development was severely delayed as she could not hold her head nor stay at 12 months. Karyotype was first interpreted as a partial duplication 2p. Further analyses using FISH defined the rearrangement as an inverted duplication with terminal deletion 2p. Thus the karyotype is: 46,XX, inv dup del(2) (:p23.1→p25.2::p25.2→qter). The duplication is estimated to be 31 Mb, the deletion spans at most 170Kb as the BAC RP11-356M6 localized at 2p25.3 between 0.17 and 0.33 Mb is present in a single copy just proximal to the deleted 2ptel. Phenotype is compared to the trisomy 2p and the phenotypic effect of the telomeric deletion is discussed. 1.117-P Evaluation of a new kit for automated detection of Y-chromosome microdeletions G. Scarano (1) G. Cantalupo (1) M. Ciavarella (1) M. Della Monica (1) C. Lombardi (1) M. Maioli (1) L. Masella (1) F. Lonardo (1) (1) A.O.R.N. Gaetano Rummo Infertility is a significant problem, affecting up to 15% of couples of reproductive age. For many years, it was assumed that most reproductive problems could be attributed to the female partner but now about 30– 50% of infertility is attributed to a male factor. Microdeletions in the Y chromosome is one of the most common known genetic causes of spermatogenetic failure. Most of the microdeletions that cause azoospermia or oligospermia occur in three regions of the long arm of the Y chromosome (Yq11.23), the azoospermia factor (AZF) regions, known as AZFa, AZFb, and AZFc. Screening for these microdeletions in azoospermic or severely oligospermic men should be warranted. Detection of various subtypes of these deletions has a prognostic value in predicting potential success of testicular sperm retrieval for assisted reproduction.

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The microdeletions of the Y chromosome are too small to be detected by karyotyping but can be easily identified using polymerase chain reaction (PCR). The aim of the current study was to evaluate a new kit developed for diagnosis of deletion in the AZF regions. The Devyser AZF kit (DevyserAB, Stockholm, Sweden) is a CE-labelled kit based on PCR amplification of 14 sequence-tagged sites (STS) in AZF a-c and control regions on the Ychromosome. All 14 markers are co-amplified in one single PCR reaction. By the use of fluorescently labelled primers the visualization and identification of the PCR products can be automatically performed using a Genetic Analyzer. The new kit was compared to another commercial kit (AZF-MK, AB Analitica, Padova, Italy), which is based on three multiplex PCR amplifications including a total of 11 STS markers and two internal controls. Reaction products are separated and identified using EtBr agarose gel electrophoresis. A total of about 80 infertile males were included in the study. The two kits showed fully comparable results. The Devyser AZF kit demonstrated to be an efficient and simple method for detecting microdeletions of chromosome Y. 1.118-P High resolution chromosomal breakpoints mapping of a Xpterp11.3 deletion and a Xq24qter duplication in a girl with ovarian failure S. Toujani (1) S. Brisset (2) J. Young (3) A. Le Dû (1) O. Aubry (4) G. Rousseau (1) A. Berheim (5) G. Tachdjian (6) (1) hôpital Antoine Béclère (2) hôpital Antoine Béclère (3) Hôpital Bicêtre (4) Hôpital Bicêtre (5) Institut Gustave Roussy (6) hôpital Antoine Béclère Background: X chromosome abnormalities are associated with ovarian failure. Cytogenetic studies of these chromosomal anomalies have been usually performed using a resolution of 5–10 Mb. Oligonucleotides array comparative genomic hybridization (aCGH) provides actually the highest potential resolution for unbalanced chromosomal analysis. Methods: A Whole-genome 244 K Oligonucleotide aCGH was carried out in a young patient with ovarian

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failure, short stature, obesity and hypothyroidism and presenting a der(X)del(Xp11.2)dup(Xq25qter) detected by conventional cytogenetic. Array CGH results were confirmed by Fluorescence in situ hybridization. Results: Oligonucleotide aCGH showed a 43.66 Mb deletion at Xp11.3 region and a 36.87 Mb duplication at Xq24qter region. At Xp11.3, the chromosomal breakpoint disrupted SLC9A7 gene while at Xq24, the chromosomal breakpoint was located through a noncoding DNA sequence. SLC9A7 (solute carrier family 9 isoform 7) plays a role in maintaining the cation homeostasis and function of the trans-Golgi network. SLC9A7 is widely expressed, but a high level of SLC9A7 transcript has been detected in brain, skeletal muscle and secretory tissues. This gene was deleted in X-linked mental retardation. Among genes located to Xq24Xqter duplicated segment, FMR1 may be a gene of interest. The premutation of FMR1, is a known genetic factor associated with premature ovarian failure. Conclusion: In this study we reported the first fine mapping of chromosomal breakpoints of a Xpterp11.3 deletion associated with a Xq24qter duplication in a girl with ovarian failure. We also discuss the contribution of our case in refining genotype-phenotype correlations in cases with ovarian failure.

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a ring 17 case and found that the breakpoints were close to the telomeric ends. METRNL is the sole gene located on the q arm terminal end, while two open reading frames and the RPH3AL gene are located on the terminal p arm. To detect possibly unrevealed small deletions involving the transcription units, we used subcloned FISH probes obtained by long range PCR, which showed that the investigated regions were preserved. Comparing our findings to other reports it emerges that different breakpoints, involving (or not) large genomic deletions, present overlapping clinical aspects. In conclusion, our data suggest that a mechanism based on gene expression control besides haploinsufficiency should be considered to explain the common phenotypic features found in the mild ring 17 syndrome. 1.120-P Cytogenetic analysis of the mesenchymal stem cells derived from the subendothelium of the human cryopreserved umbilical vein D. de Medeiros Duarte (1) D. Afonso Cornélio (1) S. Batistuzzo de Medeiros (1) (1) UFRN

1.119-P Mild ring 17 syndrome shares common phenotypic features irrespective of the chromosomal breakpoints location C. Surace (1) S. Piazzolla (1) P. Sirleto (1) M. Digilio (2) M. Roberti (1) A. Lombardo (1) G. D’Elia (1) A. Tomaiuolo (1) S. Petrocchi (1) A. Angioni (1) ( 1 ) O s pe da l e Pe di a t r i c o “B a m b i n o G e s ù ” (2) Ospedale Pediatrico “Bambino Gesù” Ring 17 syndrome is a rare disorder with clinical features influenced by the presence or deletion of the Miller-Dieker Critical Region. Presence of the MillerDieker Critical Region is associated with a mild phenotype, including growth delay, mental retardation, seizures, cafè au lait skin spots and minor facial dysmorphisms. Previous studies have been mainly focused on this locus providing poor information about the role of other genes located on the p and q arms. Here, we used BAC/PAC and fosmid clones as FISH probes to perform a cyto-molecular analysis of

Mesenchymal stem cells (MSCs) are known as a population of multipotent progenitors cells able to proliferate and differentiate into multiple mesodermal tissues. The process of cryopreservation is essential for the maintenance of cell cultures, since the cell line is frozen, it can be maintained in liquid nitrogen for an indefinite period and then thawed for therapeutic or experimental purposes. Furthermore, concerns that adult human MSCs may be prone to malignant transformation have been recently raised. The aim of this study was to isolate a population of MSCs derived from the subendothelium of the umbilical vein human (MSCsSUVH) to assess by cytogenetic analysis the possibility of appearance of chromosomal changes in MSCsSUVH after the process of cryopreservation. Human umbilical cords from full-term deliveries (n=3) were collected after informed consent at the Januário Cicco’s Hospital (Brazil, Natal). MSCs-SUVH were isolated by using enzymatic digestion and cultivated in alfa-MEM supplemented with 10% FCS. Immunophenotypically, MSCs-SUVH were defined as cells expressing CD29, CD73 and CD90 and lacking hematopoietic lineage

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markers, such as CD14, CD34 and CD45. They also demonstrated capacity for osteogenic differentiation. The chromosomes obtained from the primary culture of MSCs-SUVH before and after the cryopreservation were analyzed by GTW banding, and results are described afollowing the guidelines to International System for Human Cytogenetic Nomenclature (2005). A total of 245 metaphases were analyzed. There was no emergence of clonal chromosomal aberrations in the MSCs-SUVH in different situations analyzed. However, only after the cryopreservation, 14 metaphases showed nonclonal chromosomal aberrations, including monosomies and structural changes. These findings should be further investigated, since the use of MSCs for clinical approaches requires that the biosafety of these cells be carefully investigated through appropriate and sensitive tests. 1.121-P Therapy-Resistant Iron Deficiency Anemia In a Patient With Shprintzen Goldberg Syndrome U. Cetincelik (1) H. Aniktar (2) O. Oztürk (3) F. Borlu (4) (1) Sıslı Etfal Training and Research Hospital (2) Sıslı Etfal Training and Research Hospital (3) Sıslı Etfal Training and Research Hospital (4) Sıslı Etfal Training and Research Hospital

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1.122-P With specific chromosomal regions to evaluate the angle between centromere F. Özen (1) N. Koçak (2) H. Acar (3) (1) Cumhuriyet University Medical Faculty (2) Konya Training and Research Hospital Faculty (3) Selcuk University Meram Medical Faculty Background: Despite karyotype analysis of basic structure of chromosomes often remain the same, the size change. Considered to change the basic structure of chromsomes is proportional,seems to be possible to find some specific numeric identifiers for the chromosomes. specific regions of chromosomes to determine the angle between each other may provide a hint. Hard to reach the original value of the chromosome is important in the evaluation. For this purpose, specific regions of chromosomes among them tried to Comparison (angle, distance, ratıo). Method: Karyotype examples obtained from 50 different people in our work was used. Measurements were made between them in chromosome regions (telomere, centromere, etc). After, was to determine fixed and variable. Conclusion: Our work is still continuing Key words: Karyotype, chromosomes, telomere

The Shprintzen Goldberg Syndrome (SGS) is a rare autosomal recessive disorder characterized by neurological, cardiovascular, skeletal anomalies as well as and connective tissue dysplasia, craniosynostosis and marfanoid habitus. The syndrome was first defined by SGS (1982) in two unrelated boys. At least 40 cases have been reported. A male patient 28 year old was having treatment for therapy-resistant iron deficiency anemia. As he was showing a particular phenotype and seemed to have a mental retardation, physicians of Department of Internal Medicine decided to transfer him to our department. This patient came to our department under these circumstances. His physical examination suspected SGS. The cytogenetic analysis showed normal male karyotype. Nothing was found explaining therapy-resistant iron deficiency anemia in laboratory testings. The case of SGS accompained with therapy-resistant iron deficiency anemia seems was not previously reported.

1.123-P Male carriers of structural alterations in the Y chromosome: consequences for reproduction M. Martínez (1) C. Méndez (2) I. Campos-Galindo (3) M. Nicolás (4) L. Fernández (5) J. Landeras (6) (1) IVI-Murcia (2) IVI-Murcia (3) IVI-Murcia (4) IVI-Murcia (5) IVI-Murcia (6) IVI-Murcia Introduction: Alterations which affect the Y chromosome are present in approximately 7% of males with a severe defect in their spermatozoa production. This malfunction is due fundamentally to two events: either the correct pairing of the XY bivalent necessary for the progression of meiosis is prevented or deletions occur (a loss of genetic material) in the AZF locus (Deleted in Azoospermia) in Yq11.23, where genes (USP9Y,

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RMBY, DAZ, DBY, etc), which participate in male gametogenesis, are located. Our aim in this study was to evaluate the most suitable treatment for reproduction for these patients, depending on the clinical significance of each alteration and the risk it implies for the offspring in each case. Materials and methods: 1. Karyotype using GTG (> 550 bands per haploid set) and QFQ high resolution bands. 2. Fluorescent In Situ Hybridization (FISH) analysis on metaphase and interphase lymphocytes. 3. Classification of the alterations into five categories to evaluate and to analyze the results in groups. Results: Spermatozoa were obtained via testicular biopsy from two of the 7/20 patients who presented secretory azoospermia. This indicated that in 25% of the cases (5/20) germinal cells were not observed either in the ejaculate or in the testicle so as to be able to offer some form of assisted reproduction treatment (ART). In the 13/20 cases with spermatozoa in the ejaculate: 5/20 of them were cryptozoospermic (< 100.000mill / mL, 3/20 were oligozoospermic (< 20mill/mL) and 5/20 were normozoospermic (> 20 mill/mL). In all cases, Intracytoplasmic Sperm Injection (ICSI) was the treatment of choice. Additionally, preimplantation cytogenetic diagnosis was offered to groups at risk of transmitting chromosomal imbalances to their offspring and of producing malformations and/or suffering miscarriages. A pregnancy rate of 56.25% was achieved in 16/20 couples who underwent ART treatment, following an average of 2.2 cycles, and with a miscarriage rate of 18.75%. Two couples are still under treatment not having managed to obtain a successful pregnancy. Conclusion: We propose an action protocol with reproduction options suited to the type of anomaly. 1.124-P A girl with mos45,X/46,XY/47,XYY: Genetic and clinical findings T. Çora (1) H. Acar (0) E. Atabek (0) Ö. Balasar (0) S. Nergis (0) (1) Selcuk Üniversity (2) Selcuk Üniversity (3) Selcuk Üniversity (4) Selcuk Üniversity (5) Selcuk Üniversity

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Disorders of sexual differentiation have been associated with numerical and structural aberrations on chromosomes X and Y. In this study, we present a paitent who had female external genitalia, with minimal phallic enlargment and also had some stigmata of Turner syndrome including short stature. Chromosome analysis revealed a 45,X/46,XY/47,XYY karyotype and cytogenetic results were confirmed by using fluorescence insitu hybridization (FISH) with chromosome X and Y spesific probes. Her both parents had normal karyotypes. The existence of the sex-determining region Y (SRY) and also two loci microdeletion (sY233 and sY147) of the azoospermia factor (AZF) regions were detected by multiplex polymerase chain reaction (PCR). The genetics basis and clinical manifestations of this patient are discussed and compared with the similar cases with Y chromosome aneuploidy and AZF region alterations published in literature. 2.1-O Genomic Duplications involving a Conserved Non-Coding Regulatory Element Downstream of BMP2 are Associated with Brachydactyly Type A2 E. Klopocki (1) K. Dathe (1) A. Brehm (2) K. Kjaer (3) P. Meinecke (4) C. Ott (1) P. Seemann (5) S. Mundlos (1) (1) Charité Universitätsmedizin Berlin (2) MaxPlanck-Institute for Molecular Genetics (3) Wilhelm Johannsen Centre for Functional Genome Research (4) Altonaer Kinderkrankenhaus (5) Berlin-Brandenburg Center for Regenerative Therapies (BCRT) Autosomal dominant brachydactyly type A2 (BDA2, MIM112600) is a limb malformation characterized by hypoplastic middle phalanges of the 2nd and 5th fingers caused by mutations in the Bone morphogenetic protein receptor 1B (BMPR1B) or in its ligand Growth and differentiation factor 5 (GDF5). By linkage analysis in a large Brazilian kindred of German origin negative for BMPR1B and GDF5 mutations we identified a novel locus for BDA2 on chromosome 20p12.3 encompassing the Bone morphogenetic protein 2 (BMP2) gene. Since sequencing detected no point mutations in BMP2 a high density array covering the critical interval of ~1.3 Mb was designed. Array CGH analysis detected a microduplication of ~5.5 kb in a non-coding sequence

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~110 kb downstream of BMP2. Screening of other BDA2 patients by qPCR revealed a similar duplication in a second family. The duplicated region contains conserved non-coding sequences which likely function as cis-regulatory element regulating BMP2 expression in the limb. Several studies have identified such elements as essential regulators of developmental gene expression, that have the potential to switch genes off and on in particular types of cells/tissues during certain developmental time points. Given the importance of gene regulation in development it is to be expected that a large number of developmental defects are caused by mutations affecting such regulatory elements. Using a transgenic mouse model we are able to show that this sequence indeed is able to drive expression of a lacZreporter construct exclusively in the limbs. The lacZ expression pattern resembles that of endogenous Bmp2 supporting the hypothesis of a limb-specific Bmp2 enhancer within the identified duplication. Our results reveal deregulation of the BMP signaling pathway as a novel molecular basis for the pathogenesis of BDA2 and identify duplications of regulatory elements as a novel mutational mechanism for developmental defects. 2.2-O Genomic changes detected by array CGH in human embryos with developmental defects E. Rajcan-Separovic (1) Y. Qiao (1) C. Tyson (2) C. Harvard (1) C. Fawcett (2) D. Kalousek (1) M. Stephenson (3) T. Philipp (4) (1) University of British Columbia (2) Royal Columbian Hospital (3) University of Chicago (4) Donauspital Developmental abnormalities of human embryos can be visualized in utero using embryoscopy. Our previous embryoscopic and genetic evaluations detected developmental abnormalities in the majority of both euploid (74%) and aneuploid or polyploid (90%) miscarriages. Since we found the pattern of morphological changes to be similar in euploid and noneuploid embryos, we proposed that lethal submicroscopic changes, not detected by standard chromosome testing, may be responsible for miscarriage of euploid embryos. In this study, we used whole genome oligo and BAC array CGH (0.02Mb and 1Mb resolution, respectively) to screen for submicroscopic chromosomal changes in 17 euploid embryonic miscarriages, which were found to

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have a range of developmental abnormalities (growth disorganization, multiple or isolated defects) documented by embryoscopy. Six unique submicroscopic gains and losses (DNA copy number variants or CNVs), previously not reported, were identified in 5 (29%) of the 17 embryos. All 6 unique CNVs were less than 250 kb in size and were identified by higher resolution Agilent whole genome array CGH. Based on parental array CGH analysis, a de novo origin of a CNV was determined for one embryo (at 13q32.1) and suspected for another (at 10p15.3). Three CNVs, at Xq28, 1q25.3 and 7p14.3, were inherited and a CNVat 17p13.1 was of unknown origin because maternal DNA was unavailable. The CNV breakpoints were refined and further compared between the embryos and parents using a custom array (Agilent) with a high resolution coverage of the chromosomal regions with unique CNVs. The genes that are contained within these unique CNVs will be discussed, with special reference to syntaxin and WD repeat genes which showed recurring rearrangements in studied embryos. Our report describes for the first time, de novo and inherited unique CNVs and, therefore, potential “miscarriage genes” associated with specific developmental defects, in euploid embryonic miscarriages. 2.1-P SNP Arrays: first experiences with a genomewide screening technique A. Koehler (1) A. Hahn (2) J. Kreuder (3) B. Neubauer (2) U. Mueller (1) (1) Justus-Liebig-University (2) Justus-LiebigUniversity (3) Justus-Liebig-University (4) JustusLiebig-University We recently introduced SNP array diagnostics using the Illumina Infinium HD assay and report on our findings of small interstitial duplications in two dysmorphic children using the Human610-Quad BeadChip. Case 1: a six year-old male patient with normal chromosomes presented with developmental delay and mild dysmorphism. We detected a small duplication of 530 kb within the terminal band of the short arm of chromosome 3 (3p26.3). This region (about bp 1,862,885-2,410,741) includes several small

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CNVs and the gene CNTN4 (contactin 4). The protein encoded by CNTN4 is a member of the immunoglobulin superfamily that may play a role in the formation of axon connections in the developing nervous system. Case 2: a 9 year-old boy presenting with tetralogy of Fallot, hypotonia, and mild dysmorphism. We identified a small interstitial duplication of 270 kb in the long arm of chromosome 22 (22q11.2–q12.1). This region (from bp 23,931,520-24,197,568) includes many CNVs, the gene IGLL3 (immunoglobulin lambda-like polypeptide 3), and two members of a gene cluster CRYBB2 and CRYBB3 (crystallin, beta B2 and beta B3). The Crystallin genes may play a role in eye development. Currently we investigate the parents of these two patients. A pathogenetic function of these duplications will become even more likely if the parents do not carry these duplications. 2.2-P Microsatellite Null Alleles in paternity testing F. Rim (1) G. Moez (1) H. Dorra (1) A. Labiba (1) B. Ilhem (1) M. Ons (1) M. Amira (1) Taheni (1) S. Ali (1) (1) CHU Farhat HACHED Sousse 2.3-P Idiopathic Silver-Russell syndrome patients should be tested for (sub)microscopic chromosomal aberrations

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hypomethylation in 11p15, a further 10% carry a UPD (7)mat. In several cases, conventional cytogenetic aberrations have been reported, but often the clinical picture was ambiguous. Furthermore, a uniform aberration pattern was not obvious. Due to the recent identification of two cases with submicroscopic duplications in 11p15 we decided to screen a cohort of 20 SRS patients without 11p15 epimutation or UPD(7)mat for submicroscopic imbalances by genomic microarray analysis using the Affymetrix GeneChip® Human Mapping 500K Array Set. The detected imbalances were surveyed in respect to their gene coverage and overlaps with registered copy number variations (CNVs) by online databases queries. Apart from numerous apathogenic CNVs we identified 13 so far unregistered copy number alterations (CNAs) in 10 patients. We classified the majority of them as apathogenic because they were either detectable in one parent or because they did not affect genes. However, in one SRS patient with normal intellectual capacities we detected a de-novo 1 Mb deletion in 12q14 for which recently a new microdeletion syndrome has been described; one of the 12q14 microdeletion carriers showed SRS features, however all patients were mentally retarded. In an additional patient from routine diagnostic screening for the 11p15 epimutation we detected a cytogenetically invisible 11p15 duplication by MLPA which was retrospectively diagnosed as a 11;15 translocation by FISH. In conclusion, our findings show that a significant number of 11p15 epimutation and UPD(7)mat negative patients carry chromosomal abnormalities. We therefore suggest to routinely test idiopathic SRS(-like) patients for submicroscopic chromosomal aberrations. 2.4-P

T. Eggermann (1) N. Schönherr (1) B. Denecke (2) S. Singer (3) E. Rossier (3) G. Binder (4) M. Baudis (5) (1) RWTH Aachen (2) RWTH Aachen (3) University of Tübingen (4) University of Tübingen (5) University of Zürich Silver-Russell syndrome (SRS) is a clinically and genetically heterogeneous disorder. The phenotype is characterized by intrauterine and postnatal growth restriction and additional morphologic abnormalities including a typical triangular face, relative macrocephaly and asymmetry. In about 50% of SRS cases (epi)genetic alterations can be detected: >38% show a

The crucial role of mdr1 (ABCB1) gene polymorphisim in abdominal aortic aneurysm: preliminary results of a pilot study Hande Kucuk Kurtulgan1, Sinasi Manduz2, Atilla Uslu3, Nurkay Kantarcioglu2, Oguz Karahan2, Fazilet Yildiz1, Ocal Berkan2, Oztürk Ozdemir1 Department of Medical Genetics1, Heart and Vessel Surgery2, Cumhuriyet University, Faculty of Medicine 58140-Sivas and Department of Physiology3, Istanbul University, Istanbul Faculty of Medicine, 34039 Capa, Istanbul, Turkey

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Summary Objective: The aim of this study was to assess the influence of multidrug resistance (MDR1) gene polymorphisms on Turkish patients with abdominal aortic aneurysm(AAA). METHODS: The comman polymorphic G2677T/A region in MDR1 was determined by PCR based StripAssay revers hybridisation thecnique in a total of 116 individuals(58 patients with AAA, mean age 62.94±6.59 and 58 healthy controls, mean age 58.82±11.60).RESULTS: The G2677T/A polymorphism of MDR1 gene was significantly higher in patients with AAA when compared to the control group individiuals (X2 = 11.47; P<0.0001 for homozygous and X2 =5.80; p= 0,016 for heterozygous mutations), (p<0,05). CONCLUSION: The preliminary results of current pilot study demonstrated that the G2677T/A single nucleotide polymorphism in MDR1 gene is associated with AAA. These gene polymorphism may play an curicial role in initiation and/or progression aneurysm in human with combine effetcs of other ethiological parameters. Key words: AAA, MDR1 gene polymorphism 2.5-P Screening for Genomic Imbalances in Autism Spectrum Disorders (ASDs) using Array-based Comparative Genomic Hybridization (array-CGH) A. Bremer (1) B. Anderlid (1) M. Nordenskjöld (1) M. Giacobini (1) J. Schoumans (1) (1) Karolinska Institutet Autism spectrum disorders (ASDs) are characterized by impairments in communication and social interaction, accompanied by stereotyped behaviors and interests. It’s a highly heritable and heterogeneous group of disorders with a complex genetic etiology. Until recently, G-banded karyoptyping was the standard method for the detection of cytogenetic aberrations in patients with developmental disorders. The development of whole-genome screening methodologies such as array-CGH has enabled screening of the whole genome at much higher resolution than provided by karyotyping, leading to the identification of novel microdeletion- and microduplication syndromes. In our present ongoing study, we use array-

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CGH to screen for genomic imbalances in ASD patients in order to identify genetic aberrations and genes that cause or increase susceptibility for autism. So far ~200 patients have been screened and clinically significant imbalances have been identified in 18 cases (~9%). Some of these aberrations are recognized as recently identified recurrent aberrations associated with autism. We also observed that duplications seem more frequent in ASD patients than in patients with mental retardation where deletions are more frequently observed. An identified genetic cause gives the family and the patient an explanation of the disorder. In hereditary cases, genetic counseling and prenatal diagnosis is also possible. 2.6-P MLPA: A new tool for detection of delection in FVIII gene R. Santacroce (1) V. Longo (1) V. Bafunno (1) N. Bukvic (1) M. Chetta (1) G. D’Andrea (1) M. Sarno (1) F. Sessa (1) M. Margaglione (1) (1) Genetica Medica, Università Degli Studi Di Foggia; (2) Unita’ Di Emostasi e Trombosi, I.R.C.C. S. “Casa Sollievo Della Sofferenza”, S. Giovanni Rotondo; Italy Haemophilia A is an X-linked bleeding disorder caused by mutations widespread in the human coagulation FVIII gene. Most of the mutations in the FVIII gene are detectable by routinely screening methods such as sequencing analysis or DHPLC. However, large deletions or deletion encompassing the entire gene F8 can go undetected. Moreover, in heterozygous females the diagnosis proves difficult as the presence of a normal allele does not recognize the partial or complete loss of the F8 gene. In this study we analyzed 25 patients, affected by haemophilia A which resulted mutation negative after complete sequencing of F8 gene. However, PCR failed to amplify one or more exons in some of these patients. This work aimed to confirm the conjectured deletions and the diagnosis of carriers thanks to the Multiplex Ligation-dependent Probe Amplification (MLPA) Test. Recently, MLPA has been broadly applied to detect mutation as deletions and duplications, especially in X-linked disease (Duchenne/

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Becker muscular dystrophy, Haemophilia A). Actually, MLPA test allowed to detect 7 deletions in haemophiliacs and the carrier status was identified in 2 female, relatives of our patients. According to the results, MLPA is a helpful tool to detect the mutation in haemophiliacs with large deletions, and the status of carriers in heterozygote related female. The only way to correlate genotype with observed phenotype is to detect the gene mutation and MLPA provides an important tool for the detection of its complete mutational spectrum. 2.7-P Identification of a novel microdeletion syndrome in 17p13.3 distinct from isolated lissencephaly sequence and Miller-Dieker syndrome

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sequence (ILS). We used a targeted custom array created in Agilent’s web portal eArray (https://erray.chem. agilent.com/earray) with an effective resolution of 60–1000 bp across the region to refine the breakpoints. Detailed molecular analyses of the 17p13.3 microdeletion cases identified a minimal region of overlap spanning 220 kb and encompassing five genes, including two likely candidates of clinical significance (YWHAE encoding 14-3-3ε and CRK encoding Crk). Copy number instability in chromosome region 17p13.3 and the range of specific pathological consequences which correlate to the underlying genomic abnormalities will be presented. 2.8-P A comparative, high resolution analysis of DNA— a technological challenge or a real breakthrough in the diagnostics of genomic diseases

A. Lindstrand (1) B. Anderlid (1) D. Bruno (2) J. Lundin (1) C. Lese Martin (3) C. Nowak (4) H. Slater (2) J. Schoumans (1) (1) Karolinska Institute (2) University of Melbourne, (3) Emory University (4) The National Birth Defects Center (8)

B. Kałużewski (1) &. Sieczkowski (2) I. Płowaś (2) M. Constantinou (1) (1) Medical University of Lodz (2) Hospital No. 3 in Łódź

Microarray analysis for detection of chromosomal abnormalities in patients with developmental problems has brought a new era of cytogenetic testing, which in turn has significant impact on clinical practice. An important outcome of large scale testing of patients has been the identification of several ‘new’ microdeletion/duplication syndromes through so called ‘reverse dysmorphology’, that is, using a genotype to phenotype approach. Today, a large number of genetic diagnostic centres across the globe offer whole genome copy number analysis at high resolution. However, the clinical interpretation of the high number of copy number variants (CNVs) that are detected in each sample is still challenging. Genotype as well as phenotype information can be shared through online databases such as DECIPHER and ECARUCA in order to facilitate the interpretation of CNVs and allow identification of novel microdeletion and microduplication syndromes. In collaboration with genetic departments in Europe, USA and Australia, we have identified a, novel microdeletion syndrome in 17p13.3, distally located of Miller Dieker Syndrome (MDS) and Isolated Lissencepahly

In the literature, approximately 5000 loci have been described with structural differences of human genome, regarding the number of copies—large-scale copy number variants (LCVs). The functional consequences of LCVs are still unidentified. The majority of LCVs covers the coding regions of known genes or involves the whole genes. Therefore, we assume that they may significantly influence predispositions to the development of certain genetic diseases which, according to the valid knowledge, have been classified to non-hereditary diseases, while the risk of their recurrence has been estimated as low. LCVs may control the pathogenesis of some genetic diseases by means of two mechanisms. On one hand, a decrease or an increase of gene dose may induce an analogous change of expression of the genes critical for development, being, in this way, a cause of disease, while on the other, LCVs may predispose, via nonallelic homologous recombination (NAHR), to the development of chromosomal rearrangements, being the direct cause of the disease. This paper discusses the relationships between patients’ genotypes and their phenotypes. Additionally, taking into account our studies, the role of LCVs is approached in the pathogenesis of genetic diseases. We examined 100

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subjects by means of high resolution hybridisation for micromatrixes, the group including 50 patients and their parents. Approximately 300 LCVs were identified in the studied group. The obtained results have been compared with available literature data. On the basis of literature data and our studies, we assume that LCVs are at the base of individual differences in genome in total magnitude of approximately 10– 20 Mbp, what exceeds the number of assumed differences regarding SNP (5–10 Mbp). The identified number of LCVs is but the tip of the iceberg and the currently performed studies of more and more numerous population groups, different from the ethnic point, may unquestionably bring about a much higher total number of LCVs. 2.9-P Novel loci and candidate genes for autism spectrum disorder detected by SNP array based segmental aneuploidy screening E. van Daalen (2) N. Verbeek (1) H. Özgen (2) J. Vorstman (2) M. de Jonge (2) R. van ’t Slot (1) E. Brilstra (1) C. Freitag (3) R. Hochstenbach (1) M. Poot (1) (1) University Medical Center Utrecht (2) University Medical Center Utrecht (3) University Medical Center UtrechtUniversity Medical Center Utrecht (4) University Medical Center Utrecht (5) University Medical Center Utrecht (6) University Medical Center Utrecht (7) University Medical Center Utrecht (8) GoetheUniversität Frankfurt am Main (9) University Medical Center Utrecht (10) University Medical Center Utrecht Autism spectrum disorders (ASD) are neurodevelopmental conditions characterized by concomitantly impaired reciprocal social interaction, communicative deficits, and restricted and repetitive behavioral patterns. ASD occurs in syndromic forms (e.g. FRAX, del (22q11), Rett), and as non-syndromic cases frequently involving segmental aneuploidies. Recently, arraybased genome-wide screens have demonstrated frequent copy number variation in non-syndromic ASD. Yet, it remains largely unknown whether these represent truly pathogenic genome rearrangements. Screening 56 patients (8 girls and 48 boys) with ASD and additional major or minor anomalies with the Infinium HumanHap300 SNP platform (Illumina, Inc., San Diego, CA)

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we found in 8 patients 4 regions with hemizygous losses and 4 with gains containing brain-expressed genes. One gain (in 22q11.2) was paternally inherited; another gain (in 8p23.1) was maternally inherited. All other rearrangements had occurred de novo and were not flanked by segmental duplications. Only one locus has previously shown significant association with ASD. In two families overlapping aneuploidies for part of the MCPH1 gene (in region 8p23.1) have been found. This suggests that changes in copy number of MCPH1 are a susceptibility factor for ASD. In two unrelated boys with Asperger syndrome we detected maternally inherited aneuploidies of the proximal part of region 15q11.2. We conclude that SNP array-based screening of ASD patients uncovers an appreciable number of novel segmental aneuploidies containing brainexpressed genes that may represent clues toward etiological pathways of perturbed brain development likely to result in ASD. 2.10-P Genomic imbalances detected by array-CGH in patients with congenital eye malformations and mental retardation A. Delahaye (1) P. Bitoun (2) N. Chassaing (3) M. Gérard-Blanluet (4) A. Toutain (5) L. Faivre (6) C. Francannet (7) J. Elion (8) B. Benzacken (9) . Pipiras (9) (1) AP-HP-University Hospital Jean Verdier, Paris XIII (2) AP-HP-University Hospital Jean Verdier, Bondy, France (3) Hôpital Purpan, University Hospital of Toulouse (4) AP-HP-University Hospital Robert Debré, Paris VII (5) Hôpital Bretonneau, University Hospital of Tours (6) University Hospital of Dijon (7) University Hospital of Clermont-Ferrand, Univ Clermont1, UFR Médecine (8) UMR 763 Inserm-Paris VII,AP-HP-University Hospital Robert Debré (9) APHP-University Hospital Jean Verdierand Robert Debré, Paris XIII, UFR 676 Inserm Genomic imbalances are a major cause of mental retardation and developmental delay. With the advent of whole genome array-CGH analysis, the number especially of interstitial genomic imbalances increased dramatically. Hereditary diseases of the eyes are a frequent cause for blindness in early childhood with complex etiology. The exact frequency of

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chromosomal deletions and duplications causing congenital eye abnormalities is unknown. Considering the high frequency of microscopically visible imbalances associated with eye disorders we hypothesize that the sensitivity of array-CGH could improve the genetic diagnosis in patients with idiopathic eye anomalies associated with mental retardation. Fifty patients with unexplained congenital eye malformations and mental retardation were analyzed using 105K oligonucleotide arrays (Agilent). We detected 8 possibly pathogenic de novo imbalances (16%) among analysed patients. Seven are deletions and 1 is duplication. No recurrent abnormality was identified. FISH analyses confirmed the chromosomal abnormalities in 5 cases and real time quantitative PCR in the remaining cases. In 5 patients, the genetic imbalance was shown inherited from a phenotypically normal parent (1 deletion and 4 duplications). Further analyses to confirm chromosomal abnormalities suspected using CGH array are still under investigation in 13 cases. These results demonstrate that array-CGH is able to provide a high diagnostic yield in patients with congenital eye malformation and mental retardation. Interestingly, only 2 of the 8 possibly pathogenic de novo imbalances encompassed genes already known to be involved in eye development(FOXC1 and OTX2). Besides their importance for diagnosis and genetic counselling, these data may pave the way to the search of genes involved in eye development. 2.11-P FISH at a kilobase-resolution: a direct visualization approach for straightforward diagnostics of FSHD and (epi-)genetic exploration of the FSHD locus

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present on the subtelomere of chromosome 10q. Disease alleles carry 1–10 copies on a 4qA chromosome, one of two equally frequent variants of the 4qter subtelomere. 4qA arrays with greater than 10 repeat units, as well as 4qB and 10q arrays, are non-pathogenic. The specificity of this CNV-related disease is the broad range of repeat numbers, and above all the importance of the chromosomal location of these repeats. This precludes use of common technologies for analyzing CNVs, such as aCGH or qPCR. Only Southern blotting may, to some extent, provide sufficient insight on the genotype in order to diagnose FSHD, but it falls short of reliably accounting for recombination events, translocations between 4q and 10q, somatic mosaicism, which are all quite frequent. To bypass these heavy limitations, we have developed a test based on Molecular Combing, which allows direct visualization of probes hybridized on individual, stretched DNA molecules at kilobase-resolution. This technology shares most of the advantages of FISH, but with a hundred-fold improved resolution. In the case of FSHD, our results on reference cell lines and patient samples demonstrate the ability of Molecular Combing to precisely visualize the haplotype and precise repeat unit count of all four 4q- and 10q- repeat arrays in a single experiment. We also have identified unexpected rearrangements in the D4Z4 loci. This test thus should allow for genetic testing, as well as provide better understanding of the pathophysiology of FSHD. Our efforts are now directed to the implementation of this test for routine diagnostics in a hospital lab, opening the way for novel approaches to the diagnosis of large rearrangements- or CNV-related diseases. 2.12-P

P. Walrafen (1) K. Nguyen (2) A. Vannier (1) E. Renard (1) C. Vovan (2) R. Bernard (2) A. Bensimon (1) N. Levy (3) (1) Genomic Vision (2) Hopital d’enfants La Timone (3) Hopital d’enfants La Timone and INSERM Facioscapulohumeral dystrophy (FSHD) is the third most common muscular dystrophy, with autosomal dominant transmission. It is associated to the contraction of a repeat array which comprises 1–150 copies of the 3.3 kb repeat unit, D4Z4, in the subtelomeric region of chromosome 4q (4q35). A virtually identical array is

Microdeletion of the 5q14 region, encompassing the MEF2C gene, is associated with severe mental retardation, poor eye contact and seizures N. Le Meur (1) S. Jaillard (2) P. Saugier-Veber (3) M. Holder-Espinasse (4) D. Bonneau (5) H. Journel (6) T. Frebourg (3) C. Dubourg (7) A. Goldenberg (3) J. Andrieux (8) (1) Efs-Normandie (2) Chu Ponchaillou (3) Chu de Rouen (4) Chru Lille (5) Chu D’Angers (6) Ch Bretagne Atlantique (7) Chu Ponchaillou (8) Chru Lille

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Over the last few years, array-CGH has remarkably improved the ability to detect cryptic unbalanced rearrangements in patients presenting with syndromic mental retardation. Using whole genome oligonucleotide array-CGH, we detected 5q14.3 microdeletions ranging from 216 kb to 8.8 Mb in 5 unrelated patients showing striking phenotypic similarities, namely severe mental retardation, poor visual contact, absent speech, stereotyped behaviour, facial dysmorphic features, epilepsy and/or cerebral malformations. The minimal common deleted region of these 5q14 microdeletions encompassed only MEF2C, known to act in brain as a neurogenesis effector which regulates excitatory synapse number. Therefore, we suggest that 5q14.3 microdeletion may represent a novel clinically recognizable condition caused by haploinsufficiency of the MEF2C gene. 3.1-O Galliera genetic bank as a part of telethon network of genetic biobanks M. Mogni (1) C. Baldo (1) M. Catagnetta (1) F. Dagna Bricarelli (1) (1) E.O. Ospedali Galliera, Genova The network of telethon genetic biobanks (TGB) interconnects and coordinates biobanks already supported, as a core facilities, by Italian telethon foundation in order to promote research into genetic diseases improving the access to human biological resources ensuring legal, ethical and quality standards. The TGB Network includes the following Biobanks: – – – – –

Cell Line and DNA Bank from patients affected by Genetic Diseases-Genova (M. Filocamo) Galliera Genetic Bank: DNA, cell lines and tissue bank—Genova (C.Baldo) Human Genetic Bank of patients affected by Parkinson Disease and Parkinsonisms-Milano (S. Goldwurm) Cell line and DNA Bank of Rett Syndrome and other X-Linked Mental Retardation-Siena (A. Renieri) Neuromuscular Biobanks composed by: “Neuromuscular Bank of Tissues and DNA samples”Padova (C. Angelini), “Bank of DNA, Cell lines and Nerve-Muscle-Cardiac tissues”-Milano (M. Moggio), and “Cell, tissues and DNA from

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patients with Neuromuscular Diseases”-Milano (M.Mora). Each Biobank collects samples from patients affected by genetic diseases and their respective family members. At present the Network collection consists of about 37.000 samples from approximately 450 different genetic diseases. In particular the Galliera Genetic Bank (GGB) is the only Network biobank that stores a large number of cell lines (399 amniotic cells, 44 trophoblast cells, 311 fibroblasts, 222 lymphoblasts) and foetal tissues with several chromosomal aberrations. The “Galliera Genetic Bank” of the Human Genetics Laboratory—Galliera Hospitalin Genoa, was established in 1983 and since 2008 has become a partner of TGB Network. Thanks to the experience of lab staff in cytogenetic, FISH and CGH analysis the majority of samples are well characterized and represent a precious resource for researchers. GGB samples are available only for research purpose free of charge. All the information about Network activities and samples catalogue, including GGB collection, can be found at web address: www. biobanknetwork.org. 4.1-P Chromogenic in situ hybridization (CISH) study of HER2 and Topo2α amplifications in metastatic breast cancer N. Todorovic-Rakovic (1) Z. Neskovic-Konstantinovic (2) D. Nikolic-Vukosavljevic (1) (1) Institute for Oncology and Radiology of Serbia (2) Institute for Oncology and Radiology of Serbia HER2 (Human epidermal growth factor receptor 2) is the most frequently amplified oncogene in breast cancer, associated with unfavorable tumor phenotype, but there are conflicting results regarding its prognostic significance. Potential reason for this could be the fact that although HER2 is considered as target gene for amplification at chromosome 17, the HER2 amplicon harbors several closely located genes. Among them, Topo2α (Topoisomerase 2α) is the most frequently coamplified gene. The aim of this study was to determine the relationship between amplification of HER2 and Topo2a genes and their

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influence on prognosis in metastatic breast cancer (MBC) patients. The study included 71 MBC patients, initially diagnosed at different stages of the disease. Median follow-up period for the cohort at the time of analysis was 5 years. We used chromogenic in situ hybridization (CISH) on paraffin embedded primary tumor samples to study amplification of both genes. CISH is easily acceptable in clinical practice and has several advantages over FISH. More than 6 signals per cell is considered as cut-off value for amplification, as recomended. Although there was significant correlation (p<0,011) between HER2 and Topo2a amplification, Topo2a amplification was not strictly related to HER2 amplification. Neither HER2 or Topo2a amplification showed correlation with available clinicopathological parameters. Follow-up of patients showed that there was no difference in MBC survival, when stratified according to HER2 status i.e. between HER2nonamplified and HER2-amplified patients for subgroup as whole. There was significant difference in MBC survival (p<0,006) when patients are stratified according to Topo2α status. Topo2α-amplified cases have poorer survival than Topo2α-nonamplified, with coresponding median survival time 24 vs. 54 months. In conclusion we imply that, unexpectedly, Topo2α amplification that is not strictly related to HER2 gene amplification seems to be more promising and independent biomarker for risk evaluation in MBC than HER2. 4.2-P High resolution analysis of chromosomal abnormalities in Burkitt’s lymphoma

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Materials and methods:A whole-genome oligonucleotide array CGH analysis correlated with karyotype and FISH was performed in a set of 27 Burkitt’s primary tumors and cell lines. Results: More than half of the 145 CNAs <2 Mb were mapped to Mendelian CNVs, including GSTT1, and BIRC6, an anti-apoptotic protein, possibly predisposing to some cancers. Somatic cell line-specific rearrangement of IGloci, were consistently observed with the 244 K aCGH platform. Among 136 CNAs > 2 Mb, gains were found in 1q (12/27), 13q (7/27), 7q (6/27), 8q(4/27), 2p (3/27), 11q (2/27) and 15q (2/27). Losses were found in 3p (5/27), 4p (4/27), 4q (4/27), 9p (4/27), 6p (3/27), 17p (3/27), 6q (2/27), 11pterp13 (2/27) and 14q12q21.3 (2/27). Twenty minimal critical regions (MCR), ( range 0.04–71.36 Mb), were delineated in tumors and cell lines. Three MCRs were localized to 1q. The proximal one was mapped to 1q21.1q25.2 <142.87–177> with a 6.3 Mb amplicon (1q21.1q21.3) harboring BCA2and PIAS3. In the other 2 MCRs, 1q32.1 and 1q44, MDM4 and AKT3 appeared as possible drivers of these gains respectively. An amplification of 13q31.3q32.1 contained ABCC4, a multidrug resistance gene. A 40 Kb 2p16.1 MCR specifically encompassed the RELoncogene, already implicated in B cell lymphomas. The most frequently deleted region was 3p14.1 <60.43–60.53>. This deletion removed the fifth exon of FHIT. Conclusion: BL appears to exhibit a non-random genetic heterogeneity with new genes. Further investigations which combined gene expression and functional studies are essential for understanding the lymphomagenesis mechanism and for the development of more effective, targeted therapeutic strategies. 4.3-P

A. Bernheim (2) S. Toujani (1) P. Dessen (3) J. Wiels (4) N. Ithzar (5) Y. Vassetzky (6) V. Ribrag (7) C. Patte (8) (1) Institut Gustave Roussy (2) Institut Gustave Roussy (3) Institut Gustave Roussy (4) Institut Gustave Roussy (5) Institut Gustave Roussy (6) Institut Gustave Roussy (7) Institut Gustave Roussy (8) Institut Gustave Roussy Background: Additional chromosomal abnormalities are currently detected in Burkitt’s lymphoma (BL). Although they play major roles in the pathogenesis of BL and in prognosis, the genes involved remain elusive.

Deciphering of chromosome 3 rearrangements in MCF7 breast cancer cell line : from SKY to G-PET analysis. k. Hana Utami (1) L. Ukil (1) Y. Fei (2) A. Hillmer (2) Y. Ruan (2) E. Liu (1) V. Cacheux (1) (1) genome institute of singapore (2) genome institute of singapore MCF7 breast cancer cell line is known for its complex chromosomal rearrangements and model role to understand cancer anomalies. SKY techniques have

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shown many chromosomal anomalies in MCF7 but the limitations of the technique are the resolution and the complexity of interpretation for highly rearranged chromosomes. Recently at Genome Institute of Singapore, the Genome Paired-End-diTag (G-PET) high throughput sequencing technology has been developed as a major technique to identify fusion genes in cancer genomes. This new technology has been applied to MCF7, and has allowed the description of all the breakpoints which occurred in this cell line. In order to validate these breakpoints and to precisely reconstruct the chromosomal map of MCF7, we compared the results obtained for chromosome 3 by FISH, SKY and G-PET technologies. Among the 30 breakpoints between chromosome 3 and other chromosomes identified by G-PET analysis, we validated 28 of them by FISH and /or PCR. We then have reconstructed the chromosomal map of chromosome 3 in MCF7 and showed that the chromosome rearrangements are usually more complex than expected by SKY technique. Furthermore, this technique is able to directly pinpoint the precise breakpoints between chromosomes and to identify potential fusion genes. We here identified and validated a new fusion gene between exon 30 of TEX14 and exon 2 of PTPRG genes in MCF7. We show here that the combination of different technologies permits a more complete deciphering of chromosomal anomalies in cancer cell lines and offers an improvement in resolution especially for balanced anomalies, and might ultimately allow for the identification of new genomic rearrangements.

dramatic evidence for this is that the majority of prostate cancers have fusions of TMPRSS2 to ERG or another gene of the ETS family. We have been analysing the genome rearrangements of breast cancer cell lines in the search for gene fusions, using a combination of ‘array painting’, array-CGH and ‘paired end reads’. We mapped all translocations in four breast cancer cell lines to 1Mb resolution by ‘array painting’ (i.e. isolating individual chromosomes by flow-sorting and hybridizing them to DNA microarrays) [1]. Balanced breakpoints were then mapped using custom oligonucleotide arrays to about 1 kb, revealing two expressed fusion genes [1]. Unbalanced breaks were subsequently located from array-CGH data on Affymetrix SNP6 arrays provided by the Sanger Institute Cancer Genome Project: from these we have found a further four fusions in the two cell lines HCC1187 and HCC1806, suggesting that these cell lines may typically express several fusion genes. In ‘paired end reads’, the genome is broken into fragments of defined size, e.g. 500 bp, and short sequence tags are read from both ends using the Illumina (originally Solexa) sequencing system [2]. Translocation breakpoints are detected as paired reads from different chromosomes. Amplicons are particularly easy to analyse as multiple reads are obtained from them. We have begun to analyse breakpoints using this approach. [1] Howarth KD et al, Oncogene 2008;27;3345-59 [2] Campbell PJ et al Nature genetics 2008;40;722-9

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Specific chromosomal regions affected by fuel oil in fishermen who participated in the clean-up of Prestige oil spill

Fusion genes caused by chromosome rearrangement in breast cancer cell lines P. Edwards (1) J. Pole (1) E. Batty (1) K. Howarth (1) S. Newman (1) J. Beavis (1) I. Schulte (1) G. Bignell (2) C. Greenman (2) C. Caldas (3) (1) University of Cambridge (2) Sanger Institute (3) Cancer Research UK Cambridge Research Institute It has recently become clear that fusion genes, caused by chromosome translocations, inversions and deletions, play a role in the development of the common epithelial cancers, just as they do in leukaemias, lymphomas and sarcomas. The most

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G. Monyarch Gros (1) J. Zock (2) G. Rodríguez-Trigo (2) F. Gómez (2) F. Pozo-Rodríguez (2) J. Barberà (2) M. Coll (1) C. Fuster (1) (1) Universitat Autonoma de Barcelona (2) Grupo SEPAR-Prestige Several studies have shown the existence of genotoxic effects in subjects of the exposure to oil spills during the clean-up tasks. The aim of the present study was to analyze if this cytogenetic damage persists 2 years after the exposure and whether any chromosomal regions are especially affected in fishermen who

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participated in the clean-up of the Prestige oil spill. The fishermen who were non smokers were included in the exposed (E) group (>15 days of cleaning-up tasks at least 4 h per day) and non-exposed (NE) group. We analyzed 91 E and 46 NE. The samples were analyzed two years after the exposure. Breakpoints implicated in chromosomal abnormalities (lesions and structural alterations) were identified by G-banding. For each subject, a minimum of 100 uniform stain metaphases and 25 karyotypes were examined respectively using 72 h cultures peripheral lymphocytes. Comparison of cytogenetic data between the E and NE groups showed a slightly more chromosome lesions in E individuals (100 chromosome lesions in 9520 metaphases analyzed) than NE (35 /4859) (P=0,06). However, there was a positive association, between frequency of structural chromosome alterations and fuel exposure (196 structural chromosome alterations in 2448 karyotypes analyzed) vs NE (33 /1285) (P<0,0001). Statistical analysis of the 203 breakpoints implicated in these chromosomal abnormalities showed that eleven bands were specially affected in the E group: 2q21, 3q27, 5q31, 7p15, 9q34, 13q11, 14q24, 16q22, 17p12, 18q11.2 and Xq21 (each of them having three or four breakpoints). In addition, in the NE group 61 breakpoints were found, and of those the most affected bands were: 1q21, 11q13 and 14q32 (with two breakpoints). In conclusion, participation in clean-up tasks of oil spills results in prolonged genotoxic effects lasting 2 years after the exposure and some specific chromosome regions affected however their human health consequences are unknown. Acknowledgments: This work was supported by FIS (PI07-0086) and CIRIT (2005, SGR-00495) 5.2-O Genotoxic effects in a population exposed to Prestige oil during the cleaning tasks: a review of literature F. Reis (1) G. Monyarch Gros (1) C. Fuster (1) (1) Universidad Autonoma de Barcelona In 2002, the wreck of the Prestige oil tanker spilled near 63,000 tons of crude oil off the Galician coast. This accident was a big ecological disaster and

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exposed thousands of people who participated in the clean-up to the complex mixture of potentially dangerous substances. Genotoxic studies in population exposed to crude fuel during an oil spill are scarce. The objective of this work was to review the genotoxicity effects in subjects exposed to Prestige oil, including our results. The first study carried out in samples obtained from volunteers exposed to fuel during the cleaning tasks applied the sister chromatid exchanges tests (SCE), micronucleus test (MN), and comet assay (Perez-Cadahia et al. 2006). The results showed a significant increase in the genotoxic damage, being the comet assay the most sensitive biomarker to detect it. Following studies, carried by the same research group, showed that CYP1A13’UTR, EPHX1 codons 113 and 139, GSTP1, GSTM1 and GSTT1 metabolic polymorphisms, and XRCC3 codon 241 and XPD codon 751 repair polymorphisms influenced cytogenetic damage levels (Perez-Cadahia et al. 2008a). The relationship between exposure to heavy metals present in fuel and genotoxic parameters (SCE, MN and comet assay) was investigated in these subjects detecting that cadmium and zinc showed an inverse relationship with the levels of DNA damage detected by the comet assay and that lead influenced significantly the strand break levels (PerezCadahia et al. 2008b). Moreover, our results, in fishermen that had participated at least 15 days in clean-up activities for on average > 4 h per day, revealed an increase in the chromosome alterations to 1–2 years after exposure (Monyarch et al. in ECA communication 2009). In view of these results, it seems essential to pay more attention to the human health effects of exposure to oil after accidental spills. Acknowledgments: This work was supported by FIS (PI070086), and a grant from Universitat Autònoma de Barcelona (PS-456-01/08) 5.3-O Genomic Instability in hESCs revealed by Molecular Cytogenetic Techniques M. Dekel (1) A. Aviram-Goldring (1) I. Laevsky (2) M. Amit (2) J. Itskovitz-Eldor (2) T. Litmanovitch (1) J. Shamash (1) E. Guetta (1) E. Schiff (3) S. Rienstein (1) (1) The Sheba Medical Center (2) Institute of Technology (3) The Sheba Medical Center

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The multipotency and proliferative capacity of human embryonic stem cells (hESCs) makes them a promising source for transplant therapies. However, in order to use hESCs in a therapeutic context, they must maintain long term genomic stability in culture. Until recently, cytogenetic analysis using G-banding was considered to be the gold standard for detecting chromosomal abnormalities in general and specifically in ESCs. The introduction of molecular cytogenetic techniques (such as FISH, CGH, aCGH), enable us to examine the human genome with an ever finer resolution. In this study, two hESC lines, line I3 (at p.98) and line I4 (at p.59 and at p.90), were karyotyped using Gbanding. While line I4 (p.59) was found to be normal (46, XX), line I3 (p.98) and line I4 (p.90) showed an unexplained segment amplification of chromosome 2p. CGH analysis revealed an amplification of chromosome 1p32–34.1. FISH analysis with whole chromosome 1 probe verified these findings, and also located this interstitial amplified region to the short arm of chromosome 2. Surprisingly, CGH analysis of line I4 p.59 also revealed an amplified region, located at chromosome 1q21–43. By using DNA fingerprinting, we confirmed a cross-contamination between line I3 p.98 and line I4 p.90 samples. Finally, aneuploidy rate was evaluated at different passages number, using FISH probes (x, y, 13, 18, 21). Aneuploidy was noted for late-passage hESC lines, implying a direct correlation between number of passages and increased aneuploidy rate. This study demonstrates the phenomenon of genomic instability of hESC lines, which is common in tumorogenic processes. These findings emphasize the importance of the use of molecular cytogenetic methods for authentication and determination of the genomic stability of hESC lines. In order to integrate these methods into a plan for routine cell line quality control, it is crucial to optimize, validate and assess the advantages and limitations of these cutting edge techniques. 5.1-P Genotoxicity of formaldehyde in human chromosomes M. Pongsavee (1) (1) Faculty of Allied Health Sciences, Thammasat University, Rangsit Campus

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Formaldehyde is a clear, colorless, volatile liquid which has a pungent odor and slightly irritating taste. It is useful for industry and medicine but it is the forbidding material for the vegetable and seafood preservation. When formaldehyde is accumulated in human body, it causes eyes and skin irritation, suffocation, chest pain and death. The heparinized blood from thirty healthy Thais were studied for the impact of formaldehyde genotoxicity through human lymphocyte culture and G-banding technic. The formaldehyde concentrations of 0.036, 0.072, 0.15, 0.3, 0.576, 0.8 and 1.152 mg/ml were used in this experiment. The results showed that the numbers of metaphase chromosomes in the 0.036 and 0.072 mg/ml formaldehyde concentration groups were different significantly comparing with the control group (P < 0.05). The numbers of metaphase chromosomes were decreased when the concentration of formaldehyde increased. Loss of the human chromosomes in group B, D, E were observed in the formaldehyde concentrations of 0.036 and 0.072 mg/ml groups. No metaphase chromosomes were observed in the formaldehyde concentrations of 0.15, 0.3, 0.576, 0.8 and 1.152 mg/ ml groups. It can be concluded that formaldehyde effects to human genotoxicity. Loss of chromosome induced by formaldehyde may increase risk of carcinogenesis in human. 5.2-P Detection of mutagen impact of Arsenic upon children A. Zedginidze (1) M. Antelava (1) K. Gvimradze (1) M. Gagoshidze (2) N. Mandgavidze (2) (1) Georgian Institute of Hematology&Transf. (2) Tbilisi State Medical University Presently great attention is paid to studying of the impact of mutagen polluting environment upon human health. Among chemical mutagens heavy metals are very important In one of the mountainous regions of Georgia, which for many years was mined and processed arsenic (As), increase of general morbidity and congenital anomalies between children were registered. In this region content of As in the environment considerably exceeds the admissible norms.

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The aim of this work was revealing the As impact on genetic apparatus of children from this region. Cytogenetic disorders of 50 children (7–15 years old) from mentioned region (group1) and 50 children living far (>50 km) from territory of As mining (group II) were compared. The chromosomal aberrations in peripheral blood cells, the level of micronuclei (MN) in binuclear lymphocytes and buccal exfoliative cells were studied. Simultaneously the level of As in the blood and urine was defined. Structural chromosomal aberrations (acentric fragments, chromosomal exchanges and singular dicentric chromosomes) only in group 1 were revealed. In the same group increase of MN as in lymphocytes, since in buccal cells were stated. In group 1 it was 0.24± 0.001 per cell in lymphocytes and 0.022±0.0012 in buccal cells, while in group II it was respectively 0.017±0.0007 and 0.0043±0.0005. By our own data the background level of MN in Georgia composed 0.014±0.008 in lymphocytes and 0.0016±0.0012 in buccal cells (interval of statistical confidence was 95%) Index of MN in lymphocytes was higher then in exfoliative cells. The same correlation in investigated group was revealed. The increase of chromosomal aberration and of the level of MN, also the contents of As in blood and urine of investigated children of group 1, gives account the ecological background witnesses about the mutagen impact of As

translocation frequencies in men and women. Present study comprised twenty-one generally healthy male and female subjects between the ages of 65 and 76. Individuals included have no record of suffering any inflammatory and/or autoimmune disease, malignancies or psychological disorders or any other state that would require chronic medical treatment. Examinees were matched with control subjects by age (68.26±3.74 vs. 45.90±6.77, respectively), sex (11 females; 10 males), and alcohol consummation (8 alcohol consumers; 13 non-alcohol consumers). Persons reporting alcohol consummation higher than one unit per week and smokers were omitted from the study. We analyzed metaphase lymphocytes by fluorescent in situ hybridization using paint probes for chromosomes 1, 2 and 4. For each subject not less than 1000 metaphases were analysed. Translocation frequencies were calculated according to Lucas et al., 1992. Genomic frequency of translocations per cell was significantly elevated (0.0228 ±0.0065 vs. control 0.0039±0.0017). Although, distribution of translocations among chromosomes 1, 2 and 4 slightly differed from the control subjects it did not significantly vary from DNA fractions for selected chromosomes. The frequency of chromosome 1 increased by 3.15%, for chromosome 2 decreased by 8.95% and for chromosome 4 increased by 5.81%. Preliminary results are interesting and further research on larger population should be conducted involving inclusion of some other chromosomes into evaluations of genome stability on healthy elderly individuals.

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Translocations yield and its distribution among chromosomes 1, 2 and 4 in healthy elderly population

Bleomycin-expressed hidden chromosome instability in peripheral blood lymphocytes of unexposed and irradiated persons in different terms following radiation exposure

M. Mladinic (1) N. Kopjar (1) M. Milic (1) D. Dasovic (2) D. Zeljezic (1) (1) Institute for Medical Research and Occupational Health (2) Medical practice

M. Pilinskaya (1) S. Dibskiy (0) Y. Dibskaya (0) L. Pedan (0) (1) Research Centre for Radiation Medicine

The accumulation of damage to cellular biomolecules, including DNA, over time may play a significant role in the aetiology of the ageing process. Studies have shown a significant increase in chromosome aneuploidy with age but none have investigated translocations in healthy elderly populations. The aim of the study was to investigate whether the age-related changes influence

At the cytogenetic level the important role in human genome destabilization take place hidden chromosome instability that expressed as increase of human somatic chromosome sensitivity to the mutagenic exposure— both in vivo and in vitro. With the help of modifying “G2-bleomycin sensitivity assay” (treatment of human peripheral blood lymphocytes culture in late G2 phase

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of mitotic cycle by radiomimetic bleomycin in concentrations 0.05 аnd 5.00 mcg/ml) the voluntary investigation of hidden chromosome instability in 9 unexposed donors as well as in 44 persons with different intensity and duration of radiation exposure (mainly external) had been fulfilled. Three exposed groups—patients recovered from acute radiation syndrome (ARS) transitory exposed to high doses of ionizing radiation ~ 21 years ago because of Chernobyl accident; clean-up workers exposed to prolonged irradiation of middle intensity due to liquidation of Chernobyl accident consequences in 1986–1987 years; personnel of 30 kmalienation zone (Shelter’s staff) exposed to chronic irradiation of low intensity during 2006–2008 years had been observed. The significant exceeding of chromosome aberrations individual frequencies under their mean-group level had been considered as the basic criterion of increased chromosome sensitivity to the testing bleomycin exposure. It had been determined that in all examined groups the individual levels of the chromosome injuries induced by identical concentrations of bleomycin varied in wide range and didn’t depend on their initial values in intact cultures (without testing bleomycin exposure). Among control donors as well as in liquidators and Shelter’s personnel ~ 33% persons hypersensitive to one or both bleomycin concentrations had been identified that can be considered as genetically caused phenomenon. Among ARS patients ~ 58% persons expressed hidden chromosome instability. The data received permit to assume that high doses of ionizing radiation can modify the inherited human chromosome susceptibility to mutagen exposure.

peripheral blood lymphocytes had been carried out. The frequency of chromosome aberrations had been studied with G-banding cytogenetics in recently proposed by us model system—“mixed human peripheral blood lymphocytes culture” consisted of cells differed on cytogenetic sex markers (XX, XY) and some morphological chromosome peculiarities. Under joint incubation of in vivo irradiated (in doses 350–690 mGy) and intact lymphocytes, it had been shown that the level of chromosome aberrations in bystander cells (4.67± 0.56per 100 metaphases) was significantly (р<0.01) higher than the spontaneous level (1.73±0.65 per 100 metaphases). The mean-group frequency of chromosome aberrations in targeted cells received from Chernobyl accident liquidators (4.11±0.66 per 100 metaphases) is significantly elevated over the background level. Cytogenetic effect in the targeted cells (4.23±0.69) cultivated jointly with unirradiated ones, didn’t change to compare with separate cultures. The difference between spectrum of aberrations in exposed and intact cells had been established—in targeted cells specific cytogenetic markers of irradiation dominated (unstable and stable chromosome exchanges) just as in bystander cells simple aberrations (chromatid breaks that can be consider as the markers of chromosome instability) mainly induced. The data confirmed the induction of chromosome instability in nonirradiated human peripheral blood lymphocytes (untargeted cells) jointly incubated with irradiated ones (targeted cells) as the result of bystander effect and the lack of the vice versa influence of nonirradiated lymphocytes to irradiated ones under their joint cultivation in vitro.

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Cytogenetic investigation of radioinduced bystander effect in mixed human lymphocytes culture

Calibration curves for cytogenetical indicators of ionizing radiation exposure in low doses

O. Shemetun (1) O. Talan (0) M. Pilinskaya (0) (1) Research Centre for Radiation Medicine

E. Djomina (1) Y. Demchenko (0) (1) R.Ye.Kavetskyi Institute of Experimental Pathology, Oncology and Radiobiology of NAS of Ukraine

For understanding the mechanisms of origin, both nonstochastic and stochastic abnormalities in persons exposed to ionizing radiation the investigation of radioinduced bystander effect is quite necessary. With this purpose the study of possible influence on chromosome stability of joint cultivation of intact somatic human cells within vivo irradiated (received from Chernobyl accident liquidators, 22 years following irradiation)

The purpose of the study was investigation of calibration curves characteristic for cytogenetical parameters after in vitro testing exposure of cultured human peripheral blood lymphocytes by ionizing radiation in the range of low doses. The irradiation of lymphocytes received from healthy donors had been done taking in account the individual radiosensitivity on therapeutic apparatus

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“Rockus” (with 6°Co source) in the range 0.1–0.5 Gy in late G2 phase of a mitotic cycle. The metaphase analysis of chromosome aberrations had been carried out analysing karyotypes. For the approximation of dependences of radiation-induced cytogenetic effects three models of regression—linear, linearly-square and spline—had been used. It had been shown that linear components had advantage for radiation-induced aberrant cells and total frequency of chromosome aberrations curves. Exchange chromosome aberrations (dicentrics mainly) better approximated by linearlysquare model. In the range of doses 0.1–0.3 Gy the curve for dicentrics had plateau—dose-independent field, but with increasing of dose till 1.0 Gy the level of dicentric chromosomes regularly grew. Thus, in the range of radiation doses 0.1–0.3 Gy it is impossible to evaluate correctly absorbed doses on the frequency of dicentric chromosomes which considered as most objective markers of radiation exposure. The analysis of standard curves “dose-effect” which had been constructed with the help of spline regression model compared with linear and linearly-square regressions had been shown that all type of dependences had the similar form, but with the greatest accuracy they had been approximated by spline regression model so far as the error of this model had least value for all cytogenetical indicators in all observed donors. So, for the purpose of further improvement of biological dosimetry of radiation-induced damages in human it is recommended to construct calibration curves on the basis of spline regression model and to consider individual sensitivity to radiation exposure. 5.7-P Accuracy assessment of Diepoxybutan test in differential diagnosis of Fancony anemia patients in Serbia M. Guc-Scekic (1) S. Cirkovic (1) D. Vujic (1) D. Micic (1) N. Krstovski (2) (1) Mother and Child Health Care Institute (2) University Children’s Hospital M. Guc-Scekic1, S. Cirkovic1, Vujic. D1, Micic. D1, N. Krstovski2 1 Mother and Child Health Care Institute of Serbia “Dr Vukan Cupic”, Laboratory of Medical Genetics, Belgrade, Serbia 2 University Children’s Hospital, Department of Hematology and Oncology, Belgrade, Serbia

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Fancony anemia (FA) is clinically and genetically heterogenous autosomal recessive disease, with familial AA, congenital anomalies, chromosomal breakage and increased predisposition to cancer. Chromosomal instability of FA cells reflects the hypersensitivity towards the DNA cross-linking agents, such as diepoxybutane (DEB); this test was used for screening of FA among Serbian patients with bone marrow failure syndromes (BMFS). From February 2004. to January 2009, 54 patients with BMFS, diagnosed and treated at the Mother and Child Health Care Institute and University Children’s Hospital in Belgrade, were provided for DEB hypersensitivity examination. Chromosome breakage analyses were carried out on control and DEB treated cultures of peripheral blood according to a standard method. DEB in concentration of 0.1μg/ml was added to DEB treated cultures, 24 h. before harvesting. 100 metaphases were examined from control and DEB treated cultures, for each patient. In a group of 54 DEB-tested patients, seven of them (13.2%) were found to have increased DEBinduced chromosome breaks. The ranges of DEBinduced chromosome breaks were: for DEB-insensitive patients 0.00–0.20 and for DEB-sensitive patients 0.58–4.39 break per cell. No overlap between DEBsensitive and DEB-insensitive group was found, while the ranges of spontaneous breaks for DEB-insensitive and DEB-sensitive patients were overlapping (0.00– 0.27 vs. 0.00–0.20). Our results suggest the importance of DEB test for differential diagnosis of FA among BMFS patients in Serbia. This work is supported by the Ministry of Science and Environment Protection of the Republic of Serbia (Grant N0 143046). 5.8-P Cytogenetic parameters in the evaluation of human lymphocytes sensitivity to low doses of ionizing radiation N. Ryabchenko (1) E. Diomina (1) (1) R.Ye. Kavetsky Institute of experimental pathology, oncology and radiobiology of NASU Evaluation of radiation induced cytogenetic abnormalities in human peripheral blood lymphocytes is known to be the “gold” standard in biological

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dosimetry. The aim of the presented study was estimation of chromosomal radiosensitivity of human lymphocytes to low doses of ionizing radiation. Materials and methods. Frequency of chromosomal aberrations was evaluated in the peripheral blood lymphocytes of healthy individuals (n=34) after the exposure of cell cultures in G2 phase of cell cycle to γ-rays in the dose range of 0,1–0,7 Gy on the bases of G2-assay. Metaphase plates were prepared using standard cytogenetic method. Results. It was shown that in G2 phase, which is characterized by the high chromosomal radiosensitivity, the interindividual variations in the levels of radiation induced chromosomal breaks are determined since the dose of 0,1 Gy. Coefficient of variation (CV) for aberration levels induced by 0,1 Gy radiation dose was 15%, 0,3 Gy—12%, 0,5 Gy—9%. Using 90th percentile as cutoff criterion it was founded that 15% from the examined cultures had enhanced chromosomal sensitivity to 0,1 Gy irradiation while 9% showed the aberration levels of unexposed cultures at the same dose. The enhanced radiosensitivity to 0,1Gy correlated with enhanced radiosensitivity to 0,5Gy irradiation. We assume three different types of chromosomal outcome to low dose irradiation. Thus cytogenetic patterns observed may reflect cell sensitivity to low doses of radiation and G2 assay can be applied to the estimation of genome instability induced by low dose ionizing radiation. 5.9-P

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level was also established in normal embrionic and fetal cells and in the short-term lymphocyte cultures of patients with chromosome instability syndromes (NBS, ataxia-telangiectasia), reproductive losses and children suffering from environmental pollutions (heavy metals and fluor). In acute stage of hemoblastoses more than 10% cells revealed premature division of 3–20 chromosomes with following regression to control indices in full remission: 0– 3% metaphase with partial PCS of 1–2 chromosomes of C and E groups. The positive correlation with aneuploidy frequency and blasts cells level was also noted. Except that, increased partial PCS level seems to be typical for the cases of chromosome instability syndromes and reproductive disorders. We found significant evidences, that the partial PCS is correlated with induction of chromosome aberrations and may cause chromosome’s losses (aneuploidy), whereas the total PCS is associated either with induction of apoptosis or endoreduplication in following mitotic cycle. The obtained results show regularity of PCS appearance resulting from exogenous or endogenous DNA damage, tissue differentiation processes, malignant transformation and cell’s death. We recommend evaluation of total and partial PCS for diagnostics and prognosis of prevalent hemoblastosis, chromosomal instability syndromes, in cases of a history of reproductive failure and for cytogenetic monitoring of genotoxic influence. We also suggest the probability of heterozygous carrying of mutations of the BUBR1, NIPBL and ESCO2 genes in the certain cases of increased PCS level.

Premature chromatid separation in diagnostics and prognosis of human pathology

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H. Akopyan (1) (1) Institute of Hereditary Pathology of Academy of Medical Sciences of Ukraine

Genotoxical monitoring using micronucleus assay and chromosomal aberrations in hospital staff exposed to Formaldehyde

The spontaneous level of premature chromatid separation (PCS) was established in lymphocytes, bone marrow cells, chorion villi cells and amniocytes in vitro. The significant increase of total PCS level was revealed in bone marrow and blood cells of both ,children and adult patients, suffering from prevalent hemopoietic malignancies (ALL, AML, CML, non-Hodgkin lymphoma) with following regression to control indices (0–1% cells) in full remission. The significant increase of total PCS

S. Bouraoui (1) A. Brahim (2) F. Tabka (2) N. Mrizek (2) A. Saad (1) H. Elghezal (1) (1) Farhat Hached University Teaching Hospital (2) Farhat Hached University Teaching Hospital Genotoxic nature of formaldehyde in vivo and in vitro was reported in many previous studies. In this study, chromosomal aberrations (CAs) and micronuclei (MN) were used as a biomarker to evaluate the genotoxic effect of professional exposure to formal-

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dehyde, in pathology laboratory workers (Farhat Hached hospital of Sousse—Tunisia). Chromosomal damage was analyzed in peripheral lymphocytes of 31 exposed subjects (19 women and 12 men) and compared with 30 controls matched for gender, age, smoking and life style. Two thousand cells per sample were analyzed for the presence of micronuclei after a cytochlaasin B treatment inducing cytokinesis block. The frequency of binucleated micronucleated cells was significantly higher in exposed population (42, 27‰±18, 75 versus 7,08‰±4,626, p<0,005). A clear effect of the time of contact with formaldehyde was found, with significantly increased frequencies of micronuclei in binucleated cells (p<0, 05). CAs were explored using cytogenetic analysis of cultured lymphocytes in 25 ng/ml Mitomicyn C. The frequency of CAs was also significantly higher exposed group than in control (6, 97%±14,634 versus 1, 77%±1,543, p<0,005). Our results support the genotoxic effect of professional exposure to formaldehyde in pathology laboratory workers and we recommend the use of cytogenetic monitoring to evaluate the severity of genetic damage on the way to develop a global and specific safety programs. 5.11-P Estimation of chromosomal translocations frequency in transoceanic air crew members compared to a control population M. Prieto (1) M. Moreno (1) L. Zapata (1) R. Dominguez-Mompell (2) R. Herranz (4) (1) Hospital General Gregorio Marañón (2) IBERIA Air crew members are exposed to ionising radiation of cosmic origin, so they are considered to be a radiation exposed occupational group in the last report of the International Commission for Radiation Protection (ICRP 90). Chromosome translocations are considered a biomarker of cumulative exposure to external ionising radiations better than chromosome dicentrics, we analysed the frequency of translocations from peripheral blood of a group of 50 air crew members and 50 controls matched for the age, smoking habits and sex.

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Metaphases spreads were obtained after incubation of peripheral blood lymphocytes in the presence of PHA for 48 h at 37° (all procedures were done under laboratory routine procedures). Chromosomes 1 and 2 were painted red and a pancentromeric probe was used simultaneously to coloured all centromers green, to distinguish translocations and dicentrics, this procedure gives an efficiency of 28% when extrapolating to the full genome. There were analysed 2000 metaphases per person, that means 560 cell equivalents. There are not statistically significance differences in translocations frequency between both populations but the statistical analyses is in progress, (it will be present in the final work at the Congress). 5.12-P Tau protein and chromosome stability G. Rossi (1) E. Panzeri (2) D. Conconi (2) F. Crosti (3) S. Lissoni (3) E. Piccoli (1) F. Tagliavini (1) L. Dalpra’ (2) (1) Neurological Institute “Carlo Besta” (2) University of Milano-Bicocca (3) S. Gerardo Hospital Tau is a microtubule associated protein that promotes microtubules assembly and stabilization. It is mainly expressed in neuronal and glial cells, but is also present in fibroblasts and lymphocytes. An altered tau leads to cytoskeleton disruption in the central nervous system, causing neurodegenerative diseases such as tauopathies. It has been suggested that tau might play further functions, given its presence in different cellular compartments such as ribosomes, the area near the plasma membrane, and nucleoli. We demonstrated in normal human fibroblasts the presence of tau in the nucleus during interphase and in the periphery of mitotic chromosomes, assuming that tau may have a nuclear role. Thus, we evaluated the presence of chromosomal abnormalities in lymphocytes and fibroblasts of patients with tauopathies caused by tau mutations. We also investigated whether mutated cells were more sensitive to genotoxic agents. Several chromosomal aberrations and alterations in chromatin structure have been found in cells of patients bearing the P301L mutation. Lymphoblasts challenged

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with bleomycin showed a reduced mitotic index. Our results prompted us to believe that tau plays a role in chromosome stability, most likely through interactions with both microtubules and chromosomes. 5.13-P Chromosome fragility in Romanian river buffalo females I. Nicolae (1) M. Paraschivescu (1) M. Enculescu (1) D. Vidmichi (1) A. Bota (2) (1) Research and Development Institute for Bovine Breeding (2) Research and Development Station for Buffalo Breeding Karyotype analyses were performed on a group of 16 female river buffaloes undergoing hormonal stimulation and on a control group of 16 female river buffaloes. Peripheral blood lymphocytes were cultured for about 72 h at 38,5°C in Minimal Essential Medium (Sigma) supplemented with 15% fetal bovine serum (Sigma) and Con A as mitogen. Two types of cell cultures, without (normal cultures) and with addition of 5-bromodeuxiridine (BrdU) 30 h before harvesting, were carried out. Slides from both cultures were stained with acridine orange. At least 30 metaphase plates per animal were observed under a fluorescence Aristoplan Leitz microscope, captured with a Photometrics Cool Snap camera, transferred on PC and processed by a specific image software. The preliminary results revealed that three of the treated females were found to carry a total number of breakages/cell (7.86± 2.65) that was higher than that of the control group (2.34 ±1.23). The mean numbers of SCE/cell in the three treated females were higher (X =11.8) than that of those observed in the control group (X = 7.8). Further cytogenetic studies are needed to verify if there is a relationship between the chromosomal fragility and the hormonal treatment or the effect of the environmental toxic agents. Acknowledgements This study was supported by the Sectorial Project 351/2006. We are grateful to Prof.L.Iannuzzi for the scientific and technical support.

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5.14-P Chromosome instability analysis in parents of fetus with aneuploidies using CBMN test. J. Garcia-Sagredo (1) A. de Leon Rodriguez (1) M. Arias (1) K. Yanowsky (1) E. GarciaGalloway (1) C. Villalon (1) M. Talavera (1) P. Cabello (1) M. Ferro (1) (1) University Hospital Ramon y Cajal Objective: To analyze if parents with aneuploidic fetus diagnosed in the prenatal diagnosis clinic, have an increment in the rate of chromosomal instability. Methodology: Cytokinesis block micronucleus test (CBMN) to analyze MN rate in binucleated cells and distribution of MN among FISH positive and FISH negative using a pancentromérica probe. Material: Couples recruited in the prenatal diagnosis clinic after a diagnosis of aneuploidy in their fetus. Couples with two or more abortions without chromosome aberration. Control couples. Results and conclusions: Preliminary results did not show any significant differences among parameters analyzed with CBMN test in the three groups analyzed: MN rate 6.66±2.2 in parents of aneuploidy, 7.78±2.6 in couples with abortions, and 6.49±2.6 in controls. FISH distribution using a pancentromeric probe shows slight but non-significant increase of MN FISH+ in couples with an aneuploidic fetus: 53.5% MN FISH+ in parents of aneuploidy; 49% MN FISH+ in abortion couples, and 40% MN FISH+ in controls. 6.1-O Patterns of meiotic recombination on chromosome 16 in human oocytes R. Garcia Cruz (1) M. Brieño (1) P. Robles (1) I. Roig (2) M. Garcia Caldés (1) (1) Universitat Autònoma de Barcelona (2) Memorial Sloan-Kettering Cancer Center Trisomy 16 is the most common aneuploidy among clinically recognized pregnancies. Since it is not compatible with life, trisomy 16 is the leading chromosomal cause of miscarriages. Linkage studies of affected fetuses have established that all the cases

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originate in the maternal germ line. In addition, trisomy 16 is unique in the sense that the nondisjunction event originates exclusively in the maternal first meiotic division. Indirect studies have associated abnormal patterns of meiotic recombination in the origin of the nondisjunction event in many trisomies. Reduced but not absent recombination has been associated with the origin of trisomy 16. However, although the exclusively maternal origin, no direct analyses of the recombination process on chromosome 16 have been reported in human oocytes. Such studies could provide data about the susceptibility of the control population to conceive a trisomic 16 fetus. Herein, we present data from a direct analysis of meiotic recombination on chromosome 16 on human prophase oocytes. The data is compared to the recombination pattern of chromosome 17, a similar sized chromosome not involved in the most commonly recognized aneuploidies. Number and distribution of the crossover marker MLH1 have been analyzed in both chromosomes by immunofluorescence and consecutive fluorescent in situ hybridization. Results show that the percentage of oocytes presenting a single CO in chromosome 16 is threefold higher compared to chromosome 17. Based on these results and on the information from indirect studies on trisomy 16, we hypothesize that these oocytes might be at risk of nondisjuntion during the first meiotic division occurring in the adult ovary, leading to unbalancies in the resulting fertilizable oocyte.

throughs enable new clinical applications including preimplantation and prenatal genetic diagnosis of simple and complex traits on a genome-wide scale. Furthermore, the technology is invaluable for genetic research not only in the field of human gametogenesis, embryogenesis and tumorigenesis, but also in studies of mosaicism, chromosomal instability and studies towards the origin of constitutional genetic aberrations. We have generated proof-of-principle by applying this novel methodology to diverse cell types, including single blastomeres of human cleavage stage embryos, single fertilized oocytes and single lymphoblastoid cells. This enabled us (1) to detect and confirm by genome-wide SNP-analysis whole chromosome and segmental aneuploidies as well as uniparental disomies in single cells, (2) to do SNPbased parent-of-origin analyses on detected chromosomal aberrations and (3) to reconstruct haplotypes of single cells using different genotyping and Merlin phasing algorithms. These methods are of interest to both a scientific and clinical community.

6.2-O

Genomic disorders are caused by meiotic chromosomal rearrangements involving unstable regions characterized to be flanked by Low Copy Repeats sequences (LCR). LCR act as substrates for Non Allelic Homologous Recombination (NAHR) leading to different chromosome reorganizations such as deletions, duplications and inversions that in some cases are the cause of human diseases. The aim of this study was to assess the basal frequency of deletions and duplications of the 15q11q13, 7q11.23 and 22q11.2 regions in spermatozoa from control donors to check differences in the susceptibility to generate anomalies in these hot-spots. Semen samples from ten control donors were processed and analyzed by triple-color FISH as standardized in our laboratory. A customized combination of probes (Vysis Inc.) was used to assess each

Haplotyping of single human cells T. Voet (1) E. Vanneste (1) H. Peeters (1) P. Konings (2) J. Fryns (1) T. D’Hooghe (3) Y. Moreau (2) J. Vermeesch (1) (1) KULeuven (2) KULeuven (3) University Hospital Gasthuisberg Determining chromosomal anomalies and genotypes in single human cells has vast applicative value, but is technically not straightforward. We have developed novel microarray-based methods that type simultaneously genome-wide copy number and single nucleotide polymorphisms (SNPs) in single cells with unprecedented accuracy. These methodological break-

6.1-P Incidence of de novo deletions and duplications at 15q11q13, 7q11.23 and 22q11.2 regions in spermatozoa Ò. Molina (1) J. Blanco (1) F. Vidal (1) (1) Universitat Autònoma de Barcelona

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region allowing the discrimination between normal, deleted and duplicated sperm genotypes. A minimum of 10000 sperm were assessed per sample and region. As a whole, no significant differences were observed in the frequency of deletions and duplications (del/dup) between the 15q11q13, 7q11.23 and 22q11.2 regions (0.46%±0.07; 0.37%±0.02 and 0.27%±0.07 respectively) (Friedman test; P=0.122). However, by using hierarchical cluster analysis two groups of individuals were clearly identified regarding to the NAHR pattern. One group showed a higher frequency of 15q11q13 and 7q11.23 del/dup while the second one tended to have a higher frequency of 22q11.2 del/dup. The frequency of deletions was not different than the frequency of duplications in 15q11q13 (0.22% vs 0.24%) and 7q11.23 (0.21% vs 0.17%) regions, thus suggesting a predominant interchromatid-NAHR. In the 22q11.2 region the frequency of deletions was significantly higher (0.17% vs 0.11%, P<0.05) and point to the involvement of intrachromatid-NAHR. In summary, our results reveal a similar basal level of del/dup in these three hot-spots, but a different behavior between individuals and hot-spots, suggesting that particular haplotypes could be the main source of variability in NAHR rates. Keywords:Deletions, duplications, sperm-FISH. Aknowledgements: Universitat Autònoma de Barcelona (PIF/2007). 6.2-P Segmental UPD(7q)mat is more frequent in Silver Russell syndrome than expected K. Eggermann (1) S. Spengler (1) G. Binder (2) T. Eggermann (1) N. Schönherr (1) (1) RWTH Aachen (2) University of Tübingen Segmental UPD, e.g. UPD affecting only segments of a chromosome, is generally considered to be rare in humans. As reviewed by Kotzot in 2008 (J Med Genet 45), 26 cases have been described in the literature, among them two carrying UPD(7q)mat. We recently reported on two additional Silver-Russell syndrome (SRS) patients with this aberration (Eggermann et al., Clin Genet 2009), and we now have identified a third case ascertained by routine molecular diagnostic

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testing for SRS. In our study on the molecular etiology of SRS and in our routine diagnostic screening we have meanwhile identified 18 UPD(7)mat carriers, including the three carriers of segmental UPD(7q)mat. Thus a frequency of segmental UPD of >15% among UPD(7)mat patients can be delineated. We assume that segmental UPD(7q)mat is much more frequent in SRS than expected. Our data show that it is important to analyse both arms of chromosome 7 by microsatellite typing and/or methylation specific PCR and not to rely only on single loci. The finding of segmental UPD restricted to 7q prompts us to speculate that a SRS relevant gene is localised on the long arm. In contrast to this putative involvement of 7q genes in SRS, 7p encoded factors were so far in the focus of research because of the identification of maternal 7p duplications. Our findings bring more confusion than enlightment into the role of chromosome 7 in the aetiology of SRS. 6.3-P The relation between consanguineous marriage and mental retardation of the offspring M. Ghanbari (1) M. Hasanzadeh Nazarabadi (2) A. Arghami (3) (1) Mashhad University of Medical Sciences (2) Mashhad University of Medical Sciences (3) Mashhad University of Medical Sciences Introduction: Consanguineous marriages are traditionally favored in most of Asian and African countries especially in Muslim countries. However, it is well known that this type of marriage is a major factor responsible for some genetic disorders inherited in an autosomal recessive pattern. On the other hand, consanguinity among parents as a cause of mental retardation (MR) in their children is debatable. Although consanguineous marriage is quiet common in Iran, the information on its prevalence is too poor. The present study was conducted to find out the risk ratio of having a mentally retarded child by consanguineous married couple to that of unrelated couples. Another objective of this study was to provide an acceptable estimate of consanguineous marriage rate in Mashhad (Khorasan province, Iran). Material and methods: This project was carried out in two parts: Part I: The rate of consanguineous marriage

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in Mashhad was estimated from the data collected in a sample survey in Mashhad using stratified two stage cluster sampling with a grand total sample size of 1142 families by the research team just for this purpose. Part II: Rate of consanguinity among couples who had at least one mentally retarded child was estimated from the data supplied by a simple random sample from records of patients in Behzisty center (total sample size = 1270) and by cluster sampling from the records of the school for MR students (total sample size = 669). Results: Base on the collected data, the rate of consanguineous marriage in Mashhad was estimated to be 30.5%. The estimate of the same rate for families who had at least one mentally retarded child was 38% in Behzisty center and 44% in disability schools. Using statistical methods for calculating risk ratio in indirect sampling we obtained the risk ratio to be equal to 1.4 for Behzisty data and 1.8 for disability schools data. Discussion: I review of the results, the rate of consanguineous marriage among children in Behzisty and disability schools was greater than that in general population in Mashhad; and the difference was statistically significant (p-value=.000). Thus it is important to take action through educating young adults in this regard. Key words: Consanguineous marriages, Mental Retardation, Offspring, Mashhad 6.4-P Full monosomy 21: new cytogenetic and molecular perspectives M. Martinez-Garcia (1) E. Ainse (1) A. BustamanteAragones (1) M. Garcia-Hoyos (1) B. GomezDominguez (1) D. Diego-Alvarez (1) R. Cardero (1) M. Rodriguez de Alba (1) C. Ramos (1) I. LordaSanchez (1) (1) Fundacion Jimenez Diaz Introduction: Based on molecular studies of spontaneous miscarriages in which culture fails, the observed frequency of chromosomal anomalies could be higher than expected. Coventional cytogenetic studies have revealed that 60% of first trimester spontaneous

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abortions are chromosomally abnormal. Full monosomy 21 (FM21) is a very uncommon cytogenetic finding and only a few cases have been previously reported. Here, we report a FM21 identified by MLPA and confirmed by array-CGH as part of a molecular study of 310 miscarriages. Case report: A sample from a miscarriage of 7 weeks of gestation was referred to us from a 33 year old primigravida. No cell culture was established due to the advanced stage of maceration of the sample. Chorionic villi were sampled for molecular assays only. MLPA for subtelomeric regions (P070 & P036 probe mixes) was performed. A significant decrease peak pattern involving the 4 probes included for chromosome 21 was detected. Due to the absence of karyotype and in order to distinguish between a monosomy 21 versus ring chromosome, further analyses with D21S263, D21S266, D21S1414 and D21S1411 STRs were performed. Results revealed the loss of maternal alleles for all those markers. Finally, FM21 was confirmed by 1Mb resolution array CGH. Conclusion: Although few full monosomy 21 cases have been previously reported, the use of molecular techniques might reveal that the frequency of this anomaly could be likely underestimated. Cell culture may yield normal karyotypes and select those with certain chromosomal anomalies that allow cell proliferation in vitro. Molecular assays could be a complement to cytogenetic studies to solve culture limitations. Furthermore, latest advances in molecular genetics could open up new perspectives in our understanding of the cause of spontaneous miscarriages. 6.5-P On the origin of constitutional aneuploidy M. Hulten (1) E. Iwarsson (1) (1) Warwick University (2) Karolinska Institutet The current consensus is that the oigin of constitutional aneuploidy is complex, involving both genetic and environmental factors. We have recently challenged this dogma. Thus, we suggest that the main reason for the origin of constitutional trisomies is different degrees of fetal ovarian trisomy mosaicism in normal females (Hulten et

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al 2008, Mol Cytogenet 1(1):21). At the meiotic reductional division (taking place just before ovulation) the extra chromosome in the oocyte will then lead to an extra chromosome (or chromatid) in either the oocyte or the first polar body. We here highlight the implications of our proposal in relation to previous work using FISH/ CGH arrays of oocytes and polar bodies.

preimplanation diagnosis by polar body analyses to select oocytes with aneuploidies. More than 50% of the tested polar bodies showed nondysjunction for chromosome 21 or chromosome 21 chromatids. In summary, these data indicate strongly an intrachromosomal effect in the meiosis of the inversion carrier, resulting in an increased risk for trisomy 21.

6.6-P

7.1-P

Pericentric inversion (21)(p13q21) increase the risk for maternal meiosis errors

High-resolution imaging of mitotic instability

M. Stumm (1) O. Schmid (2) I. Zastrow (3) H. Tönnies (4) M. Blöchle (5) R. Wegner (1) R. Wegner (6) M. Stumm (7) (1) Center for Prenatal Diagnosis (2) Practice for Prenatal Diagnosis (3) Practice for Genetic Counselling (4) Institute for Human Genetics (5) Fertility Center at Kaiser Wilhelm Memorial Church (6) Institute for Human Genetics (7) Institute for Human Genetics We report the case of a 36 year old woman referred to our Center for prenatal diagnosis on chorionic villi. Karyotyping of chorionic villi cells showed a free trisomy 21, including one structural aberrant chromosome 21 showing a pericentric inversion (21)(p13q21). Cytogenetic follow up studies demonstrated that the mother herself was carrying the inversion as de novo aberration. There is no phenotypic effect in the majority of pericentric inversion heterozygote carriers, when it is a balanced rearrangement. However, infertility, miscarriages and/or chromosomally unbalanced offsprings can be observed in carriers of a pericentric inversion (Gardner and Sutherland, 2004). Therefore, the patient requested invasive prenatal diagnosis in her following pregnancy. Chorionic villi sampling was performed, and cytogenetic analysis detected a free trisomy 21, including again the inversion chromosome inv(21) (p13q21). Microsatellite analyses demonstrated meiosis 1 non-disjunction in both pregnancies. A correlation between the inversion chromosome 21 and the recurrent pregnancies with trisomy 21 (intrachromosomal effect) could not be excluded. Because of this putative risk, the patient chose for

D. Gisselsson Nord (1) (1) Lund University Hospital Abnormal chromosome segregation is one way by which neoplastic cells accumulate the many genetic abnormalities required for tumour development. Routine chromatin stains are sufficient to detect many such alterations in chromosome dynamics. However, the precise location of individual chromosomes can be difficult to discern by routine staining. Fluorescence in situ hybridisation (FISH) on mitotic figures is one efficient strategy to allow simultaneous visualisation of several chromosomes at different stages of the mitotic process. In particular, FISH on anaphase or telophase configurations allows the scoring of sister chromatid segregation patterns. FISH can now be efficiently combined with immunofluorescence, making it possible to visualize simultaneously the dynamics of chromosomes and the movement of key proteins in the mitotic machinery. This methodology can be efficiently combined with confocal microscopy and other techniques for studying mitotic topography in three dimensions. There are now also several fluorescence-based and/or laser-enhanced systems available to study the movement of chromosomes in real time. Using these methods, chromosome segregation errors in cancer can be broadly sub-divided into abnormalities in spindle symmetry (spindle multipolarity and size-asymmetry of ana-telophase poles) and abnormalities in sister chromatid segregation (chromosome bridges, chromatid bridges, chromosome lagging, acentric fragment lagging). Often, these categories of errors must be combined to accurately describe the events in a single abnormal mitotic

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cell. Further characterisation of the molecular alterations leading to abnormal chromosome segregation together with the current developments in nano-level and real-time imaging will undoubtedly lead to an improved understanding of chromosome dynamics in cancer cells. 7.2-P Common Fragile Sites and Genomic Rearrangements in Neuroblastoma A. Blumrich (1) M. Schwab (1) L. Savelyeva (1) (1) German Cancer Research Center (2) German Cancer Research Center (3) German Cancer Research Center Common fragile sites (cFS) represent chromosomal regions that are prone to breakage after partial inhibition of DNA synthesis. They are associated with various forms of DNA instability in cancer cells and cFS activation is thought to be an initiating event in the generation of DNA damage in early stage tumorigenesis. DNA amplification is one type of genomic alteration in cancer cells, but its molecular basis remains poorly understood. Neuroblastoma provides a prototypic example for studying the mechanism of DNA amplification due to the fact that the proto-oncogene MYCN on chromosome 2p24 is amplified in about 20% of all tumors. The main focus of this study is the exact mapping of the genomic region FRA2C distal of chromosome 2 and to analyze its correlation to MYCN amplicon boundaries in neuroblastoma cell lines and primary tumors. The application of six-colour FISH allows us to identify the DNA sequence of FRA2C extending over two genomic regions of 500 kb (proximal) and 700 kb (distal) at 2p24.3 and 2p24.2, respectively. Fine-Tiling Array CGH was performed at an ultra high resolution (~200 bp) to determine genomic rearrangements on the short arm of chromosome 2. We showed that MYCN amplicon borders are clustered in the telomeric region of FRA2C in 63% of analyzed neuroblastoma cell lines and primary tumors and propose that initiation of MYCN amplification might be a consequence of FRA2C activation.

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7.3-P Nuclear positioning of genes and chromosome territories in lymphoma cells with the t(14;18) translocation. E. Garimberti (1) J. Bridger (1) S. Tosi (1) (1) Brunel University Chromosomes occupy specific territories within interphase nuclei. The localization of these territories is non-random with gene-poor chromosomes positioned at the nuclear periphery, and gene-rich chromosomes towards the interior of nuclei. This spatial organization is influenced by the interaction between chromatin and the proteinaceous structures of cell nuclei and is thought to have a relevant role in the regulation of gene expression. We are interested in the study of the behaviour of chromosome territories and specific genes in cancer cells that harbour chromosome rearrangements. In this study we used fluorescence in situ hybridization (FISH) on a non-Hodgkin lymphoma derived cell line to study the interphase spatial positioning of the t(14;18) translocation. Our aim was to investigate possible changes in the nuclear positioning of the chromosome 14 and 18 territories and IgH (on 14q32) and Bcl2 (on 18q21) loci directly involved in the rearrangement. We predicted two main possible scenarios: (i) chromosome territories would change nuclear position to facilitate the fusion of relevant loci or (ii) the bulk of chromosome territories would not change nuclear position, but relevant loci would come in contact due to formation of chromatin loops. We have revealed that chromosome 14 territories are dissimilar to control cells and are not located at the nuclear centre, but occupy a more peripheral position in the cell nucleus. The distribution of FISH signals for the IgH loci within the lymphoma cell nuclei is bimodal. This could suggest that one of the two alleles is found towards the nuclear interior whereas the other allele extends towards the nuclear periphery. Further experiments will explore the nuclear localization of both derivative chromosomes and their spatial relationship with other chromosomes and the nuclear environment. More comprehensive studies on the genome organisation in lymphoma cell nuclei will lead to a better understanding of the mechanisms of diseases due to chromosomal aberrations.

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7.4-P

7.5-P

Genome organization during hepatocarcinogenesis

Characterisation of chromosomal aberrations involved in acquired docetaxel resistance in human breast cancer cell lines

L. Parada (1) L. Espinosa (0) F. Royo (0) M. Martínez-Chantar (0) J. Mato (0) (1) CICbioGUNE-CIBEREHD Hepatocellular carcinoma (HCC) is one of the most common human malignancies worldwide. Decreased activity of Methionine AdenosylTransferase 1A (MAT I y Mat III), the liver specific form of MAT, has been demonstrated in various liver diseases, including HCC. MAT1A knockout mice develop hepatic steatosis (HS) after 3 months, non alcoholic steatohepatitis (NASH)after 8 months, and HCC after 12 moths in a step wise fashion. We investigated the organization of the genome during hepatocarcinogenesis in this metabolic deficiency scenario. Hepatocytes from 4, 8 and 12 months-old wild type (WT) and MAT1A-KO male mice were subjected to metaphase and interphase genome analysis. Spectral karyotyping (SKY) from short term cultured hepatocytes showed clonal losses of chromosomes 17, 18, 19 in 50% of animals with HS. These numerical abnormalities were present in all mice with NASH, accompanied by recurrent chromosome structural changes and unidentifiable markers denoting chromosome fusion. 3-D analysis of the nuclear architecture showed that the fraction of the nuclear volume occupied by chromocenters in hepatocytes from MAT1A-KO mice increased with the progression of the disease, contrasting with age matched WT mice in which decreased with age. Telomere length measurement on metaphase chromosomes showed that it decreases significantly in animals with NASH compared to HS, but elongation seems to happen after HCC onset(68,8 Kb in HS; 13,Kb in NASH; 59,6 Kb in HCC). However, volume measurements revealed that the fraction of the nuclear volume occupied by telomeres in interphase increases. Finally, comparison of the histone H3-K9 acetylation level of hepatocytes from mice at different stages of liver disease demonstrated a trend of hyperacetylation during tumour development. Taken together, these data indicates that the malignant progression of liver disease, in MAT1A-KO mice, involves global genome organization changes affecting centromeres, telomeres and chromosome arms.

J. Turbitt (1) L. Smith (1) K. McAuley (1) D. Massie (1) A. Schofield (2) D. Stevenson (1) (1) Cytogenetics (2) University of Aberdeen Characterisation of chromosomal aberrations involved in acquired docetaxel resistance in human breast cancer cell lines Julie Turbitt1, L.Smith1, K. McAuley1, D. Massie1, A. Schofield2, David Stevenson1 1 Cytogenetics Department, North of Scotland Regional Genetics Service, Aberdeen 2School of Medicine, University of Aberdeen, Aberdeen Docetaxel (Taxotere®) is one of the most active cytotoxic drugs available for the treatment of advanced breast cancer. However docetaxel resistance is a major clinical problem, limiting its effectiveness for breast cancer treatment. To date the precise mechanisms of docetaxel resistance are poorly understood and therefore require further investigation. Previous studies by our group used docetaxelresistant human breast cancer cell lines, MCF-7 (oestrogen-receptor positive) and MDA-MB-231 (oestrogen-receptor negative), and metaphase CGH in order to identify chromosomal aberrations involved in docetaxel resistance. A common amplified region on chromosome 7q21 and loss of chromosome 10q was observed in both MCF-7 and MDAMB-231 docetaxel-resistant cell lines, suggesting that these regions may be indicative of resistance to docetaxel chemotherapy in vitro. Metaphase CGH is not without limitations, and hence other techniques in addition to metaphase CGH should be employed to determine mechanisms of chemotherapy resistance. Therefore this current study aims to further characterise chromosomal aberrations in docetaxel-resistant cell lines using a combination of conventional cytogenetics and SNP array analysis. This data confirms and extends those of metaphase CGH, helping to enhance our understanding of the mechanisms involved in taxane resistance in breast cancer.

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Julie Turbitt: [email protected] Preference: Poster presentation 7.6-P Molecular characterization of double minutes/ homogeneously staining regions in selected tumors cell lines. C. Storlazzi (1) A. Lonoce (1) D. Trombetta (1) M. Guastadisegni (1) P. D’Addabbo (1) G. Daniele (1) G. Macchia (1) K. Kok (2) M. Carella (3) M. Rocchi (1) (1) University of Bari (2) University of Groningen (3) RCCS Casa Sollievo della Sofferenza Hospital DNA amplification in cancer is a common genetic mechanism by which cells can up-regulate gene expression in amounts exceeding the transcriptional capacity of a single copy gene. Double minutes (dmin) and homogeneously staining regions (hsr) represent the cytogenetic hallmarks of genomic amplifications in tumors. Little is known on their genomic structure, gene expression pattern and possible mechanisms for their genesis. Previous results obtained on 34 patients affected by acute myeloid leukemia (AML) with MYC (8q24)-containing dmin provided evidence in favor of the episome model for the formation of dmin (Storlazzi et al., 2006). Following the same rationale as in leukemia patients, we have performed oligo array-CGH, FISH, and PCR analyses in order to disclose the molecular architecture of amplicons in dmin/hsr in six and eleven tumor cell lines with MYC and MYCNamplifications, respectively. The results obtained, regarding the definition of the amplicon junction sequences, indicated that the episome model is the favorite mechanism behind 8q24- and 2p24-dmin/ hsr formation in solid tumor cell lines. We found that the whole amplicons were arrayed in a “head to tail” fashion in the majority of the cell lines, generated by non homologous end joining mechanisms. Interestingly, in some of the cell lines investigated, the sequencing of the whole amplicons disclosed the extreme complexity of their architecture, as they were composed by multiple, non-colinear, segments from bands 8q24 or 2p24, or fused with further fragments surprisingly mapping outside the MYC- or MYCN- region. The results so far obtained clearly indicated that the dmin/hsr genesis in solid tumor cell lines with 2p24 or 8q24 amplifications is far more

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complicated than what predicted by the episome model. Moreover, our results showed that unexpected genes were co-amplified with MYC or MYCN. We have tested the expression of such genes in order to show if they could be important targets of the genomic amplification in the cell lines under study. 7.7-P Comparative analysis of HER2 and TOP2A amplification in breast cancer U. Gamerdinger (1) (1) University Hospital Gießen and Marburg In tumorigenesis up-regulation of gene expression can be achieved by DNA-amplification. In breast cancer amplification of ERBB2 (HER2/neu) gene and TOP2A gene, located nearby on chromosome 17, is of particular interest because they are supportive biomarkers in the management of patients with regard to therapy regimen. A series of 90 breast cancers, 38 negative cases (0 and 1+), and 52 positive ones (2+ and 3+, n=32 and n=20, respectively) according to results of immunochemistry (IHC) analysis (HercepTest, DAKO), were analyzed by HER2- and TOP2A- FISH (pharmDx Kit, DAKO), both kits combining a gene-specific (GS) and a centromere-specific (cen) probe. For finer stratification of outcome beside ratio evaluation (positive ≥ 2.0) for cases scored 2+ by IHC, the FISH results were grouped as disom, aneusom (i.e. monosomy, trisomy), polysom (> 4 cen-/GS-signals), low amplification (modest degree of amplification: elevated number of GS-signals compared to number of cen-signals), and high amplification (large clusters of GS-Signals and 2 to few cen-signals). According to this classification, by HER2-FISH the IHC-negative cases maximally showed low amplification (n= 4) but mostly being disom or aneusom. The most heterogeneous results were found in the 2+ cases exhibiting all patterns from disomy to high amplification. In the 3+-group all but one with polysomy had low or high amplification. TOP2A- FISH revealed comparable pattern to HER2 in most of the cases. Discrepant results were

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almost exclusively stated in the cases with high amplification, i.e. being HER2 amplified but nonamplified for TOP2A, in half of the latter even having a relative loss of TOP2A compared to the number of cen-signals. Only one case from the 2+ group showed an equivocal result, being aneusomic and showing more TOP2A than HER2-signals. In conclusion, these results seem to reflect different ways of gaining elevated HER2- and TOP2A numbers. On one hand these are most likely mitotic segregation errors which may be combined with structural aberrations (i.e. isochromosome formation, translocation) plus polyploidization in the further course of tumorigenesis ending “out of control”. On the other hand this seems to be “true” amplification in an early stage of tumorigenesis involving HER2, but not mandatorily accompanied by TOP2A amplification. 7.8-P Determination of microRNAs involved in genomic instability in prostate cancer cells. Z. Demir (1) S. Sevli (1) O. Bayrak (1) M. Ozen (1) (1) Yeditepe University (2) Baylor College of Medicine Genomic instability is believed to be one of the earlier events in tumorigenesis. Critical markers for genomic instability are translocations and chromosomal breaks. Recent studies demonstrated that microRNAs are located in chromosomally instable regions. In order to determine important microRNAs involved in genomic instability, karyotypes of commonly used prostate cancer and immortalized prostate cell lines including DU145, PC3, LNCaP with derivatives and PNT1a are compared and common regions of genomic instability are noted. Chromosome 5 contained one of the most consistent regions of instability. In order to analyze the role of microRNAs on this chromosome, we have searched for microRNAs located on instable regions of chromosome 5 as previously determined in prostate cancer. These include miR-579, miR-580, miR1274a, miR-581, miR449a, miR-449b, miR-1974, miR-583, miR-1244, miR-584, miR-143, miR-145, miR-378. Among these miRNAs, miR145 has been further studied in prostate cancer cell lines. miR145 has been transfected in PC3 and DU145 cells

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meanwhile an anti miR-145 has been transfected in PNT1a cells. In order to show the effect of the selected microRNAs in chromosomal instability, chromosome spreads were prepared and any break or chromosomal aberrations were recorded. Verification of effects of these microRNAs in prostate cancer cells are in progress. Our preliminary results showed that selected microRNAs might have an important role in chromosomal instability. Our studies might help to delineate microRNAs crucial for genomic instability. 7a.1-P Telomeres in hepatitis C A. Amiel (5) L. Goldberg-Bittman (1) Y. Kitay-Cohen (2) R. Hadari (3) M. Fejgin (4) (1) Meir Hospital (2) Hospital (3) Tel-Aviv University (4) Bar-Ilan University Background and aims: Telomeres are nucleoprotein structures located at the termini of chromosomes that protect the chromosomes from fusion and degradation. Their natural shortening precedes cell apoptosis. In HCV infection faster hepatocyte cell-cycle turnover was found to be a primary mechanism of telomere shortening compared to control hepatocytes. Short telomeres, telomere’s capture and telomere’s aggregates are biological parameters representing genomic instability that appear in malignant and premalignant conditions. The aim of this study was to estimate the telomere length, telomere’s aggregates (TA’s) and telomere’s capture (TC) rates in two groups of HCV patients, patients with chronic disease (HCV group) and patients who eradicated the virus by antiviral treatment (SVR group), compared to matched healthy controls. Methods: The different parameters were estimated on patient’s leukocytes. We used PNA telomere kit (Dako) to evaluate the telomere length and TA’s formation, and two Cytocell probes: 15qter and 13qter for estimating the TC rate. Results: We found shorter telomeres in chronic HCV patients (HCV group) but not in the SVR group. Higher TA’s and TCs rates were observed in both HCV groups compared to control group.

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Conclusions: Our results showed the presence of genomic instability parameters in HCV infection, which is a predisposing factor to primary liver cancer and lymphoma. Reduced telomerase activity has been reported in chronic HCV infection and may explain our finding of shorter telomeres. Higher TC’s rate, as shown in this study, is probably an alternative mechanism of repairing shorter telomeres. According to our results, HCV patients, even after viral eradication, carry some cytogenetic changes that are considered to be involved in the cascade of events leading to malignancies. 7a.2-P JAK2 V617F is associated with 5q- syndrome in Chinese

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5q- syndrome is characterized by isolated del(5q) in an elderly female with macrocytic anaemia, normal or raised platelet count, and monolobulated megakaryocytes, and is associated with a favorable prognosis and a low incidence of leukaemic transformation. It has been reported that patients with RARS-T and JAK2 mutation had a more favorable prognosis compared with those without the mutation and leukaemic transformation is also rare, suggesting that JAK2 mutation is a relatively benign proliferation signal and its effect is negated by the ineffective erythropoiesis. Of interest, JAK2 mutation was not detected in the two patients with 5q- syndrome and leukaemic transformation. It is therefore tempting to speculate that the occurrence of JAK2mutation in 5qsyndrome may have ameliorated the dysmegakaryocytopoiesis and reduced the leukaemic risk. 7a.3-P

K. Wong (1) W. Wong (1) L. Siu (1) T. Lau (1) N. Chan (2) (1) Queen Elizabeth Hospital (2) Prince of Wales Hospital JAK2 V617F mutation is mainly found in BCR/ ABL-negative myeloproliferative neoplasms. Among other myeloid neoplasms, only refractory anemia with ring sideroblasts associated with marked thrombocytosis (RARS-T) shows remarkably high frequency of its occurrence. It has also been reported in occasional cases of refractory cytopenia with multilineage dysplasia (RCMD) and isolated del (5q). We studied JAK2 mutation in Chinese patients with myeloid neoplasms and isolated del(5q), and showed an association between JAK2 mutation and 5q- syndrome. During the period 1990 to 2008, ten cases of myeloid neoplasms with isolated del(5q) were diagnosed in the QueenElizabethHospital,Hong Kong. All were female and mostly elderly patients (mean age: 67 years) who presented with a normal or raised platelet count (mean: 409×109/L). The diagnosis at initial presentation was: 5q- syndrome, 2; 5q- syndrome in leukemic transformation, 2; refractory anaemia with excess blasts, 4; and RCMD, 2. Allele specific-polymerase chain reaction showedJAK2 mutation in three patients (5q- syndrome, 2; RCMD 1). It was, however, not detected in the two patients with 5q- syndrome in leukaemic transformation.

Novel biomarkers located at 3p22.1 and 10q22.3 enable a non-invasive diagnosis of lung cancer in induced sputum samples by Fluorescence in situ Hybridization (FISH) M. Daniely (1) A. Guber (2) J. Greif (5) T. Kaplan (1) B. Pal (1) R. Rona (3) E. Fireman (5) Z. Yemini (5) M. Gottfried (4) R. Katz (6) (1) BioView Ltd (2) Meir Hospital (3) Meir Hospital (4) Meir Hospital (5) Tel Aviv Medical Center (6) The University of Texas M. D. Anderson Cancer Center Background: Lung cancer results from a series of genetic and epigenetic alterations. Recently, two biomarkers located at 3p22.1 and 10q22.3, were found to be altered in early stage lung cancer. At the present, most patients with lung cancer are diagnosed at advanced stage mainly because symptoms in the early stages are rarely seen. If detected early, about 60% of patients can be cured following surgical resection. Therefore, an early detection assay is highly warranted. Objectives: To evaluate a new assay combining FISH and cytology for detection of lung cancer by induced sputum (IS). Methods: We blindly tested 83 IS samples from advanced and early stage lung cancer patients and from healthy smokers and non-smoking controls (19, 18, 36 and 10 samples, respectively). Samples were

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evaluated using the Duet system (BioView, Rehovot, Israel). Cells were stained in Papanicolaou and scanned automatically. Following the scan, “target cells” defined as normal and atypical cells derived from the lower airways, were selected. Subsequently, cells were hybridized to a 3-color probe mixture containing locus-specific home-brew probes for chromosomes 3p22.1 & 10q22.3 and a centromeric 10 probe. Then, the slides were scanned again and target cells were analyzed by their FISH pattern. The cutoff for malignancy was 7% of cells showing genetic abnormalities by FISH. Results: 36/37 lung cancer patients (97.37% sensitivity) were detected, with a specificity of 82.22%. The mean level of genetic abnormalities detected in early and advanced stage lung cancer patients was significantly higher than controls (13.09% and 5.56%, respectively, p=0.0035). Conclusions: Combined analysis of cytology and FISH in IS samples can be used as a non-invasive diagnostic test for lung cancer. The high levels of genetic abnormalities found in patients with stage I disease point out the potential of using this assay for early detection in high-risk population.

scope and cytogenetic analysis.A minced schwannoma biopsy was cultured in culture medium supplemented with either autologous human plasma (AHP), general human plasma (GHP), or fetal calf serum (FCS). Results: Schwannoma cells were observed in all cultures. They were the majority of the cells seen in the cultures supplemented with AHP and GHP; in the FCS culture, many fibroblasts were also seen. Cytogenetic analysis revealed random numerical changes in all cultures, and a clone bearing t(2;13) was found only in the culture supplemented with AHP. Conclusion: Our results demonstrate that AHP supports tumor cell growth and improves the ability to obtain more reliable cytogenetic results. AHP is relatively simple to obtain and can be used as a tumor-specific growth factor for in vitro culturing. Further studies are needed to confirm our findings.

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I. Ouahchi (1) A. Bennour (1) N. Chaftar (2) M. Elloumi (3) B. Meddeb (4) A. Khélif (5) H. Sennana (1) A. Saad (1) (1) CHU Farhat Hached (2) Faculty of Medecine (3) CHU Hedi Chaker (4) CHU Aziza Othmana (5) CHU Farhat Hached

The effect of autologus human plasma on growth and karyotype of schwannoma cells E. Manor (1) L. Bodner (2) (1) Soroka University Medical Center, Ben Gurion University of the Negev (2) Soroka University Medical Center, Ben Gurion University of the Negev Objective: Cytogenetic analysis is important in the diagnosis and prognosis in many tumors, and is also a valuable tool in understanding the neoplastic process. The main reasons for failure in karyotyping solid tumors are the poor growth of tumor cells and the overgrowth of normal cells in vitro. We assume that autologous plasma (AP) might contain tumor-specific growth factors that are essential for tumor cells to thrive in vitro. In the present study we examined the possible use of AP as a source of schwannoma-specific growth factors. Materials and Methods: The effects of AP on the growth of schwannoma cells were studied by inverted micro-

7a.5-P Cytogenetic characterisation in Tunisian patients with B-chronic lymphocytic leukemia: a retrospective study on 77 cases

Chromosomal abnormalities play an important role in diagnosis and prognosis of B-cell chronic lymphocytic leukemia (B-CLL). The aim of this study was to retrospectively explore the frequency of chromosomal abnormalities in Tunisian patients with CLL using conventional cytogenetic, in order to provide an evidence for diagnosis and therapy. R-banding karyotype analyses were performed on bone marrow samples from 77 patients with B-CLL (52 men, 25 women; mean age 65), 45 (58%) cases had normal karyotype, and 32 (42%) cases had abnormal karyotype. The most frequent aberration was trisomy 12, which was detected in 13 (17%) patients: in11 patient as a sole abnormality; in one patient, it was accompanied by nullisomy Y and translocation t(14;19), and in another patient, it was escorted by trisomies 18 and 19. Trisomy 12 was followed by aberrations at 14q32 in 4 (5%) patients, 13 q deletions with other chromosomal

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abnormalities were detected in 3 (4%) patients, deletion on chromosome 6 were found in only one patient, and 11 (14%) patients showed other chromosomal abnormalities. Our study showed that conventional cytogenetic study is useful tool for identifying chromosomal abnormalities associated with B-CLL. However, due to the low mitotic activity of leukemic cells in vitro, it may underestimate the frequency of specific chromosome aberrations. New molecular approaches, such as fluorescent in situ hybridization (FISH) may circumvent problem. 7a.6-P Telomere length in neuroblastoma: a prognostic factor? G. Lundberg (1) I. Öra (2) D. Nord, Gisselsson (1) (1) Laboratory medicine Lund Aim: To assess possible correlations between telomere lengths in neuroblastoma (NB) and clinical outcome. Background: NBs are tumours of the sympathetic nervous system, occurring predominantly in early childhood and accounting for 8–10% of all paediatric cancers. Multiple studies have shown that NB has a heterogeneous pattern of somatic chromosome changes and previously we have found abnormally short telomeres in five NB cell lines, concurrent with chromosome segregation disturbances such as anaphase bridging and mitotic multipolarity. Materials and Methods: Quantitative fluorescence in situ hybridisation was carried out on 18 NB-biopsies. The fluorescence intensity in NB cell nuclei was compared to that of non-neoplastic cell nuclei, mainly endothelium, to obtain a relative measure of telomere length. The results were correlated with clinical and histopathological data, and cytogenetic abnormalities. Result: Of the 18 cases, six had longer, six shorter and six had the same median telomere length as that of the endothelium. Biopsies with shorter or unchanged telomere length showed a higher frequency of prognostically unfavourable cytogenetic abnormalities, including1p deletion, 17q gain and MYCN amplification; however these patients tended to have a better clinical outcome than those showing long telomeres. Conclusion: The results of this investigation suggest that telomere length might be a prognostic parameter

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of clinical relevance in a sub-group of patients with NB. Further investigations are needed to asses whether telomere length should be included in the recommended diagnostic investigations for patients with neuroblastoma. 7a.7-P A cytogenetic study of 310 chronic lymphocytic leukemia (CLL) cases in greece K. Manola (1) M. Karakosta (1) M. Kalomiraki (1) P. Diamantopoulou (1) M. Margariti (1) D. Pantelia (1) G. Pantelias (1) C. Sambani (1) (1) NCSR Background: Cytogenetics is an important diagnostic and prognostic factor in Chronic Lymphocytic Leukemia (CLL), although its prognostic value is limited by the cytogenetic diversity of the disease. Aim: The aim of this study was the detection of chromosome abnormalities and their incidence in BCLL greek patients by conventional cytogenetics. Patients and methods: Chromosome studies were performed on unstimulated and stimulated with tetradecanoyl phorbol acetate (TPA) bone marrow cells, derived from 310 CLL patients, aged 16–88 years. Patients were karyotyped at diagnosis or during the course of the disease between 1998 and 2008. Results: Two hundred and twelve patients were male and 98 were female. The median age was 64.61 years. Cytogenetic analysis was successful in 288 patients (93%). Normal karyotype was found in 174 patients (~60%), and abnormal in 114 patients (~40%). Chromosome analyses showed a complex karyotype in 32 cases (28%), +12 in 33 cases (29%), -Y in 20 (17.5%), abnormal (abn) 14q in 18 (15.8%), abn 17 in 17 (14.9%), del(6q) in 11 (9.6%), abn 3 in 10 (8.7%), abn 18 in 9 (7.9%), del(13q) in 7 (6.1%), add(12p) in 3 (2.6%), del(7q) in 6 (5.3%), del(11q) in 6 (5.3%), t (11;14) in 3 (2.6%), t(11;18) in 1 (0.9%), -X in 6 (5.3%), abn 19 in 5 (4.4%), +8 in 5 (4.4%), del(5q) in 3 (2.6%), chromosome markers in 13 (11.4%), and other abnormalities in 51 cases (44.8%). Conclusions: The sex ratio was 2.2M/1F, similar to that reported in the literature. Trisomy 12 was the most common abnormality. Surprisingly, -Y was the second most common abnormality, either as a disease associated aberration or as a consequence of advanced

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age. The low percentage of abnormal karyotypes, due to the low mitotic in vitro activity of B-CLL cells, indicates the need for new B-CLL cell stimulators. The cytogenetic complexity and heterogeneity of CLL emphasizes the value of conventional cytogenetics which permits an overview of all microscopically visible chromosome abnormalities.

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remaining TEL allele. Taking together the patients outcome, our finding suggests that increased copy number of oncogenes and fusion genes may contribute to the appearance of an aggressive phenotype. The amplified genes could be easily identified by FISH. Recognition of these genes may help in choosing optimal treatment and may be applied to monitor minimal residual disease.

7a.8-P 7a.9-P Oncogenes amplifications and multiple copy of fusion genes in childhood acute lymphoblastic leukemia I. Haltrich (1) M. Csóka (1) G. Kovács (1) G. Fekete (1) (1) Semmelweis University Gene amplifications and multiple copies of fusion genes occur rarely in hematological malignancies and its pathogenic implication is still elusive. We report three cases of gene amplifications and four cases of copy number increase of fusion genes in childhood ALL. In the last three years 58 newly diagnosed and 6 recurrent childhood ALL bone marrow samples were analyzed for chromosomal abnormalities with conventional G-banding and interphase–fluorescence in situ hybridization using probes to detect BCR/ABL fusions, cryptic TEL/AML1 and MLL rearrangements and p16 (9p21) tumor suppressor gene deletions. The interphase screening of specific childhood ALL rearrangements discovered 1–5 copies of ETV6-RUNX1 fusion gene in a newly diagnosed three years old boy and two copies in a 13 years old recurrent ALL female patient. Two exemplars of Philadelphia chromosome and BCRABL1 fusion gene respectively, were found in a new and in another, recurrent ALL cases. In a 16 years old patient diagnosed with T-ALL, without analyzable metaphases, the t(9;22)(q34;q11) specific FISH probe did not show BCR-ABL1 fusion signals, but detected multiple, uncountable ABL1 signals indicating ABL1 amplification in 60% of the cells. One other, newly diagnosed, 4 years old girl presented unusual 11q23 Gbanding pattern and the MLL specific FISH hybridization revealed 4–6 copies of red-green signals corresponding to intrachromosomal amplification of MLL gene. In a third patient amplification of AML1 gene on der(21) was combined with TEL/AML1 fusion of the homologue AML1 gene and deletion of the

Molecular genetic investigations of breast carcinoma I. Urbanovska (1) B. Kubova (1) R. Skalikova (1) M. Uvirova (1) J. Dvorackova (1) (1) CGB laborator a.s. Poster presentation Aims: Breast cancer is a heterogeneous disease resulting from the acquisition of probably multiple static mutations which, in combination, define the characteristics of the tumour. Methods of molecular genetics have become an essential part of diagnostic and prognostic methods in oncology of solid tumours. Trastuzumab (Herceptin) is the cornerstone of molecular biology treatment of breast cancer women with HER2/NEU gene amplification. Accurate assessment of HER2/NEU status is therefore critical for identifying of patients who may benefit from trastuzumab-based therapy. HER2/NEU gene amplification and overexpression were evaluated by IHC and FISH. Methods: Breast carcinomas were investigated by imunohistochemistry (IHC), fluorescence in situ hybridisation (FISH) and comparative genomic hybridisation (CGH). Results: IHC was performed with DAKO Cytomation HercepTest and FISH with PathVysion Probe Kit. A total of 213 (10,56%) of 2017 patients were positive by IHC. These positive IHC results (2+, 3+) were verified by FISH. HER2/NEU gene amplification was confirmed in 138 cases (70%). CGH was performed at 29 cases to screen breast tumors for copy number changes. We obtained successful results in 26 of 29 cases. 22 cases were positive for copy number changes, 4 cases were negative for copy number changes probably due to analysis of non tumour tissue.

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The most frequent DNA sequence copy number changes were gains of 1q (14 cases), 8q (13 cases) and loss of 1p (14 cases), 16q (12 cases). Conclusions: Our results indicie that especially borderline results of IHC (2+) should be interpreted with caution using both IHC and FISH with standardised metodology. The prevalence of the most common copy number aberrations detected by CGH was roughly similar to that reported in the literature. 7a.10-P Segmental duplications and evolutionary plasticity at tumor chromosome break-prone regions

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satellite repeats and above average retrotransposed sequences as well as other segmental duplications. We propose that the instability of these regions stems from specific features of the chromatin structure associated with the presence of TBSDs and “instability elements” like satellite repeats and retroviral sequences. 7a.11-P Conventional cytogenetic analysis of chromosomal aberrations in B-CLL using CpG oligonucleotide stimulation.

E. Darai Ramqvist (1) A. Sandlund (2) S. Muller (3) G. Klein (2) S. Imreh (2) M. Kost-Alimova (2) (1) Karolinska University Hospital (2) Karolinska Institute (3) Institute for Anthropology and Human Genetics

J. Sobotka (1) M. Brejcha (1) Y. Brychtova (2) V. Holubova (1) A. Tovarysova (1) B. Drevojankova (1) M. Stoklasova (1) M. Doubek (2) M. Radina (1) J. Mayer (2) (1) J.G. Mendel Cancer Center (2) Internal Hematooncological Clinic (3) *CELL, The Czech Leukemia Study Group-for Life

We have previously found that the borders of evolutionarily conserved chromosomal regions often coincide with tumor-associated deletion breakpoints within human 3p12–p22. Analysis of a frequently deleted region at 3p21.3 showed associations between tumor breaks and gene duplications. (In the present study,) Now we have studied the distribution 54 chromosome 3 breaks by multipointFISH (mpFISH) in 10 carcinoma derived cell lines. In lines with highly complex karyotypes, breaks were clustered within known common fragile sites at 3p14.2 (FRA3B), at 3q25 (FRA3D) and at 3q26–q27 (FRA3C) and in two other regions: 3p12.3–p13 (~ 75 Mb position) and 3q21.3 (~ 130 Mb position). The later were involved in 3 out of 4 chromosome 3 inversions during primate evolution. Regions at 75, 127 and 131 Mb positions carry a large (~ 250 kb) segmental duplication (tumor break-prone segmental duplication, TBSD). TBSD homologous sequences were found at 15 sites on different chromosomes. They were located within bands frequently involved in carcinoma-associated breaks. Thirteen of them have been involved in chromosomal inversions during primate evolution; 10 were re-used by breaks during mammalian evolution; 14 showed copy number polymorphism in the human population. TBSDs showed an average 10-fold increase in

Background. Conventional cytogenetic analysis in B-cell chronic lymphocytic leukemia (B-CLL) is hampered due to the low mitotic activity of most CLL cells even in the presence of B-cell mitogens. Recently Dicker et al. (Blood, 2006) have reported immunostimulatory CpG oligonucleotides and IL-2 method to induce metaphase generation in CLL. Aims. Identification of chromosomal aberrations by conventional cytogenetics analysis in B-CLL patients using CpG oligonucleotide and IL-2 stimulation. Methods. We performed conventional cytogenetic and I-FISH analysis on group of 125 selected B-CLL patients. Conventional cytogenetic analysis was performed on peripheral blood samples using 72 h cultivation with CpG oligonucleotide and IL-2 stimulation. I-FISH analysis was done using the DNA probe set designed to detect 17p, 11q and 13q deletions, trisomy 12 and 14q32 rearrangement on peripheral blood. Results. By cytogenetic analysis 97% of samples were successfully cultivated, more than 75% of samples had enough and good quality metaphases. Chromosomal aberrations by conventional cytogenetics were detected in 60% of patients and by FISH in 79% of patients. Conventional cytogenetics and I-FISH gen-

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erated these groups of patients: (a) 17% of cases have no aberration detected by FISH and chromosome analysis, (b) in 23% of cases with FISH aberrations no chromosomal aberrations were detected, in all cases the submicroscopic 13q14 deletion was present, (c) in 7% of cases with no FISH abnormalities conventional cytogenetics revealed chromosomal aberrations, (d) in 33% of patients with FISH aberrations conventional cytogenetics revealed additional chromosomal aberrations, complex karyotype was detected in 18% of patients. Summary. In conclusion, our results confirm, that cultivation technique with CpG oligonucleotide and IL-2 stimulation is an efficient method for metaphase induction in CLL which increases a detection rate of chromosomal aberrations. Detection of the additional chromosomal aberrations can provide the new prognostic information in B-CLL patients. 7a.12-P Classic and molecular cytogenetic analysis of splenic marginal zone lymphoma (SMZL)

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FISH approaches (probes: bcl 6, p53). Clonal chromosome aberrations (CCA) were identified in 14/22 cases (63.6%) and were mainly structural. Hypodiploidy/hyperdiploidy were detected in 6 and 3 cases, respectively. Eight of 14 CCA+ cases (57%) exhibited complex karyotype (CK). The most frequent CCAs involved chromosomes #8/17/1/3/4/ 6/13/14/7/12. One case carried t(14;18); she was tested PCR negative for bcl-2 rearrangements. Another case showed coexistence of trisomy 3q and del7q. FISH analysis identified 4/18 cases with additional bcl-6 and 7/19 cases with p53 deletion (all with U-IGHV). Of note, 3/4 cases with additional bcl-6 also exhibited p53 deletion. All eight patients with deleted p53 or del17p required treatment; 4/8 had no response. The precise prognostic impact of the aforementioned aberrations was difficult to assess due to the small number of cases. In conclusion, our results underscore the heterogeneity of SMZL at the cytogenetic level. The frequent detection of CK and the coexistence of trisomy 3q with del7q indicate the need for additional studies in order to define cytogenetic subtypes of SMZL.

A. Athanasiadou (1) M. Gaitatzi (1) G. Papaioannou (1) Z. Lazarou (1) N. Stavroyianni (1) R. Saloum (1) N. Laoutraris (2) T. Papadaki (3) A. Anagnostopoulos (1) (1) G. Papanicolaou Hospital (2) Nikea General Hospital (3) Evangelismos Hospital

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SMZL is not characterized by a recurrent genetic aberration. Published series are relatively small and report heterogeneous findings. The largest series so far has confirmed the existence of two distinct subtypes, associated with trisomy 3q and del7q, respectively. In the present study we performed a detailed cytogenetic analysis of 22 SMZL patients. Study group: M:F = 16:6; Δm age: 70 (52–82); splenectomy (SPL): 9/22 cases; villous lymphocytes: 16/22 cases. Immunophenotype: CD5+: 3/22 cases/ CD23+: 1 case/ CD5+CD23+: 0/22 cases. Eleven of 18 cases (61%) carried unmutated IGHV genes (U-IGHV; ≥98% identity to germline). Six of 9 SPL+ patients required additional treatment; among 13 non-SPL patients, 10 received various therapeutic regimens. With a median follow-up time of 23.5 months (7–155), 20/22 patients are alive. The cytogenetic study was based on G-banding and

A. Bennour (1) H. Sennana (1) B. Achour (2) Y. Ben Youssef (2) M. Zaier (2) A. Khelif (2) A. Saad (1) (1) CHU Farhat Hached (2) CHU Farhat Hached

Translocation t(7;12) in a patient with chronic myeloid leukemia receiving Imatinib Mesylate therapy

The treatment of chronic myeloid leukemia (CML) has considerably evolved since imatinib mesylate (IM) has been introduced as a new therapeutic weapon for this disease. However, little is known about its long-term efficacy and toxicity. One of the important issues pertaining to the biology and treatment of CML is the emergence of chromosome abnormalities in Philadelphia positive cells in patient resistant to IM, these abnormalities may be presented on repeated occasions suggesting the possibility of development of new malignant clones and/or evolution to blastic crisis.

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We describe a novel chromosomal aberration t(7;12)(q11; p13), acquired in a patient affected by Philadelphia-positive CML. Conventional cytogenetic studies at onset showed a classic t(9;22)(q11;q11) in all bone marrow cells, Fluorescence in situ hybridization (FISH) with BCR-ABL probes revealed the BCR-ABLfusion gene, and neither obvious deletions on 9q34 nor on 22q11.2, reverse transcriptionpolymerase chain reaction analysis revealed the b3a2 transcript. After 36 months with IM treatment, the malignant clone developed a new additional cytogenetic abnormality consisting on a translocation t(7;12) (q11; p13), confirmed by FISH with whole chromosome painting probes. To our knowledge, this is the first case of t(7;12) reported in the literature associated with Philadelphia chromosome in CML. The pathogenesis of this phenomenon still remains unclear, and different hypothesis include evolution of Philadelphiapositive clone, or toxicity effect of logterm follow-up IM. Also clinical relevance of these new clones remains to be clarified for new alternative therapeutic strategies and a better understanding of the molecular mechanisms underlying disease progression. 7a.14-P Genetic instability of common fragile site genes in colorectal cancer

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It has been shown that such genomic DNA damage, represented mainly by deletions and amplifications, occurs particularly at specific, non-random loci that are prone to DNA double-strand break formation. These loci are present in all human individuals and are referred to as common fragile sites (CFS). This study aims to identify regions of profound genomic instability in colon cancer cells and to examine the DNA sequences affected by genomic aberrations upon fragile site activation. Genomic DNA isolated from colon cancer cell lines is analysed by fine-tiling CGH in order to identify DNA copy number changes on chromosomes 8 and 1p, as well as on a selection of 21 known CFS. Fluorescence in situ Hybridization (FISH) is carried out to detect structural chromosomal rearrangements, such as inversions, insertions and translocations, and furthermore serves as a means of validation of CGH array data. Initial results indicate genomic instability within several fragile sites, a common example being FRA3B. Moreover, genomic aberrations on chromosome arm 8p as well as gains of 8q appear to be frequent features in colorectal tumour cells. Aiming to provide further insights into the molecular mechanisms underlying cancer development this study could make a significant contribution towards prevention, early diagnosis, and more efficient treatment of cancer. 7a.15-P

L. Brueckner (1) M. Schwab (1) L. Savelyeva (1) (1) German Cancer Research Center (preferred presentation type: Poster Presentation) Genetic instability of common fragile site genes in colorectal cancer Lena Brueckner, Manfred Schwab, and Larissa Savelyeva. Division of Tumor Genetics, German Cancer Research Center, 69120 Heidelberg, Germany Colorectal cancer represents one of the leading causes of cancer death worldwide. The identification of genes that influence cancer susceptibility and progression is still a crucial goal of basic and clinical research. Several recent studies have demonstrated that at the earliest stages, cancer development is associated with DNA replication stress, which leads to DNA doublestrand breaks and consequently to genomic instability.

A novel chromosomal translocation t(11;14)(q24.1; q32) involving IGH in childhood B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) E. Tassano (1) M. Acquila (1) E. Tavella (1) C. Rosanda (1) C. Panarello (1) C. Morerio (1) (1) Istituto Giannina Gaslini Rearrangements involving IGH gene on chromosome 14q32.3 are well known in mature B-cell malignancies and have been more recently described in BCP-ALL. IGH translocations are usually reciprocal and bring genes on other chromosomes into close apposition with the IGH locus, where their expression is deregulated due to the presence of potent B-cell-specific transcriptional enhancers. A two-year-old girl was diagnosed

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with an ALL common type and treated according to AIEOP-ALL-00 Protocol. Death occurred after four months due to haematological toxicity. Cytogenetic analysis of PB and BM blasts revealed a t(11;14) (q24∼32;q32). FISH analysis with IGH break-apart probe confirmed the rearrangement of the IGH locus between chromosomes 11 and 14. Cloning by LDI-PCR localized the breakpoint on chromosome 11q24.1 within the intronic region 1 of BC089451, a non coding gene. Quantitative realtime PCR showed over-expression of BLID mRNA, located 14 Kb downstream the BC089451 gene. BLID codes for a protein containing a BH3-like domain essential for apoptosis. FISH studies performed with 11 close BACs to confirm the breakpoint junction identified a 585Kb deletion on der (11), with complete SORL1 loss. SORL1 controls intracellular trafficking of amyloid precursor protein and is causally linked to the pathogenesis of Alzheimer disease. Moreover SORL1 is downregulated in high-grade astrocytoma. Deletion of SORL1 could play a role in leukemogenesis, like the IGH-ID4 association in the t(6;14)(p22;q32) with PAX5 and CDKN2A deletions. The functional consequence of BLID over-expression due to IGH enhancer juxtaposition is currently unknown. Mutational analysis of BLID BH-3 like domain is ongoing. The translocation to the Ig locus may result not only in deregulated expression of the incoming oncogene, but also in mutations due to the action of the Ig somatic hypermutation mechanism. In our case, a mutation of BLID in BH3 domain could result in a protein not inducing apoptosis.

counts. BM aspirate showed no blasts or myelodysplasia; bone biopsy showed reduced cellularity and megakaryocyte number. Her height was <3rd centile due to partial GH deficiency. MR showed Chiari I malformation. Slight somatic dysmorfisms were observed. DEB tests and cell cycle analysis on peripheral blood (PB) lymphocytes and fibroblasts were negative. NBS1 protein was present in western blot and the lymphoblastoid cell line was not sensitive to ionizing radiation. Nijmegen Breakage Syndrome was excluded. c-Mpl gene associated with amegakaryocytic congenital thrombocytopenia, is not mutated. The Q-banded karyotype of PHA-stimulated PB lymphocytes was 46,X,i(X)(p10)[6]/46,XX[43]. Dual color FISH analysis using Xp- and Xq-arm partial paints confirmed the staining of the entire two isochromosome p-arms and revealed a subtle q-arm signal in the centromeric region. Compared to the normal X, the size of centromere signal (DXZ1 coupled with STS Xp22.3) was larger, suggesting an isodicentric chromosome. FISH with subtelomeric Xpter (RP11-215A12) probe and RP13-216E22 BAC probe, which specifically hybridizes to the XIST locus on Xq13.2, showed the absence of the inactivation center on i(X)(p10). Subtelomeric Xpter FISH analysis on BM cells demonstrated three signals in 11/300 nuclei. Fibroblast karyotype was 46,XX. Uncommon acquired i(X)(p10) associated with hematologic disorders as well as one case of constitutional mosaic i(X)(p10) in a girl with failure to thrive and thrombocytopenia have been reported in the literature. Our case seems to confirm the i(X) (p10) involvement in bone marrow failure.

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Mosaic isochromosome Xp in a girl with short stature and bone marrow failure

The evolution of karyotype in children with t(4;11) (q21;q23)

C. Morerio (1) E. Tavella (1) A. Casalaro (1) E. Cappelli (1) C. Dufour (1) C. Panarello (1) E. Tassano (1) (1) IRCCS Istituto Giannina Gaslini

A. Pastwinska MSc (1) E. Chmarzynska-Mroz MSc (2) A. Stefaniak MSc (2) K. Pawelec MD (3) A. Wasiutynski MD PhD (2) M. Matysiak MD PhD (3) B. Pienkowska-Grela MD PhD (4) (1) Medical University of Warsaw; Centre of Oncology M. Sklodowska-Curie Memorial Institute (2) Medical University of Warsaw, The Chair of Pathological Anatomy (3) Medical University of Warsaw (4) Centre of Oncology M. Sklodowska-Curie Memorial Institute

A 18-month girl, second daughter of unrelated parents, was diagnosed with bone marrow (BM) failure and treated with cyclosporine ad steroids, with partial response. At 6 years of age, she was referred to us for anemia, low white bood cell and platelet

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Acute lymphoblastic leukaemia (ALL) is a childhood disease characterized by clonal proliferation and accumulation of neoplastic cells. The recurrent chromosomal abnormalities, such as t(4;11)(q21; q23), t(9;22)(q34;q11) or t(1;19)(q23;p13) are well known in children with ALL. Reciprocal translocation t(4;11)(q21;q23) is associated with fusion of AF4 and MLL genes and affects more than 60% infants and about 20% children in 1 to 9 years of age. This translocation predicts the worst prognosis of all subtypes ALL. We examined four children with pre-B ALL. All of them had t(4;11) with additional karyotype changes. Patient 1 with additional aberration i(7)(q10)had early relapse. When the child achieved remission, he had allogeneic bone marrow transplant (allo-BMT) with unrelated donor. Patient 2 had hyperdiploid karyotype with additional marker chromosome and non-typical involvement of MLL: ins(4;11)(q21;q13q23). When she was in remission she had an allo-BMT with unrelated donor. Patient 3 with a lot of additional aberrations in karyotype had early relapse. The quality of his chromosomes was so poor in second examination, that we could not recognize all of changes, except of t(4;11). Patient 4 had never achieved remission. Third examination showed hyperdiploid karyotype with numerous aberrations, next to t(4;11): add(3)(q25), del(6)(q21), t(6;13)(p23;q13), inv(7)(p15q11), +8, +13, add(19)(q13), mar1 and mar2. The analysis of karyotype during fourth examination revealed additional changes to these in last examination: t(1;10) (p22;p13), dup(1)(q21;q32), t(2;5)(q37;q11.2), + 5, +6, add(7)(q36), der(12)del(12)(p13)add(12)(q24) and mar3. Patients 3 and 4 died because of rapid and aggressive development of disease, no response to therapy and involvement of central nervous system. Two children after allo-BMT are in very good condition. Patients with early relapse had numerous chromosomal aberrations and died quickly, without response to therapy. Although, two children with single additional changes achieved remission and they could now have bone marrow transplant.

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7a.18-P Abnormal Clone in Acute Promyelocytic Leukemia (APL) Patient Treated With ATRA and Chemotherapy h. bar-el (1) Z. Borochowitz (2) T. Tadmor (3) Z. Vadas (4) T. Aloni (5) M. Ziv (6) D. Attias (7) (1) bnai-zion medical center (2) bnai-zion medical center (4) Rappaport Faculty of Medicine (5) Research Institute Abnormal Clone in Acute Promyelocytic Leukemia (APL) Patient Treated With ATRA and Chemotherapy H. Bar-El1,3,4, Z. U. Borochowitz1,3,4,5, T. Tadmor2,3, Z.Vadas2,3,T.Aloni1,3, M. Ziv1,3, D. Attias2,3. The Simon Winter Institute for Human Genetics1 and the Hematology Institute2, Bnai-Zion Medical Center3, Rappaport Faculty of Medicine4 and Research Institute5, Technion-Israel Institute of Technology, Haifa, Israel. Translocation (15;17) is a remarkably specific APL associated cytogenetic abnormality. We present a case of a 58-year-old female, diagnosed as APL. FISH technique showed translocation PML-RARA (Vysis) in 30% interphase cells of her bone marrow. She was treated with ATRA and chemotherapy (antracyclines) with complete response. She continued her treatment according to the PETHEMA protocol. During her treatment serial PCR and cytogenetic examinations of the bone marrow were normal. At the end of her treatment, bone marrow examination revealed relapse with the presence of blasts (about 10%) which were different on both histology and cytometry than the original cells. PML-RARA translocation was negative, however a new reciprocal translocation appeared in all metaphases of bone marrow (11;15)(q23;q22), seen by G-Banding cytogenetic technique. FISH technique, Using MLL Split Probe (DAKO) showed breakpoint at this gene. PML-RARA probe ensure that PML gene is distal to the breakpoint of chromosome 15, and moved to the translocated chromosome 11q+. This translocation developed in cells that are not carrying the primary abnormality. MLL gene (11q23) abnormalities are known to be evolved in M4, M5 and other AML, also as secondary to genotoxic exposure. There is a wide range of chromosomes involvement in reciprocal translocations with MLL gene. This one is less reported. This genetic finding leads to a secondary diagnosis and another treatment.

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7a.19-P Essential thrombocythemia without JAK2 V617F mutation in a patient with constitutional Robertsonian translocation der (13;14)(q10;q10) B. Todoric Zivanovic (1) (1) Military Medical Academy Essential thrombocythemia without JAK2 V617F mutation in a patient with constitutional Robertsonian translocation der (13;14)(q10;q10) Biljana Todorić Živanović, Milica Strnad, *Tatjana Kostić, *Sonja Pavlović, Željka Tatomirović, Dragana Stamatović Military Medical Academy, *Institute for molecular genetics and genetic engeniring, Belgrade, Serbia Essential thrombocythemia (ET) is a chronic myeloproliferative disorder characterized by excessive production of platelets, thrombohemorragic and vasomotor symptoms with a long median survival, and a low risk of transformation to leukemia. Up to 50% of ET patients carry JAK2 V617F mutation. The patient was presented with peripheral blood cell counts: Hb 128 g/L, WBC 6.13×109/L, RBC 4.65 ×109/L, Plt 1228× 109/L. The bone marrow aspirate showed hiperproductive megakaryocytes with clumps end sheets of thrombocytes. Spontaneous megakaryocyte colonies were seen in the culture of bone marrow progenitors. Bone marrow cells were cytogeneticaly analized after direct and 24 h culture preparation of chromosomes by standard GTG method. We detected 45,XY, der(13;14)(q10;q10) karyotipe in all 20 analyzed metaphases. PHA stimulated limphocytes from peripheral blood (PB) of the patient were analyzed for proving congenital nature of this aberration. In all 20 metaphases we detected the same 45,XY,der(13;14) (q10;q10) karyotipe. The presence of bcr-abl gene was excluded by RTPCR method. The presence of JAK2 V617F mutation in DNA samples from PB myeloid cells was analized and the result was negative. Constitutional chromosome aberrations have been reported in hematologic disorders. Robertsonian translocations, although relatively common as a constitutional aberration, are rarely reported in chem-

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atological malignances. Further studies and reports of such cases and their families is necessary. 7a.20-P Constitutional RUNX1 deletion in non-syndromic thrombocytopenia with myelodysplasia; 21q22 ITSN1 as a candidate gene in mental retardation A. Buijs (1) E. van Binsbergen (1) M. Ausems (1) M. Poot (1) M. Bierings (1) S. van der Crabben (1) (1) University Medical Center Utrecht We present a developmentally normal patient with isolated thrombocytopenia and myelodysplastic features associated with a constitutional submicroscopic 21q22 deletion (including RUNX1). In contrast, four cases were recently published of syndromic thrombocytopenia with 21q22 deletions and variable degrees of dysmorphic features and mental retardation (MR), suggesting a dosage-sensitive causative role of gene (s) on 21q22 in MR. One patient had developed myelodysplastic syndrome/acute myeloid leukemia (MDS/AML) at the age of 6 years. A 5-year-old boy was referred with a history of frequent haematoma caused by a relatively mild thrombocytopenia. Growth and psychomotor development was normal as well as dysmorphologic evaluation. Bone marrow (BM) revealed an active, atypical megakaryopoiesis with slight dysplastic features. G-banding on BM showed a 46,XY karyotype. FISH analyses demonstrated two constitutional microdeletions of one chromosome 21; a 21q22 interstitial deletion with loss of RUNX1 and a terminal 21q deletion. High-resolution oligo arrayCGH (105K Agilent) showed a 1.60 Mb interstitional deletion 21q22.11–12, including RUNX1, and a 2.22 Mb terminal 21q22.3 deletion. Several genes at 21q22.12 have been proposed as candidate genes to contribute to MR in syndromic thrombocytopenia. We compared the genotypes and phenotypes of the published cases with our case. This approach pointed to an association with MR of a minimally deleted region of 0.4 Mb containing the ITSN1. ITSN1 is a scaffold protein and has a role in endocytic vesicle trafficking in neurons. It has been implicated in Alzheimer’s disease and Down syndrome, and could play a role in MR in these patients.

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We hypothesize that haploinsuffiency of ITSN1 is implicated in MR in syndromic thrombocytopenia with 21q22 deletions. Our case emphasizes the necessity of cytogenetic and molecular analyses of 21q22 for RUNX1 status in diagnosing patients with isolated or syndromic thrombocytopenia and supports RUNX1 haploinsufficiency in FPD/AML. 7a.21-P Prognostic signification of copy number changes of MYCN and MYCC genes studied by I-FISH method in childhood medulloblastoma H. Filková (1) K. Zitterbart (2) L. Tomášiková (1) V. Vranová (3) D. Žežulková (1) D. Kantorová (2) P. Kuglík (3) Z. Pavelka (2) J. Štěrba (2) R. Veselská (3) (1) University Hospital Brno,Czech Republic (2) University Hospital Brno, Czech Republic (3) Institute of Experimental Biology Medulloblastomas are highly malignant primitive neuroectodermal tumors of the cerebellum that display a wide variety of histopathological patterns. However, these paterns do not provide an accurate prediction of clinical-behavior. For this reason genetic alterations may provide additional diagnostic information. Our work presents analyses of survival of the patients suffering from medulloblastoma (patients treated at the Clinic of children’s oncology in University Hospital Brno) relating to quantitative changes of proto-oncogenes MYCC, MYCN studied by means of I-FISH method and CGH. The informative result was obtained from 26 or 25 examinations. Period of monitoring of the patients after the medication is sufficient and it exceeds median of the period to relapse / progression of this disease (the patients were diagnosed in 2000–2006). Detection of five or more copies of the MYCN gene was documented in retrospective analysis as a negative prognostic marker for total and asymptomatic survival. Detection of five or more copies of the MYCC gene was also connected with the tendency to worse total and asymptomatic survival. Our results confirm that gain of the genetic material at locuses MYCC and MYCN is the negative prognostic marker for medulloblastoma. The practical meaning is obvious—based on these results it will be possible to detach group of the patients who can profit

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from intensive therapy and application of biological treatment. This work was supported from IGA NR9125-4/ 2006 and VZ MSM 0021622415 grants. 7a.22-P Translocation t(8;17;16)(p11;q21;p13), a new variant of t(8;16)(p11;p13) H. Podgornik (1) M. Fink (1) T. Pajič (1) P. Černelč (1) (1) University Medical Center Acute myeloid leukemia (AML) with translocation t(8;16)(p11;p13) is rare leukemia subtype with characteristic clinicobiological features. It is characterized with monocytic differentiation or AML M4/M5 by FAB classification, frequent extramedullary involvement, severe coagulation disorder, and a poor outcome with a median survival of less than 1 year. At the molecular level, translocation t(8;16) fuses MYST3 and CREBBP. The transcript produced by fusion gene is believed to be of importance in the leukemogenic process. A 64-years-old previous healthy woman was presented in November 2008 with a 2–3-week history of pleural pain. Bone marrow aspirate as well as flow cytometry analysis revealed a blast population (89%) expressing CD45, CD34, CD13, CD15, HLA-DR, and lacking CD14 expression. These findings support a diagnosis of acute myeloid leukemia with maturation (AML M2). By cytogenetic examination of bone marrow cells rearrangement of both chromosomes 8 was found. Chromosome 1 was translocated to the first chromosome 8 resulting in partial trisomy for the long arm of chromosome 1. The other homologous chromosome 8 was rearranged in a three-break translocation with partner chromosomes 16 and 17. The observed rearrangements were confirmed by FISH using WCP probes. Combining the results of standard and molecular cytogenetics the karyotype was interpreted as: 46,XX, +1,der(8)t(1;8)(q21;q24),t(8;17;16)(p11;q21;p13). Since the breakpoints of partner chromosomes 8 (8p11) and 16 (16p13) corresponded to that of the recurrent translocation t(8;16) we tested expression of MYST3/CREBBP protein. Using RT-PCR we were able to confirm it. Here, we report for the first time t(8;17;16)(p11;q21; p13) with MYST rearrangement. In our case the subtype

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of AML (M2) as well as clinical course of disease (no extramedullary involvement or coagulation disorder) were distinct from the earlier reports. Our patient achieved a complete remission after the induction therapy. Therefore, also the prognostic value of the novel variant t(8;16) should be studied in the future. 7a.23-P Deletion of chromosome 5q as a rare but non random abnormality in chronic lymphocytic leukemia (CLL) M. Karakosta (1) I. Korantzis (2) A. Tsakiridou (3) G. Pantelias (1) C. Sambani (1) K. Manola (1) (1) NCSR (3) General Hospital of Patras, Aghios Andreas Background: Chronic lymphocytic leukemia (CLL) is associated with cytogenetic abnormalities at diagnosis or during the course of the disease. However, most of them have not been completely determined due to the low mitotic in vitro activity of B-CLL cells. Aim: We present a conventional and molecular cytogenetic study of two CLL cases with deletion 5q [del(5q)] as a sole abnormality in order to contribute to the identification of rare recurrent aberrations and their prognostic impact in CLL. Patients and methods: Patient 1, a 66-year-old man, and patient 2, a 59-year-old woman were diagnosed with B-CLL in September 2008 and in October 2007 respectively. Both received no therapy and are still in excellent clinical condition 5 and 17 months later correspondingly. Cytogenetic studies were performed on unstimulated and stimulated bone marrow cells at diagnosis. Fluorescent in situ hybridization (FISH) studies were performed on the same specimens using commercial DNA probes for detection of deletions of 5q31 (EGR1/D5S23), 5q33–q34 (CSF1R/D5S23, DS5721), 17p13 (TP53), 11q22.3 (ATM), 13q14/ 13q34 (D13S319/13q34) and trisomy 12 (CEP 12). Results: Chromosome analysis revealed del(5q) as a sole abnormality and FISH demonstrated normal hybridization pattern for 13q14, 13q34, ATM, p53 and CEP12 regions in both patients. Furthermore, in patient 1, FISH showed a normal hybridization pattern for 5q31 but a single signal for 5q33-q34 region. FISH results for del(5q) were not obtained for patient 2 due to lack of cytogenetic material.

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Conclusions: This study presents the first case reports of del(5q) in CLL and demonstrates that del(5q) as a sole aberration, is a rare but non random abnormality, possibly not associated with an adverse prognosis. It also indicates that 5q33–q34 could be the potential critical region. Further studies are required to delineate the prognostic value of del(5q) in CLL, and to identify candidate genes with potential role in the pathogenesis of the disease. 7a.24-P Cytogenetic changes in breast cancer with polysomy of chromosome 17: significance for diagnosis and treatment V. Palková (1) M. Čížková (2) R. Trojanec (1) L. Radová (1) K. Bouchalová (1) S. Mlčochová (1) B. Melichar (2) Z. Kolář (3) M. Hajdúch (1) (1) Palacky University, Faculty of Medicine (2) Palacky University, Faculty of Medicine (3) Palacky University, Faculty of Medicine Amplification and/or overexpression of the Her-2/neu gene has been reported in approximately 20% of patients with breast cancer. These changes are associated with poor prognosis and higher tumor aggressiveness. Humanized monoclonal antibody trastuzumab was developed for treatment of breast cancer patients with ratio Her-2/neu : chromosome 17 (CH17) copy number ≥ 2.2 and/or immunohistochemically positive 3+. Roughly 5–7% of breast cancer patients are not indicated for trastuzumab therapy because they do not match criteria due to CH17 polysomy. The efficacy of trastuzumab in polysomic cases has not yet been confirmed. Apart from Her-2/neu the most frequently altered genes in breast cancer are TOP2A (topoisomerase 2α), C-MYC and CCND1 (cyclin D1). We focused on the status of genes C-MYC and CCND1 and corresponding chromosomes 8 and 11 detected using FISH. For the pilot study we chose 280 patients: 112 (40%) with confirmed chromosome 17 polysomy (CH17 copy number ≥ 2.5) and 168 (60%) with diploid status. Amplification of CMYC, resp. CCND1 was determined in 48.2% (54/112), resp. 47.3% (53/112) cases with CH17 polysomy vs. 10.7% (18/168), resp. 23.2% (39/168) without CH17 polysomy. Extra copies of chromosome 8 and 11 (≥ 2.5) were seen in 34.8% (39/112) and 23.2% (26/112) cases

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with CH17 polysomy vs. 2.4% (4/168) and 4.2% (7/168) without CH17 polysomy. Our data demonstrate more frequent genetic alterations in CH17 polysomic tumors. Their clinical relevance is being analyzed. Acknowledgement: Project was supported in parts by grants MSM6198959216, LC07017 and GACR 303/09/H048. Special thanks belong to all cooperating clinical departments and local laboratories. 7a.25-P Experience with Her-2/neu (c-erbB-2) gene status evaluation in breast cancer samples with unusual FISH results R. Trojanec (1) Z. Kolář (2) V. Palková (1) J. Berkovcová (1) B. Braunerová (1) M. Tichý (2) K. Bouchalová (1) V. Krejčí (2) B. Melichar (3) M. Hajdúch (1) (1) Palacky University, Faculty of Medicine (2) Palacky University, Faculty of Medicine (3) Palacky University, Faculty of Medicine Background Determination of Her-2/neu status is crucial for effective indication of trastuzumab (Herceptin®) treatment. In the CzechRepublic, Her-2/neu status is evaluated by FISH and immunohistochemistry at six Reference Centres (Laboratories of Predictive Medicine). However, some samples cannot be evaluated by FISH due to DNA degradation and those tumors were evaluated by quantitative real-time PCR for Her-2/neu gene. Moreover, due to polysomy of chromosome 17 (CH17) at least 5–7% of patients are not indicated for trastuzumab treatment as they do not fulfill criteria of Her-2/neu: CH17 ratio >2.2. The efficacy of trastuzumab in polysomic patients has not yet been confirmed. In the CzechRepublic, such patients are indicated for trastuzumab treatment only when they are immunohistochemically positive (3+). Results More than 2818 breast cancer samples were evaluated in our institution over a period of seven years. Overall, 148 (5.25%) cases failed to be concluded by FISH. Absence of cancer cells and/or DNA degradation in the tumor biopsy were the major causes of the failure. For such cases, quantitative real-time PCR comparing the Her-2/neu gene status to reference genes dck, gcs1 and epn2 was established. Among 148 cases which failed

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using FISH technique, we have successfully investigated 77 patient samples by qRT-PCR achieving unambiguous results in 78% (60/77). In 13.8% (368/2670) cases, polysomy of CH17 was detected by centromeric probe (CEP17). Using locus specific probe mapping of 17p11.2 region, we found that 57% (212/368) of such a “polysomic cases” contain only 2 copies of CH17. We found in some instances, the hybridization of centromeric probe was not specific enough and the probe also hybridized to centromeres of other chromosomes or the cells showed complex cytogenetic rearrangements which misrepresent the number of CH17. Acknowledgements. Project was supported by grants MSM6198959216 and LC07017. Special thanks go to all cooperating departments, and health insurance companies. 7a.26-P Molecular cytogenetic characterization of translocation t(9;12)(q34;p13) and ETV6/ABL1 fusion in patients with acute lymphoblastic leukemia L. Lizcova (1) Z. Zemanova (1) E. Malinova (1) J. Brezinova (2) S. Izakova (2) J. Zuna (3) E. Mejstrikova (3) P. Smisek (4) K. Michalova (1) (1) General Teaching Hospital and 1st Faculty of Medicine, Charles University in Prague (2) Institute of Hematology and Blood Transfusion (3) CLIP— Childhood Leukaemia Investigation Prague, University Hospital Motol (4) University Hospital Motol Translocation t(9;12)(q34;p13) leading to ETV6/ ABL1 fusion is a rare event in patients with lymphoid as well as myeloid leukemia. Several chromosomal rearrangements resulting in ETV6/ABL1 fusion were described and it was shown that at least three chromosomal breaks are necessary to generate functional ETV6/ABL1 fusion. Cytogenetically, this aberration is difficult to detect and can be confirmed only by molecular cytogenetic or molecular genetic methods. During the years 2006–2008 we examined bone marrow cells of three patients (two boys and one adult woman) with acute lymphoblastic leukemia (ALL) and ETV6/ABL1 fusion. In all of them, fusion of ETV6 exon 5 to ABL1 exon 2 was confirmed by RT-PCR.

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Using conventional cytogenetic analysis, translocation t (9;12)(q34;p13) has not been found but in two patients, other chromosomal rearrangements were detected. For molecular cytogenetic characterization of ETV6/ABL1 fusion we performed FISH with locus-specific, centromeric and subtelomeric probes (Abbott-Vysis). In two patients insertion of part of the ABL1 gene into 12p13 region was detected and no rearrangement of ETV6 gene and subtelomeric region of chromosomome 9 and 12 was proved. In one patient the rearrangement of the ETV6 gene also has not been found and even no split of the probe spanning the ABL1 gene was observed. In this case distal end of 12p was shown to be translocated on long arm of chromosome 9. Detailed molecular cytogenetic analysis will be presented on the poster. Molecular cytogenetic analyses of present cases demonstrate distinct chromosomal aberrations leading to ETV6/ABL1 fusion and so provide further evidence for complex nature of this rearrangement. Additional comprehensive cytogenetic studies of lager series are needed to delineate mechanisms which are responsible for this rare event. Supported by grants MSM 0021620813, MSM LC535, NR/9227-3 and MZO VFN2005 7a.27-P Molecular cytogenetics, telomere length and other molecular and immunophenotypic features in patients with B-chronic lymphocytic leukemia J. Brezinova (1) S. Vcelikova (2) A. Berkova (3) Z. Zemanova (3) S. Izakova (1) I. Sarova (1) L. Lizcova (3) E. Cmunt (4) J. Schwarz (1) K. Michalova (1) (1) Institute of Hematology and Blood Transfusion (2) Institute of Hematology and Blood Transfusion (3) General Teaching Hospital and 1st Faculty of Medicine, Charles University in Prague (4) General Teaching Hospital and 1st Faculty of Medicine, Charles University in Prague Interphase fluorescence in situ hybridization (I-FISH) has an important role in diagnosis, classification and managment of patients with the B-cell chronic lymhocytic leukemia (B-CLL). During the last decades new prognostic markers such as IgVH gene mutational status, ZAP 70 and CD38 expression levels

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or telomere length enabled clinicans to identify patients with high risk B-CLL. Aim of the study was to assess the frequency of chromosomal aberrations and to correlate the results of molecular and immunophenotypic prognostic markers. During the years 2007–2008 we examined peripheral blood samples of 182 patients with B-CLL (107 male, 75 female, mean age 66.5 years). I-FISH analyses were performed using CLL Probe panel for regions 17p13.1 (gene p53), 11q22.3 (gene ATM), 13q14.3, 13q34, 12p11.1–q11 (Abbott-Vysis) and Poseidon Repeat Free IGH (14q32) Break probe (KREATECH). Deletion of 13q14 was the most frequent aberration proved in 97 patients (53.3%), as a sole aberration in 50 patients (27.3%). Trisomy 12 was found in 26 patients (14.3%), aberrations with poor prognosis were proved in 35 cases: deletion of ATM gene in 22 patients (12%), deletion of p53 gene in 13 patients (7.1%). In 8 cases (4.4%) rearrangement of IgH gene was ascertained. In 77 patients telomere length, telomerase activity, IgVH gene mutational status, ZAP-70 and CD38 expression analysis were evaluated. Reduced telomeres were identified in 48 patients (62.3%). A correlation between telomere length and IgVH mutational status was confirmed with IgVH mutated patients showing long and IgVH unmutated short telomeres (p=0.001). Conclusion: The prognostic impact of I-FISH is important in B-CLL, however, new parameters such as telomere length and telomerase activity complete the risk profile of these patients. This work was supported by grant IGAMZCR NR9244-3, MZOVFN2005, MSM 0021620808, LC535. 7a.28-P Genome aberrations and their relation to prognosis in childhood acute myeloid leukemia G. Armengol (1) A. Canellas (1) M. Caballin (1) J. Sanchez de Toledo (2) P. Bastida (2) J. Estella (3) M. Perez (3) E. Tuset (3) S. Knuutila (4) (1) Universitat Autònoma de Barcelona (2) Hospital Materno-Infantil Vall d’Hebron (3) Hospital Sant Joan de Deu (4) Helsinki University Central Hospital Childhood acute myeloid leukemia (AML) is a heterogenous disease that comprises approximately 20% of all pediatric leukemias. Despite improvements in clinical

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management, the probability of 5-year disease free survival (DFS) is about 50%. Therefore, an appropriate risk-group stratification based on a better knowledge on the biology of pediatric AML is needed. To study genetic features of this disease, we analyzed 24 childhood AML cases by array comparative genomic hybridization (CGH). This method is useful in the detection of submicroscopic DNA copy number changes and has been applied to the study of several neoplasms. Array CGH has so far been applied to the analysis of adult AML but not childhood AML. In this study, a genome-wide screening was carried out using commercial oligonucleotide microarrays (Human Genome CGH 244A microarrays, Agilent Technologies). All cases but one showed one or several regions with gains and/or losses of genetic material. Overall, few alterations were observed and the majority were not recurrent. The only recurrent changes were present in very few cases: gain of 8q24.3 and of 11p15.5–p15.4 (4 cases) and gain of 2p21 and losses of 4p16.3, 7q31.1 and chromosome Y (2 cases). The genes RUNX1, TNF, LTB, SDC1, and RUNX1T1, involved in regions with gains or losses of genetic material, have been reported to be involved in AML leukemogenesis. The number of aberrations per tumor detected by array CGH was tested for prognostic significance. DFS and overall survival (OS) rates were estimated with the Kaplan-Meier method. The survival curves were statistically compared by the log-rank test. Cases with four or more genomic aberrations had a worse prognosis. In conclusion, DNA copy number changes measured by arrayCGH showed a statistically significant impact on childhood AML survival curves.

domestic animals and, more specifically, cat mammary tumours (CMT) are considered good models for their human counterpart. However, data regarding CMT cytogenetic characterization is very scarce, most especially at the molecular level. Genetic aberrations, such as gene amplification, deletions, and loss of heterozygosity are hallmarks of cancer and are thought to be major contributors for the neoplastic process. The comparison of total genomic DNA extracted from a tumour with total genomic DNA obtained from normal cells - Comparative genomic hybridization technique (CGH) allows the detection of individual genomic changes of high value for differential diagnosis and prognosis. On this respect the CGH technique demonstrates an evident clinical impact in oncology. In the present work, we present the study of a spontaneously occurring CMT by Comparative Genomic Hybridization analysis. As far as we know, this is the first time that a GGH analysis is described in a CMT. The CMT sample (comprising four mammary glands) presented a multinodular growth mass that was chirurgically removed from a 14 years old female cat and was histologically classified as being a tubulopapillary carcinoma. Simultaneously, genomic DNA was extracted for a CGH evaluation. The CGH analysis, with 95% of confidence, allowed the detection of deletion regions in chromosome X (p11p12, q31q33). We also identified gains in the telomere regions of chromosomes B1 (p13) and D3 (p15), and at the interstitial region of chromosome E1 (p14).

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Burkitt leukemia of donor cell origin after allogeneic stem cell transplantation

Analysis of a Cat Mammary Tumour (multinodular tubulopapillary carcinoma) by Comparative Genomic Hybridization: a case report S. Santos (1) A. Borges (1) F. Adega (1) F. Gärtner (2) H. Guedes-Pinto (1) R. Chaves (1) (1) Institute for Biotechnology and Bioengineering, Centre of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro (IBB/CGB–UTAD) (2) Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP) An interesting opportunity for comparative oncology has been recognized in spontaneously occurring tumours in

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N. Nadal (1) J. Cornillon (2) F. Solly (1) B. Regeffe (1) E. Tavernier (2) D. Guyotat (2) L. Campos (1) (1) CHU Hopital Nord (2) ICL We report a case of donor derived Burkitt Leukemia/ Lymphoma (BL) occurring in a 48-year-old man after allogeneic stem cell transplantation (SCT) for Diffuse Large B-Cell Lymphoma (DLBCL). In 1998, the patient was diagnosed with a DLBCL of parieto-frontal localization with mediastinal and abdominal dissemination. No cytogenetic studies were performed. He was treated with chemotherapy, radiation and an autologous peripheral-blood-SCT

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(PBSCT). In 2002, he relapsed and underwent an allogeneic PBSCT from an HLA-matched sister in 2003. FISH studies with X and Y probes after BMT, revealed a full donor chimerism. In December 2008, he developed a BL with no feature suggesting an Epstein Barr Virus (EBV) origin. The bone marrow karyotype was //46,X,-X,6,t(8;22)(q24;q11),add(9)(p13), dup(12)(q12q21), add (13)(q22),-16,+3mar[5]/46,XX[18] FISH analyses confirmed (1) cMYC implication in 21/50 mitoses and 19/200 nuclei and (2) a female sex chromosome complement (XX) in all cells, with no evidence of a Y chromosome, consistent with a donor bone marrow status. Donor Cell Leukaemia (DCL) was confirmed by simultaneously hybridizing cMYC probe with X/Y probes. The patient died of massive pulmonary embolism during the induction treatment. The donor is alive and with no evidence of malignant disease. Usually early leukaemia post BMT is a recurrence of the host initial disease when DCL tends to occur later. Thus it is possible that most late relapses are DCL (Cooley, 2000). Their etiologic mechanism remains unknown and is probably multifactorial. Our case is of interest because of the type of leukaemia involved. DCL are mainly acute myeloid leukaemias. Most lymphoproliferative disorders of donor cell (DC) origin have an early onset and are associated with immunosuppression and the presence of EBV. Thus an original pathogenic mechanism is likely to be implicated in our observation. 7a.31-P Polysomies and lack of ALK rearrangement in a pseudosarcomatous myofibroblastic proliferation of the heart P. Caria (1) T. Dettori (1) D. Frau (1) S. Nemolato (2) G. Faa (2) R. Vanni (1) (1) University of Cagliari (2) University of Cagliari The precise diagnosis of most of soft tissue tumors, including the classification of myofibroblastic proliferations, may benefit from a joint morphological and molecular approach. We report the characterization of a polypoid lesion, incidentally found at autopsy in the septal wall of the left ventricle of a 75-year old male, died from a heart attack. The histopathological examination of the polypoid mass revealed a general architecture

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characterized by a fibrous stroma with focal myxoid areas, and a prominent vascular pattern characterized by delicate, thin-walled, branching blood vessels, that was reminiscent of inflammatory myofibroblastic tumors (IMT). Nevertheless, rare lymphocytes and plasma cells were occasionally detected scattered in the stroma, in contranst to the prominent lymphoplasmocytic infiltrate characterizing IMT lesions. No mitosis was detected. Moreover, the myofibroblastic proliferation showed a positive immunohistochemical reaction for vimentin and smooth muscle actin in the large, atypical star-shaped stromal cells, similar to those previously reported in a subset of nasal polyps with aneuploidy. Based on this observation and on the reported ALK gene rearrangement in IMT, cytogenetic investigation by fluorescence in situ hybridization was performed on interphase nuclei from formalin-fixed paraffin-embedded tissue in order to check both possible ALK gene rearrangement and presence of aneuploidy. Cytogenetic investigations revealed aneuploidy restricted to nuclei particularly large in size: trisomy 7 in about 60%, half of which also showed trisomy 9. All other tested chromosomes (7,8,9,10,11,12,17,20) showed disomy. These findings support the diagnosis of a pseudosarcomatous myofibroblastic proliferation (PMP). Conclusion: the present case of PMP showed scarce lymphoplasmacytic infiltrate, lack of ALK gene rearrangement, and presence of polisomy 7 and 9. Although the lesion showed evidence of histological findings overlapping those of IMT, the above mentioned features were in favour of a pathological/cytogenetic diagnosis of PMP. Supported: FIRBMIUR project No. RBIP0695BB and “Fondazione Banco di Sardegna”. 7a.32-P Significance of chromosome 3 breakpoint in a patient with Familial Renal Cell Carcinoma, a balanced chromosome 3 translocation, and a VHL mutation J. McGowan-Jordan (1) G. Graham (1) (1) Children’s Hospital of Eastern Ontario Acquired chromosomal translocations causing overexpression of an oncogene or inactivation of a tumoursuppressor gene are well recognized as mechanisms involved in tumour initiation and progression. In Familial Renal Cell Carcinoma (RCC), constitutional chromo-

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somal translocations have been implicated in predisposition to development of bilateral renal tumours; translocations involving chromosome 3 have lead to the identification of a number of RCC-related genes (reviewed by Bonné et al., 2004). We report a 45-yr-old male with a history of bilateral clear cell renal carcinoma whose deceased father was diagnosed with renal cell carcinoma of unknown histology at the age of 50. Chromosome analysis on peripheral lymphocytes from the proband revealed an apparently balanced reciprocal translocation “t(3;11)(q21;q13.5)”. Concurrent testing for mutations in the VHL gene also identified an apparent splice-site mutation (IVS1+5 G>C) interpreted as a pathogenic by the reporting laboratory on the basis of its presence in a single family (Zbar et al., 1996). The proband is asymptomatic. In addition to bilateral renal cell carcinoma he had a 1.1×1.4 cm simple cyst of the pancreas on abdominal imaging. An opthalmology exam was negative for ocular angiomata. MRI of the brain and spinal cord are pending. There is no additional family history to suggest VHL. Thus, it is unclear whether the translocation or the VHL “mutation” has contributed to the RCC in this patient, an important element in genetic counseling and risk assessment for his family. FISH on metaphases is required to determine whether any of the previously identified RCC-related genes on chromosome 3 are involved in the rearrangement. 7a.33-P Karyotypic and molecular cytogenetic findings in childhood t-cell acute lymphoblastic leukemia: the Schneider medical center of isreael experience M. Jeison (1) J. Heker (1) J. Mardoukh (1) G. HalevyBerko (1) D. Luria (2) G. Avrahami (3) J. Stein (3) I. Yaniv (3) B. Stark (3) 1 Cancer Cytogenetic Laboratory,2 Flow Cytometry Laboratory, 3 Pediatric Hematology Oncology, Schneider Children’s Medical Center of Israel, Petah Tikva; Sackler Faculty of Medicine, Tel Aviv. Introduction T-cell acute lymphoblastic leukemia (T-ALL) is a neoplastic disorder which represents 15% of pediatric ALL. It is characterized by specific chromosomal abnormalities and additional secondary aberrations which may contribute prognostic information for treatment stratification.

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Aim of the work We used classical and molecular cytogenetics to detect recurrent combinations of cytogenetic/ Fluorescence In Situ Hybridization (FISH) findings, and to study their correlation with immunophenotype and outcome. Patients and methods Between 1990 to 2008, 79 newly diagnosed T-ALL patients, age 0.7–19 years, were treated at the SCMCI. Cytogenetic analysis and FISH on cultured fresh BM specimens (part from archival material) was performed. Results Cytogenetic analysis was successful in 77 patients. Karyotype was abnormal in 59 patients. In 18/59 patients 14q11 was involved. By FISH analysis, TCRB (7q34) showed split in 6/61 samples; TLX1 in 2/57 samples; TLX3 in 8/59 samples; SIL/TAL1 in 5/59 samples; MLL in 6/63 samples and TCRAD (14q11) in 9/59 samples. 21/58 patients showed mono/bi allelic deletion of 9p and 2/66 cases with episomal ABL1 (9q34) amplification were detected. Conclusions The findings of nonrandom primary translocations and the combinations with recurrent secondary genetic aberrations, suggest specific multistep pathways in leukemogenesis of T-Cell leukemia. In the context of the present intensive treatment only the TLX3 split group fared worse, while TLX1 split group has no relapses. A larger study is needed to evaluate the prognostic significance of the various cytogenetic subgroups in TALL. 7a.34-P Discordance between morphological and cytogenetical remission in a patient with AML W. Kroes (1) E. den Ouden (1) A. Baasten (1) J. Kerkhoffs (2) R. Willemze (3) (1) LUMC (2) Haga Hospital (3) LUMC We report here on a 58 year old male presenting with biphenotypical acute myeloblastic leukemia .Conventional cytogenetic techniques on bone marrow at initial diagnosis, before treatment, showed the following karyotype: 46,XY,t(2;11)(p21;q23),del(5)(q21),add(12)(p12)[9]/ 46,XY,t(2;11)(p21;q23)[5]/46,XY[1].FISH analysis with a DNA probe for the MLL-gene demonstrated that

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the breakpoint at 11q23 was telomeric to the MLL gene. T(2;11) (p21;q23) is a rare but recurrent translocation observed in MDS and AML. This translocation is specifically associated with a deletion of the long arm of chromosome 5. After induction treatment the patient was in complete morphological remission. However, cytogenetic studies revealed the t(2;11) in all metaphases, without the del(5q) and the add(12p). Evaluation after the second therapy cycle showed again a complete morphological remission but persistence of t(2;11) in all analyzed metaphases. To investigate the possibility of a constitutional chromosome aberration chromosomal analysis was performed on skinfibroblasts and peripheral blood. The skin-fibroblasts revealed a normal karyotype in all 100 analyzed metaphases. PHA stimulated blood showed a mosaic karyotype : 46,XY,t(2;11)(p21;q23)[11]/46,XY[21]. This suggests that the t(2;11) is an acquired aberration that persists in spite of morphological remission. The patient underwent a bone marrow transplantation and is still in complete morphological remission after 10 months. To our knowledge this is the first description of an AML case with a t(2;11) showing discordant morphological and cytogenetical remission. 7a.35-P Molecular Cytogenetic Studies of Complex Chromosomal Aberrations in Myelodysplastic Syndromes (MDS) Z. Zemanova (1) K. Michalova (1) J. Brezinova (2) L. Lizcova (1) S. Izakova (2) D. Bystricka (1) I. Sarova (2) M. Siskova (3) O. Cerna (4) J. Cermak (2) (1) General Teaching Hospital and First Faculty of Medicine, Charles University in Prague (2) Institute of Hematology and Blood Transfusion (3) General Teaching Hospital and First Faculty of Medicine, Charles University in Prague (4) Faculty Hospital Kralovske Vinohrady, Prague Complex chromosomal aberrations (CCA) are present in 10–20% patients with myelodysplastic syndromes (MDS) and are associated with treatment resistance. For precise analyses of chromosomal regions and breakpoints involved in CCA we used various modifications of molecular cytogenetic techniques: FISH with locus specific

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probes (Abbott Vysis), CGH, mFISH/mBAND (MetaSystems) and/or array CGH (BlueGnome, NimbleGene). During 2002–2008 we examined bone marrow of 680 adults at diagnosis of MDS and in 86 of them (12.6%) CCA were found. Primary MDS was diagnosed in 74 (86.0%), in 12 cases therapy related MDS was ascertained. Deletion of 5q31 region was proved in 75 patients (87.2%). In 35 cases mFISH analyses showed that parts of deleted No.5 were translocated into other chromosomes. Translocations were nonbalanced and the most recurrent partners were chromosomes 17 (11x), 3 (6x), 7 (5x) and 12 (5x). Monosomy of No.5 was confirmed in one case only, thus showing that it is not separated diagnostic entity in MDS. The most frequent breakpoints on chromosome 5 were 5q33 (31x), 5q13 (25x), 5q14 (7x) and 5q12 (5x). Except No. 5 the most frequently involved in CCA were chromosomes 7 (37x), 3 (33x), 12 (31x), 17 (31x) and 11 (24x), the most recurrent breakpoints were at regions 7p11 (6x), 7q11 (6x) and 12p11 (7x). CCA in bone marrow at diagnosis was in this cohort sign of poor response to therapy and short survival (median 5 months). Using combination of molecular cytogenetic techniques we found a wide variety of cryptic aberrations not detectable by conventional cytogenetics, however, karyotypes evaluated by all molecular methods showed highly corresponding results. Finding of new recurrent rearrangements and breakpoints might lead to discovery of genes, involved not only in origin but also in progress of malignancy and to better understanding of molecular etiology of MDS. Supported by: IGAMZCR NR9227-3, MZO VFN2005, MSM0021620808 and MSMTLC535. 7a.36-P Screening of TERC gene amplification as an additional genetic diagnostic test in detection of cervical pre-neoplastic lesions N. Kokalj Vokac (1) T. Kodric (1) A. Erjavec Skerget (1) A. Zagorac (1) I. Takac (1) (1) University Medical Centre Maribor (2) University Medical Centre Maribor (3) University Medical Centre Maribor (4) University Medical Centre Maribor (5) University Medical Centre Maribor

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The aim of the current study was to present TERC gene amplification as possible diagnostic marker for use in routine cytological screening to improve the accuracy of conventional screening procedure in detection of cervical pre-neoplastic lesions. Cervical smears were screened and classified as low-grade squamous intraepithelial lesions (LSIL) and high-grade squamous intraepithelial lesions (HSIL). From the same specimens FISH procedure using TERC-specific DNA probe was performed. Copy number enumeration for TERC gene was evaluated. More than 2 signals per cell were considered as TERC positive case. In cervical smears graded after conisation as CIN1, no TERC positive cases were found in either LSIL or HSIL. Neither was TERC amplifications found in LSIL cases with histological results CIN1 and CIN 2. Amplifications of the TERC gene first appeared in HSIL cases with CIN2 histology. In the group of CIN3, TERC positive cases were present in LSIL and HSIL. In these, there were no statistically significant differences between TERC positive and TERC negative cases. Statistically significant differences in TERC positive cases were found between LSIH and HSIL without regard to the CIN grade. From the results obtained, it can be concluded that TERC gene amplifications inevitably lead to a high risk of CIN3 in both LSIL and HSIL after cytological smear examination. A high CIN is not necessarily correlated with TERC amplification, but a positive TERC result certainly demands a high CIN classification. 7a.37-P Molecular diagnostics of multiple myeloma using oligonucleotide array-based comparative genomic hybridization (array-CGH) R. Zaoralova (1) V. Vranova (1) J. Smetana (1) D. Kyjovska (3) B. Sablikova (4) R. Kupska (2) R. Hajek (4) P. Kuglik (1) (1) Masaryk University, Faculty of Science (2) Masaryk University, Faculty of Medicine (3) Masaryk University, Faculty of Medicine (4) The Faculty Hospital Brno

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Multiple myeloma (MM) is a malignant disease caused by malignant transformation of the B-cell, its clone proliferation and its accumulation in a bone marrow. Despite of improvements in treatment, MM is still considered as an incurable disease. Median of patients’ overall survival ranges from 3 to 4 years, only about 10% patients survives at 10 years. Generally MM is characterized by high clinical and genetic heterogeneity. From view of diagnostic improvement and effective targeted MM therapy, there is expected a radical breakthrough based on detailed genetic subclassification, leading to detection of novel reliable prognostic markers, which can exactly characterize the disease. The clinical implementation of array-CGH analyses allows us to do a whole-genome screening and sensitive identification of new genetic markers important for diagnosis, classification and prediction of prognosis of many tumor diseases including MM. Main aim of our work was molecular diagnostic of multiple myeloma focused on detailed genomic profiling of this disease using 60-mer oligonucleotide array based comparative genomic hybridization (Agilent Human Genome CGH Microarray Kit 44K). We performed array-CGH on 18 MM patients. The results confirmed the previous FISH findings (+1q, −13, hyperdiploidy) and showed up another frequent copy number changes, e.g. del(8p), del(16q) or +19. Biallelic deletion in cytoband 11q22 occurred in two cases. We observed a clear difference in genome profile between hyperdiploid (H, N=8) and non-hyperdiploid (NH, N=7) group of MM patients: in hyperdiploid samples, mostly whole-arm gains were present, and occurred repeatedly (19 regions, 10 in3 or more patients in H group vs. 6 regions, one in 3 patients NH). For non-hyperdiploid samples, smaller and unique deletions were the typical aberrations (more than 30 in NH vs. 17 in H). Analyses of repeating genetic changes might lead to a better classification of stage and risk, providing the basis for improved clinical management and tailored treatment of MM. Supported by grants and projects LC06027, MSM0021622434, MSM0021622415 and IGA NR9317

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7a.38-P Cytogenetic and molecular analysis in a series of 68 childhood acute myeloid leukemia

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tions. Moreover, technologies such as CGH and expression microarrays could be useful to identify new genetic alterations. 7a.39-P

A. Canellas (1) G. Armengol (1) J. Estella (2) M. Perez (2) E. Tuset (2) M. Camos (2) J. Sanchez de Toledo (3) P. Bastida (3) M. Caballin (1) (1) Facultat de Biociencies. Universitat Autonoma de Barcelona (2) Hospital Sant Joan de Deu de Barcelona (3) Hospital Materno-Infantil Vall d’Hebron de Barcelona Childhood acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease. The detection and characterization of recurrent chromosomal rearrangements, that include inv(16), t(8;21), t(15;17) and rearrangements of MLL, have provided the means to identify distinct biologic and prognostic subgroups. However, a large number of patients have no detectable chromosome abnormalities at diagnosis (25% of childhood AML). Recently, a number of gene mutations (FLT3, NPM1, etc.), have been identified, in particular in AML cases exhibiting a normal karyotype; internal tandem duplication (ITD) of the FLT3 gene is the most frequent (15% of childhood AML) and correlates with poor prognosis. It is particularly common in cytogenetically normal (CN) AML (55%) and in acute promyelocytic leukemia (25%). In this study, we present a series of 68 childhood AML patients. Conventional cytogenetic (CC) and FISH analysis with MLL, PML/RARα, AML1/ETO and CBFβ probes (Vysis) were applied. PCR assay for the detection of FLT3-ITD and NPM1 mutations was performed. After CC and/or FISH analysis, 46% of cases presented a recurrent rearrangement. FISH enabled to identify alterations in patients CN or with no cytogenetic results. The incidence of FLT3ITD was 14.5% (8 out of 55 patients). Of these, the major incidence of FLT3-ITD was found in patients CN (50%) or with presence of t(15;17) (25%). NPM1 mutation test was performed on some of the CN patients and the wild-type allele was found in all patients. Clinical parameters and genetic alterations detected by cytogenetic, FISH or PCR did not show any correlation with survival. Overall, 20% of cases did not show any of the tested genetic aberrations. Some of these cases could have other gene muta-

Array-CGH analysis of acute myeloid leukemia patients with high hyperdiploid karyotypes C. Veigaard (1) E. Kjeldsen (1) (1) Aarhus University Hospital, Aarhus University Background: Identification of chromosomal abnormalities by conventional cytogenetic analysis is an essential component of the multidisciplinary approach to diagnosis, classification and risk-stratification of patients with acute myeloid leukaemia (AML). A rare prognostic subgroup of AML patients characterized by high hyperdiploidy (>=49 chromosomes) was recently identified. Data from treatment and survival statistics suggested that this group of AML patients may define a novel entity. In childhood acute lymphoblastic leukaemia patients with high hyperdiploidy arraybased comparative genomic hybridization (aCGH) identified novel cryptic chromosomal aberrations. Aims: 1. Identification of submicroscopic chromosomal abnormalities in AML patients with high hyperdiploid karyotypes using aCGH analysis in a population based retrospective study design. 2. Correlating aCGH findings with data from conventional cytogentics, survival and treatment statistics refining the otherwise unfavourable prognosis of this AML subgroup. Materials and Methods: 23 AML patients admitted to our department in the period from 1988 to 2008 had high hyperdiploidy. DNA could be extracted from stored material from 19 of these patients. aCGH analysis was performed with sex-matched genomic reference material using the BAC-clone array from BlueGnome. Results: aCGH data confirm the high hyperdiploidy found by conventional karyotyping but in many cases also identify novel submicroscopic chromosomal abnormalities.

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Perspectives: High hyperdiploid AML patients may be better classified into different prognostic groups depending on the presence or absence of additional cryptic aberrations identified by aCGH analysis. 7a.40-P Correlation between chromosomal abnormalities detected by fluorescent in situ hybridisation (FISH) in sputum and bronchial dysplasia in patients at risk for lung cancer N. Ruiz-Xivillé (1) A. Rosell (2) E. Monsó (3) I. Granada (1) F. Andreo (3) R. López-Lisbona (2) F. Maria Teresa (4) C. Eva (4) L. Mariona (4) F. Millá (1) (1) Institut Català d’Oncologia - Badalona (2) Hospital Bellvitge-IDIBELL (3) Hospital Germans Trias i Pujol (4) Hospital Germans Trias i Pujol Background and aim: Although multiple genetic markers have been identified in lung cancer, there is few information regarding bronchial dysplasia. The aim of this study was to analyse 4 possible genetic markers (c-MYC, LPL, p53 and p16) by FISH in sputum samples in patients with risk for lung cancer, and to establish a correlation with dysplasia. Patients and method: Subjects with smoking history of > 30 pack-year, between 45 and 75 years old, without chronic respiratory failure, FEV1%>35% and chemo or radiotherapy naïve were included. Induced sputum and autofluorescence bronchoscopy were performed and all abnormal spots were biopsed. Sputums were analysed by conventional cytology and by FISH using LSI c-MYC, LSI LPL, LSI p16 and LSI p53 probes (Vysis). Cut off values for losses and gains for each probe were established with control sputums. Results: Eighty five patients (93% male), median age 63 years, smokers 35.6% and former smokers 64.4%, median tobacco 48 pack-year. Biopsy: 52 of 85 (61%) show dysplasia. Cytology: 6 of 85 (7%) sputums show atypia. FISH: 7 of 85 (8.2%) samples show genetic aberrations with a percentage of cells close to cut off value. Gains: p16 (n=2), p53 (n=1), c-MYC (n=1). Losses: LPL (n=2), p16 (n=1). Only one case with genetic aberrations also shows sputum atypia (p=0.413). Five cases with genetic aberrations also show dysplasia in bronchial biopsy (p=0.441).

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Conclusions: In this study, there is not a correlation between cytology and FISH results in the sputum samples studied. Genetic aberrations have been observed more frequently in cases with dysplasia but it is not statistically significant. The follow up of this cohort will determine the importance of these chromosomal abnormalities. Funded by: FIS (051715), SOCAP, FUCAP and CibeRes 7a.41-P Characterization of centlein, a novel common fragile site gene S. Zahedi (1) . Arnaudova M. Schwab L. Savelyeva DKFZ Germ-line mutations of the BRCA2gene account for a large proportion of familial breast cancer cases in females and the majority of familial breast cancers in males. In our previous study, we found that heterozygous BRCA2 mutation carriers exhibit high degree of constitutional instability on the distal portion of the short arm of chromosome 9. The existence of a far unknown fragile site in the vicinity of the observed chromosomal aberrations was shown. A novel common fragile site FRA9G has been identified and this newly discovered fragile site spans the major part of the 369 kb genomic sequence of C9orf39, which is the only gene encompassing FRA9G. Breakage at FRA9Gmight lead to disruption of the C9orf39 gene, the function of which has recently been assumed to be a centrosomal protein. This led to call the protein of this gene, centlein (CNTLN). In our current study we are focusing on characterizing different aspects of the centlein gene. The overall expression of the gene in normal and some different cancer tissues was evaluated at RNA and protein level. The primary experiments indicate that centlein is ubiquitously expressed in almost all normal human tissues and most of the cancer cell lines. Immunostaining studies in several breast cancer cell lines showed scattered localization of centlein in the nucleus. Using ultrahigh resolution FRA chip array technology, we analyzed in great details the FRA9G region.

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To clarify more the role of this protein in cell function and survival, and its possible contribution to tumorigenesis, further overexpression / knockdown experimments are being performed. 7a.42-P 12p12 amplification in one case of acute monoblastic leukemia F. Viguié (1) A. Tabet (2) M. El Khoury (1) A. Joseph (3) L. Heuberger (4) L. Roda (4) S. Haiat (1) J. Marie (1) B. Rio (1) A. Aboura (2) (1) Assistance Publique Hôpitaux de Paris/ University Paris 5 (2) APHP, Hôpital R. Debré (3) Société PerkinElmer (4) Centre Hospitalier de Polynésie Française Introduction Gene amplification > 4 copies is infrequent in leukemia, it involves mainly cMYC, MLL or AML1, located on HSR or double-minute chromosomes. Other amplifications are very rare and may give informations on leukemogenesis. Here is described a large amplification involving several cancer genes at 12p12.1, detected by CGH array. Observation The patient was a 67-year old Polynesian female. She was admitted for pancytopenia, fever and abdominal pain. Bone marrow analysis showed 80% of monoblasts CD33+, CD34+, CD13+, CD117−, MPO+, JC1−, diagnosed as acute monoblastic leukemia (FAB AML-M5A). Bone marrow karyotype was hypodiploid complex in 13/16 mitoses: 42–44,XX, del(4)(q1?3),add(6)(p2?2),−7, add(12)(p12),add(17)(q2?4),add(18)(p11),−19, +mar1,+mar2, +mar3,+variations. Patient was included in the MyloFrance 3 protocol. After induction chemotherapy involving daunorubicin and cytarabin, a cytological and cytogenetic remission was obtained. Because of infectious complication, treatment was deferred and carried on in Polynesia. A relapse occurred at 6 months post diagnosis, patient refused to be treated again and died. In order to define more precisely the quantitative chromosomal changes, a CGH array analysis was performed from diagnosis peripheral blood DNA, with a PerkinElmer 4900 BACs clones array. Dele-

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tions were detected from 4q12 to 4q26, 5q14.3 to 5q31.3, 6p22.2 to 6p25.3, 7q11.2 to 7qter, 8q23.3 to 8q24.13, 12p12.1 to 12p13.2, 16q12.1 to 16q24.3, 17p12 to 17p13.3, 17q21.32 to 17q23.2 and 19q12 to19q13.43. A trisomy was found for whole chromosome 18 and for 8q24.13 to 8q24.3, 17q23.2 to 17q25.3, 19q13.12 to19q13.34. Finally a very large amplification was observed from 12p11.21 to 12p12.1. The size of the amplicon was estimated to approximately 2Mb, with a maximum amplification localized to a region incompassing 3 locus closely involved in cancer genesis: LRMP, CASC1 and KRAS. Without current FISH confirmation, the amplification was provisionally located on an HSR at 12p12. Discussion The amplicon contains a number of genes whose respective role in the malignant process is not easy to define. KRAS is frequently involved in every type of malignancy as point mutation. LRMP and CASC1 are considered as genes of tumor susceptibility. None of these genes has been previously noted in amplification process. Other genes of the amplicon may be also considered. To delineate the pattern of amplification, a FISH study with fosmid probes is in progress. 7a.43-P Cervical intraepithelial neoplasia in five carriers of constitutional balanced reciprocal translocations involving 11q23 I. Bache (1) A. Silahtaroglu (1) L. Nazaryan (1) B. Norrild (2) J. Olsen (3) Z. Tümer (4) N. Tommerup (1) (1) ICMM, University of Copenhagen (2) ICMM, University of Copenhagen (3) Danish Cancer Society (4) Kennedy Center Persistent infection with high risk human papillomavirus (HPV) and stepwise (epi)genetic changes in the host is important factors in cervical carcinogenesis. A chromosomal region believed to be involved in early stages of cervical carcinogenesis is 11q23, as loss of heterozygosity (LOH) at 11q23 is a common genetic alteration in both cervical intraepithelial neoplasia (CIN) and in cervical cancer. During re-examination of carriers of constitutional balanced reciprocal translocations by questionnaires

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and nationwide registries (The Danish Cancer Registry and The Danish Cytogenetic Registry), we identified five unrelated women with a 11q23 breakpoint who had developed CIN. Three of the patients had the recurrent translocation t(11;22), while the two others had a t(11;15)(q23.3;q26.3) and a t(11;18) (q23.3;q21) translocation respectively. We hypothesise that constitutional breakpoint of 11q23 in these five women is a predisposing factor to CIN and that we can obtain new insight in early cervical carcinogenesis by studying these breakpoints. We mapped the chromosome 11 breakpoints by FISH and found that the same BAC clone spanned the der(11) breakpoints in the three t(11;22) carriers as expected, and that all the five breakpoints were located centromeric for the known 11q23.3-LOH region. We have analysed DNA from the five translocation carriers for additional constitutional alterations by Affymetrix Genome-Wide Human SNP Array 6.0 and are currently performing global DNA methylation analysis by high throughput sequencing (Illumina Genome Analyzer (Solexa)) to study short- and long-range epigenetic modification of the genes in the 11q23-LOH region. Furthermore, we are obtaining CIN tissue from the translocation carriers for HPV typing and for LOH studies. 7a.44-P Karyotype instability evaluated by array-CGH and risk of myeloid malignancy in Shwachman diamond syndrome F. Pasquali (1) R. Valli (1) B. Pressato (1) C. Marletta (1) C. Morerio (2) M. Zecca (3) C. Panarello (2) F. Lo Curto (1) E. Maserati (1) (1) Università dell’insubria (2) IRCCS Istituto G. Gaslini (3) Università di Pavia, Fondazione IRCCS Policlinico S. Matteo Shwachman Diamond syndrome (SDS) is a bone marrow (BM) failure syndrome, inherited as an autosomal recessive trait, in which the risk to develop myelodysplastic syndrome (MDS) and/or acute myeloid leukaemia (AML) is evaluated 19% at 20 years of age, and 36% at 30. Karyotype instability in BM cells was repeatedly recognized, and the relevance of consequent acquired chromosome changes, most

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frequently involving chromosomes 7 and 20, for the development of MDS/AML is a topic of debate. Comparative genomic hybridization on microarray (a-CGH) performed with a genome-wide system (Agilent, 244K) on DNA from BM of 7 patients led to the following results: The isochromosome i(7)(q10), which is the most frequent change in BM, does not include short arm material, contrary to previous evidence obtained by fluorescent in situ hybridization (FISH); The presence of the i(7)(q10) was detected in one case also on peripheral blood, while this evidence was not reached by chromosome analysis; In one patient with the i(7)(q10) in a small BM clone, a-CGH showed that also an interstitial deletion of the long arm of chromosome 20 was present; In one case, with a clonal deletion of the long arm of a chromosome 20, and a subclone with a subsequent rearrangement of this abnormal 20 into a ring chromosome, the latter was defined precisely, with lacking segments of both the short and the long arm, but also with partial duplications; Other 4 possible cryptic deletions (involving chromosome 13 in3 cases, and chromosome 5 inone case), detected in small clones or very thin, are being confirmed by FISH. a-CGH demonstrated that the degree of karyotype instability in SDS, with the establishment of clonal anomalies in BM, is definitely higher than previously evaluated on the basis of chromosome analysis and FISH assays. Possible new recurrent chromosome anomalies, previously unrecognized, were found, with both theoretical and practical relevance in relation to MDS/AML development. 7a.45-P Cytogenetic study: our experience of 10.000 analysis A. Schmid-Braz (1) T. Borgonovo (1) V. Jamur (1) L. Wuicik-Merfort (1) L. Veiga (1) R. Pasquini (2) J. Zanis Neto (2) I. Cavalli (1) (1) Universidade Federal do Paraná, Hospital de Clinicas (2) Universidade Federal do Paraná The Bone Marrow Transplant Program of the Universidade Federal do Paraná was founded 30 years ago, in 1979 and the Cytogenetics Laboratory started its activities six years later, in 1985. The laboratory

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develops activities of diagnosis evaluation and follow up of hematological patients pre and post clinical treatment as well bone marrow transplantation. The aim of the work is to present a brief review of the cytogenetics analysis obtained in the last 15 years. We used samples of bone marrow cells (to conventional cytogenetics) and peripheral blood (to chromosomal instability tests) cells. During this time 10.875 tests were done, 3.774 (34,7%) showed a normal karyotype. More than 100 different types of chromosomal aberrations were identified, the more frequents were: t(9;22) in 20.2%, +8 in 1.91%, −7 and del(7q) in 1.1%, del(11q) in 1.04%, +21 in 0.98%, t(15;17) in 0.74%, del(12p) in 0.49%, −5 e del(5q) in 0.33% e t(8;21) in 0.29%. The cytogenetics evaluation, with other laboratory tests, has been of great significance concerning the clinical diagnosis, with prognostic and therapeutic effects. 7a.46-P Do we need to do FISH analysis in MDS as often as we do? D. Costa (1) S. Valera (1) A. Carrió (1) A. Arias (1) B. Nomdedeu (2) M. Belkaid (2) E. Campo (1) (1) Hospital Clinic (2) Hospital Clinic

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with de novo MDS. One hundred seventy four FISH studies were performed in 60 patients. Reasons for FISH studies were: normal karyotype (n =77), confirmation of a chromosomal abnormality (n = 77) and when a cytogenetic result was not obtained (n=20). Five different regions were tested: 5q31 (n =44), 7q31 (n= 40), CEP 8 (n= 41), 20q12 (n =32), and 11q23 (MLL) (n =17). FISH studies using the 5 probes confirmed either normal or abnormal conventional cytogenetics results in 153 out of 154 (99.4%) cases. Only one discrepancy was found (0.6%) in a case with normal karyotype by conventional cytogenetics (16 metaphases) and 20q12 deletion in 11% (cut off=10%) of the studied nuclei. FISH studies allowed testing for the chromosomes most frequently involved in the prognosis of MDS, in 20 cases without cytogenetic result. In 5 of these 20 cases, a chromosomal abnormality was found involving deletion of 5q31 and 7q31 regions in one case respectively, and a trisomy of the centromeric region of chromosome 8 in 3 cases. In conclusion, FISH studies were extremely useful in cases lacking a conventional cytogenetic result and also confirming chromosomal abnormalities involving the tested chromosomal regions. However, no extra information was provided in normal karyotypes. 7a.47-P

Myelodysplastic syndromes (MDS) are a group of clonal haematopoietic stem cell diseases characterized by cytopenia(s), dysplasia in one or more of the major myeloid cell lines, ineffective haematopoiesis, and increased risk of development of acute myeloid leukaemia (AML). The role of cytogenetic studies as a prognostic indicator in MDS has been recognized and codified by the International Myelodysplastic Syndrome working group where three major risk categories of cytogenetic findings have been defined: i) good risk- normal karyotype, isolated del(5q), isolated del(20q), and –Y; ii) poor risk—complex karyotypes (≥3 abnormalities), or abnormalities of chromosome 7; and iii) intermediate risk—all other abnormalities. The aim of this study was to assess the use of FISH studies in the detection of chromosomal abnormalities. From January 1997 to April 2008, cytogenetic studies were carried out in 318 bone marrow samples in patients diagnosed

The evaluation of the genomic instability by micronucleus and sister chromatid exchange in myelodysplastic syndrome E. Nazlıgül (1) &. Palanduz (1) &. Öztürk (1) A. Bayrak (1) A. Uçur (1) G. Bağatır (1) K. Cefle (1) M. Yenerel (2) M. Nalçacı (2) (1) Istanbul University, Istanbul Medical Faculty, Division of Medical Genetics. (2) Istanbul Unıversity, Istanbul Medical Faculty, Division of Hematology Myelodysplastic syndrome (MDS) is a clonal disorder of hematopoietic stem cells, and has an increased risk of leukemic transformation. According to its etiology; it is classified as primary and secondary. The primary MDS may be related to viral agents or genetic tendency may also be responsible. The secondary MDS appears related to environmental toxins, chemotherapy and irradi-

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ation. Sister chromatid exchange (SCE) and micronucleus (MN) are considered to be the biomarkers of DNA damage. Material and method: In our study, the frequency of SCE and micronucleus were analyzed at the bone marrow samples of 20 patients who had no treatment yet. The control group consisted of 13 individuals being prepared as donors for bone marrow transplantation. Result: We observed a statistically significant increased frequency of both sister chromatid exchange(SCE) and micronucleus(MN) in our patients compared to the control group. Conclusıon: The increased frequencies of sister chromatid exchange and micronucleus support the role of genomic instability in the development of MDS. 7a.48-P A Myeloproliferative Disorder Patient with Chromosome 20q Deletion, Trisomy 9 and BCR-ABL Negative H. Acar (1) K. Acar (2) Ö. Balasar (3) T. Çora (4) M. Balasar (5) (1) Selcuk University The myeloproliferative disorder (MPD) is a heterogeneous range of clonal hematological malignant diseases at the time of their initial presentation. MPD is classified three main categorized as polycythemia vera, essential thrombocythemia and primary myelofibrosis. These are closely related in terms of both pathogenesis and clinical phenotype. All three are characterized by the presence of JAK2V617F at variable mutational frequency. Here we describe the case of a 66-yearold woman, presenting with MPD-primary myelofibrosis (MP). At diagnosis, bone marrow biopsy examination showed a hypercellular example and an increasing of reticulin fibers. Genetic analysis was done by using conventional cytogenetic analysis for karyotyping, FISH anlysis for BCRABL fusion and chromosome 20q deletion, and also molecular genetic analysis for JAK2V617F mutation. Patient had 46,XX,del(20q) [3]/47,XX, +9,del(20q)[6] and BCR-ABL negative, chromosome 20q deletion. This case was discussed in

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clinical features and relation to the genetic abnormalities. 7a.49-P Expression of Bcl-2 protein, amplification of c-myc oncogene and presence of additional chromosomal alterations in patients with chronic myelogenous leukemia M. Strnad (1) B. Todoric-Zivanovic (1) N. Strelic (2) Z. Tatomirovic (1) D. Stamatovic (3) G. Brajuskovic (4) Z. Magic (2) (1) Military Medical Academy (2) Military Medical Academy (3) Military Medical Academy (4) Faculty of biology Background: Chronic myeloid leukemia (CML) represents the malignant myeloproliferative disease developed out of pluripotent hematopoietic stem cell that contains fusion bcr-abl gene. The mechanisms that lead to these changes at molecular level aren’t still well known as well as the mechanisms that increase proliferative capacity of these cells. Within our study we carried out the analysis of the presence the amplification of c-myc oncogene as well as the analysis of the changes in expression of Bcl-2 in patients with CML and presence of additional chromosomal alterations. Methods: Our study included 30 patients with CML. Using immunohistochemical APAAP method we analyzed the expression of Bcl-2 protein in mononuclear bone marrow cells. By way of differential PCR method we followed the presence of amplified c-myc gene in mononuclear peripheral blood cells. Cytogenetic studies were carried out on Gimsa-banded chromosomes obtained directly or from 24-hour unstimulated bone marrow cultures. Results: The level of expression of Bcl-2 protein is considerably higher in bone marrow samples of patients undergoing blast transformation of disease. The amplification of c-myc gene has been detected in 30% patients in blast transformation of disease. The evolution of karyotypes was detected in 13 (32%) patients. In 8 patients we detected mitosis with more than 60 chromosomes, in 4 patients additional structural chromosomes alterations [46,XX,t(9;22)(q34;q11),t(3;21)(q21;p11);

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46,XX,t(9;22)(q34;q11),t(6;9)(q15;p24);46,XY,t (9;22)(q34;q11),i(17)(q10);46,XY,t(9;22)(q34;q11), t(9;22)(q34;q11) and in one patient presence of additional marker chromosome (47,XX, t(9;22) (q34;q11),+mar). Conclusion: The results of this work have shown that determine of expression of Bcl-2 protein and correlation with the amplification of c-myc gene and evolution of karyotypes give us important informations about of disease progression. Key words: CML, apoptosis, cytogenetic evolution, Bcl-2, c-myc 7a.50-P Correlation of Her-2/neu Gene Amplification with Other Prognostic and Predictive Factors in Female Breast Carcinoma S. Brahem (1) S. Mougou (1) M. Yacoubi (2) H. Elghezal (1) A. Saad (1) (1) CHU Farhat Hached Sousse (2) CHU Farhat Hached Sousse The purpose of this study was to determine if any relationship exists between Her-2/neu gene amplification and estrogen receptor (ER), progesterone receptor (PR), grade and age in female breast cancer. Twenty female patients with invasive breast carcinoma, in which assessment of Her-2/ neu amplification by Fluorescence In-Situ Hybridization (FISH) has been performed, were included in this study. Each patient was further assessed for ER, PR, grade and age at diagnosis. Her-2/neu gene was amplified in 2 out 11 ER positive cases and in 8 out of 9 ER negative cases. Her-2/neu was amplified in 1 out of 10 PR positive cases and in 9 out of 10 PR negative cases. So there is an inverse relationship between Her-2/neu amplification and ER or PR expression. Amplification was seen in 5 out of 7 grade III tumors, 4 out of 10 grade II tumors and in any case of grade I tumors. Her-2/neu was amplified in 10 out of 15 woman 45 years and older, and in any women 44 years and younger. Comparison of these frequencies revealed statistically significant correlation between Her-2/neu amplification and hormone receptor negative tumors,

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high histological grade and high age of the patients (older than 45 years). 7a.51-P Chromosomal imbalances in laryngeal carcinomas and its invasive front E. Ambrosio (1) M. Coelho (0) V. Sacomano (0) M. Domingues (0) R. Coudry (0) J. Tagliarini (0) L. Kowalski (0) S. Rogatto (0) (1) UNESP/ AC Camargo Hospital (2) UNESP/ AC Camargo Hospital (3) UNESP/ AC Camargo Hospital (4) Faculty of Medicine, UNESP, Botucatu (5) AC Camargo Hospital (6) Faculty of Medicine, UNESP, Botucatu (7) AC Camargo Hospital (8) UNESP/ AC Camargo Hospital Laryngeal squamous cell carcinoma (LSCCs) is the second-most common malignancy of the head and neck. When detected early, 75% of the patients present a 5-year survival rate; however, most of them develop metastatic disease at the time of diagnosis, which reduces survival rate to 35%. In LSCCs, the tumor aggressiveness is unpredictable and prognosis depends of several clinical and pathological factors. It has been reported that invasion is a crucial parameter related to prognosis and that distinct genes are differentially expressed by cancer cells at the borders of tumors and invasive front. In the present study, it was used a genome-wide screening by high-resolution comparative genomic hybridization to identify chromosomal gains and losses in 26 formalin fixed paraffin embedded samples of LSCC. It was used a laser microdissection in order to obtain cells from border of tumors and invasive front. Although common chromosomal imbalances were detected in both areas, gains were more frequently detected in the border of tumors and losses in the invasive front. The distinct chromosomal imbalances found in this study suggest that there are particular genes involved in different tumoral areas, possibly related to invasiveness and outcome. In order to validate the losses at 3q26 and 18q23, it was selected microsatellite markers mapped on these regions to evaluate loss of heterozygosity in a

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subset of independent cases. Three markers mapped at 3q26 confirmed losses in 25% of LSCC. Two markers mapped at 18q23 confirmed losses in 15% of the cases. The present study demonstrated that specific genomic alterations are frequently observed in tumoral and invasive front cells. The chromosomal regions involved in gains or losses provide a basis for further functional validation and may lead to the identification of novel candidate genes associated with prognosis.

SKY as a translocations, two of them with the chromosome X. In regard to numerical abnormalities, trisomy 9 was the most frequent cytogenetic abnormality, followed by +3,+7,+11,+15,+19 and +21. Our results support that there is a cytogenetic heterogeneity in the hyperdiploid MM that must be related to the different clinical behaviour. Specific cytogenetic and molecular prognostic factors are still to be unmasked.

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Cytogenetic heterogeneity in hyperdiploid multiple myeloma

Array-CGH used to monitor dysplastic/neoplastic disorders with unbalanced chromosome anomalies may detect as low as 8% abnormal cells

R. Alfaro (1) Á. Pérez-Granero (1) J. Rosell (1) M. Durán (2) G. Puget (2) P. Galán (3) J. Besalduch (2) M. Bernués (1) (1) Hospital Universitari Son Dureta (2) Hospital Universitari Son Dureta (3) Hospital General Mateu Orfila Multiple myeloma (MM) is an incurable malignant plasma cell neoplasia characterized by the accumulation of malignant plasma cells in the bone marrow. Hyperdiploid MM constitutes 50–60% of MM and little of its biology is known. Overall survival is favourable but some patients clearly have a more aggressive behaviour. We present a cytogenetic characterization (conventional and spectral karyotype) of a series of 14 hyperdiploid MM. We observed that cytogenetics was heterogeneous. Some patients presented a high amount of numerical alterations, particularly trisomies, and a very few structural rearrangements. Other patients, conversely, showed a high number of structural abnormalities and only a few numerical alterations. A third group of patients presented a similar amount of both numerical and structural chromosome abnormalities. While numerical alterations were recurrent, structural abnormalities were not. However, some chromosome regions appeared mostly involved. Chromosome 8q rearrangements were the most frequent abnormality followed by alterations of chromosomes 1p, 6q and 16. Three different deletions of chromosome 6q were redefined by

E. Maserati (1) R. Valli (1) C. Marletta (1) B. Pressato (1) F. Lo Curto (1) F. Pasquali (1) (1) Università dell’insubria Comparative genomic hybridization on microarray (a-CGH) provided a powerful tool for the study of unbalanced chromosome anomalies, both constitutional and acquired. A problem seldom faced is the definition of the sensitivity of a-CGH to detect chromosome unbalances when the DNA is extracted from a pool of cells with normal and abnormal karyotype: this concerns constitutional mosaics, but even more relevant is in dysplastic/ neoplastic disorders and in their monitoring, as may be the case of myelodysplastic syndromes and leukaemias, or of predisposing diseases, as Shwachman syndrome. In an experimental approach to define a-CGH sensitivity, we used DNA from three cases of unbalanced constitutional anomaly, defined with chromosome analyses and FISH, and more precisely by whole-genome a-CGH: a 14.1 Mb deletion of the long arm of chromosome 7; a 14.3 Mb deletion of the long arm of chromosome 4, inwhich a-CGH showed the presence also of a 5.5 Mb duplication of the short arm of chromosome 9; a pericentric inversion of chromosome 21, in which aCGH showed an unbalanced pattern, with a 46.6 Kb duplication of part of RUNX1 gene, a 37.5 Kb duplication of a segment including WRB gene, a 1.4 Mb deletion, always in the long arm, and a 161.6 Kb duplication to be considered a copy

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number variation. In total, seven different unbalances of different size. We mixed the DNA under study with DNA from cells with normal karyotype in the following percentages: 15%, 10%, 8%, 7%, 6%, and 5%. We tested the DNA pools obtained by a-CGH, with standard and customized platforms (standard and higher density of oligomers for the regions to be analyzed), and with the different tools offered by the analytical software. The results demonstrated the possibility to detect as low as 8% abnormal cells in the cases with the three largest unbalances; in two other more subtle unbalances the threshold was 10%, but, varying the stringency of the algorithm applied, it was 8%, and even 6% in a further case. 7a.54-P High-dose Chemotherapy with Peripheral Blood Stem Cell Transplantation for Patients with Multiple Myeloma: Prognostic Impact of Chromosomal Aberrations A. Jauch (1) K. Neben (2) D. Hose (2) F. Cremer (7) C. Heiss (4) T. Hielscher (4) U. Klein (2) U. Bertsch (2) A. Ho (2) H. Goldschmidt (2) (1) Institute of Human Genetics (2) University Hospital Heidelberg (3) Zentrum fuer Humangenetik (4) German Cancer Research Center Multiple Myeloma (MM) is a malignant lymphoproliferative B-cell disease characterized by the accumulation of monoclonal plasma cells in the bone marrow. Genomic aberrations have been shown to significantly influence response to chemotherapy and survival in MM. The aim of this study was to evaluate the clinical relevance of specific chromosomal aberrations in 315 MM patients treated with high-dose chemotherapy (HDCT) and peripheral stem cell transplantation (PBSCT). All patients underwent frontline HDCT and PBSCT according or in analogy to the GMMG-HD3- or GMMG-HD4trials. Interphase FISH analysis on CD138 enriched plasma cells detected gains of chromosomes 1q21 (36%), 9q34 (62%), 11q23 (48%), 15q22 (55%), and 19q13 (55%), as well as deletions of chromosomes 6q21 (11%), 8p21 (19%), 13q14 (46%), 17p13 (10%), and 22q11 (15%). Furthermore, the IgH

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translocations t(11;14), t(4;14), and t(14;16) were observed in a frequency of 19%, 13%, and 2%, respectively. For the entire group, the median overall survival (OS) and progression-free survival (PFS) after HDCT was 6.4 and 2.2 years, respectively. First, we analyzed the prognostic impact of each individual chromosomal aberration on PFS and OS. In the univariate analysis deletions of chromosomes 8p21, 13q14 and 17p13, translocation t(4;14) as well as gains of chromosomes 1q21, 11q23 and 19q13 preserved significant impact on PFS, while deletion 17p13, translocation t(4;14) and gain of 1q21 were of statistical significance for OS. In the multivariate analysis only deletion 17p13 showed a significant result on OS and deletion 13q14 and/or gain of 1q21 were significant for PFS. In conclusion, our results show that the heterogeneity seen in the clinical course of MM patients after HDCT can be correlated with distinct chromosomal aberrations. These results may have implications for the risk-adopted management of patients with MM. 7a.55-P Identification of t(17;22)(q22;q13) COL1A1/ PDGFB in dermatofibrosarcoma protuberans. A comparative study using FISH and multiplex RT-PCR techniques B. Espinet (1) R. Salgado (1) B. Llombart (2) A. Toll (3) A. Fernández (2) O. Sanmartín (2) S. Segura (3) C. Serra (2) C. Barranco (1) C. Guillén (2) E. de Álava (4) F. Solé (4) R.M. Pujol (4) J.A. LópezGuerrero (4) (1) IMIM-Hospital del Mar (2) Instituto Valenciano de Oncología (3) IMIM-Hospital del Mar (4)? Introduction: Dermatofibrosarcoma protuberans (DFSP) is a rare cutaneous tumor with infiltrative growth and a marked tendency to recur locally after surgical excision. However, distant metastases may develop in up to 5% of cases. Cytogenetically, DFSP is characterized by either supernumerary ring chromosomes containing 17q22 and 22q13 sequences or translocation t(17;22). These chromosomal rearrangements lead to the formation of a COL1A1/PDGFB chimeric gene and a constitutive activation of the PDGFR. This phenomenon provides a rationale for targeted inhibition of PDGFR as a treatment strategy

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for patients with unresectable locally advanced or metastatic DFSP. Our aim was to compare the efficiency of FISH and RT-PCR techniques to detect COL1A1/PDGFB. Patients and Methods: Sixty-three patients with DFSP (37M/26F, mean age 45 yrs) were analyzed in formalin-fixed, paraffin-embedded tissues. FISH was performed using a dual colour dual fusion COL1A1/ PDGFB non-commercial probe (Children’s Oakland Research Institute library BAC clones) and multiplex RT-PCR employing specific primers that flank the whole exons of COL1A1 and a primer at the exon 2 of PDGFB. Sequencing analysis was used to identify the specific breakpoints. Results: FISH technique was successful in 59/63 (94%) cases, from which 51 (86%) were positive for the translocation (simple translocation or multiple copies of the translocation plus amplifications of COL1A1 and/or PDGFB). Regarding RT-PCR, it was succesful in 49/63 (78%) cases, 39/49 (80%) being positive for COL1A1/PDGFB. Only 2 cases were negative using both techniques. Conclusions: FISH appears to be the most effective technique to detect COL1A1/PDGFB rearrangement in DFSP, as FISH probes cover all the possible breakpoints, and overcome the problem of extracting RNA from paraffin embedded tissues. The identification of such rearrangement in DFSP could be of interest as a targeted therapy using imatinib mesilate has been used. Aknowledgments: “2a Beca José Ma Buesa de GEIS” and Novartis. 7a.56-P The PMS2 Locus Is a Site of Chromosomal Rearrangements that Predispose Humans to Colon Cancer E. Valtorta (1) G. Marra (1) (1) University of Zurich (2) University of Zurich Background: Genetic alterations in the mismatch repair gene PMS2 are associated with hereditary nonpolyposis colon cancer. The gene is located in chromosome 7 as well as 15 PMS2 pseudogenes. In particular, the PMS2gene and one of its pseudogenes, (PMS2CL) are located in two duplicons with more than 99% similarity. Duplicons serve as

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substrates for nonallelic homologous recombination that may result in deletion, inversion, or more complex rearrangements. We hypothesized therefore that the duplicons that include PMS2 and PMS2CL, being in the same orientation, might predispose to rearrangements leading to deletions, whereas rearrangements involving duplicons with opposite orientation (also present in this chromosomal region) might lead to inversions. Using FISH analysis, we intended to identify genomic alterations of the PMS2 locus in the germline of patients operated on for PMS2-deficient colon cancer. Methods: Metaphase and interphase FISH analysis of chromosome preparations from peripheral blood samples of patients was performed. We searched first for deletions using a single probe and then for inversions using different combinations of three probes. Results: A 125 kb deletion involving the PMS2 gene was found in 1 out of 13 patients (7,7%), whereas a 1.2 Mb inversion (0,5 Mb telomeric, and 0,7 MB centromeric to the PMS2 gene, respectively) was found in 1 out of 3 patients investigated so far for this latter rearrangement (analysis in progress for the remaining 10 cases and a series of normal controls). Conclusion: Cancer predisposing, germline mutations in mismatch repair genes are generally identified by sequencing analysis. As for PMS2, this approach is often impeded by the noise from pseudogene sequences. We show here evidence that the PMS2 locus is located in a genomic region prone to chromosomal rearrangements. Thus, we propose FISH analysis as an additional tool for the identification of germline alterations in this gene. 7a.57-P CKS1B expression and genetic analysis in cutaneous squamous cell carcinoma R. Salgado (1) A. Toll (2) F. Alameda (1) G. MartínEzquerra (2) A. Martorell (3) T. Baró (1) M. Salido (1) S. Almenar (4) C. Barranco (1) O. Sanmartín (3) R. Pujol (2) F. Solé (1) B. Espinet (1) (1) IMIM-Hospital del Mar (2) IMIM-Hospital del Mar (3) Fundación Instituto Valenciano de Oncología (4) Fundación Instituto Valenciano de Oncología

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Introduction: Cutaneous squamous cell carcinoma (CSCC) is the second most common form of skin cancer frequently developing on sun-exposed areas with occasional local invasion and metastasic potential. Around 60% of CSCCs arise from a preexisting actinic keratosis (AK) considered as a precursor lesion. From a previous arrayCGH analysis in CSCC in which 1q21.2 amplification was detected, we selected CKS1B gene which plays a critical role in cell cycle progression. Our aim was to analyze the genetic and protein status of CKS1B using fluorescence in situ hybridization (FISH) and immunohistochemical (IHC) techniques. Material and methods: A total of 71 patients were included in the study, 43 cases diagnosed of CSCC and 28 of AK. FISH was performed in paraffin sections using a BAC clone (RP11139I14) mapping the CKS1B locus on chromosome 1q21.2 from (http://bacpac.chori.org). A centromeric probe for chromosome 1 was used as control to distinguish between polysomy or amplification. For the immunohistochemical staining, the CKS1B antibody was used (Zymed, dilution 1:50). Results: We observed that 30/43 (69.7%) CSCC and 13/23 (56.3%) AK patients presented polysomies of chromosome 1. Four patients with CSCC (9.3%) presented an amplification of CKS1B gene. None of AK cases showed CKS1B amplification. Interestingly, all of them had a worse clinical course (presence of perineural invasion and metastasis). Correlation between CKS1B protein overexpression and both polysomies and amplifications determined by FISH was statistically significant (P=0.001) in CSCC patients. Conclusions: The polisomy of chromosome 1 is a frequent event in both CSCC and AK patients. The presence of amplifications of CKS1B gene is related to an aggressive behaviour suggesting that CKS1B gene amplifications could be considered as a marker of progression and aggressivity. Acknowledgements Grants PI04/1728 from Fondo de Investigación Sanitaria (FIS) and AECC (Catalunya contra el Càncer) 2007.

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7a.58-P The surveillance value of a multi-targeted in situ hybridization test (UROVYSIONTM) in patients with non-muscle-invasive urothelial cell carcinoma history. Comparison with cystoscopy A. Galván (1) J. Placer (2) M. Salido (1) B. Espinet (1) L. Pijuan (3) J. Lloreta (3) F. Solé (1) A. GelabertMas (4) (1) IMIM-Hospital del Mar (2) Hospital Universitari Vall d’Hebron (3) IMIM-Hospital del Mar (4) IMIMHospital del Mar. UAB Introduction: Non-muscle-invasive bladder cancer (BC) presents a high rate of recurrence, conditioning a long-term follow-up of these patients using an invasive technique (cystoscopy). The aim of the present study was to evaluate the performance of UroVysion™ test (UV) in the setting of patients under follow-up after a BC. Methods: A total of 230 patients in follow-up after previous history of BC were prospectively studied. We compared UV in spontaneus voided urine and flexible cystoscopy methods. Urines were collected before cystoscopy and processed using ThinPrep technology. The samples were hybridizated with UV probe according to manufacturer’s instructions. We defined recurrence if there was histological confirmation or when a second cystoscopy was positive (damaged sample). The mean follow-up was 9.5 months. Results: No differences were detected between the statistics of both techniques: Cystoscopy (n=230) UV (n=206) Sensibility

81.3%

85.7%

Specificity

95.4%

94.9%

Negative Predictive Value 76.5% (NPV) Positive Predictive Value 96.5% (PPV)

75.0% 97.4%

There were 34 recurrences: 10 low-grade and 11 high-grade and 13 not histologically confirmed. There were eight false-positive cystoscopy results and eight false-positive UV result. The median follow-up was 13 months. Longer followup will be confirmed if these false-positive UV

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results are truly false or represent anticipatory results. In nine patients, recurrences were not detected by cystoscopy (n =3), UV (n= 4) or both (n= 2). There were not differences between pathologic characteristics of the undetected tumors. The sensitivity of both was not influenced by tumor size. Conclusions: The high sensitivity and NPV of UV suggest that it could be used to delay the need of cystoscopies during the follow-up of low-risk BC patients. The high specificity and PPV of UV makes it an ideal complement to cystoscopy in the follow-up of high-risk BC patients. Acknowledgements: FIU grant from the Spanish Urological Association. 7a.59-P DDIT3 is Amplified in Atypical Lipomatous Tumours (ALT) and Dedifferentiated Liposarcomas B. Roland (1) S. Shetty (2) (1) Calgary Laboratory Services (2) ARUP Laboratories DNA sequences from chromosome arm 12q are amplified in the ring and marker chromosomes of atypical lipomatous tumours (ALTs). Studies using FISH or PCR to detect amplification of genes including CDK4 and MDM2 have been able to distinguish between lipomas and ALTs. However, some previous studies have detected no amplification of DDIT3 (CHOP) in ALTs. Because DDIT3 is near CDK4 on 12q, we investigated whether FISH for DDIT3 can also distinguish between lipomas and ALTs. Method: FISH was performed using standard procedures with a commercial probe for DDIT3 (Abbott). A labeled human BAC clone for DDIT3 (RP11258J5) was also used on some samples. The majority of samples were cultured cells, but touch preparations and paraffin-embedded tissue were also processed. A total of 47 adipocytic tumours were examined. Results: Amplification of the distal, or distal and proximal, sequences of the commercial probe was observed in 7/9 ALTs and in 2/2 dedifferentiated liposarcomas. Amplification was also observed using the BAC probe. One of 11 myxoid/round

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cell liposarcomas showed amplification. On the other hand, there was no amplification in 22 lipomas and in 3 other benign adipocytic tumours. Conclusion: DDIT3 is amplified in the majority of cases of ALT and dedifferentiated liposarcomas and is not amplified in lipomas. Therefore FISH using a DDIT3 probe may be useful in distinguishing between lipomas and atypical lipomatous tumours. 7a.60-P Alveolar Soft Part Sarcoma with a balanced X;17 translocation M. Doody (1) N. Forde (2) (1) Mater Pathology (2) Mater Pathology Alveolar Soft Part Sarcoma (ASPS) is a rare malignant tumour found in adolescents and young adults. It usually appears as a slow growing painless mass in the deep tissues of the thighs, buttocks, and the abdominal wall. The prognosis is poor, as in most instances metastases occur early in the course of the disease. Histologically ASPS is a highly distinctive tumour composed of nests or compartments of variably eosinophilic polygonal cells that often form central alveolar spaces. Electron microscopy shows characteristic membrane bound or free rhomboid crystals. A specific cytogenetic aberration der(17)t(X;17) (p11;q25), an unbalanced X;17 translocation which results in the fusion of the TFE3 transcription factor gene from Xp11 with APSL at 17q25, is a highly specific and sensitive diagnostic marker for ASPS. The same gene fusion ASPL/TFE3 is also found in a small subset of renal adenocarcinomas arising in pediatric and young adult patients, but cytogenetically abnormality is seen as a balanced t(X;17)(p11;q25). The detection of the balanced or the unbalanced form of the translocation is used as one of the criteria to differentiate ASPS from renal tumours. We present a case study of a patient with ASPS with the balanced form of the (X;17) translocation, and explore the possible relationship between Alveolar Soft Part Sarcoma and renal adenocarcinoma.

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7a.61-P

7a.62-P

Acute myeloid leukaemia following myelodysplastic syndrome

Multiple genomic anomalies detected by aCGH in acute lymphoblastic leukemia with normal karyotype

A. Divane (1) G. Paterakis (2) F. Chantzi (3) V. Kitra (3) K. Karamolegou (3) A. Sandu-Tsoutsou (3) N. Georgakopoulos (1) M. Moschovi (3) (1) Locus Medicus (2) G Gennimatas” General Hospital (3) University of Athens, “Aghia Sophia” Children’s Hospital A 3.5 years-old girl was admitted due to fever and pancytopenia. Palpable lymph nodes were found in the right axilla. She presented infectious mononucleosis like syndrome two months ago. At that time, because of concomitant anaemia and thrombocytopenia, bone marrow (BM) aspiration had been performed with immunophenotype analysis and cytogenetic studies suggesting myelodysplastic syndrome (RAEB-t). Laboratory work-up: Hb 7.9 gr/dl, Ht 24.8%, WBCs 54000/mm3, Neut 27%, Leuk 44%, PLTs 54000/mm3. The BM had 20% blasts. The cerebrospinal fluid, chest X-ray and U/S of the abdomen were normal. Serological tests for HSV 1–2, EBV, CMV IgG were positive, but IgM were negative. Immunophenotypic analysis of blasts revealed MDS with transformation to acute myeloid leukaemia. Conventional and molecular cytogenetics analysis of BM were performed. FISH analysis using LSI MLL t(11q23), LSI PML/RARA t(15;17)(q22;q21.1), LSI EGR1(5q31)/LSI D5S23, D5S721(5p15.2), LSI D7S522 (7p11.1–q11.1) and CEP8, 8p11.1–q11.1 revealed rearrangement only for the MLL gene in 96% of the nuclei that were analyzed and conventional cytogenetic analysis revealed a clone of t(6;11) (q27;q23). She received chemotherapy according to AMLBFM-2004 protocol. On day 15, in BM, blasts were <10% and FISH studies showed 42.5% MLL positive. MRD was not detected by flow-cytometry. On day 58, BM was morphologically in remission, but FISH analysis detected 2.1% MLL positive. On day 114, the BM was hypocellular, but molecular cytogenetics showed 25.3% MLL positive. Because of the recurrence, BM transplantation was decided.

v. Dev (1) J. Gore (0) G. Gu (0) (1) Genetics Associates, Inc. Conventional karyotyping, the traditional method to establishing chromosomal abnormalities in acute lymphoblastic leukemia (ALL), is considered to be the gold standard in cytogenetics, but the technique possesses several characteristics that hinder its usefulness in specialized circumstances. This study describe the application of array comparative genomic hybridization (aCGH) in identification of chromosomal abnormalities in two ALL patients whose karyotype by conventional analysis showed normal 46,XY. Fluorescence in situ hybridization (FISH) studies for a t(9;22) translocation were negative. However, the FISH study did show ABL deletion. Besides ABL deletion, aCGH showed multiple anomalies, including partial loss of 1q, 2q, 9p, 9q, 11q and 17p, partial gain of 1p, monosomy 7 and trisomy 21 in patient 1. For patient 2, aCGH additionally revealed the interstitial deletion of 1p and 11q. All these abnormalities were corroborated by subsequent FISH analysis. None of these chromosomal anomalies—most of which were large enough to be identified by conventional karyotypic analysis—were identified by karyotypic analysis of metaphases, suggested that the abnormal cells were in interphase. The absence of abnormal cells undergoing metaphase within the submitted specimen led to designation of a normal karyotype, with the chromosomal abnormalities detected only via FISH and aCGH. This may have implications for leukemic cases with cells that fail to reach metaphase in culture for detection by conventional karyotypic analysis. As the prognosis of leukemia is reflected by the presence of clonal evolution and by the presence of unfavorable prognostic markers, supplemental use of aCGH or FISH will help detect these cases. Additionally, these two cases also contained an ABL deletion that was not associated with a concomitant BCR-ABL translocation. The number of cases in the literature describing this change is limited, and its significance is unknown;

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further studies to clarify its potential effect are needed. 7a.63-P Malignant brain tumors harboring a distinct genetic heterogeneity S. Urbschat (1) S. Wemmert (2) A. Rutz (3) R. Ketter (2) K. Zang (3) W. Steudel (2) (1) Saarland University (2) Saarland University (3) Saarland University Gliomas display a wide range of histopathological features and biological behavior, and an inherent tendency to progress to a highly malignant phenotype. Molecular- and cytogenetic studies revealed that different grades of gliomas correlate with specific genetic alterations. Glioblastomas, the most malignant form of gliomas, may develop de novo (primary glioblastomas) or through progression from low-grade or anaplastic astrocytomas (secondary glioblastomas). To analyze the genetic heterogeneity, we performed different cytogenetic methods considering their methodic limitations. Cell culture analysis may be biased by clonal selection artifacts. Homogenized tissue lacks control over the tissue composition and permits contamination of the tumor specimen with preexisting and reactive non-neoplastic tissue. Moreover, gliomas exhibit a diffuse infiltrating growth pattern into normal brain so that no tumor area contains a uniform cellular composition. In order to evaluate an intratumoral genetic heterogeneity we performed FISH investigations and microdissection analysis in paraffin-embedded glioma tissue and correlated the cytogenetic data with the histomorphology of the given tumor areas. Additionally, we combined FISH with immune histochemistry of the mitotic spindle apparatus for non-disjunction of chromosomes involved in glioma development Low-grade astrocytomas most often showed normal karyotypes, by conventional cytogenetic methods. However, we were able to identify numerous alterations in low-grade astrocytomas, especially in areas with a gemistocytic appearance. Primary glioblastomas and secondary glioblastomas showed consistant as well as different genetic findings, which correlate partial with the histomorphological features of the investigated areas. Our results provide clear

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evidence of inter- and intratumoral genetic heterogeneity in gliomas independent of the applied method. In order to develop genetic prognostic criteria, the distinct genetic heterogeneity of these tumors have to be considered. 7a.64-P Cytogenetic and fluorescent in situ hybridization (FISH) studies of nodular ground-glass opacities in the lung D. Bettio (1) A. Venci (1) U. Cariboni (2) V. Errico (2) M. Di Rocco (3) M. Infante (2) (1) Humanitas Clinical Institute (2) Humanitas Clinical Institute (3) Humanitas Clinical Institute Computed tomographic screening for lung cancer has increased the detection of nodules manifesting as ground-glass opacity (GGO). The factors influencing their growth, progression and malignant potential are not well known and standard treatment strategies have not been established. Histologically GGOs can be classified in atypical adenomatous hyperplasia (AAH), bronchioalveolar carcinoma (BAC) or bronchioalveolar-type adenocarcinomas (AC). The purpose of our study was to investigate GGOs by means of classical cytogenetics (direct method and short-term cultures) and FISH (kit LAVysion, Abbott) in order to find specific aberrations that possibly allow to distinguish between lesions with malignant potential from those that will regress or remain indolent. We analysed 8 fresh GGO samples from 7 patients and 4 distal biopsies from normal tissue. All the GGO samples showed a normal karyotype both after direct method and short-term cultures. In the 4 distal biopsies no metaphases were obtained after direct method and a normal karyotype was observed after cultures. FISH was negative in all the normal tissues and in 5 out of 8 GGO cases: 2 were classified as AAH, 2 as BAC and 1 as AC. Three patients were FISH positive; two of them showed gains of all targets in around 90% of the cells, one showing a cluster of C-MYC amplification besides polisomy. The two patients were diagnosed with AC mixed type and died within one year. The third FISH positive patient, diagnosed with BAC, showed gain of the targets in 20% of the cells only. So far she is disease free (21 months follow-up) but

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had a relapse after 9 months. Although further cases need to be investigated, our study supports that “karyotyping and fishing” could be a good approach in understanding the potential invasiveness of GGOs with the same histological classification. Moreover it must be clarified why we did not observed in the GGOs’ karyotypes at least polisomy 6 detected by FISH using the CEP.

diseases, if they exist, must be located in other genes.

7a.65-P

C. Mellink (1) S. Snijder (2) N. Cerveira (2) H. van der Lelie (3) (1) Academic Medical Center Amsterdam (2) Portuguese Oncology Institute Porto (3) Academic Medical Center Amsterdam Clemens Mellink1, Simone Snijder1, Nuno Cerveira2, Manuel Teixeira2, Hans van der Lelie3 1 Department of Clinical Genetics, 3 Department of Clinical Haematology, Academic Medical Center, Amsterdam, The Netherlands 2 Department of Genetics, Portuguese Oncology Institute, Porto, Portugal

FISH screening of Receptor- and CytoplasmicTyrosine Kinase genes in BCR-ABL1 negative and JAK2V617F negative chronic myeloproliferative neoplasms (CMPNs) J. Vizmanos (1) P. Aranaz (1) C. Ormazábal (1) C. Hurtado (1) I. Erquiaga (1) M. Calasanz (1) M. García-Delgado (1) F. Novo (1) (1) University of Navarra BCR-ABL1 negative chronic myeloproliferative neoplasms (CMPNs) are a heterogeneous group of clonal haematological malignancies for which the molecular pathogenesis is not well understood. Over the last years, some genetic alterations have been described to cause these diseases, most of them activating mutations in tyrosine kinase (TK) genes. Tyrosine kinases have an important role in cell growth and oncogenesis, since gain-of-function mutations can lead to constitutive activation of the signalling pathways in which they are involved. In this study, we have searched for genetic rearrangements in all genes from the families III (PDGFRA, PDGFRB, CSF1R, KIT and FLT3) and IV (FGFR1, FGFR2, FGFR3 and FGFR4) of receptor-TKs, as well as all genes from the families Jak (JAK1, JAK2, JAK3 and TYK2), Abl (ABL1 and ABL2) and Syk (SYK and ZAP70) of cytoplasmic-TKs. All of them code for proteins with tyrosine kinase activity and some of them have been found mutated in CMPNs and in other tumor types. FISH analyses were performed on samples from 44 BCR-ABL1 negative and V617FJAK2 negative CMPN patients. Only one PDGFRA hybridization pattern was found compatible with a FIP1LIPDGFRA rearrangement not detected previously by conventional RT-PCR. For the other genes no genomic rearrangements were found in these samples, implying that genetic aberrations causing these

7a.66-P Cytogenetic and molecular characterization of T(2;11)(q37;q23) in T-MDS after treatment for acute promyelocytic leukaemia

Treatment of acute promyelocytic leukaemia (APL) with anthracycline-based chemotherapy and all-trans retinoic acid (ATRA) leads to very high rates of complete remission and survival in these patients. There are only a limited number of cases reported in which new haematological malignancies developed during follow up of APL and in only one of these a karyotype including an 11q23 rearrangement was found. Here we report a case of a 56-year old female with acute promyelocytic leukaemia with t(15;17)(q22;q21) at initial diagnosis. The patient received induction therapy with idarubicin and ATRA and consolidation with mitoxantrone. The presence of PML-RARα rearrangement was monitored by FISH and RT-PCR. Consecutive samples showed a swift and complete reduction of PML-RARα rearranged cells. However, 20 months after diagnosis conventional cytogenetics revealed a complex karyotype with loss of chromosomes 7q and Xq and a structural rearrangement involving 11q23. FISH analysis with the MLL probe identified chromosome 2q as the translocation partner of 11q. Molecular characterization of the fusion transcript showed a fusion of MLL exon 8 with SEPT2 exon 3 which was confirmed by sequencing the 312 bp chimeric pcr-fragment. Since rearrangements of 11q23 are commonly associated with anthracycline-based chemotherapy,

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and since two other cases with t-AML with t(2;11) (q37;q23) were published recently, we consider t(2;11)(q37;q23) as the primary leukemogenic event emerging during therapy in our patient. Therefore, we recommend complete karyotyping to be performed on a regular basis in all treated APL patients for early detection of chromosomal aberrations indicating development of new (therapy-related) haematological malignancies (MDS/AML). 7a.67-P Detection of genetic alterations in pap smear samples infected with Human Papilloma Virus (HPV) by FISH B. Durak (1) G. Giran (1) G. Bademci (1) H. Ozon (2) K. Peker (3) M. Ozdemir (1) S. Artan (1) (1) Eskisehir Osmangazi University (2) genetiks Laboratory (3) Istanbul University Medical Faculty Worldwide, cervical cancer is the third most common type of cancer in women. Human Papilloma Virus (HPV) is known to play a pivotal role in cervical carcinogenesis. There are actually more than 100 different strains of HPV. Of these, more than 40 exist in the genital area and they are broken down to “high”, “probable high” and “low” risk strains. HPV16 and HPV18 are high risk strains and are believed to cause about 70% of all cervical cancer. Viral DNA often integrates into the chromosomal regions where oncogenes located,suggesting a role of oncogenic alterations in cervical cancer development. In this study, copy number variations of 3q26(TERC), 8q24.1 (C-MYC) genes and chromosome 7 were determined in 50 HPV-infected PAP smear samples by the FISH analysis. Copy number alterations were seen in 18/50 patients. The C-MYC amplification were detected in 10, trisomy 7 inthree, both C-MYC and TERC amplifications in four cases whereas both C-MYC amplification and trisomy 7 were senin a case. Of the cases, 14 were infected with high-risk, two with probable high-risk and two with low risk HPV strains. Preliminary results of the study indicated that various HPV strains causes different genetic alterations. Copy number changes were commonly seen in HPV 16 and 18 strains infections. The relations of histopathological features, type of

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genetic alterations and HPV types should be determined. 7a.68-P FISH-Detected Genetic Markers in Bronchial Lavage Samples from Lung Cancer Patients M. Ozdemir (1) K. Kurtcu (1) F. Alatas (2) B. Burak (1) O. Cilingir (1) M. Muslumanoglu (1) S. Artan (1) (1) eskisehir osmangazi university (2) eskisehir osmangazi university medical faculty Despite of improving treatment protocols, lung cancer (LC) is still the most lethal type. Differences in prognosis, chemotherapy response and survival time can not be explained yet and the tests used are not sufficient to answer. The goal in this study was to prospectively determine the sensitivity and specity of FISH detected genetic markers in semi-invasive bronchial lavage samples of the patients undergoing bronchoscopy for suspected LC. The relations of EGFR, C-MYC, and HER2 genes copy number variations with the prognosis, chemotherapy response and survival time differences were also evaluated. FISH analysis were performed in bronchial lavage samples from a hundred cases with LC and in samples from 15 control cases. During the first fiberoptic bronchoscopy, the histopathological diagnosis could be revealed in 81/ 100 whereas FISH diagnosis could be determined in all. The sensitivity and specity of histopathological analysis were 81 % and 100 % whereas they were 65% and 100% for FISH analysis, respectively. The combined histopathological and FISH analyses showed cancer related positive results in 89 cases (89%). Of one hundred cases with lung cancer, 46% had EGFR, 38% had C-MYC and 35% had HER2 gene copy number abnormalities. The relations of these FISH-detected results with the prognosis, chemotherapy response and survive were evaluated. In conclusion, the samples obtained from semiinvasive bronchial lavage technique might be informative in early diagnosis, prognosis determination, even in therapy determination if different genetic markers are used.

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7a.69-P Distinct patterns of chromosomal and genomic instability characterize mouse models of Malignant Mesothelioma S. Paulien (1) D. Frau (3) A. Renier (2) J. Daubriac (2) J. Zucman-Rossi (2) M. Jaurand (2) R. Vanni (3) M. Giovannini (4) (1) INSERMU674-IBFA Université Caen BasseNormandie (2) INSERM U674 (3) University Cagliary (4) House of Ear Malignant Mesothelioma (MM) is a severe disease with poor prognosis which frequency has dramatically increased with the large use of asbestos fibers. It has been associated with loss of Neurofibromatosis type 2 (NF2) tumor suppressor gene and with genetic lesions affecting RB and p53 pathways. Experimental murine models of MM mimicked human cancer are extremely precious and have been recently developped to study initiation and progression of the tumor and for future drug development. A model was developed in wildtype (WT), hemizygous Nf2KO3/+ and Nf2 +/− ; Ink4a+/− mice exposed to abestos fibers.In this study, molecular cytogenetic analysis was used to evaluate the relative level of chromosomal instability (CIN) in eight cell lines obtained from these models and centrosomes analyses were performed to determine whether chromosomal constitution and/or ploidy changes were influenced by mitotic segregation errors.Five cell lines (WT, Nf2KO3/+) had a near-tetraploid chromosome number with centrosome amplification in 15% of cells. Three (Nf2+/− ;Ink4a +/−) were near-triploid with a higher number of double minutes chromosomes associated to a greatest percentage (30%) of cells associated with abnormal centrosomes and mitotic defects (multipolar spindles). As mutations of oncogenic and/or tumor-suppressor proteins can contribute to chromosome instability and abnormal function of centrosomes, we focused on mitotic kinases regulation: Aurora A kinase and p53, frequently associated with aneuploidy and overexpressed in several cancers, Ink4a, cyclin B1 and cyclin D1. This study suggests that chromosomal abnormalities and centrosome amplification found in all these tumors cell lines exposed to asbestos are early events and may be

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generated by different mechanisms. Moreover, our data suggest that loss of Ink4a/arf may contribute to the more aggressive tumors. Thus, cell lines from mesothelioma models can be useful for a better understanding of genetic changes involved in human mesothelioma. 7a.70-P Double translocation in a pediatric acute myeloid leukemia patient F. Sahin (1) O. Ozer (1) B. Malbora (2) Z. Yilmaz (1) N. Ozbek (2) (1) Baskent University Faculty of Medicine (2) Baskent University Faculty of Medicine Chromosome abnormalities of childhood acute leukemia are important parameters for diagnostic, prognostic and follow-up purposes. Secondary acute myeloid leukemia (AML) with chromosome abnormalities due to the drugs used in the treatment of acute lymphoid leukemia (ALL) has been reported in a wide range of latency periods (0.5–15 years). Here we report a pediatric acute myeloid leukemia patient with a double translocation who has firstly been diagnosed as T-cell acute lymphoid leukemia. A 16-year-old male patient was referred to our pediatric hematology department during his reinduction treatment for ALL (St. Jude Total Therapy Studies XIII). He had a pancreatic pseudocyst associated with chronic pancreatitis, hepatomegaly and high amilase and lipase levels. Before the re-onset of chemotherapy, a bone marrow biopsy and aspiration were performed. Pathological and flow cytometric examinations of the bone marrow were consistent with AML M2. Cytogenetic evaluation of the bone marrow displayed a double translocation reported as 46,XY,t(7;12)(q34;q22),t(9;11)(p22;q23). Fluorescence in situ hybridization analyses with probes detecting structural abnormalities t(15;17), t(8;21), inv(16), 5q31, 7q31 and t(9;22) were all normal except for a split signal detected after hybridization with the MLL probe for the 11q23 region. The patient started receiving treatment (BFM 2004 AIE protocol) after which, first evaluation results of peripheral blood samples revealed 46,XY,t(9;11) (p22;q23)[2]/46,XY[14] karyotype suggesting the

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loss of the t(7;12)(q34;q22) clone. The patient is now taking the second round chemotherapy. As we don’t have the patient’s cytogenetic results at the time of diagnosis, we cannot conclude whether the rearrangements are due to the chemotherapy that he received previously. 7a.71-P A novel pedigree with heterozygous germline RUNX1 mutation causing familial MDS related AML - Can these families serve as a multistep model for leukemic transformation in MDS and AML? D. Steinemann (1) T. Ripperger (1) G. Göhring (1) C. Niemeyer (2) B. Strahm (2) B. Schlegelberger (7) (1) Medical School Hannover (2) University of Freiburg Background: Heterozygous germline mutations in RUNX1 are causative genetic alterations in familial platelet disorder with a propensity to myeloid malignancy (FPD/MM). We report here on a father and daughter with familial MDS-related AML (MDRAML) caused by a heterozygous germline mutation in RUNX1 differing in clinical course. Subjects: Following a brief history of symptomatic anaemia, the female index patient presented with MDS-related AML (MDR-AML) at age 13. While her twin brother, her one younger and one older brother, and her mother are clinically healthy, MDR-AML was diagnosed in her 47-year-old father six months earlier. No history of thrombocytopenia or platelet defects was reported in the family. Results: In both patients, a heterozygous nonsense mutation was detected in RUNX1 (NM_001001890.2) by direct sequencing. The mutation c.520C>T (p.Arg174X) causes a premature truncation at the end of the runt homology domain (RHD). Chromosome analysis of bone marrow cells from the index patient showed a deletion of 5q (del(5q)) and a structural aberration of 2q. In addition, array-based comparative genomic hybridization (aCGH) confirmed the del(5q) and pointed to an unbalanced translocation t(2;6)(q36;q23), finally confirmed by fluorescence in situ hybridization using a specific probe for MYB. In contrast, the only aberration

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identified in bone marrow cells of the diseased father was a loss of the Y chromosome (–Y). Conclusion: While heterozygous mutations in RUNX1 are not sufficient for leukaemogenesis, somatically acquired secondary events may promote transformation leading to overt MDS and AML. The recruitment of different secondary alterations may partially explain the variable penetrance and clinical heterogeneity seen in FPD/MM. Consequently, rare FPD/MM-related myeloid malignancies may serve as a model for multistep leukaemogenesis in MDS/ AML and, as illustrated here, aCGH may lead to the identification of candidate genes involved in malignant transformation in familial and sporadic myeloid malignancies. 7a.72-P Role of Bcr/abl genetic amplification in imatinib-resistent acute lymphoblastic leukemia (ALL) J. Gaillard (1) J. Bureau (0) T. Lavabre-Bertrand (0) (1) University hospital Philadelphia chromosome positive (Ph1+) ALL and chronic myeloid leukaemia (CML) share partially the same pathophysiology based on the expression of a chimeric protein, resulting from t(9;22)(q34; q11). They are usually treated by a tyrosine kinase inhibitor like imatinib. The challenge is now to optimize the treatment and fight against three major mechanisms of resistance: acquired mutations in the kinase domain of the BCR-ABL protein, genetic amplification and transcript overexpression of BCR-ABL rearrangement. Between 2003 and 2009, we observed 67 CML and 6 ALL with Philadelphia chromosome becoming resistant to imatinib treatment. Resistance was defined by either an increase in BCR-ABL level of transcripts, or failure to obtain a complete cytogenetic response. We performed, on one hand FISH analysis to detect bcr/abl genetic amplification (defined as the presence of more than two fusion signals in interphasis or mitotic cells), and on the other hand bcr/abl kinase domain sequencing to detect point mutation (mRNA sequences from 156S to 520V, i.e. p-loop upstream to the c-terminal).

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In the CML subgroup, 47/67 (70.1%) displayed no anomaly whereas in ALL subgroup, all cases presented either a point mutation in tyrosine kinase domain (n =4), or a genetic amplification (n= 3). Two ALL patients had genetic amplification at diagnosis, and relapsed earlier (10 months versus 25 months) combined to the deleterious T315I mutation. Among the four others, one of them relapsed with both genetic amplification and point mutation. This study shows the importance of genetic amplification as a resistance mechanism in Ph1+ ALL, and emphasizes the relevance of FISH analysis at diagnosis and during follow-up. Further studies are necessary to evaluate the relevance of genetic amplification detection at diagnosis as a risk of earlier relapse with point mutation.

there are additional genomic rearrangements. Using different array-CGH approaches we aim to more clearly assess the relationship between genetic abnormalities and clinicopathological characteristics in a series of oligodendrogliomas and glioblastomas. Critically, the identification of additional regions of copy number change with arrayCGH may have important implications for rapid diagnosis, assisting prognosis and determining the most appropriate treatments for these patients.

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M. Shago (1) O. Abla (2) J. Hitzler (2) S. Weitzman (2) M. Abdelhaleem (1) (1) The Hospital for Sick Children, University of Toronto (2) The Hospital for Sick Children, University of Toronto

Characterisation of the molecular heterogeneity of oligodendrogliomas and glioblastomas using microarray-CGH F. Bergin (1) C. Smith (2) E. Maher (1) F. Sharkey (1) (1) South East Scotland Cytogenetics Service (2) Western General Hospital Brain tumours are the most common solid tumours in children and the sixth most common tumour group in adults. Neuropathological diagnosis of brain tumour subtypes is essential for clinical management, and this is currently achieved using histological criteria. Recent studies have identified genetic abnormalities in a range of brain tumours, many of which are of clinical prognostic and therapeutic significance. An oligodendroglial component may be seen in a number of glial brain tumours ranging from oligodendroglioma (WHO grade II) to glioblastoma with an oligodendroglial component (WHO grade IV). Conventional cytogenetic diagnosis and comparative genomic hybridisation have shown that these brain malignancies are characterised by complex structural and numerical abnormalities. Co-deletions of chromosomal arms 1p and 19q have become an established genetic signature of cases with oligodendroglioma. However, the limited resolution of these techniques have not permitted whole genome profiling and if

7a.74-P Rearrangements of the CIZ(ZNF384) gene with the E2A and EWSR1 genes in pediatric acute lymphoblastic leukemia with CD10-low/negative immunophenotype

The CIZ (ZNF384) gene, a putative zinc finger transcription factor located distal to the TEL (ETV6) gene on the chromosome 12 short arm, is recurrently rearranged in acute leukemia. To date, twenty-two patients with CIZ rearrangements have been reported. Most of the patients with CIZ rearrangements are children or young adults with B-precursor acute lymphoblastic leukemia (ALL). The CIZ gene has three known partners: TAF15 (17p13; 16 cases), EWSR1 (22q12; 4 cases) and E2A (19p13; 2 cases). We present five additional pediatric ALL patients with CIZ gene rearangements. Three of the patients had E2A-CIZ rearrangements and one had an EWSR1-CIZ rearrangement. The remaining patient had a CIZ gene rearrangement involving a novel region on chromosome 22, suggestive of a fourth CIZ partner gene. As with ETV6(TEL)-RUNX(AML1) rearrangement, the t(12:19)(p13;p13.3) and t(12;22) (p13;q12) mediating the E2A-CIZ and EWSR1CIZ translocations are cryptic by traditional karyotyping analysis. Identification of the rearrangements was facilitated using dual colour breakapart probes for the E2A, CIZ and EWSR1 loci. The patients, four females and one male, ranged in age

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at diagnosis from 2 to 15 years. All of the patients presented with a CD10-negative or CD10-low immunophenotype with concurrent expression of myeloid antigens CD13 and CD33, similar to the antigenic profile seen in MLL gene-rearranged ALLs. Follow up on the patients ranges from 19 to 29 months, and none of the patients have relapsed. Since CIZ gene rearrangement may be associated with a more favorable prognosis than MLL rearrangement, FISH analysis with probes to detect CIZ gene rearrangements is recommended in patients with CD10-low/negative ALL. (Poster presentation) 7a.75-P High rate of genetic abnormalities of neoplastic cells in pseudofollicles of CLL Z. Balogh (1) L. Reiniger (1) H. Rajnai (1) J. Csomor (1) A. Szepesi (1) A. Balogh (2) L. Deak (1) E. Gagyi (1) C. Bodor (1) A. Matolcsy (1) (1) Semmelweis University (2) TÁRKI Social Research Institute/Eötvös Loránd University Several lines of evidences suggest that lymph node microenvironment is critical for survival of chronic lymphocytic leukaemia (CLL) cells. In lymph nodes neoplastic cells (prolymphocytes and paraimmunoblasts) form pseudofollicles (PFs) which are also known as proliferation centers. To reveal whether PFs play a role in generation of genetic alterations in CLL we compared deletion at p53, ATMand RB-1 loci and trisomy 12 by fluorescent in situ hybridization (FISH) technique in PFs versus surrounding small lymphocytes in 12formalin-fixed paraffin-embedded (FFPE)lymph nodes. The FFPE sections were stained with methylene blue and PFs were marked by laser. Subsequent FISH analysis was performed relocalizing the previously defined regions. Loss of 11q was detected in 5 cases, loss of 13q in 2 cases, loss of 17p in 2 cases and trisomy 12 was found in 1 case. In 7 cases PFs contained significantly higher ratio of cells with genetic alterations than the surrounding areas. Our results revealed higher rate of genetic abnormalities in PFs suggesting that these sites play a role in the generation or accumulation of cells with genetic abnormalities.

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The accumulation of genetic lesions in PFs denotes that genetic progression and transformation of CLL may begin in these proliferation centers. 7a.76-P Array based characterization of relevant aberrations in common cancers of DNA-repair deficient Fanconi Anemia cancers: AML and HNSCC H. Tönnies (1) K. Konrat (2) E. Klopocki (3) S. Vahs (1) J. Richter (1) J. Weimer (4) R. Siebert (1) H. Neitzel (2) (1) UK-SH, Christian-Albrechts-Universtity (2) Charité Universitätsmedizin Berlin, Berlin, Germany (3) Charité Universitätsmedizin Berlin, Berlin, Germany (4) UK-SH, Christian-Albrechts University Kiel, Kiel, Germany Fanconi anemia (FA) is a recessive chromosome instability disorder characterized by a variety of congenital anomalies, a high incidence of bone marrow failure, and an increased rate of malignancies as acute myeloid leukemia (AML) and head and neck tumors (HNSCC). In contrast to sporadic AML and HNSCC, which are supposed to have a multifactorial origin, the biological background of cancers in FA patients relies on a monogenic defect in one of the 13 genes of the FA/BRCA complex facilitating the recognition and processing of DNA damage. During bone marrow failure in FA-patients, clonal chromosomal imbalances are detectable in bone marrow (BM). The most common prognostic relevant aberration in FA-BM cells, displaying a high risk for AML and high mortality rates in carriers, is a partial gain of chromosome 3q, now routinely investigated by early single cell I-FISH screening. We currently started to reinvestigate 20 FA-BM probes with and without formerly detected imbalances by high resolution array CGH, which gives us a much higher resolution than conventional CGH. The same array and molecular cytogenetic based strategy, which we used for the BM probes, we have chosen for the detailed characterization of FA-HNSCC. Up to now, molecular cytogenetic and array based data for FA-HNSCC are not available in the literature. We investigated three extremely rare FA-HNSCC cell lines using by SKY-FISH, locus-specific interphase and metaphase FISH, conventional CGH and BAC-, SNP- and high resolution array analysis. Additionally, we

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addressed the question of gene silencing by hypermethylation using bisulfite pyrosequencing of specific target gene CpG islands. On this meeting, we will present our genetic data on these rare cancers, based on a monogenic FA/BRCA pathway defect. Main goal of these studies is, as we did successfully for FA-BM before, to establish a sensitive single cell approach for the early detection of primary lesions in the tumorigenesis of HNSCC in Fanconi anemia patients.

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also identified regulatory regions and/or evolutionary constrained sequence elements located very close (< 300 bp) to APC breaks in five additional complex CML cases. These findings suggest that in up to 90% of CML cases with complex BCRABL1 rearrangements, expression of genes located at or near to the APC breakpoint may be altered due to the chromosome recombination. Additional gene involvement has potential to influence the biology and pathology of the leukaemic disease, with further study required to extend these novel findings.

7a.77-P 7a.78-P Complex BCR-ABL1 gene rearrangements in chronic myeloid leukaemia S. Benjes (1) P. Ganly (2) R. Spearing (2) C. Morris (1) (1) University of Otago at Christchurch (2) Canterbury Health Laboratories Leukaemia often happens when DNA that encodes genes at different chromosomal sites spontaneously recombines in progenitor cells of the haematopoietic system. In one subtype, chronic myeloid leukaemia (CML), the BCR-ABL1fusion protein is responsible for the clinical phenotype, and is expressed after recombination occurs between the BCRgene on chromosome 22 and the ABL1gene on chromosome 9. In about 90% of CML patients, this recombination results in a cytogenetically visible, simple reciprocal exchange involving the long arms of chromosome 9 and 22. In the remaining cases, recombination between BCRand ABL1 can be more complex involving additional chromosomal sites that may be visible cytogenetically or cryptically concealed within a normal appearing chromosome complement. We have isolated BCR gene fragments linked to additional partner chromosomes (APC) from eleven patients with complex BCR-ABL1 rearrangements. Unexpectedly, coding regions are found disrupted at the additional partner chromosome breakpoint sites in five cases. In silico analysis predicts genes disrupted at the APC-BCR junctions to result in truncated proteins in three cases and form APCgene-BCR gene fusions with functional read trough transcripts in two cases. In silico analysis

Cytogenetics findings in a group of patients with Myelodysplastic disease of Chtmad, Portugal R. Pinto Leite (1) M. Souto (2) M. Inácio (2) M. Guerra (2) M. Cunha (2) E. Ribeiro (3) (1) Centro Hospitalar de Trás-os-Montes e Alto Douro (2) Centro Hospitalar de Trás-os-Montes e Alto Douro (3) Centro Hospitalar de Trás-os-Montes e Alto Douro The myelodysplastic syndromes (MDS) comprise a spectrum of stem cell malignancies that give rise to ineffective hematopoieses involving one or more myeloid lineages. Generally, this disease is idiopathic and occur primarily in individuals over 60 years of age, with an annual incidence of 4 / 100 000. The cytogenetic evaluation of bone marrow samples from patients with MDS should be performed whenever possible. A cytogenetic analysis not only confirms the diagnosis but it also offers critical information regarding the prognosis and therapeutic strategy. Approximately 40–50% of patients with de novo, and as many as 90% of patients with secondary/treatment-related MDS have clonal cytogenetic abnormalities. The frequency of cytogenetic abnormalities increases with the severity of disease, as does the risk of leukemic transformation. Chromosomal deletions are common in MDS: del(5q), del(7q), del(20q). Loss of portions of chromosomes 3,11,12,13 and the Y chromosomes have been described with less frequency. Trisomy of chromosome 8 is also frequent and +21 may also occur.

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The authors present the cytogenetic results of 40 MDS samples reported to the Service of Genetics of Centro Hospitalar de Trás-os-Montes e Alto Douro (CHTMAD), between January 2006 and December 2008. The technical procedures have been optimized in order to obtain a maximum of metaphases. GTL bands were applied and, in some cases, the technique FISH (Fluorescent in situ hybridization) were performed. The analysis and classification of chromosomes followed the international standards. In 40% of cases were detected chromosomal abnormalities, the most common the monosomy of chromosome 7 and trisomy of chromosome 8. In one case a complex karyotype were observed. Despite the small number of cases the results obtained are consistent with those described in the literature and were important to the prognosis of the disorder.

frozen (n=7) tumor samples and primary cell cultures (n=45) derived of 37 patients were analyzed. Small, high level amplifications at chromosomes 7p12 (harbouring the gene locus for the EGFR) and 4q12 (with the gene loci for PDGFRa and c-kit) were frequently detected in original tumor samples by conventional CGH, but rarely in the corresponding cell cultures. To investigate the dynamics of small amplicons during in vitro culture at a higher resolution, we performed in selected cases oligonucleotidebased array CGH analyses. Our data demonstrate selective loss of amplicons on chromosomes 7p12, 7p15 and 4q12 frequently containing receptor tyrosine kinase (RTK) genes (EGFR, PDGFR, c-kit) whereas other amplicons (e.g. at chromosomes 12q13–14 and 17q12) remained unchanged in the identical cell cultures. These results imply that small gene amplifications encoding RTK genes are selectively lost under in vitro cell culture conditions.

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Selective Loss of Small RTK Amplicons during Establishment of Human Glioblastoma in Vitro Cell Cultures

Genomic Changes of Glioblastoma Stem Cell Subpopulations are Widely Identical with those of the Original Parental Cell Line

C. Pirker (1) D. Loetsch (1) S. Spiegl-Kreinecker (2) M. Grusch (1) J. Fischer (2) M. Micksche (1) W. Berger (1) (1) Medical University Vienna (2) Wagner Jauregg Hospital, Linz, Austria

D. Loetsch (1) C. Pirker (1) S. Spiegl-Kreinecker (2) H. Koppensteiner (1) M. Grusch (1) J. Fischer (2) M. Micksche (1) W. Berger (1) (1) Department of Medicine I, Medical University Vienna (2) Wagner Jauregg Hospital, Linz, Austria

Glioblastoma multiforme (GBM) is one of the most aggressive tumors, with an average survival time of less than 12 months. GBMs are morphologically and genetically highly heterogenous and display a high degree of chemoresistance. Consequently, the use of reliable biological models/methods to better understand GBM development and progression represents an important issue in brain tumor research. The quality of glioma cell cultures for biological/genomic analyses is currently a matter of debate, as GBM cell cultures nearly always alter the genotype found in the original tumor tissue during in vitro culture. For example amplifications of the EGFR gene locus disappear regularly during establishment of in vitro cell culture. In this study, we focused on the presence of small gene amplifications in GBM tumor tissue and the corresponding primary cell cultures as detected by (array)CGH. Paraffin-embedded (n=35) and snap

Glioblastoma multiforme (GBM) is characterized by bad prognosis and limited treatment response. Stemcell like GBM subpopulations are believed to cause tumor initiation and treatment resistance. Aim of this study was to clarify whether the GBM stem-cell like subpopulations (GSCLC) present in GBM cell cultures are characterized by specific genomic alterations. Fourteen GBM primary cultures and 6 cell lines were analyzed for the presence of GSCLC compartments by investigating their ability to grow in serum-free neurobasal medium. Growth in neurospheres was compared to the expression of the stem cell markers CD133 and/or nestin. Consistently, only two GBM samples expressed considerable amounts of CD133 while all samples were at least weakly positive for nestin. 11/14 primary cultures and 4/6 cell lines formed neurospheres which could be

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maintained in long-term culture. Both CD133-positive but also the majority of CD133-negative cell models potently formed neurospheres and tumors in SCID mice. Genomic changes in selected primary cultures (N=8) and a GBM cell line grown in parallel for 6 to 60 weeks in spheres and monolayer cultures were comparably analyzed by (array)CGH. Typical genomic changes for GBM were found including gains of chromosome 7 and losses of 9p and 10, while EGFR amplicons of the original tumor were rapidly lost during both kinds of in vitro culture. In all cases analyzed, GSCLC grown in neurospheres contained widely the identical chromosomal gains and losses as the respective parental cell cultures. Occasional differences observed concerned random low-level gains/losses of large chromosomal regions or whole chromosomes. In contrast, smaller and distinct amplifications/deletions were in all GSCLC cultures retained from the original parental cell cultures while no indications for presence of EGFR amplicons of the original tumor were found. In conclusion, we demonstrate that most GBM cell cultures/cell lines harbor stem cell-like subpopulations with widely identical genomic alterations as the respective monolayer cell cultures.

marker chromosome derived from a translocation t (8;18), resulting in a partial trisomy 8 and a partial monosomy 18. FISH procedure confirmed this translocation, causing loss of bcl-2 gene and gain of c-myc with three copies. Fanconi anemia diagnosis was confirmed using chromosome instability MitomycinC test. The molecular analysis showed that this patient belonged to the complementation group of FANCC. Case No. 2 A 6 years old child with thrombopenia. Chromosome instability Mitomycin-C test was positive for Fanconi anemia, and confirmed by molecular analysis as FANCA. Bone marrow karyotype showed a clone with a translocation t (1;18)(q12;p11) resulting in a partial trisomy of the long arm of chromosome 1. Discussion unbalanced translocations in bone marrow are frequent in patients with Fanconi anemia. These two patients have clonal abnormalities in which normal chromosome 18 has been replaced by a derivative. In the former case, this derivative results in a partial trisomy of the long arm of chromosome 8 and in the second in trisomy 1q. The significance of chromosome 18 abnormalities in Fanconi anemia are not known.

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Genetic heterogeneity in a chronic lymphatic leukemia patient

Bone marrow cytogenetic abnormalities in Fanconi Anemia M. Ferro (1) M. Talavera (1) D. Rey (1) C. Villalon (1) P. Cabello (1) P. Garcia-Miguel (2) J. Sevilla (3) J. Garcia-Sagredo (1) (1) Medical Genetics University Hospital Ramon y Cajal (2) Medical Genetics University Hospital La Paz (3) Hospital del Niño Jesus, Madrid Fanconi anemia is an autosomal recessive disorder characterized by multiple congenital abnormalities and bone marrow failure. Bone marrow karyotype analysis in Fanconi anemia showed the presence of abnormal clones, temporary in many cases and having different meanings. We report two Fanconi anemia patients in which bone marrow chromosomal analysis showed different abnormal clones having in common structural abnormalities of chromosome 18. Case No. 1 2 years old child with myelodysplastic disease in which bone marrow karyotype showed a clone with a

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E. Garcia-Galloway (1) M. Talavera (1) C. Villalon (1) P. Cabello (1) D. Rey (1) J. Garcia-Sagredo (1) J. Garcia-Laraña (2) M. Ferro (1) (1) Medical Genetics, University Hospital Ramon y Cajal, Madrid (2) Hematology, University Hospital Ramon y Cajal, Madrid Introduction: Chronic lymphocytic leukemia (CLL) is characterized by clonal proliferation of mature B lymphocytes. This disease has a very variable evolution, with survival ranging from few months to over a decade. Presence and type of chromosomal abnormalities are among the different prognostic parameters described. We report a patient diagnosed of B-CLL, who throughout his illness showed a genetic heterogeneity and a very infrequent chromosomal abnormalities within the hematologic malignancies. Case report: A 31 years old male was diagnosed of non-Hodgkin’s lymphoma of high grade malignancy in 1989, he remains in remission after treatment.

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In 2002 was diagnosed of B-CLL, receiving various chemotherapy treatments and autologous peripheral blood progenitor cells transplantation. Survival was 5 years from diagnosis of B-LLC. Cytogenetic studies and FISH were done at the time of diagnosis of lymphoma and two times during CLL disease. First cytogenetic study was normal. Second study was developed when the LLC was established, it was found an abnormal clone with a translocation between the two chromosomes 22: t (22;22)(q11;q13), confirmed by FISH. The third chromosome and FISH study were carried out one year later, showing an abnormal clonal but different, consisting in a translocation between the whole long arms of chromosomes 17 and 18, resulting in loss of p53 gene. Conclusion: The cytogenetic abnormalities discovered in this patient are extremely rare, the t(22;22)(q11;q13) has been described as a single abnormality in the CML, and the translocation t(17;18)(q10;q10) has been reported in 10 cases, 8 with myeloid disease and 2 with CLL. It should be noted that this patient developed different chromosomal changes throughout their disease, with loss of the first cytogenetic clone and the appearance of a new different. To the current knowledge, the latter was of worse prognosis, resulting in the loss of p53. 7a.83-P New advances in Cat Mammary Tumours: Comparative Genomic Hybridization profiles in a case of tubulopapillary carcinoma A. Borges (1) S. Santos (1) F. Adega (1) F. Gärtner (2) H. Guedes-Pinto (1) R. Chaves (1) (1) Institute for Biotechnology and Bioengineering, Centre of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro (IBB/CGB–UTAD) (2) Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP) The advent of Comparative Genomic Hybridization (CGH) has opened a reliable way for detection of all genomic imbalances in each tumor through a single analysis. Its utility is based on the concept that chromosome regions with increased copy-number reveal sites that may contain dominantly acting oncogenes, whereas regions with decreased copy-number may harbor putative suppressor genes.

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Spontaneously Cat Mammary Tumours (CMT) offer a unique opportunity as cancer models for human breast oncology. Data regarding CMT cytogenetic characterization is very scarce, most especially at the molecular level. Moreover, the cytogenetic analysis of solid tumours has been a challenge area to work on; some of the technical hitches are difficulties in chromosome preparation and the complexity of the karyotypes displayed by these cells. In the present work, we analyze a CMT case by CGH that allowed the detection of most significant genomic imbalances. As far as we know, this is the first time that a GGH analysis is described in CMTs. Two mammary mass respectively with 1,5 cm and 4,5 cm diameter, were chirurgically removed from an 15 years old female cat. The tumour masses were histopathologically classified as a tubulopapillary carcinoma with identification of some solid pattern and necrosis areas. Simultaneously, gDNA was extracted for a CGH evaluation. The CGH analysis allowed the identification of several gains in all chromosomes with 99,99% of confidence. Chromosomes B2 (p12p15, p12q11, q31, q33, q33q36), C2 (p11, p23p24, q24q25, q26q28), D3 (p11p12, p13p14, p15, q22, q22q23), E1 (p12p14, q11, q13q14), E2 (p12q11, q13q14), E3 (p13, q11q13, q13) and F1 (q11q13, q23, q24) were the ones showing to be more prone to this type of genomic imbalances. In the future, it will be important to extend the analysis to a higher number of cases in order to correlate the genomic imbalances detected with cancer critical genes. 7a.84-P Cytogenetic Studies of Venereal Granuloma of Domestic Dog R. Deshmukh (1) R. Deshmukh (2) H. Purandarey (3) D. Suryavanshi (4) S. Chilgunde (1) V. Pawar (1) U. Umrikar (1) (1) Bombay Veterinary College, Mumbai (2) University of Copenhagen (3) Centre for Genetic Health Care (4) Bombay Veterinary College Chromosome investigations have revealed characteristic and tumor-specific cytogenetic rearrangements in various human cancers. However, research is still needed in the area of canine cancer cytogenetics to extend clinical applications. Four samples of venereal granuloma tumor tissues from male dogs (Breed nondescript) were collected in RPMI 1640 medium

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(HIMEDIA ltd). Direct and long term cultures were performed in RPMI 1640 medium with 20% Fetal Bovine Serum (GIBCO ltd.) at 37°C in a 5% CO2 incubator. Long-term cultures were successfully maintained for 8 to 25 days, whereas the direct cultures were harvested on the same day. One hour before harvesting, cells in culture were synchronised in metaphase by treating them with 0.005% colchicine (LOBA chemie). Metaphases were prepared by tissue disaggregation (0.25% trypsin EDTA, 3 min, 37°C), subsequent hypotonic treatment (0.05%, 0.075M KCL 12 min, 37°C) followed by fixative washings (3:1 methanol:glacial acetic acid) of the cells. Direct cultures were found to be inefficient for numerical and morphological analyses of the chromosomes due to poor metaphase preparations. Metaphases obtained from long-term cultures, however were subjected to GTG-banding, and 20 metaphases from each tumor were evaluated. The four samples showed aneuploid, hypodiploid and hyperdiploid metphases (chromosome number 65–88); however, the normal canine karyotype is 78 XY/XX. The modal chromosome number of metaphases were 77 in three cases and 79 in one case. Other chromosomal abnormalities, such as increased number of biarmed chromosomes, presence of ring chromosomes, telomeric arrangements, and double minutes were also found. In the present study, long-term culture of venereal granuloma tissue was standardized successfully. In dogs, in general, individual chromosome identification with GTG banding is difficult, particularly the small chromosome pairs (22 to 38) with only a few bands and in condensed state. Results, theref-ore based on identification of only numerical and simple structural changes. 7a.85-P Selection of genetically characterized clones in a patient with chronic myeloid leukemia sequentially treated with tyrosine kinase inhibitors G. Calabrese (1) D. Fantasia (1) R. DiGianfilippo (2) F. Pompetti (3) E. Morizio (3) D. Romagno (1) P. Guanciali Franchi (1) A. Spadano (3) G. Palka (1) (1) University of Chieti/Center for Ageing (2) Pescara Hospital (3) Pescara Hospital

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Treatment based on tyrosine kinase inhibitors (TKIs) is considered the gold standard for chronic myeloid leukemia (CML). As relevant part of the therapeutic strategy, genetically targeted therapies need an appropriate monitoring of efficacy, and equally a careful evaluation of drug toxicity. We report on a Ph-positive CML patient, sequentially treated with imatinib, nilotinib and dasatinib, and monitored by conventional cytogenetics, FISH and RQ-PCR. TKIs-based therapy resulted poorly effective, and early the patient showed additional genetic alterations, preceding disease progression. During therapy with imatinib the predominant blast population showed no ABL kinase domain (KD) mutations, but a double Phchromosome was displayed in 7% cells; under nilotinib therapy, the screening for mutations allowed to detect a population bearing an alternatively spliced BCR/ABL KD. Later on, after dasatinib therapy start, the KD mutated clonal population disappeared and an isochromosome 17q-positive cell clone became prevalent. The patient deceased in blastic crisis 3 months after commencing dasatinib treatment. In this patient, CML probably derived from a highly unstable Phpositive stem cell, that produced a large number of mutated subclones. These abnormal clones were sequentially selected on the basis of the proliferative/survival advantages and drug sensitivity. Every tyrosine kinase inhibitor possibly acted through a selective pressure on the leukemic blast population allowing specific resistant clones to stand out. 7a.86-P Identification of tumour related chromosomal rearrangement in BLOOM mouse cancer model using high resolution CGH and M-FISH N. Conte (1) R. Banerjee (1) O. Dovey (2) B. Ling Ng (1) N. Carter (1) A. Bradley (1) (1) Wellcome Trust research Campus (2) University of Leicester Our project aim to perform a comprehensive analysis of the “cancer genome” in a cancer prone mouse model: irradiated bloom deficient mice. Loss of the DNA helicase Bloom function has been described in human Bloom syndrome and leads to a breakdown in the maintenance of genome integrity, in particular hyper-recombination and cancer predisposition. Mice

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deficient for this gene are cancer prone in a wide variety of different cell types including carcinomas, sarcomas and lymphomas. In order to produce a wide range of tumours of different types, a cohort of 1000 BLOOM deficient mice has been irradiated. Mice were sacrified when terminally ill and tumours collected In order to identify gene/regions recurrently rearranged, we subjected tumour samples to a complete cytogenetic analysis, including high resolution array CGH (aCGH) and Multicour fish (MFISH). – –

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We have harvested thousand of tumours samples from different cell type. Thirty-tow primary culture derivated from T lymphoma have been analysed using high resolution CGH and M-FISH. These 2 technologies allowed us find already known candidates cancer region/genes (chromosome 11: Ikaros; Chromosome 15: c-Myc; chromosome 19: Pten) as well as a region on chromosome 12 which is found often involved in translocation and/or deleted. We were able to identify a 50 kb sub region on chromosome 12, within a genomic desert, which seemed particularly targeted. We are currently investigating the relevance if this region in cancer using chromosome engineering. Solid tumour samples from other cell origins are currently being characterised.

The BLOOM deficient mice allowed us to harvest hundreds of tumour from different origins generating the tumour panel which is subjected to genetic analysis (aCGH and MFISH). The strength of MFISH is in defining translocations and marker chromosomes in complex karyotypes, whereas array CGH can reveal hidden deletions and amplifications. In combination, this promises a very powerful approach to cancer gene discovery.

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A 22 years old female patient, undergone to BMT because of b-thalassemia major when she was six from her HLA-identical brother, was found to have Acute Myeloid Leukemia. Cytogenetic analysis indicated the presence of 2 cell lineages: the first one, the most represented, had complex and several rearrangements, the second one had normal male chromosomes. The karyotype was designed as: 44-46,X,der(3),der(4),der(5),+11,-14,-17,-19,der (21),-22,-X?-Y?,+mar[17]/46,XY[3]. Because of the loss of sex chromosome, conventional cytogenetic analysis made it impossible to establish the origin of the leukemic clone. No cytogenetic polimorphisms were evident. FISH analysis, with centromeric probes of X and Y and painting of chromosome 11, confirmed the total absence of sex chromosome in all the aberrant metaphases “tagged” with the additional chromosome 11, but it was not helpful to assigning aberrant cell lineage. In addition it showed the presence of 3 clones: leukemic one, 46,XX and 46,XY. We coudn’t clearly assign the 46,XX one to the recipient, because of the possibility of donor’s X duplication. Donor bone marrow analysis was also performed and showed a normal karyotype 46,XY. One month later, after complete clinical and cytogenetic remission (46, XY), leukemia relapse occurred with the same complex cytogenetics findings described above. Microsatellite analysis on DNA donor (peripheral blood) and recipient (oral mucose and bone marrow) were performed, but neither cytogenetic standard methods, (absence of “sex markers”), nor molecular techniques (presence of three cell lineages), allowed to identify the origin of leukemic clone. Further molecular investigations will be necessary to elucidate the possible mechanisms of leukemia onset. 7a.88-P Chromosomal gain: from MGUS to MULTIPLE MYELOMA

7a.87-P AML post BMT: donor or recipient? A. Azzena (1) L. Martorana (1) R. Murru (1) M. Licheri (1) S. Deidda (1) M. Virdis (1) A. Di Tucci (2) S. Orrù (1) C. Carcassi (1) (1) University of Cagliari (2) Businco Hospital

L. Martorana (1) A. Azzena (1) R. Murru (1) S. Deidda (1) M. Licheri (1) M. Virdis (1) R. Floris (2) S. Orrù (1) C. Carcassi (1) (1) P.O. R. Binaghi (2) P.O. R. Binaghi (3) P.O. R. Binaghi (4) P.O. R. Binaghi (5) P.O. R. Binaghi (6) P. O. R. Binaghi (7) CTMO P.O. R. Binaghi (8) P.O. R. Binaghi (9) P.O. R. Binaghi

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Multiple Myeloma is a malignant uncontrolled monoclonal proliferation of bone marrow plasma cells (PCs). In most cases of MM a preexisting condition of excessive plasma cells growth, called Monoclonal Gammapathy of Undetermined Significance (MGUS), is almost always present, but not associated with symptoms or tumoral masses. We describe a case of a 59 years old female patient who was diagnosed with MGUS in 1999; clinical parameters showed a monoclonal spike of IgG kappa chains in serum immunoelectrophoresis, plasma cells infiltration < 5%, skeletal RX unremarkable, normal urine immunofixation, normal serum IgG dosage and beta-2 microglobulin. This clinical condition lasted stationary for 9 years. On may 2008 PCs infiltration rised to 20%, IgG to 2.5g/dl with no other significant clinical changes; cytogenetic analysis on bone marrow biopsy showed a normal karyotype 46,XX. Six months later plasma cells infiltration rised to 50% and bone marrow cytogenetic analysis revealed the presence of an aberrant clone in 3 out of 20 metaphases analysed. MGUS evolved definitely in Multiple Myeloma at 1st stage according to Durie-Salmon staging system. The karyotype was 46,XX[17]/53,XX,+3,+5,+7,+11,+15, +19,+21[3]. No monosomy or deletion on chromosome 13 were detected by FISH analysis on 200 cells scored (metaphases and interphases nuclei). The patient didn’t require therapy from 1999 until now. The hyperdiploidy observed, with the specific gain of chromosomes 3, 5, 7, 9, 11, 15, 19, 21, is well described in literature and is associated with a significantly better overall survival; the absence of monosomy or deletion on chromosome 13 also represents a good prognostic factor but the unexpected and rapid transformation from MGUS to MM could predict a quick evolution and a strong aggressiveness of the disease. 7a.89-P Variant Ph translocations in CML—cytogenetic differences and clinical similarities O. Haus (1) E. Duszeńko (2) Z. Salamanchuk (3) A. Klonowska (4) B. Mucha (1) A. Jaśkowiec (2) M. Jakóbczyk (3) I. Kardaś (4) K. Skonieczka (1) M. Iliszko (4) (1) Collegium Medicum, Nicolaus Copernicus University (2) Medical University (3) Collegium Medicum, Jagiellonian University (4) Medical University

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Variant tranlocations, t(9;22;v), represent 2–10% of all chromosome Ph translocations in chronic myeloid leukemia (CML). They are formed by one-step or two-step mechanisms, and more often than standard Ph translocations are associated with deletions of whole or part of a fusion gene: BCR/ABL on chromosome der(9) or BCR/ABL on der(22). The prognosis associated with variant translocations was poor in the interferon era, but now, due to implementation of imatinib treatment in CML, it is generally almost equal to prognosis of standard translocations. However, the prognosis associated with specific chromosomes or specific breakpoints involved in variant translocations may be different. Here, we present the results of cytogenetic and FISH examination in 66 patients, 39 women and 27 men, aged 22– 83 years, diagnosed in four different Polish hematologic and genetic centers. The cytogenetic examinations were carried out in untreated patients at clinical diagnosis of CML. The material for cytogenetic examinations were bone marrow cells from 24 to 48 cultures. After harvesting and fixation 20 GTGbanded metaphases were analyzed for each patient. Then FISH with painting (wcp) and BCR-ABL specific probes was used. Among 1000 consecutive Ph-positive CML patients diagnosed between 2000 and 2008, there were 66 with variant Ph translocations. The most often involved in t(9;22;v) chromosomes were: 3, 10, 12–7x each, and 1, 2, 5, 15, 17–5x each. The most frequently involved breakpoints were: 3p21 and 10q22–4x each, and 1p36, 3q21, 12p13, 12q13–3x each. Four of them; 1p36, 3p21, 10q22, 12p13 are known as the most frequent breakpoints in variant Ph. 53 patients had one-step variant („true variant”), 9 of them had in addition a deletion at 9q34, 9 patients had two-step variant with ABL/BCR signal on der(9), and 4 had two-step variant with ABL/BCR on the third chromosome. Fifty-eight patients had threeway, 7 patients—four-way, and one patient—a five-way variant translocation. All patients were treated with imatinib in standard dose 400 mg/d. The achievement of complete cytogenetic response (CCR) was comparable in different cytogenetic groups, however, with a slightly better prognosis for the group with one-step variant without deletion (NS). There wasn’t any correlation between localization of breakpoints and prognosis concerning CCR achievement or survival time, but the groups with the same breakpoints were small. The larger groups of patients with identical breakpoints are needed

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to strictly evaluate prognostic significance of Ph variants. 7a.90-P Quality assessment in leukemia cytogenetics using viable cells for interlaboratory tests H. Rieder (1) H. Hager (2) C. Haferlach (3) L. Harder (4) G. Prescher (5) B. Moehlendick (1) (1) University Hospital Duesseldorf (2) University Hopsital Heidelberg (3) MLL Munich Leukemia Laboratory (4) University Hospital Kiel (5) Laboraties Eberhard and Partners Cytogenetic investigations are an integrated part of the diagnostic panel in hematological neoplasias, because the chromosome findings may directly influence the choice of treatment and, thus, the prognosis of the patients. The quality of the chromosome investigations depends on several factors reaching from the type of the leukemia and duration of probe shipment, over the cultivation and preparation procedures to the banding methods and the experience of the investigator. To test if there are differences in the performance between laboratories engaged in leukemia cytogenetics, we established an interlaboratory test (IT) which included the complete procedure of chromosome banding analysis. Surrogate leukemic blood sample were prepared by mixing a whole blood sample of a healthy volunteer with a lymphoblastoid cell line with normal karyotype and and with a leukemia cell line with aberrant karyotype. The samples were sent to the participating laboratories by mail. The laboratories were requested to process the sample and to report the findings according to their routine protocols. A review committee evaluated the cytogenetic results and reports according to a catalogue for minimum standards contained in the ISO15189 and for the formal correctness of the karyotypes. Target aberrations were definded which had to be detected by the laboratories. Up to now, five interlaboratory tests have been performed with 20–40 participating laboratories. Overall delivery time of the sample did not afffected the results. The minimum standards of reports were fulfilled from the majority of the laboratories. A high degree of inaccuracies in formal karyotype descriptions was found which indicated the need of an internal or external review system of karyotypes. The target aberrations for each cell line

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were found by 8–75% of the laboratories. In summary, our results demonstrate that the IT using viable cells is a useful instrument to compare the quality of cytogenetic investigation between laboratories. Supported by the BMBF, grant 01GI 9974 7a.91-P New chromosomal translocation in B-cell chronic lymphocitic leukemia A. Carrió (1) D. Costa (1) C. López (2) A. Arias (1) A. Varela (2) N. Villamor (1) D. Colomer (1) M. Rozman (1) E. Campo (1) (1) Hospital Clinic (2) Fundació Clinic B-cell chronic lymphocitic leukemia (B-CLL) is the most common type of leukemia in the Western countries and is characterized by the clonal expansion of morphologically mature small lymphocytes. The clinical course of B-CLL is highly variable with survival times ranging from months to decades. To predict prognosis and to decide the treatment strategy, prognostic factors are of immense importance. The prognostic markers related to the biology of B-CLL, such as chromosomal abnormalities are increasingly being evaluated for their prognostic value. Clonal chromosome abnormalities in B-CLL are detected in 40–50% of cases by conventional cytogenetics after mitogen stimulation. By FISH these rate is about 80% of B-CLL cases. We report four novel chromosomal translocations identified by conventional cytogenetic in four patients with B-CLL. All patients were male. Clinical, laboratory and inmunophenotipic data were consistent with B-CLL diagnosis. Cytogenetic studies were carried out in peripheral blood (PB) using 12-Otetradecanoylphorbol-13-acetate (TPA) as mitogen. Cultures were maintained for 3 days in CO2 atmosphere. The karyotypes were: Case 1: 46,XY,t(1;7)(q34;q11)[2]/46,XY[18]. Case 2: 45,XY,der(7,14,15)t(7;14)(p10;q32)t(7;15) (p10;q10),del(11)(q22q23)[2]/44,idem,der (13;14)(q10;q10)[6]/46,XY[11]. Case 3: 46,XY,-8,der(17)t(8;17)(q10;p13)[8]/46,XY [6]. Case 4: 46,XY,t(15;22)(q26.1;q11.2)[13]/46,XY[7] A search in the National Cancer Institute Mitelman Database of Chromosome Aberrations in Cancer

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revealed that none of the translocations reported herein has previously been described in B-CLL. Cytogenetic analysis of B-CLL patients, despite inherent technical limitations, has provided important information on disease biology and clinical outcome. Thus, it could be successful perform conventional cytogenetics, combined with routine FISH, to identify new chromosomal translocations that could pinpoint subgroups of patients with B-CLL who have different prognoses, and other candidate genetic lesions susceptible to be targets for new therapies.

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chemistry and cytogenetic techniques. The improvement of the assumption of responsibility and the forecast rests on a multidisciplinary collaboration between anatomic-pathologists, ORL, medical neurosurgeons, radiotherapists and oncologists. Key words: Ewing’s sarcoma, maxillary, sinus, cytogenetic, translocation. 7a.93-P Variant philadelphia translocations in patients with chronic myeloid leukemia

7a.92-P Primary Ewing’s sarcoma of the maxillary sinus: a Tunisian case F. Farah-Klibi (1) L. Hila (2) O. Elamine (0) r. Zermani (0) S. Ben jilani (0) (1) FMT (2) EPS Charles Nicolle Primary Ewing’s sarcoma of the maxillary sinus: a Tunisian case. F. Farah-Klibi, L. Hila, O. El Amine, R. Zermani, .S.Ben Jilani . Service d’anatomie et de cytologie pathologiques de l’hôpital Charles-Nicolle de Tunis.Tunisia Abstract Ewing’s sarcoma (SE) is the second most common malignant bone tumour of childhood, yet it is a rare tumour. Primary maxillary localization is unusual and occurs in only 1–2% of cases. The prognosis of Ewing’s sarcoma has been improving considerably since the introduction of combined modality treatment. We report one case of Ewing’s sarcoma localized on the maxillary sinus: a 13 year old girl presented with a right facial tumefaction, successfully treated by combined modality treatment radiotherapy and chemotherapy. Diagnosis was established at microscopic examination with immunohistochemistry (anti-FLI-1; NSE; EMMA), cytogenetic and molecular biology (RTPCR). Cytogenetic and molecular analyses, on fresh or frozen material are fundamental argument for the diagnosis of the SE and have also a prognostic role. Identification of a chromosomal rearrangement T (11; 22) (q24; q12) was a decisive step in the knowledge of the pathogenesis of the disease. The SE of the maxillary sinus is a rare tumour whose diagnosis is difficult, resting on immunohisto-

N. Satkin (1) S. Palanduz (1) B. Karaman (3) G. Bagatir (1) S. Ozturk (1) K. Cefle (1) A. Ucur (1) A. Bayrak (1) M. Nalcaci (2) M. Yenerer (2) (1) Istanbul University, Istanbul Medical Faculty (2) Istanbul University, Istanbul Medical Faculty (3) Istanbul University, Istanbul Medical Faculty Chronic myeloid leukemia (CML) is a haematological malignancy with a relatively favorable course. It is characterized by a chronic phase followed by an accelerated phase and blastic transformation occurs eventually. During the chronic phase, almost all patients with Chronic Myeloid Leukemia (CML) show the classical Philadelphia translocation, t(9;22) (q34;q11), or a variant translocation on conventional cytogenetic anaysis. Variant philadelphia (Ph) translocations can be simple translocations or complex rearrangements.We present six variant translocations found in CML patients. Cytogenetic study was performed on Giemsabanded metaphases obtained through short-term synchronized culture of bone marrow cells supplemented by direct harvest.FISH tecnique by using VCFS N25 (D22S75/N85A3), subtelomeric, wcp and bcr/abl probes were applied to identify the breakpoints of the translocations. Out of 6 translocations, 3 were simple and 3 were complex rearrangements.While chromosome 22 were involved in all translocations, chromosome 1, 4, 9, 10, 20 were found only in one translocations. The clinical course and impact of variant translocations on long-term outcome are the reports on conflicting. Some studies have suggested that patients with variant Ph translocations may have an adverse prognosis, while others no impact on prognosis. It has been reported that after Imatinib

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Hairy-cell leukaemia (HCL) is a distinct B-cell disorder accounting for about 2% of all cases of leukaemia. Hairy-cell leukaemia-variant (HCL-variant) is a rare B-cell disorder which accounts for 10% of HCL cases. It affects elderly or middle-aged males. The main features are splenomegaly, lymphocytosis and cytopenias without monocytopenia. The circulating cells have a morphology intermediate between prolymphocytes and hairy cells. Cytogenetic studies in hairy cell leukemia (HCL) are rare. In our study, cytogenetic investigations were performed on blood cells obtained from one HCL variant male patient. Cytogenetic study was performed on Giemsa-banded metaphases obtained through short-term synchronized culture of peripheral blood cells supplemented by direct harvest. Fluorescsence in situ hybridization (FISH) tecnique by using subtelomeric, wcp probes were applied to identify the breakpoints of the translocations. We found a clonal chromosome abnormality; 48,XY, t(1;14)(q31;q31),+3, +18. This is not defined before. We report clonal chromosome abnormalities found in our patient. If it is found in other patients, the prognostic value may be clasified.

In the classical Stebbins’s book Chromosomal Evolution in Higher Plants, he presented some cases where the chromosomal morphology, number and size strongly correlate to floral specialization. A similar case occurs in the genus Crotalaria (Leguminosae-Papilionoideae), where the species with floral specialization present shortest chromosome and genome sizes, while the species without floral specialization have bigger and symmetrical ones. The use of chromosome banding and FISH with 45S and 5S rDNA probes allowed to investigate news aspects of chromosome organization in addition to classical karyotypes. Every species studied present pericentromeric heterochromatin. However, those with floral specialization have bright CMA bands (GC-rich) that quenched in combination with distamycin A (CMA/ DA) while those without specialization do not have binding sites to any fluorochrome, and the heterochromatic blocks can be seen only after chromosome denaturation. This heterochromatic variation occurred early in the group diversification, probably as a result of changes in the nature and composition of repetitive DNA families that usually reside in the heterochromatin. All species presenting 45S and 5S rDNA sites at chromosome 1 have been considered as ancient. Secondary inversion, translocation and transposition events involving the rDNAs are associated with diversification inside the botanical groups. Moreover, an inversion changing the positions of 45S and 5S rDNA detected in the chromosome 1 discriminates the specialized from the non-specialized groups. Two polyploids from the specialized group showed typical characteristics of polyploid evolution, like 45S and 5S rDNA reduction size, number and inactivation, chromosomal length reduction and structural rearrangements. Altogether, the results showed a close association between floral specialization and chromosome diversification.

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Chromosome diversification between floral specialized and non-specialized groups of the genus Crotalaria (Leguminosae-Papilionoideae)

Development of a Real Time PCR method based on SYBR Green dye for the evaluation of sex ratio in Bovine semen

M. Mondin (1) (1) Universidade de São Paulo—Escola Superior de Agricultura

V. Khazaeli (1) F. Mahjoubi (2) A. Salehi (1) B. Shamsi (2) E. Arbabi (2) (1) Abooreihan (2) NIGEB

Mesilat treatment, prognosis is similar in patients with variant translocations and patients with classic Ph translocations. We report variant translocations found in our series and impact of the IM treatment on prognosis. 7a.94-P Translocation (1;14) in hairy cell leukemia variant A. Bayrak (1) A. Ucur (1) B. Satkin (1) S. Palanduz (1) M. Karan (2) S. Ozturk (1) K. Cefle (1) (1) Istanbul University, Istanbul Medical Faculty (2) Istanbul University, Istanbul Medical Faculty

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Sex preselection in bovine offspring has an important role in dairy and beef cattle herds’ improvement. One of the best ways to preselect the offspring is to use sexed semen. The sexed sperm demand is intensively related to the sexed semen purity, therefore after the semen was sexed, a sex ratio determination must be accomplished. In this work, we aimed to develop a fast, reliable, reproducible and economic procedure to be used for the evaluation of the sexed semen employing Real Time PCR. We also used SYBR Green dye instead of probes for more decrease in expenses. Real Time PCR was performed using primers specific for SRY (Y chromosome specific sequence) and B-actin (as an endogenous gene, located on chromosome 25) genes on both semen and blood (reference) specimen. The percentages of Y and X chromosome bearing sperms were determined using ΔΔCt formula (ΔCt Sperm –ΔCt Blood). The method introduced in here is reliable, simple and economical and can be easily used for evaluation of natural and commercial sexed semen. 8a.2-P Negative Impact of somatic Aneuploidy in bivalve’s growth rate: ten years of case studies J. Teixeira de Sousa (2) S. Joaquim (1) D. Matias (1) R. Chaves (3) A. Leitão (1) (1) INRB/L-PIMAR (2) Universidade Lusófona de Humanidades e Tecnologias (3) Institute for Biotechnology and Bioengineering The aneuploidy phenomenon (mainly hipodiploidy) has been observed in several species of marine bivalves. Furthermore, a negative relationship between growth rate and this phenomenon was already verified for the species of oyster Crassostrea gigas, C. angulata and interspecific hybrids. This negative relationship is of particular importance, since the variability of growth rate is one of the biggest problems faced by bivalve producers. The clam Ruditapes decussatus is one of the most important bivalve species in Southern Europe (Portugal, Spain and Italy). However, despite the high commercial and social importance of the production of this clam, very few studies of selection and genetic improvement were made so far. Moreover, local Portuguese

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producers have observed, in the last years, a decrease in the maximum size reach by individuals of this species. In this study, and in order to establish the potential link between aneuploidy and growth rate in R. decussatus we determined the aneuploidy rate of two types of individuals of the same cohort, the socalled “fast-growers” and “slow-growers.” These two types are present despite the fact that all individuals are raised under the same conditions of culture. In order to try to clarify the nature of the aneuploidy phenomenon in bivalves (and its negative relationship with growth rate) we applied the molecular cytogenetic technique of banding with restriction enzymes for the identification of the missing chromosomes in aneuploid karyotypes of R. decussatus and other bivalve species. Joana Teixeira de Sousa1,2, Sandra Joaquim1, Domitília Matias1, Raquel Chaves3, Alexandra Leitão1* 1 INRB/L-IPIMAR. Avenida 5 de Outubro, 8700-305 Olhão (Portugal). 2 Universidade Lusófona de Humanidades e Tecnologias. Campo Grande, 376, 1749-024 Lisboa (Portugal). 3 Institute for Biotechnology and Bioengineering, Centre of Genetics and Biotechnology (IBB/CGBUTAD), 5001-801 Vila Real (Portugal). * Corresponding author ([email protected]) 8a.3-P Radial nuclear position of specific loci is conserved in Primate lymphocytes C. Federico (1) R. Picciotto (1) M. Galvagno (1) S. Motta (1) S. Saccone (1) (1) University of Catania - Italy Human chromosomes, in the cell nuclei, are located on specific large subdomains according to their genedensity. However, since most chromosomes are composed by non-contiguous gene-dense and genepoor regions, the radial nuclear organization of each chromosome territory involves the gene-dense/GCrich and the gene-poor/GC-poor bands being localised towards the interior and the periphery, respectively. By using human BAC probes from a number of compositionally different chromosomal bands, we investigated the level of conservation of the radial nuclear position on cell nuclei from Primate species.

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We analyzed a number of loci that, during karyotype evolution, were relocated along the chromosomes by translocation/inversion/neocentromerization events. An example is represented by the human chromosomal 6q24 band that in Macaca mulatta corresponds to the centromere region of the homologous chromosome MMU4. Our results indicate that, homologous loci show similar radial nuclear location in the cells of the Primate species investigated, in spite of their different position along the mitotic chromosome (telomeric, centromeric, etc.). Our findings show that the radial nuclear position of a specific locus is preserved during chromosomal evolution. As a consequence, specific intra- and inter-chromosomal interactions between loci endowed with similar expression pattern are maintained.

as WHO-TEQ) than those permitted (6.0 pg/g of dioxins+furans+PCBs as WHO-TEQ) and the results were compared with samples from 16 dairy cows (1.75 pg/g of fat as WHO-TEQ) used as control. Significantly (P<0.01) higher mean number of SCE/ cell (7.10±2.8) were found in dioxin exposed cows compared to those measured in the controls (SCE/ cell=5.24±2.51). The same test was applied on 26 river buffaloes showing average values of WHOTEQ over (21.79 pg/g of fat as WHO-TEQ) than those achieved in the milk mass of 26 river buffaloes (1.30 pg/g of fat as WHO-TEQ) used as control. Opposite to cattle, no significant differences were found between mean number of SCE/cell in exposed vs. non exposed river buffaloes (8.73±3.32 vs. 8.79±3.40).

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Chromosome stability test in both cattle and river buffaloes exposed to dioxins

Sequencing and organization of Nomascus Leucogenys (gibbon) centromere

G. Di Meo (1) A. Perucatti (1) V. Genualdo (1) D. Incarnato (1) V. Peretti (3) A. Caputi-Jambrenghi (2) R. Rasero (4) C. Nebbia (5) L. Iannuzzi (1) (1) National Research Council (2) University of Bari (3) University Federico II (4) University of Turin (5) University of Turin

A. Cellamare (1) C. Alkan (2) F. Antonacci (2) M. Rocchi (1) E. Eichler (2) M. Ventura (1) (1) University of Bari (2) University of Washington School of Medicine

Polychlorodibenzodioxins (PCDDs) and related substances including polychlorodibenzofurans (PCDF) and the dioxin-like polychlorobiphenyls (DL-PCBs) are powerful mutagens that can enter the food chain as the result of a number of industrial processes, waste incineration or illegal waste burning. Cytogenetic tests could be useful to reveal the presence of such mutagens in the food chain by simply monitoring food producing species. In previous studies we studied sheep exposed to dioxins and found a pronounced chromosome fragility in exposed herds, compared to unexposed one (control) by using both SCE and chromosome abnormality (AC) test. In this study we applied the SCE-test in both cattle and river buffaloes reared in dioxincontaminated areas, although the WHO-TEQ levels of bulk milk were much lower than those earlier detected in sheep milk. Peripheral blood cultures were performed in samples from 15 dairy cows showing average milk values over (18.56 pg/g of fat

Alpha-satellite is a family of tandemly repeated DNA found at the centromeric regions of all human and primate chromosomes. The human α-satellite has been classified in two types of α-satellite DNA according to its organization and sequence proprieties: high order alpha-satellite and monomeric alphasatellite. In the higher-order α-satellite the monomers are organized as highly similar multimeric repeat units mainly located at centromeric side. The monomeric α-satellite, instead, has not any detectable higher-order periodicity and its monomers are far less homogeneous than the higher-order repeat units. Further, the monomeric alphoid sequences have pericentromeric localization. Phylogenetic analysis suggested that the higher order α-satellite DNA emerged more recently than the monomeric repeat. The African Green Monkey (AGM) α-satellite DNA appears to have the simpler organization than the other primates and has been considered the ancestral form. The existing alpha satellite organization has been hypothesized as the result of AGM monomer evolution, through the

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molecular drive. In this work we have fully characterized α-satellite sequences of the white-cheeked gibbon (Nomascus leucogenys, NLE), in order to better understand the α-satellite evolutionary dynamics. Alphoid cloning, hybridization experiments and bioinformatics analysis were performed to define the NLE alphoid organization. Our results showed that: i) the NLE alphoid structure has a monomeric structure characterized by units with high similarity percentage; ii) the NLE alphoid sequences have not high order repeats (HOR) or subfamily organization and iii) (for first time) the α-satellite sequences have a telomeric localization sharing the same organization of the centromeric alphoid sequences. Our data strongly suggest, according to the evolutionary distances between gibbon species and human, the hypothesis of the more recently higher order α-satellite DNA emergence respect to the monomeric structure.

male dog. Different techniques of classical and molecular chromosomal differentiation were implemented. Cytogenetical female sex was confirmed by the consistent presence of both X chromosomes. We compared the results obtained in these different techniques of chromosomal analysis as tools to describe the possible case of dog Hermaphroditism, since there isn’t much information on cytogenetic analysis in this area. Molecular genetics—DNA extraction and SRY gene search—was applied to further identify the origin of the intersex. In this work the main objective was to fully describe a case of real Hermaphroditism in Canis familiaris, through genetic and histological findings.

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Cytogenetic characterization of species of two genus of venerid clams

About a case of Hermaphroditism in Canis familiaris R. Sousa (1) R. Marcos (2) J. Carrilho (1) B. Porto (1) I. Malheiro (1) (1) Instituto de Ciências Biomédicas Abel Salazar— Universidade do Porto (2) Instituto de Ciências Biomédicas Abel Salazar—Universidade do Porto Hermaphroditism or intersex is a general term that includes several congenital anomalies in the genital system, used to define animals that have ambiguous sexual characteristics (MICKELSEN and MEMON, 1997). It is characterized in pseudo Hermaphroditism and real Hermaphroditism, the last one defines a testicular tissue and ovarian tissue combined into a single gonad (ovotestis) (BEARDEN and Fuquay, 2000). This paper describes the case of a female dog that arrived at the veterinary clinic of the Institute of Biomedical Sciences Abel Salazar, University of Porto (ICBAS—UP) for a consultation, and in which was concluded, according to macroscopical and histological exams, that real Hermaphroditism (ovotestis) was present. A blood sample for cytogenetic analysis was drawn. Cell cultures were established from the patient and from a control

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J. Carrilho (1) J. Pasantes (2) P. Morán (2) I. Malheiro (1) (1) Instituto de Ciências Biomédicas Abel Salazar— Universidade do Porto (2) Universidade de Vigo Species belonging to the class Bivalvia, namely from the family Veneridae, have high commercial value, being harvested all over the world. Despite their economical interest, little is known about the cytogenetics of these species, most studies being done using classical techniques and only a few using fluorescent in situ hybridization (FISH). Our study focused on the chromosomal mapping of ribosomal and histone gene clusters in species from the genus Venerupis and Ruditapes. As banding techniques are not well developed in these species, physical mapping of known genes is an important way of identifying certain chromosomes and thus more fully describe the karyotype of these species. In order to do so, spreads of both mitotic and meiotic chromosomes were obtained from gills and gonads of juvenile individuals, after colchicine exposure, hypotonic treatment and fixation with ethanol: acetic acid. Surface spreadings of synap-

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tonemal complexes of male mature individuals were also done. Species specific probes for histone genes, 28S, 18S and 5S rDNA genes were obtained by Polimerase Chain Reaction (PCR). Differences found in the location of those genes among the species can help to understand evolutionary processes in venerid clams. 8a.8-P Repetitive sequences: a possible source of genome remodelling A. Paço (1) F. Adega (1) H. Guedes-Pinto (1) R. Chaves (1) (1) Institute for Biotechnology and Bioengineering, Centre of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro (IBB/CGB– UTAD) An interesting observation emerging from comparative genomic studies is the fact that breakpoint regions (chromosome regions prone to breakage and reorganization) are rich in repetitive elements, suggesting a probable relation between the occurrence of chromosomal rearrangements and the location of repeats. Besides, according to several works, it is also suggested that repetitive sequences could be hotspots for chromosomal rearrangements. In an attempt to understand the dynamics of mammalian genome evolution, we performed an in silico analysis between the location of evolutionary breakpoint regions and the distribution of tandem repeats in two chromosomes of Mus musculus (MMU17 and MMUX). For this analysis we used the data available in public databases (NCBI genome and Ensembl Mouse SyntenicView) and the software “Tandem Repeats Finder”. Our results confirm previous investigations, being observed a striking correlation between the location of evolutionary breakpoint regions and the distribution of tandem repeats throughout the two analysed mouse chromosomes. These observations provide further evidence that the tandem repeat sequences could be involved in the plasticity of genomes, showing that genome reorganization tends to occur at, or close to, regions rich in repetitive elements.

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8a.9-P Telomeric repeats (TTAGGG)n as footprints of evolutionary chromosome rearrangements S. Meles (1) F. Adega (1) H. Guedes-Pinto (1) R. Chaves (1) (1) Institute for Biotechnology and Bioengineering, Centre of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro (IBB/CGB– UTAD) Telomeric repeats, (TTAGGG)n, are among the most important sequences with regard to mammalian chromosome evolution. These sequences are present mainly at the telomeres of all mammalian chromosomes investigated so far, but relatively large blocks of this sequence are also found at non-telomeric sites, known as interstitial telomeric sites (ITS). Several studies considered that such atypically located telomeric sequences can be related to chromosome rearrangements, representing mirror steps in karyotype evolution. The order Rodentia, in particular Muridae rodents, presents high rates of chromosome evolution and remarkable chromosome diversity, most probably resulting from extensive reshufflings during the course of evolution. In this work we analysed the distribution of telomeric signals in the chromosome complement of one Muridae species, Praomys tullbergi (PTU), using fluorescent insitu hybridization with telomeric probes. We detected hybridization signals associated with either telomeric or non-telomeric sites in this genome. This approach confirmed the occurrence of pericentric inversions and tandem fusions/ fissions during the karyotype evolution of this species, already confirmed by Comparative Chromosome Painting performed in our laboratory. We believe that the telomeric repeats detected pericentromeric and interstitially in several chromosomes of PTU represent nonambiguous footprints of chromosome rearrangements. This analysis provided interesting insights about the genome organization of PTU, representing a suitable tool in reconstructing the path of karyotypic reshufflings during the evolution of Muridae genome.

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Comparative analysis of two Cricetus cricetus chromosomes with Mus musculus and Rattus norvegicus using chromosome painting

Chromosome Evolution in Bivalvia (Mollusca): Ostrea edulis and O. stentina, and the clam Chamelea gallina. Inferences by ribosomal and telomeric sequences

A. Vieira-da-Silva (1) S. Louzada (1) F. Adega (1) H. Guedes-Pinto (1) R. Chaves (0) (1) Institute for Biotechnology and Bioengineering, Centre of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro (IBB/CGB–UTAD)

J. Pereira (1) A. Leitão (2) R. Chaves (1) F. Batista (3) H. Guedes-Pinto (1) (1) Institute for Biotechnology and Bioengineering, Centre of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro (CGB–UTAD/IBB) (2) INRB/L-IPIMAR. (3) School of Ocean Sciences, Bangor University

One of the aims of analyzing mammalian genome architecture is to weave their evolutionary history. Such knowledge enables making a phylogenetic analysis of mammalian species, to establish a possible structure of ancestral karyotype for genus, families and higher taxa, and to infer probable evolutionary chromosomal rearrangements. This information is useful to evolutionary studies but also in biomedical research (e.g. to choose representative model animals to specific genetic disorders). One of the methods commonly used to make this kind of study is Comparative Chromosome Painting, which is based on chromosomal homology between genomes. Almost half of the extant mammalian species belongs to the order Rodentia, being Muroidea (where are included the index species mouse and rat) the larger and most diverse rodent superfamily. The evolutionary success of these rodents is reflected by the extensive chromosome polymorphisms (wide variation in diploid numbers, heterochromatin nature, localization and amount, etc). As more Muroidea species are analyzed, more unraveled are the evolutionary events in this taxon. Common hamster Cricetuscricetus (Rodentia, Muroidea, Cricetidae) has a diploid number of 22 chromosomes, mainly composed by metacentric/sub-metacentric chromosomes, with constitutive heterochromatin preferentially located at the (peri)centromeric regions. To establish chromosomal homologies between Cricetuscricetus (CCR), mouse (Musmusculus, MMU, 2n=40) and rat (Rattusnorvegicus, RNO, 2n=42) (Rodentia, Muroidea, Muridae), we conducted Comparative Chromosome Painting with mouse and rat chromosome-specific painting probes. Here we present the results regarding two CCR chromosomes (CCR1 and CCR2). MMU paint probes produced a higher segmentation inCricetuscricetus chromosomes. Cricetinae, Cricetidae and Muroidea specific syntenic associations were revealed. These are preliminary results of an ongoing CCR genomic architecture analysis.

Bivalves constitute a group of marine invertebrates of great economic and ecological significance, and therefore, genetic information concerning their genome is particularly important. Cytogenetic analysis may provide useful information for phylogenetic comparisons and insight on major genomic changes at chromosome level in this group. The genome of three commercially important bivalves species: the oysters Ostrea edulis and Ostrea stentina, and the clam Chamelea gallina, was characterized using molecular cytogenetic approaches such as Giemsa staining, DAPI staining and fluorescence in situ hybridization (FISH) with repetitive DNA probes [ribosomal (18S–28S rDNA, 5S rDNA) and telomeric (TTAGGG)n]. The results obtained, particularly the study of the repetitive sequences at the molecular level and their physical mapping, revealed to be a valuable tool in the illumination of the phylogenetic relationships among bivalves and their chromosome evolutionary processes. These studies are extremely useful for the implementation of bivalve species preservation or aquaculture interspecifc hybridization programs. 8a.12-P Karyotypical instability of mouse embryonic stem cells in culture T. Grinchuk (1) (1) Institute of Cytology T.M. Grinchuk, L.L. Alekseenko, K.M. Ivantsov, M.S. Lyanguzova, N.A. Pugovkina, Z.V. Kovaleva Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia

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Mouse Embryonic stem cells (ESC) are convenient model for study of a number of biological problems. The goal of the present work was to evaluate stability of the karyotype structure of mouse ESC in cultivated in vitro. For comparative analysis ESC from various sources were used: cell lines Е-14-1V, Joud-2, Rosa, CGR-8, and CG-7. The study was performed on metaphase chromosomes stained for G-bands. It was found that the mouse ESC cultivation resulted in the karyotype destabilization apparent by disturbance in homologous chromosome copies. Aneupolyploidization is accompanied by interchromosomal associations (ectopic conjugation), the appearance of atypical, randomly rearranged chromosomes and single microchromosomes (MCh) absent in normal cells. In the course of ESC cultivation the pool of chromosomal rearrangements and their diversity increase. The most frequent types of chromosomal rearrangements are Robertson translocations resulted in formation of two-arm chromosomes, isochromosomes because of non-disjunction of homologous chromosomes in mitosis, chromosome breaks. Different chromosomes are involved in the rearrangements but chromosomes 8, 10, and 15 participation is non-random. Probably, their implication in chromosome rearrangements have a selective advantage. Frequency of cells with MCh (a morphological manifestation of gene amplification) in the process of ESC cultivation increases. Revealed karyotypical modifications allow to conclude that the mouse ESC may acquire status of transformed cells during cultivation. Факс: (812) 297-03-41,[email protected] 8a.13-P Chromosomal localization of histone H3 gene in the Pacific oyster, Crassostrea gigas K. Bouilly (1) M. Fernandes (1) R. Chaves (1) H. Guedes-Pinto (1) (1) Institute for Biotechnology and Bioengineering, Centre of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro (IBB/CGB–UTAD) The Pacific oyster, Crassostrea gigas (2n=20) is an economically important mollusk species cultured throughout the world. The most frequently used technique for molecular cytogenetic studies is fluo-

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rescence in situ hybridization (FISH) which offers new opportunities for the identification of oyster chromosomes. It has been used to locate simple sequence repeats, satellite DNAs, telomeres or ribosomal DNA sequences. However, regarding chromosome identification, no study has been done with histone H3 gene. Histone H3 is among the most conserved eukaryotic proteins. Most histone H3 genes are repeatedly organized into clusters, which make them an ideal chromosomal marker. In bivalves, little knowledge is available concerning sequence DNA information or the physical mapping of histone genes. The histone H3 gene was mapped on two different pairs of chromosomes, one at an interstitial site on the long arm of chromosome pair 4, and the other on the telomeres of the smaller chromosome pair (pair 10). Polymorphism was detected on the telomeres of pair 10, once it was possible to observe single or double signals. Major ribosomal RNA genes, NORs (nucleolus organizer regions) and C-bands are also localized on the telomeres of pair 10 in C. gigas. In scallops, histone H3 gene was also clustered at two different loci or a single locus depending on the species. Comparative chromosomal mapping should improve our understanding of bivalve genome organization. 8a.14-P Mouse and rat genomes uncover the chromosome restructuring in Peromyscus eremicus (Cricetidae, Rodentia)—Chromosomes 1 and 5 in focus S. Louzada (1) A. Vieira-da-Silva (1) F. Adega (1) H. Guedes-Pinto (1) R. Chaves (1) (1) Institute for Biotechnology and Bioengineering, Centre of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro (IBB/CGB–UTAD) Rodentia are characterized by the occurrence of high rates of chromosomal rearrangements during evolution, when compared to other mammals. This fact is evidenced in the great karyotypic divergence found between rodent genomes, making the reconstruction of the rodent’s ancestral karyotype a challenging task. The Cricetidae genus Peromyscus is revealing to be an interesting model for studying chromosome evolution and speciation in Rodentia, acting as an outgroup to the murid genomes (where both index

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species, Mus musculus and Rattus norvegicus are integrated), assisting in the reconstruction of a more accurate ancestral rodent karyotype. Moreover, some Peromyscus species also revealed to constitute an important model for biomedical research. The cactus mouse, Peromyscus eremicus (PER), presents a karyotype of 48 submetacentric chromosomes. Regarding constitutive heterochromatin (CH), large amounts are found in this genome, being the p-arms of some chromosomes entirely heterochromatic. Throughout Comparative Chromosome Painting chromosomes from PER were analysed using painting probes from Mus musculus and Rattus norvegicus. In this work we present the results for PER1 and PER5. The comparison of the chromosome homologies of these genomes highlighted interesting syntenic associations, being some of them specific for Cricetidae. This preliminary approach revealed some interesting aspects regarding the genome architecture of this Peromiscine species, shedding some light on its evolutionary history.

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of non-L1 sequence throughout the genome); and by affecting gene expression. Different authors suggested that L1 are not transposed randomly and the target sites might be conserved among mammalian species, making them non-homoplasic markers, what increased the use of L1 insertions as phylogenetic markers. In order to make a phylogenetic analysis in the rodents Acomys sp. (Rodentia, Muridae, Deomyinae) and Tateragambiana (Rodentia, Muridae, Gerbillinae), we isolated a fraction of L1 sequence from these genomes and analysed its physical distribution. The same chromosome preparations were then submitted to classical C-banding, allowing its precise chromatin distribution. The isolated sequences were then sequenced and molecularly analysed and compared with LINE-1 sequences available on the NCBI Nucleotide database 8a.16-P Localization of the pentanucleotide telomeric sequence (TTAGG)n in crustacean decapods

8a.15-P Cytogenetic and molecular analysis of LINE-1 sequences in Tatera gambiana and Acomys sp. rodents F. Adega (1) A. Vieira.da.Silva (1) H. Guedes-Pinto (1) R. Chaves (1) (1) IBB/CGB-UTAD It have been demonstrated that repetitive DNA elements contribute to the evolution of Eutherian genomes. Their dynamic nature represents a major force that shapes genome’s size, configuration and plasticity. Almost half of the mammalian genome derives from ancient transposable elements, an interspersed repetitive DNA. “Long Interspersed Nuclear Elements” (LINEs) are an ancient family of autonomous non-LTR retrotransposons, being the LINE–1 (L1) the most recent lineage and the only autonomous non-LTR retrotransposons in rodents. L1 have shaped and expanded mammalian genomes through their own retrotansposition; the assistance in retrotransposition of other mobile elements (providing the machinery necessary for their retrotransposition); transduction (promoting the shuffling

E. Coluccia (1) S. Salvadori (1) F. Deidda (1) R. Cannas (1) A. Deiana (1) (1) Università di Cagliari Telomeres are highly specialized DNA-protein structures that cap eukaryotic chromosome ends. They have a number of functions allowing complete replication at the end of a linear DNA molecule, preventing chromosomal degradation and end-to-end fusion. Although the sequence of the telomeric repeat differs among different organisms, some sequences are conserved in whole taxonomic groups; in fact the human telomere consensus sequence (TTAGGG)n is highly conserved among vertebrates, and the (TTTAGGG) n sequence is frequently found in plants. Telomeres of most invertebrate species have yet to be isolated, although among arthropods different types of telomere sequences have been reported, and the pentanucleotide (TTAGG) motif seems to be widespread among insects and present in other artropods, including crustaceans. In this study, fluorescence in situ hybridization (FISH) technique was used to determine the telomeric sequence in some species of lobsters belonging to the

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Palinuridae and Nephropidae families (Crustacea, Decapoda). Mitotic and meiotic chromosomes were prepared from gonads of adult males by the air-drying technique. Both the human TTAGGG repeat and the TTAGG repeat were used as probe. No hybridization signals were detected with the TTAGGG hexanucleotide repeated sequence; whereas strong signals were observed at the end of the chromosomes with the TTAGG pentanucleotide repeated sequence. These findings are in accordance with the hypotesis that the pentanucletide TTAGG is an ancestral motif of telomeres for arthropods. In fact the TTAGG repeat is widely conserved among insect species and hybridized to DNAs from other arthropods.

This study indicated that both single and two color Fiber-FISH technique is an effective method to investigate transgene integration in transgenic tobacco plants.

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Detection of placental-derived fetal cell-free DNA in the maternal plasma for non-invasive prenatal diagnosis (NIPD) is hampered by the presence of predominant background maternal DNA. It has recently been demonstrated that chromosomal regions that are differentially methylated between placenta and maternal blood cells can be used as markers to distinguish fetal DNA from maternal DNA using methylation-sensitive restriction enzyme treatment followed by Real-time PCR. A differentially methylated region (DMR) on chromosome 18 has been found and developed as a promising marker for NIPD of trisomy 18. In order to search for fetal DNA markers on other chromosomes, we surveyed the human placentae (N=5) and female blood cells (N=5) using Illumina DNA methylation array GoldenGate Cancer Panel I which targets on 1505 CpG sites of the 23 human chromosomes. By comparing the beta-values (representing the methylation levels) of the CpG sites in the placenta and blood cells, we identified 67 DMRs (p<0.001) that are either completely methylated or completely unmethylated in the blood cells with an average of more than 30% difference in DNA methylation level between placentae and blood cells. We further confirmed some of the DMRs by bisulfite pyrosequencing. Several DMRs found here (e.g. APC, RASSF1 and SERPINB5) have been reported in previous studies which verify our study approach. The discovery of DMRs on 19 different chromosomes in this study would provide important resources for developing epigenetic fetal DNA markers for NIPD of aneuploidies.

Application of Fiber-FISH methods for Transgene Analysis in Transgenic Plants S. Aksoy (1) T. Hekimbasi (1) F. Eyidogan (3) H. Oktem (4) H. Budak (2) (1) Gebze Institute of Technology (2) Sabanci University (3) Baskent University (4) Middle East Technique University Transgene integration analysis has been carried out applying Southern Blot and/or PCR methods but these methods are often ineffective becasue of the rearrangement of delivered DNA in the host genome. Furthermore, the Southern Blot technique is time consuming and necessitates more DNA samples and expensive reagents. The fiber-FISH technique is prefered in gene mapping and transgene integration analysis, as DNA-fibers have higher resolution than metaphase chromosomes or interphase chromatin. The aim of this study is to monitor the effectiveness of the Fiber-FISH technique, and to observe transgene localization and behaviors in the host plant genome. We used a quick method to prepare DNA fibers from transgenic tobacco plants and tried to monitor the effectiveness of single and two color Fiber-FISH techniques. Transgenic regions and reference signals on DNA fibers were directly observed by fluorescence microscopy.

9.1-O A Genome-wide Search of Epigenetic Fetal DNA Markers for Non-invasive Prenatal Diagnosis of Aneuploidies R. Yuen (1) M. Peñaherrera (1) P. von Dadelszen (2) M. Kobor (1) W. Robinson (1) (1) University of British Columbia (2) University of British Columbia

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9.1-P Epigenetic alterations and oncogenes mutations in urinary bladder carcinomas Oztürk Ozdemir,1 Esin Yıldız1, Semih Ayan3, Eylem Gul2, Gökhan Gokce3, Fazilet YILDIZ2, Binnur KOKSAL2, Fahrettin Goze1, E.Yener Gultekin3 1 Medical Genetics, 2Department of Pathology, 3 Urology, Faculty of Medicine, Cumhuriyet University, Sivas,Turkey Summary Objective: Aime to investigate the tumor supressor promoter hypermethylation in urothelial neoplasms of bladder. Materials and Methods: PCR based SSCP and methylation-specific multiplex PCR techniques were used for examination of oncogene mutation and promoter methylation status of some tumor supressor genes. Additionally in a total of 83 fresh and paraffinembedded UN specimens were investigated immunohistochemically for c-myc and Ki-67 expression and correlation of these expressions with several variables (grade, stage, pathological behavior). Results: UNs yielded c-myc expression in 75 cases (90.3%) and Ki-67 expression in all of cases (%100) with UNs. Thirty two cases (89%) from non-infiltrating (pTa) 36 UNs revealed c-myc expression. Investigation of infiltrating UNs revealed c-myc expression in 19 cases (95%) of 20 pT1 tumors (n=20) and in 24 cases (89%) of 27 muscle infiltrating pT2 tumors. In this series of UNs, tumor grade for recurrence (p<0.0001) and Ki-67 expression (p<0.0001) for progression to high tumor grade were independent prognostic factors. In PCR-SSCP analysis, there were heterozygous point mutations of the exon 2 of c-myc gene in two cases with high grade, stage (pT2) and proliferation index. Mutated K-ras proto-oncogene profiles were detected in 12 (15%) tumoral specimens examined. Tumor specimens also showed fully and partially methylated pattern profiles in promoter domain for the SFRP2, MGMT and partially hypermethylated profile for the HIC1 TS genes in the current results. Conclusion: These findings strongly support that point mutation in K-ras and c-myc oncogenes and epigenetic alterations of TS promoters may be accepted as combined etiological factors in UNs.

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Key Words: c-myc, K-ras, Ki-67, urinary bladder, epigenetic alterations. 9.2-P DNA methylation profiling of 368 cases of hematological neoplasms using microarrays O. Ammerpohl (1) J. Martin-Subero (1) J. Richter (1) M. Bibikova (2) J. Suela (3) X. Agirre (4) J. RomanGomez (5) F. Prosper (4) J. Cigudosa (3) R. Siebert (1) (1) University Hospital Schleswig-Holstein (2) Illumina Inc. (3) Centro Nacional Investigaciones Oncologicas (CNIO) (4) Clínica Universitaria, Universidad de Navarra (5) Hospital Reina Sofia DNA methylation is involved in several essential cellular processes, including gene regulation and sustaining genome stability. Consequently, alterations in the DNAmethylation pattern are a typical hallmark of cancer, including hematological malignancies. We here present the analysis of the DNA methylation status of 1,505 CpG loci in 368 cases of common subtypes of hematological neoplasms (204 B-cell, 30 T-cell and 134 myeloid tumors) and 30 normal hematopoietic controls (13 normal B-cell, 5 normal T-cell and 12 PB/BM/CD34 samples) using the GoldenGate Cancer Panel I BeadArray (Illumina Inc.). Additionally, a subset of samples has been analyzed using the HumanMethylation27 DNA Analysis BeadChip allowing the analysis of 27,578 CpG loci. Interestingly, we found that lymphoid malignancies were mainly associated with higher levels of de novo DNA methylation than myeloid malignancies and that high levels of de novo DNA methylation seemed to be prevalent especially in germinal center derived B-cell lymphomas like Burkitt lymphoma, diffuse large Bcell lymphoma, and follicular lymphoma as well as precursor lymphoid neoplasias like T- and B- lineage ALL. There is a marked heterogeneity of methylation within subtypes; beside that hierarchical cluster analysis indicates that although some entities tend to cluster together (e.g. myeloid tumors or T-PLL). DNA hypermethylation was more frequent than hypomethylation in all entities analyzed but T-PLL. However, hypomethylation was a recurrent event in all hematopoietic neoplasias analyzed. In contrast to the other hematopoietic malignancies, the number of hypomethylated genes in T-PLL clearly exceeds the number of hypermethylated genes.

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Furthermore, we found a statistically significant enrichment of genes being PRC2 target genes in embryonic stem cells in all hematological tumors investigated, supporting the idea that hematopoietic malignancies might derive from a precursor cell with stem cell-like characteristics. 9.3-P Homozygous hypermethylation of tumor suppressor SFRP2 gene in a case with mucinous anal adenocarcinoma Metin Sen1, Oztürk Ozdemir2, Ahmet Colak3, Mustafa Turan1, Sema Arici4, Doğan Özcan5, Yeşim Yildirim5, Binnur Koksal2 Department of General Surgery 1, Medical Genetics2, Medical Biology3, Pathology4,and Oncology 5 Faculty of Medicine, Cumhuriyet University 58140 Sivas/TURKEY Abstract Homozygous hypermethylated promoter for SFRP2 gene was reported in the current case. Perianal mucinous adenocarcinoma is a rare disease, with poor prognosis.Here we reported a case with fistulaassociated anal adenocarcinoma and evaluated the clinical features, pathology and genetic findings.We found mutations in K-ras proto-oncogene and fully inactivated tumor supressor SFRP2 gene in both tumoral tissue and fecal sample of the proband. It ıs possible that the combined effects of somatic mutations and epigenetic alterations may play a crucial role in development of mucinous anal adenocarcinoma. Key words: Mucinous anal adenocarcinoma, K-ras mutation, epigenetic alterations.

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dependent DNA helicase 2 subunit 2 (EC 3.6.1.-), and shown that this antibody could be used in certain conditions as a proliferation marker for cells of different origin (Hybridoma. -Not available-, ahead of print. doi:10.1089/hyb.2008.0077). The subcellular localization of the Ku80 antigen had a clear dependence of the fixation methods of cells. We have compared the nonpermeabilized human melanoma cells (Bowes cell-line) fixed with 8% PFA with methanol fixed cells, and shown that in not permeabilized cells the Ku80 antigen was detected only on mitotic cells of different stages. However, in cells fixed with methanol the Ku80 antigen was seen in all stages of the cell cycle including interphase. In interphase cells it was distributed over the nuclei showing some structural heterogeneity, but no signs of cytoplasmic or surface localization could be seen; in the late prophase the Ku80 antigen was still in the nuclei, and bound to some chromosomal parts; in very late prophase/very early metaphase the Ku80 antigen filled the cytoplasm and remained still partly connected to some chromosomes. In metaphases, anaphases, and early telophases the Ku80 antigen was located in the cytoplasm. In very late telophase/early interphase the antigen was detected both in the cytoplasm and newly forming cell nuclei. In interphase cells there was no signs of the Ku80 localization outside of cell nuclei. However some authors have observed and discussed the possibility of Ku80 localization in cytoplasm and cell membrane in interphase cells before mitosis. It means that the problem needs further investigation in different cellular systems. This work was partly supported by target financing SF 0188096s08 of Estonian Ministry of Science and Education and Estonian Science Foundation grant no. 6581. 9.5-P

9.4-P The subcellular localisation of Ku80 during mitotic cycle—an immunocytochemical analysis using anti Ku80 Mab F10H2.B3 A. Mikelsaar (1) A. Mikelsaar (2) A. Sünter (1) P. Toomik (1) K. Karpson (2) R. Mikelsaar (1) E. Juronen (1) (1) Institute of General and Molecular Pathology (2) LabAs Ltd. We have described a new mouse monoclonal antibody (named F10H2.B3) which recognizes Ku80 ( ATP-

MGMT and p15 promotor methylation—prognostic parameters for glioblastoma patients? S. Wemmert (1) R. Ketter (1) J. Rahnenführer (2) K. Kammers (2) W. Steudel (1) S. Urbschat (1) (1) Saarland University (2) Technical University Dortmund Glioblastomas are the most common malignant brain tumors. Surgical cure of these tumors is virtually impossible and their clinical course is mainly determined by the biological behavior of the tumor cells

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and their response to radiation and chemotherapy. Additionally, these tumors show substantial genetic changes and a remarkable large intratumoral and intertumoral genetic heterogeneity, which obviously substantially influence the therapy success. Over the past years aberrant DNA methylation was shown to be a common molecular lesion in human tumors which had also impact on patient prognosis and treatment response. In glioblastoma patients, epigenetic silencing of the promotor gene region of the O6-methylguanine-DNA methyltransferase MGMT is shown to be predictive of response to temozolomide chemotherapy. The aim of our current study was to investigate the methylation status of candidate genes p15, p16 and 14ARF on the frequent deleted region on chromosome 9, and MGMT on chromosome 10 and correlate these results with the clinical data. Therefore, DNA was isolated from fresh frozen GBM biopsies (N=27) and modified by sodium bisulfite. Promotor methylation of p15, p16, p14ARF and MGMT was analyzed by MS-PCR. We observed promotor hypermethylation of MGMT in 56%, and of p15 in 37% of the tumors, whereas hypermethylation of p16 and p14ARF was rare. Interestingly, hypermethylation of p15 emerged as a significant predictor of a shorter overall survival (16,9 vs. 23,8 months, P=0,025; Log-rank test), whereas MGMT hypermethylation had no effect on median overall survival in our patient cohort (22,5 vs. 22,1 months, P=0,49; Log-rank test). In the presence of other clinically relevant factors (age, KPS, sex, MGMT), p15 hypermethylation remains the only significant predictor in the investigated tumors (P=0,021; Cox regression). Although these results need to be confirmed in larger series, our retrospective study reveals that p15 hypermethylation can act as an additional important prognostic factor for survival in glioblastomas. 9.6-P DNA methylation pattern of metaphase chromosomes from human cytotrophoblast cells A. Kaminskaya (1) A. Pendina (2) O. Efimova (1) T. Kuznetzova (2) V. Baranov (2) (1) Saint-Petersburg State University (2) D.O.Ott’s Institute of Obstetrics & Gynecology RAMS

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DNA methylation pattern was studied in metaphase chromosomes from human placenta cytotrophoblast at 5–20 weeks of gestation with total number of 76 metaphases from 9 individuals analyzed. Samples were obtained after prenatal diagnosis or medical abortions. Indirect immunofluorescence technique with monoclonal antibodies against 5methylcytosine (Eurogеnteс, Belgium) was applied to detect regions of 5-methylcytosine-rich DNA in chromosomes. QFH/AcD-banding technique was performed for chromosome identification. All chromosomes showed band-specific methylation pattern, with 5-methylcytosine-rich DNA preferentially accumulated in R- and T-bands and in the short arms of acrocentric chromosomes. Band-specific 5-methylcytosine pattern was quite similar to MeCbanding in metaphase chromosomes from adult human lymphocytes (Pendina et al., 2005) with some exceptions. The blocks of pericentromeric heterochromatin of chromosome 9 were hypomethylated at 5–8 and at 18–20 weeks of gestation and methylated at 10– 14 week (4, 3 and 2 samples accordingly). We have also detected differences of fluorescence intensity between homologous chromosomes in number of regions: 1q32, 4p12–14 (in all samples studied), 13q32–34, 16q 11.2 (2 samples at 18–20 weeks) and 6p21 (4 samples at 5–8 weeks). Functional peculiarities of these loci, due to parental origin of homologous chromosomes could be suggested as a possible explanation of this phenomenon. 9.7-P Immunocytochemical study of acetylated histone H3 distribution in human metaphase chromosomes O. Efimova (1) J. Lezhnina (1) A. Pendina (2) A. Voskresenskaya (1) T. Kuznetzova (2) V. Baranov (2) (1) Saint-Petersburg State University (2) Ott’s Institute of Obstetrics and Gynecology RAMS Acetylation of 9th lysine residue in histone H3 (AcH3K9)—one of epigenetic modifications of human genome—is known to be involved in gene regulation and is associated with active chromatin. We have studied distribution of chromatin regions, enriched in AcH3K9, in human metaphase chromosomes. PHA-stimulated human lymphocytes were fixed with ethanol, containing 2%

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of glacial acetic acid. 35 metaphases from 4 individuals were enrolled in the study. Immunofluorescence with specific monoclonal antibodies against AcH3K9 (Abcam, USA) revealed on metaphase chromosomes some clearly distinguishable regions with bright, medium, weak or absent fluorescence. Combined DAPI and immunofluorescent staining allowed co-localizing regions enriched/depleted in AcH3K9 with Q-negative/ positive bands (or R/G-bands). AcH3K9-enriched regions corresponded to some R-bands: 1p36.3, 1p13, 1p34, 1p36.1, 2q11.2, 3p21, 5q31, 5q33, 6p21, 7q22, 8p21, 11p11.2, 11p15, 11q13, 13q12, 13q14, 15q22, 15q24, 15q26, 16p11.2, 17q21, 19p13, 19q13, 20q11.2, 22q11.2, 22q13. Average to weak signal was detected in both R- and Gbands, with a tendency of R-bands being more enriched in AcH3K9 than G-bands. In heterochromatic blocks of chromosomes 1, 9, 16 and Y as well as in the short arms of acrocentric chromosomes of D and G groups immunofluorescent signal was absent indicating no H3K9 acetylation in these regions. Thus, the distribution of AcH3K9-enriched chromatin could be treated as a functional banding pattern specific for every homologous pair and reflecting strict order of active and inactivated chromatin regions in chromosome arms.

of 2 Gy. Quantification of g-H2AX, was assessed at recovery period of time (30 min, 2 h, 5 h and 24 h) after irradiation. For each incubation time at least 200 interphase nuclei were analyzed for the presence of g-H2AX foci. The mean incidence of g-H2AX /cell in unirradiated control fibroblasts was 1.34± 0.37, wheras in unirradiated FA cells it was 1.06 ± 0.36 and in AA group 3.26 ± 0.56, respectively. In irradiated fibroblasts, the maximum of induced g-H2AX /cell was found in FA cells 30 min (22.62± 3.03) after irradiation, significantly higher compared to controls (t=3.34, p= 0.01). Similar results were obtained at other recovery periods of time (2 h, 5 h, 24 h). The level of radiation-induced g-H2AX foci in AA group was similar to control fibroblasts 30 min after irradiation, whereas at other recovery time periods the incidence of g-H2AX foci was higher compared to control, and lower as compared to FA cells. Statistically significant difference between incidence of g-H2AX foci in FA cells compared to AA and controls was observed, particulary 24 h after irradiation (t=4.4, p< 0.006; t=3.2, p< 0.04) . This finding is clearly a manifestation of delayed repair kinetics in FA cells. The g-H2AX assay in FA cells should be further examined with potential to be used as additional assay in diagnostic of FA cellular phenotyp.

9.8-P

9.9-P

Gamma H2AX foci as possible tool in diagnostics of FA cellular phenotype

Tumoral tissue specific promoter hypermethylation of

G. Joksic (1) D. Vujic (2) M. Guc-Scekic (2) A. Leskovac (1) S. Petrovic (1) I. Joksic (1) P. Slijepcevic (4) (1) Vinca Institute of Nuclear Sciences (2) Institute for Mother and Child Health Care (3) Brunel University West London

Sulhattin Arslan1, Tamer Dogan1, Binnur Koksal2, Malik Ejder Yildirim2, Cesur Gumus3, Sahenda Elagoz4, Ibrahim Akkurt1, Oztürk Ozdemir2 Department of Thorax Diseases1, Department of Medical Genetics2, Radiology3, Pathology4, Faculty of Medicine, Cumhuriyet University 58140 Sivas/ TURKEY

Fanconi cells are highly sensitive to DNA crosslinking agents but their response to ionizing radiation is still unclear. This aimed us to investigate in vitrocellular radiosensitivity of Fanconi anemia (FA) and aplastic anemia (AA) primary fibroblasts, and corresponding controls employing enumeration of g-H2AX foci. All cell lines were irradiated in vitro in exponential growth phase using 60Co g-rays, radiation dose

Summary Objective: Non-small cell lung carcinoma is an aggressive condition, and epigenetic alterations of some tumor supressor genes have been reported for different tumor types. Case Presentation: A 43 years old male with NSCLC on the lower segment of the right lung is presented. The patient underwent a diagnostic excisional

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thin-nidlee biopsy for the histological confirmation. We examined the promoter methylation status of some distinct tumor supressor genes in tumoral and blood tissues of a case after sodium bisulfite conversion and DNA amplification with methylation specific multiplex PCR technique. Both tissues were also examined for G to A transitions in codons 12 and 13 of the K-ras protooncogene. Results: Tumor specimen showed full methylation pattern profiles for the SFRP2, p16, DAPK1 and partially hypermethylated profile for the p53 and MGMT genes in a case with non-small lung carcinoma. Blood specimen showed normal hypomethylated profiles for all studied TS genes. The K-ras proto-oncogene was in normal structure both in blood and tumoral specimens examined. Conclusion: Results indicate that genes exhibit tumor suppressor activities in blood, but exhibit epigenetic inactivation in carcinoma cell. These findings strongly support the hypothesis that epigenetic mechanisms play an important role in the non-small cell lung carcinogenesis in human. Key Words: Non-small cell lung carcinoma, tumor suppressor genes, epigenetic alterations 10.1-P Odds in teaching clinical cytogenetics to first year medical students

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than to discuss in groups of 4–6 students what they learned from the articles, the difficulties encountered, what they liked and disliked. After all the 15 practicals in genetics, at the end of their practical examination (where one of the questions was to explain a paragraph from a studied article) they received questionnaires about how they perceived this different way of getting acquainted with information in cytogenetics, about how they understood the articles after receiving the same information from the teacher, and after preparing for the examination and if they found the method useful. 62.22% of students considered it useful, 24.44% considered it difficult, indicating the necessity of first teaching in a classical frontal way and only than, to deepen the knowledge through discussing different articles. This new method of teaching cytogenetics proved its advantages and disadvantages from the students’ as well as from the teacher’s point of view. 10.2-P The impact of the introduction of automation on the throughput of blood samples in the Postnatal section of the West of Scotland Regional Cytogenetics Service

R. Dragotoiu (1) (1) Medical and Pharmacy University

L. Monkman (1) J. Colgan (1) L. Crawford (1) M. Campbell (1) G. Lowther (1) (1) NHS Greater Glasgow and Clyde

There is a continuous interest in achieving a high quality teaching process in what concerns the way information is brought to students and also how this is appreciated for being memorized and than used in future activities. Quality assessment is carried out through a series of transparent actions of designing, planning and implementing educational programs which comply with quality standards. The mechanisms for quality assurance and assessment in education are mainly focused on the learning outcomes and their application is based on the qualifications awarded, but students themselves have to adapt to new teaching techniques and develop new skills in dealing with a high amount of information. From the 106 students included in the study 90 answered questionnaires. The students received articles on cytogenetic studies during their first 3 workshops of practical training (“practicals”). They were asked to read about the subjects before coming to the practicals and

The West of Scotland Regional Cytogenetics Service provides a service for approximately 3 million people and processes around 6500 analyses per year. Of these, around 2800 are postnatal referrals. Over the last year, several automated techniques have been introduced into the postnatal section. These have included the use of: a banding machine, coverslip machine, and scanning and image capture system. The impact of these new technologies has been measured by comparing the quality, reporting times, and failure rates over the initial 6 month period after the implementation of the new systems and the same 6 month period from the previous year. An automated harvesting machine and a humidity and temperature controlled chamber for slide making are currently being integrated into the postnatal system which we anticipate will further improve the service to our population.

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10.3-P Determination of distribution of chromosomes in the spreads n. Koçak (1) F. Özen (2) H. Acar (3) (1) Konya Training and Research Hospital (2) Cumhuriyet University Medical Faculty (3) Selcuk University Meram Medical Faculty Background: Classical cytogenetic applications are inflated cells with hypotonic liquids then by using fixative liquids make cell membrane is stable.These cells are then multiplied by the high drop spreading on a thin plate is providedIn this way chromosomes are spread on the slide. At first glance, it is believed that the spread of the random. The position of the cell core of the chromosome is not randomly know. random distribution of chromosomes on the slide so Can I? Purpose: chromosomes to determine the position of and away from each other and are together to calculate the probability. Methods: We have used examples of karyotype patients. Obtained from 100 different people have used karyotype examples.Arms of chromosomes pq and centromere regions consider measurements were made by. Results: Our work is still continuing Key Words: Chromosomes, karyotype, centromer 11.1-O Chromosomal rearrangements, mosaicism and heteromorphism in children with idiopathic autism Y. Yurov (1) S. Vorsanova (2) I. Iourov (1) V. Kravets (2) I. Demidova (2) A. Beresheva (2) A. Kolitii (2) V. Voinova-Ulas (2) N. Gorbachevskaya (1) (1) National Research Center of Mental Health, RAMS (2) Institute of Pediatrics and Children Surgery, Rosmedtechnologii Autism is one of the most devastating psychiatric disorders in childhood and is characterized with impaired social interaction and communication, restricted repetitive and stereotypic behavior, interests and activities. The condition occurs more frequently in males (2-3/1000)

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with male/female ratio about 4:1. Numerous biological and environmental factors with strong evidences for genetic background have been suggested to play a role in pathogenesis of autism. Autism is frequently associated with chromosomal abnormalities or monogenic disorders. However, the role of subtle genomic imbalances in autism has now been highlighted delineated. This study was aimed to compare the incidence of chromosomal rearrangements, mosaicism and heteromorphism in peripheral blood cells of 120 children with idiopathic autism and 60 unaffected controls. 4 autistic children (3,3%) have rare structural chromosomal abnormalities: (46,XY,t(1;6)(q42.1;q27); 46,XY, inv(2)(p11q13); 46, XY,r(22)(p11q13) and 46,XY,der(6),ins(6;1)(q21; p13.3p22.1)pat. Having studied more than 420 000 cells in 60 controls and 116 children with idiopathic autism using tricolor FISH with chromosome enumeration DNA probes, we determined that 19 (16%) of 116 children with idiopathic autism have low-level mosaicism involving chromosome X, 9,15 and 18. C-banding and quantitative fluorescent in situ hybridization (FISH) with DNA probes to variable heterochromatic regions were used to demonstrate a significant increase in the frequency of variations in the heterochromatin regions of chromosomes in children with autism as compared with a control group (48% and 16% respectively). Pericentric chromosome inversion 9phqh was not characteristic of patients with autism, while variation in heterochromatin regions 1phqh, 9qh+, and 16qh− were found significantly more frequently in children with autism. Our findings identify low-level mosaic aneuploidy and heteromorphism of heterochromatic regions as new genetic risk factors for autism. This study was supported by RGNF (060600639a, Russian Federation). 11.2-O Evidence for germline mosaicism and complex rearrangement breakpoints in interstitial 1p36 deletions M. Gajecka (1) M. Gajecka (2) L. Campbell (3) T. Shaikh (4) A. Gentles (5) K. Ciprero (3) J. Ming (3) L. Shaffer (6) S. Saitta (3) S. Saitta (4) (1) Polish Academy of Sciences (2) Washington State University Spokane (3) The Children’s Hospital of Philadelphia (4) University of Pennsylvania School of Medicine (5) Stanford University (6) Signature Genomic Laboratories, LLC

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Deletions of 1p36 occur in approximately 1 in 5,000 newborns. In monosomy 1p36 four possible classes of rearrangements have been identified: pure terminal deletions, interstitial deletions, unbalanced translocations, and complex rearrangements. Here we report two siblings with mild phenotypic features of the monosomy 1p36 syndrome, and interstitial deletions of 1p36 detected by array-based comparative genomic hybridization (aCGH). These small interstitial deletions were not detectable by conventional subtelomeric FISH analysis, and both array CGH, and oligonucleotide array analysis indicated that the recurrent deletions were identical. Parental phenotypes and molecular analysis by FISH and aCGH were normal, suggesting germline mosaicism. To further investigate the mechanism, the breakpoints were cloned and sequenced. Our results show high complexity at the breakpoint junctions. Although the causes of the chromosomal breaks that initiate the interstitial deletion formation are unknown, the joining mechanism is consistent with features of the nonhomologous endjoining (NHEJ) pathway. This is the first report of germline mosaicism for this disorder. Our findings therefore, have significant implications for diagnostic approaches and for recurrence risk counseling in families with a child with monosomy 1p36. 11.1-P Studies of numeric chromosomal abnormalities in 627 spontaneous human abortions by interphase FISH S. Vorsanova (1) A. Kolotii (1) I. Iourov (2) E. Kirillova (1) V. Monakhov (2) O. Kurinnaya (1) I. Demidova (1) A. Beresheva (1) V. Kravetz (1) Y. Yurov (2) (1) Institute of Pediatrics and Children Surgery, Rosmedtechnologii (2) National Research Center of Mental Health, RAMS Chromosome abnormalities (CA) are the most common genetic cause of spontaneous abortions (SA). It has been documented that mosaic forms of aneuploidy due to developmental chromosome instability are extremely frequent in fetal tissues (Vorsanova et al., 2007). To test these observations in context of SA, we have evaluated 627 consecutive cases of SA by interphase FISH. Chorionic villi of 627 SA of 5–15 weeks gestation (mean 8.6 weeks) from women aged from 16 to 47 years (mean

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31 years) were processed for molecular cytogenetic analysis. Interphase multiprobe FISH with arbitrarily selected DNA probes for chromosomes 1, 9, 13/21, 14/ 22, 15, 16, 18, X and Y was applied. No less than 500– 1000 nuclei for each probe/sample were analyzed. Numerics CAs were detected in 365 (58.2%) cases. Multiple aneuploidies were found in 29 (8%) cases. Trisomies involving different autosomes were found in 45%, among them for chromosome 16–18.2%; chromosomes 13 and 21–7.9%; chromosomes 14 and 22–8.9%; chromosome 18–3.1%; chromosome 15–5.5%; chromosome 9–1.4%. Aneuplody involving chromosome X was found in 27% including regular monosomy X in 9.6%. Polyploidy was found in 22.9% of cases. Mosaic forms of autosomal monosomy were found in 5.1% cases. This study revealed high incidence of mosaic form of chromosomal pathology (50.3%) in SA. Sex ratio in this group was 0.7, among cases of undetected chromosomal pathology—1.0. Mosaic forms of aneuploidy especially involved chromosome X and polyploidy more often found in females (sex ratio was 0.49). Interphase FISH could be successfully used for rapid and cost-efficient identification of regular and mosaic forms of numeric chromosomal abnormalities in about 50% of SA. However, array-based approaches might be applied for additional screening of cryptic chromosome imbalances in SA which were negative in FISH analysis. This evidences for existence of developmental mitotic instabilities that associate with spontaneous fetal death. Supported by Philip Morris USA Inc. 11.2-P Mosaic expression of chromosome instability in the ataxia telangiectasia brain I. Iourov (1) S. Vorsanova (2) A. Kolotii (2) M. Tagirova (1) T. Liehr (3) Y. Yurov (1) (1) National Research Center of Mental Health, RAMS (2) Institute of Pediatrics and Children Surgery, Rosmedtechnologii (3) Institute of Human Genetics and Anthropology The aim of study was to address chromosome instability in the brain tissue cells of ataxia telangiectasia (AT)—chromosome instability and neurodegenerative disease characterized by mutations in a gene (ATM) controlling response to double strand DNA. A paradoxical feature of AT is that progressive neuro-

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degeneration affects the cerebellum, while other brain regions are less affected. We have monitored stochastic aneuploidy and interphase chromosome damage in the normal and diseased brain by interphase FISH and interphase chromosome-specific multicolor banding scoring about 980,000 cells of the AT brain, including the frontal cortex and the cerebellum. The mean frequency of aneuploidy for arbitrarily selected chromosomes 1, 7, 8, 9, 11, 16, 17, 18, X and Y in the AT brain was 2.2±0.7% for the cerebrum and 1.7±0.8% for the cerebellum. The percentage of aneuploid neural cells was significantly lower in the diseased cerebellum versus the diseased cerebrum (p<0.001) indicating for selective loss of aneuploid cells in the degenerating cerebellum. However, aneuploidy frequency involving chromosome 14 were dramatically increased in the AT cerebellum (from 2.5 to 19% in different AT brain samples). Additionally, the AT cerebellum possessed non-balanced breaks in chromosome 14 and, to lesser extend, chromosomes 7 and X, which varied in an age-dependent manner (from to 2% to 24%). These results indicates that chromosome instability has mosaic expression in different AT brain region. Targeted neurodegeneration in AT may be associated with extended aneuploidization and chromosome 14 specific damage and aneuploidization of the cerebellum. Our findings demonstrate that mosaic genome instability is a more significant genetic factor in the pathogenesis of brain disorders than previously recognized. Supported by Philip Morris USA Inc. 11.3-P Low level mosaicism of chromosome X aneuploidy (low level gonosomal mosaicism = LLGM) in human reproduction assessed by fluorescent in situ hybridization (FISH)

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impairment. Association of this minority mosaicism with infertility has not been proved yet, because the same aberration is also detectable in healthy population. As the most frequently used chromosomal analysis of cultured lymphocytes has several inherent limitations, such as the number of cells examined and artefacts caused by in vitro culture conditions, employment of additional methods provides more complex insight into this problem. Method: Cytogenetic analysis was performed in a group of females (n=70; mean age 30; range 19–42) treated for infertility and in the control group women (n=86; mean age 33; range 25–46) with at least two spontaneous conceptions without a history of miscarriage. FISH was performed on cultured lymphocytes in 65 and 46 cases resp. and on uncultured lymphocytes in 51 and 41 cases resp. T and Mann-Whitney tests were used for statistical analysis. Results: Number of cytogenetically detected mosaic cases did not significantly differ between tested group— 18,57 % and control—14,11 % (χ(1)2 =0.056288). Frequency of cells with aneuploidy X detected by FISH was significantly higher in the tested than in the control group in samples of cultured lymphocytes (p= 0.001233) and also in samples of uncultured lymphocytes (p=0.00000). X-aneuploidy cells were more abundant in uncultured than in cultured lymphocytes in the tested group (p= 0.000139) but not in the control group (p>0.05). Conclusion: LLGM is more frequent in a population of infertile females when examined by FISH. Whether LLGM is a constitutional aberration or change gained as a consequence of physical condition of an individual is unclear. I-FISH on uncultured lymphocytes is recommended as a more reliable method, as it avoids artificial changes caused by in vitro conditions. 11.4-P

P. Capkova (1) A. Sobek jr. (2) A. Sobek sr. (2) K. Adamova (1) B. Hladikova (2) M. Krskova (3) R. Chytilikova (4) J. Santavy (1) (1) University Hospital of Palacky University Olomouc (2) Fertimed, Private Centre of Assisted Reproduction, Olomouc (3) IT Centre of Palacky University Olomouc (4) Faculty of Sciences Palacky University Olomouc LLGM is the most prevalent chromosomal aberration in the population of females treated for reproductive

Large inv dup (15) characterized by FISH and aCGH in a child with no abnormal phenotype at 2 years old A. Guichet (1) P. Boisseau (2) O. Ingster (1) P. Guardiola (3) A. Couteleau (3) D. Bonneau (1) (1) Hospital Inv dup(15) can be classified into 2 major groups according to size, determined by presence or absence

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of Prader -Willi/Angelman syndrome critical region (PWACR). Small inv dup (15), not containing PWACR seems to have no phenotypic effect where as large ones, containing 2 or more extra copies of the 15q11q13 region, are associated with abnormal phenotype. In those cases, inv dup(15) are maternal in origin. We report on a prenatal observation with a mosaic marker confirmed after birth. The proband is the second child of a young, healthy and unrelated parents. Prenatal diagnosis performed due to increased fetal nuchal translucency at 22 weeks of gestation indicated cytogenetic mosaicism for a supernumerary marker. The low level of mosaicism did not permit to identify it: 47,XX,+mar[3]/46,XX [52] .Normal karyotypes were found in both parents. After birth, the karyotype was analysed on different tissues and all confirmed mosaicism for the marker: 45% for blood sample and 25%for fibroblasts. FISH studies with commercial probes and BACs probes from 15q11q13 region allowed us to characterize the marker as an inv dup (15)(q11q12) including the Prader-willi/angelman critical region. Moreover, molecular studies for parental origin demonstrated a paternally derived inv dup (15). The observation was further validated by CGH array. Single-nucleotide polymorphisms aCGH analysis was carried out using Human 610Quad v1.0 DNA analysis (Illumina*) to precisely identify the breakpoints involved in the formation of the marker and gene contents of this duplication. At 2 years old the young girl has a normal phenotype with no dysmorphic features and no psychomotor retardation. Conclusion: we report on a young child with a large mosaic inv dup(15) whose paternal origin was rarely reported and supposed to be the explanation for the good psychomotor development of this child.

Despite the fact that double aneuploidy belongs to the first aberrations discovered in man it is a very rare phenomenon particular when observed among live newborns. In the majority it is a combination of an autosomal and a gonosomal aneuploidy, whereby some combinations do appear more frequently than others. To our knowledge, there is only one previous report of a mosaicism associating a trisomy 13 and a trisomy 21 in the same individual. Our proband was the third child of healthy parents (maternal age: 40 years). He exhibited facial dysmorphism (upslanting palpebral fissures, epicanthal folds, short nose) but initially a genetic syndrome was considered unlikely because of normal growth and development. At the age of 8.5 months developmental delay was noted and a chromosome analysis initiated. He still showed body measurements in the high normal range and had no neurological problems or inner organ abnormalities. The cytogenetic studies were performed on peripheral blood and buccal mucosa, and revealed a mosaic 47,XY,+13/47, XY,+21(43–62%/38–57%) karyotype. A normal cell line was not seen. FISH analyses with a chromosome 13/21 centromeric probe demonstrated centromeric polymorphisms in the patient and in both parents: 13cenh+mat and 21cenh-pat. Microsatellite STR segregation analysis confirmed the maternal origin of the additional chromosome 13 and the paternal derivation of the additional chromosome 21, and excluded a chimerism. Mitotic non-disjunction is a major mechanism in the causation of mosaicism. In case of a complex mosaicism more than one segregation error must have happened. Little is known about the underlying processes, but recent studies suggest that a common maternal agerelated mechanism could be implicated in single and also in double trisomy cases. Our results might confirm the hypothesis that centromeric alterations may also contribute to an abnormal chromosomal segregation.

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A rare case of autosomal double aneuploidy mosaicism—associated with relatively mild phenotype and centromeric polymorphisms of the chromosomes involved

Molecular cytogenetic characterization of rare mosaic karyotype associated with chromosome 21 monosomy clone and cell line with ring chromosome 21: three new cases

H. Schüler (1) A. Roos (1) R. Raff (2) M. Elbracht (1) S. Rudnik-Schöneborn (1) (1) Institute of Human Genetics (2) Institute of Human Genetics

A. Polityko (1) E. Goncharova (1) J. Jaroshevich (1) O. Khurs (1) L. Isakovich (1) K. Mrasek (2) T. Liehr (2) (1) National Medical Center “Mother and Child” (2) Institute of Human Genetics and Anthropology

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Mosaic combination of clones with monosomy of chromosome 21 and with partial monosomy due to ring chromosome 21 is a very rare finding in human karyotype. Here we present on two postnatal cases and one prenatal case with mentioned combined karyotype abnormality. Case 1. Proband was 3 months old female (G1;P1at term, BW=2690; BL=51 cm). Follow up examination at the age 1 year 3 months showed mental and growth retardation, multiple congenital malformations (microphthalmia, cataract, corneal opacity, cerebellum abnormality, heart defect (ASD), craniofacial dysmorphias). Cytogenetic study revealed karyotype: 45,XX,-21[160]/46,XX,r(21)(p11q22)[160]. Case 2. 3 months old male infant (G3; BW=2350; BL=46 cm). At the age 1 year 1 month he presented mental and growth retardation, neurological features, craniofacial dysmorphias, microcephaly, cardiac murmur. The karyotype was characterized as: 45,XY,-21[84]/46,XY,r(21)(p11q22)[170]. Case 3. Karyotype of the fetus with anomaly of left kidney (ultrasound examination) was studied prenatally using amniotic fluid cells culture at 20 weeks of gestation: 45,XX,-21[3]/46,XX,r(21)(p11q22)[43]. Molecular cytogenetic analysis using comprehensive FISH approaches was performed to characterize the constitution of r(21), breakpoints of the ring chromosome formation events and mosaic status of the karyotypes. We present the molecular cytogenetic data of ring chromosome constitution and mitotic stability, genotypephenotype correlation analysis and the review of literature. [Supported in parts by DAAD 325/2008, DFG WER 17/01/07]. 11.7-P Characterization of mosaic supernumerary marker chromosome using MFISH: origin from chromosome 1,16 and 17 B. Lee (1) S. Park (1) M. Lee (1) J. Kim (1) J. Park (1) J. Han (2) I. Kang (2) K. Yang (2) H. Ryu (2) (1) Cheil General Hospital & Women’s Healthcare Center (2) Cheil General Hospital & Women’s Healthcare Center

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(for oral presentation) Bom-Yi Lee1, So-Yeon Park1, Moon-Hee Lee1, Jin-Woo Kim1, Ju-Yeon Park1, Jung-Yeol Han2, Inn-Soo Kang2, Kwang-Moon Yang2, Hyun-Mee Ryu1,2 1 Laboratory of Medical Genetics, Cheil General Hospital & Women’s Healthcare Center, 2 Department of Obstetrics and Gynecology, Cheil General Hospital & Women’s Healthcare Center, Kwandong University College of Medicine, Seoul, Korea We describe four cases with varying degrees of mosaicism for supernumerary marker chromosomes (SMCs) in pre- and/or post-natal diagnosis. Individual marker chromosome of each case was analyzed by multicolor fluorescence in-situ hybridization (MFISH). Case 1, a man referring his spouse for habitual abortion had a mosaic marker chromosome in lymphocytes (53.21%), which was derived from the mostly heterochromatic region of chromosome 1 confirmed by FISH with a whole chromosome paint 1 (WCP 1) probe after MFISH analysis. In case 2 and 3, both mosaic marker chromosomes were derived from chromosome 16 identified by MFISH in prenatal diagnosis. The marker chromosome of patient 2 appeared in 90.48% of cultured amniocytes and was positive for Da-DAPI staining and FISH for all human centromere probe and was paternally inherited. Case 3 referring for high risk of neural tube defect (NTD) was ascertained two types of SMCs in amniocytes and cord blood cells. Both SMCs were distinguished from monocentric and dicentric form stained by Da-DAPI and CBGbanding. In case 4, we found de novo SMC shown to be a mosaic state in cultured amniocytes with 27.43% and in the repeated amniocentesis with 41.27%. After birth, one–month female baby with mildly developmental delay had SMC in a mosaic state (44.35%) in cultured lymphocytes, which were variable in size and shape. To confirm SMCs, we studied the combined use of comparative genomic hybridization (CGH) and FISH/MFISH. The marker chromosomes should be proximal region of chromosome 17 centromere. MFISH have facilitated to efficiently characterize the nature of marker chromosomes and would provide more accurate genetic information in diagnosis.

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11.8-P Trisomy 20: a rare case detected in CVS, fetal blood and other fetal tissues E. Cuatrecasas (1) C. Morales (3) C. Garrido (1) E. Peruga (3) L. Vila (1) V. Català (1) M. Farré (2) A. Soler (3) A. Serés-Santamaría (1) (1) Prenatal Genetics. Barcelona (2) Eugin. Barcelona (3) Servei de Bioquímica i Genètica Molecular. Hospital Clínic. Barcelona A complete trisomy 20 is not a viable trisomy and fetuses surviving beyond the first trimester are very rare, although trisomy 20 in mosaic is one of the more common trisomies detected in amniocentesis. Trisomy 20 is virtually never confirmed in newborn blood and rarely in other fetal tissues samples. We report a case of a 41 years old woman who was referred for a prenatal diagnosis at 11+5 week’s gestation. The fetal karyotypes in short-term and long-term culture of chorionic villous sample were a non-mosaic trisomy 20. A subsequent karyotype of the amniotic fluid sample at 14+6 week’s gestation was also a non-mosaic trisomy 20. However, the karyotypes from the terminated fetus were all mosaics: 3% in one culture of fetal blood, 4% in other culture of fetal blood, brain 4%, skin 68% and 96% in kidney. FISH with chromosome 20 probe showed a 7% mosaic of trisomy in fetal blood sample. Autopsy revealed a camptodactylia and right pulmonary isomerism, and the length and the weight were normal for the gestational age, no other major malformations were found. This case shows the distributions of trisomy 20 in different tissues. So far, this is one of the few cases reported with mosaic of trisomy 20 detected in CVS and fetal blood associated to minor malformations. 11.9-P Cri-du-chat Syndrome: newborn exhibiting 5p deletion and 5p deletion with an inverted duplication in mosaicism R. Almeida (1) F. Boieiro (1) C. Alves (1) B. Marques (1) C. Ferreira (1) M. Silva (1) C. Ventura (1) M. Santos (2) T. Tomé (2) H. Correia (1) (1) Instituto Nacional de Saúde Dr Ricardo Jorge (2) Maternidade Dr Alfredo da Costa

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The Cri-du-Chat Syndrome is a genetic disorder associated with a variable size deletion of the short arm of chromosome 5 (5p-). The incidence is 1:15.000 to 1:50.000 livebirths and the first diagnostic feature of this syndrome was a characteristic cat-like cry.This characteristic cry might be in association with microcephaly, round face,hypertelorism, micrognathia, prominent nasal bridge, epicanthic folds, hypotonia, and severe psychomotor and mental retardation. Deletions and duplications of chromosome 5p have been described in literature. Approximately 85% are de novo deletions, mosaicism represents 3% of the described cases, and inverted duplications with deletion are very rare. We report a case of a newborn with hipertelorism, slight axial hypotonia, upper limbs hypertonia, without Moro reflex and cat-like cry, with two abnormal cell lines: one line with a 5p deletion and another with a 5p deletion concomitantly with an inverted duplication 5p, both confirmed by molecular cytogenetic analysis. Parental karyotypes were normal. Cri-du-chat syndrome is a clinical entity that may present a great phenotypic variability.Reports of cases with 5p deletions concomitant with duplications are very rare. These two mechanisms seem to have different phenotypic outcomes and patients that present them simultaneously appear to have a relatively milder phenotype such as the present case. We hope that our results may contribute to a better understanding of this syndrome. 11.10-P Up to 6 supernumerary marker chromosomes in a child with mild developmental delay - Complexity of genetic counselling N. Le Du (1) P. Chambon (2) V. Drouin-Garraud (3) G. Joly-Helas (2) P. Mabboux (1) I. Gilles (5) S. Serero (1) A.-C. Tabet (4) A. Aboura (4) (1) Laboratoire CBCM (2) CHU Charles Nicolle (3) CHU Charles Nicolle (4) CHU Robert Debré (5) CHI Eure-Seine Supernumerary Marker Chromosomes (SMCs) are defined as extrastructurally abnormal chromosomes which origin and composition cannot be determined by conventional cytogenetics.

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Present in about 0.05% of the human population and nearly ten times more frequent in individuals with mental retardation, their clinical outcome is difficult to predict as they can have different phenotypic consequences because of differences in euchromatic DNA-content, different degrees of mosaicism, and/or uniparental disomy of the chromosomes homologous to the SMCs. The characterisation of SMCs is of the utmost importance for genetic counselling. Among SMCs, multiple small markers in the same patient are relatively rare in the literature (about 40 cases described) and majority of the reported cases had two markers. To our knowledge, there is only one previously reported case with 2–6 markers (Reddy et al, 2003). We report on a seven-year-old female with mild developmental delay, dysmorphic facial features, impaired speech, II-III toes syndactily and velar deficiency. Conventional cytogenetic analysis showed mosaicism for 1–3 supernumerary markers. Parental karyotypes were normal. Fluorescence in situ hybridization studies using allcentromere probe (Cytocell®) revealed up to six distinguishables SMCs (cryptic mosaicism) derived from chromosomes 3, 12, 13/21, 14/22 and X. Additional mosaic marker chromosomes were not seen in chromosome banding analysis because the minute size of some of the markers made them difficult to distinguish. Whole-chromosome-painting probes showed homogene hybridization onto chromosome 12 and mosaic hybridization onto chromosomes 3, 22 and X confirming that these markers contain a significant amount of euchromatin. We review the literature on SMCs and discuss the complexity of genetic counselling in these postnatal discoveries in which phenotype/genotype correlations may be further complicated by different combinations of SMCs in different cells. Key words: small supernumerary marker (SMCs), cryptic mosaicism, genetic counselling. 12.1-O Copy number variations of well-known genes detected by array CGH: a help or a hindrance? C. van Ravenswaaij-Arts (1) T. Dijkhuizen (1) R. Hordijk (1) R. Sijmons (1) T. van Essen (1) K. Kok (1) B. Sikkema-Raddatz (1) (1) University Medical Centre Groningen

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Introduction: If copy number variations (CNVs) of well-known genes are found by array CGH, three situations may arise for the counsellor. The CNV may perfectly explain the phenotype of the patient, a convenient situation; the result may be difficult to interpret and hurdles encountered; or the deletion reveals an unanticipated diagnosis, which raises further problems. Methods: In a period of 9 months, several CNVs of well-known genes were detected in a total of 400 patients with mental retardation/multiple congenital anomalies by 105K oligo array (Agilent) analysis. Patient details and counselling issues were evaluated. Results: Convenient CNVs, in which the genotype nicely explained the phenotype, included deletions of the TWIST gene in a young woman with craniosynostosis, and a unique deletion of the NR5A1 gene in an XY-female newborn. Hurdles were encountered in the following three situations. (1) An MECP2 duplication in a young boy with a straightforward cytogenetic interpretation but resulting in counselling challenges. (2) A duplication of POMGNT1, the gene involved in autosomal recessive muscle-eye-brain disease, found in a child with muscle-eye-brain phenotypic involvement. This raised the question whether duplications of this gene could result in a severe phenotype while carriers of a heterozygous mutation have no symptoms. (3) A maternal duplication of exons 45–50 of the DMD gene in a moderately retarded boy with normal creatin kinase levels. The intragenic duplication was predicted to result in a frame shift and thus a Duchenne phenotype. Further delineation of the position of the duplicated segment was needed for the correct interpretation of this CNV. More serious counselling issues were encountered when gene deletions were found that did not explain the phenotype but did predict an increased risk for malignancies (a MSH6 deletion in a mildly retarded girl) or implicated high genetic risks for the family (an AFF2 (FRAXE) deletion in a severely retarded woman). Conclusion: Although involvement of a well-known gene may facilitate the cytogenetic interpretation of the array result, for the counsellor it may raise unforeseen and challenging problems in the clinical interpretation. Oral presentation is preferred.

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12.2-O Whole genome microarray CGH vs. targeted MLPA analysis in prenatal diagnosis: a balance between sensitivity and informativity C. Pangalos (1) B. Hagnefelt (1) M. Karambela (1) C. Konialis (1) (1) Intergenetics Introduction: Our experience, using three different high resolution array CGH platforms in high risk prenatal cases, revealed the benefits but mostly the problems associated with the application of the technique during prenatal diagnosis. For 3 years, we have also applied a targeted approach for the detection of a number of known, recurrent deletion/ duplication syndromes. We present data and discuss benefits and potential shortcomings of both approaches. Material & Methods: Fetal DNA was extracted, labeled and hybridized to either an Agilent 4X44K microarray slide (4 samples), Agilent 244K microarray slide (1 sample) or 19K Human BAC Empire Genomics AccuArray (6 samples). Results were evaluated using the Agilent CGH Analytics 3.4.40 software. Also, 799 prenatal samples were analyzed by MLPA for the detection of microdeletion syndromes and subtelomeric rearrangements. Results: Multiple CNV’s were identified in 4 prenatal samples/cases, referred due to ultrasound findings and/or karyotypic abnormalities. In one case and after analysis of the parents, the variants were classified as de novo and pathogenic, involving both a subtelomeric deletion and subtelomeric duplication. Common copy number polymorphisms (CNP’s) were observed and reported as such. Following MLPA analysis, we detected 2 samples with abnormal findings, both involving well characterized microdeletion syndromes. Conclusions: Although it is obvious that highresolution array CGH will uncover more abnormalities, there are severe limitations prohibiting their routine use in a clinical prenatal setting: (a) the burden on clinicians and parents, who have to deal with ambiguous information, due to copy number variants of unclear clinical significance, (b) serious time constraints, due to the fact that prenatal testing is increasingly performed at a later gesta-

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tional age and (c) the high cost associated with the test. In contrast, our experience leads us to favor a targeted approach, by interrogating multiple, recurrent, clinically defined (and constantly updated) pathogenic regions, via standard and custom MLPA as a routine procedure. 12.1-P Validation of aCGH results using commercially available and customised MLPA kits A. Baumer (1) A. Schinzel (1) M. Riegel (1) (1) University of Zurich (2) University of Zurich (3) University of Zurich The use of arrays for the detection of deletions and duplications represents a major improvement in the diagnosis of patients with possible chromosomal imbalances. Clearly a number of aCGH investigations will result in plausible genotype/phenotype associations, by revealing the presence of deletions or duplications that, in retrospect, lead to phenotypes already described in the literature. Of particular interest are novel deletions/duplications and chromosome imbalances associated with unexpected clinical features. The aim of our analyses is to optimise the sensitivity of the detection of deletions and duplications by using a combination of aCGH and MLPA investigations. The higher the resolution of the array, the larger is the number of copy number changes that one can expect to detect. However, such short imbalances could also cause abnormal signals using arrays with lower resolution. Our strategy is to evaluate the aCGH results under non-stringent conditions. These investigations are based on the 44K Agilent arrays. All possible imbalances are tested using independent techniques: FISH and/or MLPA (commercially available MLPA kits as well as self designed kits). We will present illustrative examples of our aCGH and MLPA data, including: – – –

an intragenic deletion in a patient with GomezLopez-Hernandez syndrome: thus, defining the gene as candidate gene for the syndrome. examples of subtelomeric regions an example of a patient with a complex deletion

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Retrospective analysis of prenatal samples with unbalanced structural rearrangements using BAC array-CGH

Improved Sensitivity and Detection of Chromosomal Abnormalities Using a HighDensity, Multiplex Array CGH Platform

F. Maggi (1) S. De Toffol (1) D. Pecoraro (1) H. Raussi (2) P. Ollikka (2) B. Grimi (1) S. Milani (1) A. Borsatti (2) G. Simoni (1) F. Grati (1) (1) TOMA, Advanced Biomedical Assays (2) PerkinElmer LAS

C. Shaw (1) V. Ott (2) K. Hogan (1) K. O’Moore (1) J. Geoghegan (1) R. Selzer (1) T. Richmond (1) R. Seibl (1) (1) Roche NimbleGen (2) Roche NimbleGen

The introduction of array-CGH into prenatal diagnosis (PD) presents controversy to the clinical cytogenetic community. The advantages of aCGH are multiple: enhanced detection of fetal chromosome aberrations, shorter reporting times and need for relatively small amounts of fetal DNA. However, there is a principal challenge with its application to PD that is related to the detection of Copy Number Variations (CNVs) with questionable clinical significance. CNVs number increases with the enhancement of the platform resolution and this aspect has obvious implications for genetic counselling. On this basis many authors agree on its application limited to specific prenatal indications: i.e. fetuses with multiple structural malformations but normal karyotypes; characterization of minute structural rearrangements not definable using conventional cytogenetic and molecular cytogenetic techniques. Herein we present a retrospective analysis of prenatal samples (chorionic villi and amniocytes) with unbalanced structural rearrangements using two whole genome BAC platforms (1 Mb and 0.6 Mb resolution). The aim of this study is to verify the sensitivity in revealing CNVs related and unrelated to the cytogenetic unbalances and to draw limits and advantages coming from the application of the aCGH in these prenatal cases. All deletions/duplications were detected and the CNVs unrelated to the cytogenetic unbalances, telomeric in the majority of cases, were present in a mean number of 3 a case. Whole genome amplification products seems to be a reliable copy of the original DNA since evident bias are not present after their analysis. In conclusion, the application of the aCGH combined with conventional cytogenetic might be a useful tool for a fast and comprehensive understanding of minute cytogenetic unbalances permitting a more accurate genetic counselling.

Array CGH methods are widely used to investigate genome-wide DNA copy number variation associated with cancer and developmental disorders. The application of array CGH to genetics research has enabled the discovery and fine characterization of novel genomic disorders including 17q21 (Sharp, 2006), 15q24 (Sharp, 2007), 1q41q42 (Shaffer, 2007), 16p11.2–p12.2 (Ballif, 2007), 15q13 (Sharp, 2007; Helbig, 2009) and 1q21.1 (Mefford, 2008) microdeletion syndromes. In addition, array CGH provides an alternative to cumbersome and costly molecular techniques for the detection of exon-level deletions and duplications associated with Mendelian disease, such as the CFTR and dystrophin genes (Saillour, 2008, Hegde, 2008). We have developed new human whole-genome array designs in 3×720K and 12×135K multiplex formats (synthesized on NimbleGen’s HD2 platform), which offer improved sensitivity and reproducibility compared to earlier 385K and 4×72K arrays. These formats are particularly amenable to trio analysis (3×720K) and higher-throughput analysis of targeted loci (12×135K). The Human CGH 3×720K and 12×135K Whole-Genome v3.0 array designs are the product of multiple rounds of empirical probe optimization of the Human CGH 2.1M WholeGenome v2.0 probe set. Only the highest performing probes were chosen for inclusion on the array, to achieve a substantial improvement in the signal to noise ratio, while maintaining a reasonable distribution of probes throughout the genome. We demonstrate here the improved detection of constitutional and mosaic chromosomal abnormalities using these array designs. In addition to an improved signal to noise ratio, Roche NimbleGen’s CGH 3×720K and 12×135K Whole-Genome v3.0 arrays enable reliable, cost-effective, and high-resolution detection and characterization of chromosomal abnormalities.

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A new case of 1qter duplication explored by CGH-array helps to unveil the phenotype-genotype correlation

Two cases of Prader-Willi like syndrome: similarities and differences

M. Urbina (1) B. Pichon (1) C. Hans (1) N. De Coninck (3) Y. Sznajer (2) A. De Leener (1) (1) ULB- Hôpital Erasme (2) ULB-HUDERF (3) ULB-HUDERF Until now, several cases of terminal 1q duplication have been described but most of them were associated with the partial monosomy of another chromosome as a result of a non-balanced inherited translocation. Only one publication documented a case with a pure duplication of the 1q41-qter region (Kulikowski et al 2008). Because this chromosomal aberration appears often in combination with other anomalies and because the duplicated chromosome regions were large, no syndrome genotype-phenotype correlation has been defined yet. We report here a case of a 21 month-old girl, second child from healthy unrelated caucasian parents, who was examined for global developmental delay. The mother had a first healthy child with normal development. Pregnancy was normal and birth took place at term but dysmaturity was noted. She was referred at the age of 21 months-old for psychomotor retardation since she was unable to sit by herself, stand alone or walk. Her speech was limited to two words. At that moment OFC was measured between 75–90th centile; while height was at 25th centile and weight at 10th centile. The girl had dysmorphic features including macrocephaly, large front, a triangular face, everted inferior lip with a carp-mouth like mouth, high palate and small chin. A first standard caryotype (G banding) from peripheral lymphocytes showed a rearrangement of both chromosomes 1 in all metaphases. This result has been confirmed by High Resolution Banding. Subsequent FISH-analysis and array-CGH were performed. This is the first case of a pure duplication 1q42-1qter without any involvement of another chromosome or the subtelomeric 1q-region. This case further elucidates the genotypephenotype correlation of the 1qter syndrome by breakpoint characterization and comparison with previously reported cases.

A. De Leener (1) M. Urbina (1) C. Hans (1) S. De Saeger (1) C. Vilain (2) Y. Sznajer (3) B. Pichon (1) (1) ULB - Hôpital Erasme (2) ULB - Hôpital Erasme (3) ULB - HUDERF Interstitial 6q16 deletion is associated with a PraderWilli-like phenotype characterized by neonatal axial hypotonia, feeding difficulties, global developmental delay, mild facial dysmorphism and hyperphagic behaviour resulting in secondary obesity. Other features like cardiac defects, central nervous system (CNS) abnormalities, hypogonadism, short extremities and eye/vision anomalies have also been reported. It has been suggested that haploinsufficiency of the SIM1 gene implicated in the activation of the “food” centre of the hypothalamus is responsible for obesity. Here we describe and characterize at the molecular level two patients with overlapping interstitial 6q16 deletions. In both of them, the chromosome rearrangement was detected by standard karyotype analysis and Comparative Genomic Hybridization (CGH)microarray was used to define the deleted regions. The first patient shows a deletion of about 9 Mb removing the SIM1 gene while the second shows an overlapping deletion of 24 Mb. While the first patient shows the classical signs of Prader-Willi-like syndrome, the second one displays a more severe phenotype with the presence of cardiac (interauricular septal defect), CNS (parietal bilateral and left frontal leucomalacia and myelinisation delay) and ocular (visual acuity 20/170, ocular albinism, bilateral macular agenesia) defects. Previous reports indicate that amongst 10 patients evaluated by CGHarray, two display similar cardiac abnormality. Ocular defects are usually observed in 6q16 deleted patients but ocular albinism and bilateral macular agenesis have not been reported. Even if some CNS abnormalities were previously described, leucomalacia and myelinisation delay were never reported. It is likely that the severity of the phenotype of our second patient results from the large size of the deletion but no correlation could be established between the variability of the phenotype and the size or localization of the deletions described.

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12.6-P Alu-mediated deletions and point mutations within the FOX transcription factor gene cluster on 16q24.1 result in alveolar capillary dysplasia/ misalignment of pulmonary veins and multiple congenital ma P. Stankiewicz (1, 14) P. Sen (2, 14) S. Bhatt (1) M. Storer (3, 4) Z. Xia (1) B.A. Bejjani (5) Z. Ou (1) J. Wiszniewska (1) D.J. Driscoll (6) J. Bolivar (7) M. Bauer (7) E.H. Zackai (8) D. McDonald-McGinn (8) M.J. Nowaczyk (9) M. Murray (10) T.H. Shaikh (8) V. Martin (3, 4) M. Tyreman (11) I. Simonic (11) L. Willatt (11) J. Paterson (12) S. Mehta (12) S. Gribble (4) E. Prigmore (4) A. Patel (1) L.G. Shaffer (5) N. P. Carter (4) S. Wai Cheung (1) C. Langston (13) C. Shaw-Smith (3, 4) (1) Dept of Molecular & Human Genetics (2) Dept of Pediatrics—Nutrition, Baylor College of Medicine, Houston, Texas (3) Institute of Child Health, London, UK (4) Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK (5) Signature Genomic Laboratories, LLC, Spokane, Washington (6) Division of Pediatric Genetics and Metabolism, University of Florida College of Medicine, Gainesville, Florida (7) Dept of Pathology, Miami Children’s Hospital, Miami, Florida (8) Division of Human Genetics, Children’s Hospital of Philadelphia, Pennsylvania (9) Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada (10) Department of Medical Genetics, University of Washington, Seattle, Washington (11) Regional Cytogenetics Laboratory (12) Dept of Medical Genetics, Addenbrooke’s Hospital, Cambridge, UK (13) Dept of Pathology, Texas Children’s Hospital, Baylor College of Medicine, Houston, Texas (14) These authors contributed equally to this work Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a rare neonatally lethal developmental disorder of the lungs typically associated with multiple congenital anomalies. Using array CGH analysis, we have identified six overlapping microdeletions, ranging in size from 102 kb to 3.5 Mb in chromosome 16q24.1q24.2 in patients with histopathologically confirmed ACD/MPV (3/3 examined cases), and cardiac (6/6), genitourinary (5/ 6), and gastrointestinal (3/6) malformations. Subse-

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quent DNA sequencing of the candidate FOXF1 gene in unrelated patients with sporadic ACD/MPV and other malformations identified four different de novo mutations (frameshift, nonsense, and no-stop). Custom designed 16q24-specific high-resolution 44K microarray analysis of 14 ACD samples revealed an ~1.8 Mb microdeletion harboring FOXF1 in one sample and two microdeletions, ~524 kb and ~145 kb in size, located ~52 kb and ~259 kb upstream to FOXF1, respectively, implicating a position effect. We have identified also an ~131 kb deletion encompassing FOXL1 and FOXC2 (leaving FOXF1 intact) in a 3-year-old patient with a diastasis recti, dilated and bilaterally tortuous ureters with hydronephrosis, ASD, and developmental delay. In contrast to the association of point mutations in FOXF1 with bowel malrotation, microdeletions of FOXF1 have been found in ACD/MPV patients with hypoplastic left heart syndrome and gastrointestinal atresias, likely due to the haploinsufficiency for the neighboringFOXC2 and FOXL1 genes. In five out of seven cases, both sequenced deletion breakpoints mapped within Alu elements, indicating NAHR or FoSTeS mechanism of formation. 12.7-P Cytogenetic and Molecular Characterisation of a complex chromosomal rearrangement, insertion 4q into 1p and non-contiguous 4q deletions, associated with azoospermia S. Dahoun (1) S. Gemelli (1) C. Studer (1) I. Jenny (1) D. Marelli (1) A. Bitton (2) F. Bena (1) (1) Medical Genetics Division We report a case of a 33 years old patient referred for azoospermia. Married for two years with a 34 years old woman presenting tubal obstructions, an ICSI was envisaged. A karyotype revealed an insertion of bands q21.22q22.1 of chromosome 4 into the band p34 of one chromosome 1 [formula 46,XY,ins(1;4)(p34; q21.22q22.1)dn] confirmed by FISH. Despite some learning difficulties due to a mild mental retardation, the patient is socially well integrated and works as a pastrycook. No notable dysmorphies are observed. Array-CGH (Agilent- 244K) analyses revealed three non contiguous deletions leading to a complex

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By classical cytogenetics, large deletion 4q12q21.1 has been previously described in 8 cases. All the patients had moderate to severe retardation and minor facial anomalies. Two others cases with severe mental retardation, dysmorphic features and malformation had been characterised as CCR including 4q using Array-CGH . These CCR included deletion, duplication and inversion of several segments/clones of 4q. The mechanism of CCR’s origin in not well understood. Further FISH analyses of the parents chromosomes 1 and 4 could show some predispositions for such a rearrangement. Our patient is the first description of a 1.3 Mb non contiguous 4q deletion associated with an azoospermia. This provide that molecular cytogenetics associated with FISH techniques will increase the complexity of chromosomal abnormalities.

Here, we are reporting three cases, presenting with the phenotype of cleidocranial dysplasia (CCD), Lowes syndrome and NF2 in combination with additional symptoms. The first case, a newborn boy was suspected to suffer from a skeletal dysplasia and radiological examination suggested CCD. Sequencing of the RUNX2 gene did not show any mutations. Subsequently, the patient showed developmental delay and array-CGH revealed a deletion of 3,3 Mb on 6p12, including 25 genes surrounding the RUNX2 gene. The second patient was a prematurely born boy with phenotype of Lowes syndrome with pronounced hypotonia and cataract. Sequencing of OCRL was requested but not possible because no PCR-products could be amplified. Array-CGH revealed a 3,4 Mb interstitial deletion in Xq25, encompassing OCRL and 4 other genes. The third patient was a girl with symptoms within the CHARGE spectrum. Sequencing and MLPA of CHD7 did not show any mutations. At age of 17 she developed a severe impairment of hearing and MRI of the brain was performed, showing bilateral vestibular schwannomas, which suggested NF2. An array-CGH was performed and disclosed a 3,6 Mb interstitial deletion including NF2 and a large number of other genes. In conclusion, these three cases indicate that arrayCGH should be a method to consider when atypical signs are present in addition to those suggesting a monogenic disorder and a sequencing of a disease specific gene does not reveal any abnormality.

12.8-P

12.9-P

Array-CGH as a method for detection of monogenic disorders

Rare structural rearrangements of X chromosome affecting XIST locus; two case reports

B. Anderlid (1) G. Grigelioniene (2) J. Schoumans (1) M. Kvarnung (1) N. Ann (1) (1) Karolinska Institutet (2) Karolinska Institutet

J. Drabova (1) Z. Zmitkova (1) M. Vlckova (1) D. Novotna (1) Z. Vlckova (1) M. Koudova (1) A. Puchmajerova (1) A. Santava (2) P. Hedvicakova (1) E. Al Taji (3) (1) University Hospital Motol (2) University Hospital Olomouc (3) University Hospital Kralovske Vinohrady

chromosomal rearrangement (CCR) implicating 7 breakpoints. – – –

a 234 kb deletion in 4q21.22 between 83.845 and 84.079 Mb, (UCSC genome browser assembly march 2006) a 551 kb deletion in 4q21.23 between 86.148 and 86.699 Mb, and a 559 kb deletion in 4q22.1 between 88.498 and 89.057 Mb including the gene HSD17B11 that is a candidate on the metabolism pathway of the androgens.

During the last few years array-CGH has become widely used for the detection of genomic disorders, mainly in patients with unspecific developmental delay and malformations. As a result, several new microdeletion/duplication syndromes have been described. In rare instances array-CGH reveals a monogenic disorder by disclosing loss of one copy of a specific gene. If several other genes are lost, as in our patients, this will give rise to a broader phenotype.

We report two rare chromosomal rearrangements of X chromosome. We used G-band technique, FISH analysis and array CGH analysis to describe the rearrangements. First patient, a 2-year-old boy, was referred to array CGH testing due to abnormal phenotype and devel-

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opmental delay. The small duplication (10-14 Mb) of a segment of the long arm of X chromosome was revealed. The rearranged segment including XIST locus (Xq13.2) was diagnosed utilizing CGH microarray method at 0,5 Mb resolution and this finding was consequently confirmed by FISH studies using DNA XIST probe. This abnormal X chromosome was inherited from phenotypically normal mother. Also the probands maternal grandmother is supposed to be a carrier, by reason of two spontaneous abortions of male foetuses in her personal anamnesis. Cytogenetic analysis in second patient, a 13-yearold girl, with tall stature, mild mental retardation and suspected functional insufficiency of the ovaries revealed an isochromosome i(Xp). Isochromosomes of the short arm of the X are rare and monocentric constitutional i(Xp) with no long arm material appear not to exist, because of failure to inactivate due to the lack of the XIST locus. Array CGH with average resolution 340 bp revealed presence of material (12 Mb) of the long arm of X chromosome including XIST locus. This finding was confirmed by FISH analysis. Supported by IGA grant NR/9457-3, grant of Health Ministry of Czech Rep. 00064203 and CHERISH

showing clinical features similar to ATS-MR. The aims of this work were to compare the phenotypes of our patients with previous cases and to correlate their complex clinical manifestations with the extent of the deleted chromosomal segment. Detailed examinations showed that both Czech patients were identical in terms of clinical symptomatology and they shared the major characteristic clinical features with the originally reported ATS-MR phenotype, although the phenotypes were not completely identical with previously described patients. Oligonucleotide array-CGH performed in affected nephew showed a deletion in Xq22.3, confirmed also in the uncle and female members of the family using fluorescence in situ hybridization (FISH). The deletion had a maximum size 3.1 Mb with a proximal breakpoint in the gene COL4A6 to a distal breakpoint behind the gene DCX. Comparing the extent of deletions of all three families showed that deletion identified in Czech family extended more telomerically than two previously described deletions. This could contribute to clarification of the minor clinical differences observed and to definition of potential candidate genes involved in deleted segment. Supported by MSM0021622415

12.10-P

12.11-P

Chromosome Xq22.3 gene deletion syndrome (ATS-MR) in a Czech family: additional characterization of the clinical and molecular aspects using array-CGH

Microarray analysis of an apparently balanced translocation with phenotype reveals unexpected interstitial 6q deletion

V. Vranova (1) V. Horinova (2) A. Oltova (3) P. Kuglik (1) (1) Institute of Experimental Biology, Faculty of Science, Masaryk University (2) Genetic ambulance and counseling (3) University Hospital ATS-MR syndrome (previously AMME syndrome, OMIM #300194) is an X-linked recessive syndrome characterized by the Alport syndrome, mental retardation, midface hypoplasia and elliptocytosis. This contiguous syndrome associated with a deletion in Xq22.3 was first reported by Jonsson et al. (J Med Genet 1988, 35:273) and specified by Meloni et al. (J Med Genet 2002, 39:359) in five patients belonging to two families. We have identified a family with two affected male (maternal uncle/nephew) members

G. Lefort (1) A. Schneider (1) M. Tournaire (1) T. Le Carrour (1) D. Genevieve (1) A. Chaze (1) P. Sarda (1) J. Puechberty (1) (1) Centre Hospitalier Universitaire et Régional The young girl was first presented to our Genetic Consultation at the age of 8 years. She had mental retardation, congenital heart defect (interventricular communication and coarctation of the aorta), growth hormone deficit, ligament hyperlaxity, facial dysmorphism including hypertelorism, low-set ears and a large mouth with macroglossia, and a very friendly disposition. Chromosome studies with classical RHG and GTG banding on metaphase and prometaphase chromosomes revealed an apparently balanced “de novo” t(4;9)(q27;p23). FISH studies using subtelomeric probes for the short and long arms of

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chromosome 9 confirmed the reciprocal nature of the translocation. The patient was again seen at the age of 18 years and DNA microarray analysis (Affymetrix) was performed to investigate the translocation breakpoints for undetected microrearrangement. Molecular karyotyping did not show anomalies at the translocation breakpoints but revealed an unexpected 6.6 Mb interstitial 6q24.3–q25.1 deletion. FISH studies confirmed the deletion. This interstitial 6q deletion might have been detectable on classical chromosome studies but the discovery of an apparently balanced rearrangement probably diverts the observer’s eye from such additional small chromosome rearrangements. Although the impulse in “de novo” apparently balanced chromosome rearrangements with a phenotype is to investigate breakpoints, it is most probable that with microarray analysis a large number of these cases will show additional unrelated microrearrangements.

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duplications of the long arm of chromosome 3: 3q25q25, 3q25.3q26.2 and 3q28q29. In these families, persons with the rearrangement are either phenotypically normal or have an abnormal phenotype. These differences might be explained by imprinting, the presence of regions with dosage sensitive loci or modifications of the implicated region. To characterize this unbalanced chromosome abnormality, we are using a molecular cytogenetic technology: Affymetrix Genome-Wide Human SNP. We hope to establish a relationship between the phenotype and the karyotype and to identify a possibly more complex rearrangement. 12.13-P Array-CGH analysis of balanced chromosomal rearrangements in three patients with dysmorphic features and/or idiopathic mental retardation

12.12-P Familial proximal 3q duplication: euchromatic variant or cytogenetic abnormality causing the phenotype A. Schneider (1) J. Puechberty (1) M. Tournaire (1) A. Chaze (1) G. Lefort (1) P. Sarda (1) (1) Centre Hospitalier Universitaire et Régional Except for Cornelia De Lange syndrome 3q duplications have rarely been described in the literature. We report a proximal 3q duplication in a patient with dysmorphic traits and developmental delay. On presentation at our Genetics Consultation the following dysmorphic features were noted in the 5year-old female patient: hypertelorism, up-slanting palpebral fissures, epicanthal folds, and facial hypotony with a half-open mouth. There were no additional malformations. There was slight moderate global mental deficiency. The karyotype shows duplication of the proximal region of the long arm of chromosome 3: 46,XX,dup(3)(q11.2~13.1q13.1~13.3) .ish dup(3) (q11.2~13.1q13.1~13.3)(WCP3+). The same chromosomal rearrangement was observed in the patient’s mother who had a normal phenotype. Euchromatic variants are rarely reported in the literature. Barber JC (2005) described 3 families with

P. Mabboux (1) S. Chantot (1) B. Keren (2) L. Burglen (1) S. Marlin (1) C. Soleyan (1) B. Heller-Roussin (3) M. Portnoi (1) J. Siffroi (1) (1) Hôpital Armand-Trousseau (2) Hôpital PitiéSalpétrière (3) C.H.I. André Grégoire Background Carriers of apparently balanced reciprocal translocations and carriers of CCRs (structural chromosome anomalies involving >2 chromosomes or >2 breakpoints) are usually phenotypically normal. However, an abnormal phenotype is present in 6.1% of de novo balanced reciprocal translocations and in more than half CCRs with de novo occurrence. Conventional cytogenetics is of limited use in determining whether both types of rearrangements are balanced or unbalanced. The use of micro-array overcomes the limitations of classical cytogenetic approaches and provides the opportunity to screen the entire genome for cryptic genomic gain and losses in these apparently balanced rearrangements. Methods We tested three patients with unexplained mental retardation and/or congenital anomalies associated with de novo apparently balanced chromosomal rearrangements. Results Case 1: A 6-year-old girl was evaluated because of deafness, multicystics kidneys and aortic

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insufficiency. Conventional karyotype revealed a 46,XX,t(3;5)(p22.1;q14.2)dn. Array-CGH identified a 1.09 Mb deletion on 3p24.1p24.1. Case 2: A 2-year-old boy was referred because of psychomotor delay and craniofacial dysmorphic feature. Standard karyotype analysis suggested a reciprocal insertional translocation involving the short arm of one chromosome 1 and the long arm of one chromosome 6. Conventional cytogenetic was limited in determining whether this ins(1;6)(p22.1; q16.2q22.2),ins(6;1)(q16.2;p22.1p22.2)dn was balanced or not. Array-CGH identified a 2.24 Mb deletion on 6q15q16.1. Case 3: A 6-month-old boy was evaluated because of craniofacial dysmorphic features and psychomotor delay. Conventional karyotype revealed an apparently balanced de novo complex translocation involving chromosomes 4, 5, 9 and 21. Array-CGH identified a 11.5 Mb deletion on 5p15.1p13.3. FISH using specific BACs probes confirmed all deletions. Conclusions These cases illustrate the contribution of array CGH in investigating patients who have clinical features suggestive of chromosomal abnormalities but with apparently balanced chromosome rearrangements. Moreover, cryptic rearrangements identified are helpful to discuss genotype-phenotype correlations and genetic counseling. 12.14-P Validation of BlueGnome’s Constitutional Focus CytoChip for Application to Prenatal Diagnosis F. Togneri (1) D. McMullan (1) L. Cooper-Charles (1) L. Reali (1) S. Larkins (1) C. Patel (2) V. Davison (1) (1) Birmingham Women’s Hospital (2) Birmingham Women’s Hospital ArrayCGH analysis for the detection of submicroscopic chromosome aberrations is a rapidly developing diagnostic technique. A major challenge in the utilisation of arrays for prenatal diagnosis however exists in the poor quality and quantity of DNA samples

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available in a prenatal setting and in the difficulties faced in interpretation of genomic copy number variation (CNV) within a clinically acceptable turnaround time. BlueGnome’s Constitutional Focus CytoChip has been designed to overcome these obstacles by increased replication of well-validated BAC clones in regions of recurrent genomic disease and sub-telomeres and reduction of coverage in regions of well described CNVs. A study was conducted using samples from a total of 45 patients in three categories of; postnatal poor quality samples, follow-up samples from family members of patients with known constitutional imbalance and prospective prenatal, neonatal and fetal pathology samples. Artificial mosaics were also constructed using trisomic and microdeletion DNA samples and results were used to validate this array for use in prenatal diagnosis at the West Midlands Regional Genetics Laboratory in Birmingham, UK. Results allowed for reassurance in 6 prenatal cases and for diagnosis of a 16q duplication in a terminated fetus with holoprosencephaly. This low resolution, easily interpretable array offers a promising approach for fastturnaround, minimum validation arrayCGH Prenatal Diagnosis. Preference for presentation as a poster 12.15-P Multicolour FISH versus array CGH techniques. Which to choose for the characterization of small supernumerary marker chromosomes? J. Melo (1) N. Kosyakova (2) T. Liehr (2) L. Backx (3) J. Vermeesch (3) I. Carreira (1) (1) Faculty of Medicine University of Coimbra (2) Institute of Human Genetics and Anthropology (3) University Hospital Gasthuisberg Small supernumerary marker chromosomes (sSMC) are defined as structurally abnormal chromosomes that cannot be identified or characterized unambiguously by conventional banding cytogenetics alone, and are generally equal in size or smaller than a chromosome 20 of the same metaphase spread. The application of molecular cytogenetics, including multicolour FISH and array CGH, is very important to ascertain size, shape and content of these sSMC. We characterized 10 sSMC

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derived from different chromosomes, namely from chromosomes 1, 5, 11, 15, 16, 17 and 22, by multicolour FISH (subcenM-FISH and MCB) and array painting, after microdissection. Subcentromere specific multicolour FISH (subcenM-FISH), multicolour banding (MCB) and microdissection were done as described by Liehr et al. (2006, Cytogenet Genome Res 112: 23–34). Array CGH was done using a 32 K fulltiling chromosome specific BAC clone array. In the evaluated cases there is a strong correlation of the results obtained by the two different molecular techniques, despite some slight differences in some of the sSMC. However, array painting permits more accurate information of the breakpoints of the marker, allowing in future a better genotype/phenotype correlation. Overall, a combination of all available molecular cytogenetics techniques (including array CGH) is a valuable tool for the accurate identification of the origin, shape and content of marker chromosomes, contributing to a more informed prenatal counselling and for better genotype-phenotype correlations. The novel technique—Array Painting, combining microdissection and array CGH, is very useful for accurately mapping size and breakpoints of marker chromosomes, since sSMC are often only present in a small percentage of cells. Nevertheless, multicolour FISH is a less time-consuming technique and has showed to be also a powerful tool for sSMC characterization, especially for cryptic mosaicism. Supported in parts by DAAD (D07/00070) and Prochance 2008 of the FriedrichSchillerUniversityJena. 12.16-P An array CGH study of 150 Finnish patients with mental retardation and normal karyotype L. Siggberg (1) S. Ala-Mello (2) E. Jaakkola (3) E. Kuusinen (4) R. Schuit (5) D. Böhm (6) J. Kohlhase (6) J. Ignatius (3) S. Knuutila (1) (1) University of Helsinki (2) Hospital District of Helsinki and Uusimaa (3) Oulu University Hospital (4) Satakunta Hospital District (5) MRC-Holland (6) Center for Human Genetics Mental retardation (MR) occurs in 1% of the world population. The incidence is slightly lower in

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Finland (0.6%) due to lack of social and economical problems, such as malnourishment. Genetic factors cause 30–40% and environmental factors 10%, but around 50% of MR patients are undiagnosed. Many recent publications show that microarray-based methods detect aberrations in ~15% of MR patients, where conventional methods have failed. We have studied 150 patients with mental retardation and/or dysmorphic features and/or malformations with a normal karyotype by G-banding (300–550 bp level). Using Agilent Technologies 44K and 244K oligonucleotide microarrays, patient-genomes were screened in an effort to detect microaberrations. Confirmation of aberrations was done, when possible, by array CGH studies of parents, MLPA, Q-PCR or sequencing. Microaberrations, likely causing the clinical phenotype, were detected in 28 patients (18.6%). Aberrations relating to known syndromes were detected in 7 patients (Dandy-Walker malformation, Distal 4qdeletion, Sotos, Duplication 9q34, 16p-duplication, 17q21.3 deletion, and Bannayan-Riley-RuvalcabaSmith [BRRS] syndrome). Chromosome-X aberrations were detected in 4 male patients (one de novo, three maternally inherited). In 4 patients the detected aberration was familial (including BRRS). New aberrations were detected in 14 patients, some of which have been published previously. 12.17-P High Resolution Agilent 244K oligoarray CGH analysis in patients with MR/MCA/DD S. Kitsiou-Tzeli (1) M. Tzetis (1) C. Vrettou (1) H. Frysira (1) A. Pampanos (1) E. Kanavakis (1) (1) University of Athens Clinical characteristics of patients are not always related to specific syndromes. Array comparative genomic hybridisation (aCGH) is used to detect small copy number changes within the genome that are not always visible by conventional karyotyping (>5-10 Mb). Thirty patients with various degrees of MR, seizures, dysmorphic features and/or single or multiple congenital abnormalities and normal previous conventional

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karyotype, many of which had also received a variety of other genetic tests (FRAX, RETT, single FISH tests or metabolic screens), were analyzed with Agilent 244K oligoarrays, allowing a theoretical resolution of >50 Kb. Clinically significant submicroscopic imbalances were detected in 15 (50%) patients. We are currently in the process of confirming our findings with QPCR. The high percentage of positive patients is probably due to the strict criteria of patient selection. Array CGH is a powerful tool for the identification of novel chromosomal syndromes and for more accurate prognosis and phenotype-genotype correlations.

ID

Chromosome

1 2

cation of dysmorphic features, malformations or mental retardation does not enable to diagnose a classified syndrome of malformations. In such cases, karyotype identification is the first step of studies. Patient’s normal karyotype and rare phenotype do not provide any possibility for the use of targeted FISH technique. In turn, the CGH technique sensitivity limit is at the level chromosomal band, what is rather unsatisfactory to define genotype-phenotype correlations. Additionally, an identification of chromosomal aberration may sometimes effectively mask the actual genomic imbalance, which responsible for the disease. For all the above-mentioned reasons, new techniques are

Position start UCSC hg18 554,268

Position End 3,332,604

Length (Mb) 2.8

Known syndrome Y

Genes*

1p36.33-p36.32

Gain/ Loss L

1q41

G

220,916,807

221,288,347

0,371

N

AIDA, DISP1

Xp11.3

L

44,005,210

44,048,137

0,043

N

EFHC2

Xq21.31

L

90,917,565

91,035,630

0,118

N

PCDHX

3

2q35

L

216,986,022

217,664,090

0,68

Y

SMARCAL1

4

3p14.1

L

70,598,263

71,795,160

1,2

N

FOXP1

5p15.33

L

948,032

2,209,449

1,26

N

TERT, SLC6A3, SLC6A19

10q26.3

L

133,644,221

135,084,833

1,44

Y

NKX6, CALY

3p14.1

L

70,951,444

71,601,477

0,65

N

FOXP1

11p15.5

L

1,515,185

2,499,331

0,984

N

DUSP8, TNNI2, TGF2, TH, ASCL2, KCNQ1

11p11.2

L

43,833,775

5

12.18-P Phenotype variability in genomic diseases in the light of our studies M. Constantinou (1) &. Sieczkowski (2) I. Płowaś (2) B. Kałużewski (1) (1) Medical University of Lodz (2) Hospital No. 3 in Łódź Structural aberrations of chromosomes are frequent causes of diagnostic-prognostic tight-spots in the course of genetic counselling. In particular, this problem affects the patients in whom the identifi-

PLCH2, SKI, GABRD

searched for which may provide a number of diagnostics data at the level of gene or chromosomal locus in one study, the microarray CGH technique being an example of such a new option. This report presents an evaluation of the usability of aCGH studies in genotype-phenotype correlation building in 8 patients with disturbed sex differentiation, including 3 patients with marker chromosome from chromosome Y, 2 XX males and a true t(Xp;Yp) hermaphrodite and his “meiotic” sister—a t(Xp;Yp) woman. The aCGH technique was also used to determine genotype-phenotype correlations in case of abnormal duplication/triplication 7p and complex, apparently balanced translocation—t(7;8;14) and in 6

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cases with unspecific phenotype, suggestive of genomic disease. A relationship is discussed between the genotypes of the patients and their phenotypes vs. similar cases, either described in literature or recorded in the ECARUCA database. The application of aCGH technique for diagnostic needs is a technologically attainable challenge but a responsible interpretation of obtained results may be more difficult than originally assumed. 12.19-P Pathogenic burden of recurrent and unique copy number changes in mentally retarded children; consequences for the diagnostic workflow

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CHRNA7 gene, we found additional, etiologically relevant, genomic aberrations, such as a truncating BCOR mutation, which may explain the OculoFacio-Cardio-Dental syndrome found in one girl. These observations emphasize the need for ascertainment of the mode of inheritance of segmental aneuploidies in MCAMR patients, consideration of probable candidate genes and a genome-wide search for additional mutations that may have contributed to the observed complex MCAMR phenotype. In order to incorporate and properly interpret genome-wide CNC discoveries within the context of the workup of MCAMR patients we propose a three-step genecentered workflow scheme. 12.20-P

M. Poot (1) M. Eleveld (1) R. van ’t Slot (1) H. Ploos van Amstel (1) R. Hochstenbach (1) (1) UMC Utrecht (2) UMC Utrecht (3) UMC Utrecht (4) UMC Utrecht (5) UMC Utrecht Recent studies of patients with multiple congenital abnormalities and developmental delay or mental retardation (MCAMR) have demonstrated an appreciable prevalence of unique and recurrent copy number changes (CNCs). To determine the pathogenic burden resulting from these CNCs we performed genome-wide segmental aneuploidy profiling by BAC-based array CGH of 278 unrelated MCAMR patients and 48 unaffected family members. We found on average 38.3 CNCs per individual.In 20 patients we detected de novo CNCs composed of multiple consecutive probes. The remaining CNCs were single probe events covering 1,308 loci. Of these, 481 loci were exclusively found among the 278 MCAMR patients, and 203 loci were covered at a > 1% frequency in our patient cohort. In contrast, recurrent CNCs for 11 of the recently discovered microdeletion/microduplication syndromes comprised 76 events (0.61% of all CNCs) and were found with similar frequencies among patients (21.6%) and healthy individuals (16.6%) (P= 0.271; Fischer exact test). In addition, 7 MCAMR patients and 1 healthy individual showed losses or gains for at least 2 different microdeletion loci. Therefore, the pathogenic burden resulting from these CNCs may be modest. Analyzing three patients with a 15q13.3 microdeletions, all involving the

Interstitial duplication Xp11.23-p11.22 in a boy with severe developmental retardation U. Heinrich (1) D. Wahl (2) (1) Laboratory for Human Genetics and Laboratory Medicine Dr. Klein and Dr. Rost (2) Medical Practice for Genetic Counselling, Paediatric Genetics, and Psychotherapy X-chromosomal duplications in males are one of the major causes for X-linked mental retardation. Here we describe a boy with a 4,8 Mb duplication in Xp inherited from his mother. The boy is the second child of non-consanguineous parents, his elder sister showing normal development. In his first year, he showed a pronounced muscle weakness and a psychomotor retardation. At the age of twenty eight months, he learned independent running with ataxia, and a retarded speech development became obvious. Later on, he developed aggressive behaviour and a moderate mental retardation. Physically, he showed microcephaly, a small and long face, hypotelorism, broad nasal bridge as well as large ears. Chromosome analysis showed an inconspicuous male karyotype. Array CGH using Agilent’s Microarray Kit 44A revealed a 4,8 Mb duplication in Xp11.23– p11.22 encompassing 74 genes. FISH analysis with the clone RP11-58H17 could confirm the duplication. Analysis of the parents disclosed the mother as the carrier of the duplication. This case will be discussed with the literature cases.

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12.21-P Isolated CDH1 germline small deletion in a family with early onset breast cancer detected by Array-CGH C. Palka (1) G. Calabrese (1) A. Novelli (3) E. Morizio (1) P. Guanciali Franchi (2) D. Fantasia (1) V. Gatta (1) G. Palka (2) B. Dallapiccola (3) (1) University of Chieti/Center for Ageing (2) University of Chieti/Pescara Hospital (3) CSSMendel Institute of Rome We evaluated a 3.5-year-old boy because of psychomotor delay and dysmorphisms. Proband’s standard karyotype resulted as normal 46,XY. Therefore, we performed an array-CGH analysis, with a 75 Kb resolution, which disclosed a microdeletion of 0.24 Mb on chromosome 16q22.1. The chromosomal abnormality was confirmed with FISH analysis. Parents investigation demonstrated that his mother had the same alteration. This deletion encompasses a small chromosomal region which includes few genes, such as ZFP90, cerebrally expressed, and CDH1, coding for E-cadherin, involved in pathogenesis of lobular breast cancer (LBC). Proband’s family history was positive for tumours. In particular, his mother had a right invasive LBC combined with a carcinoma in situ, both surgically removed when she was 35-year-old. She was negative for germline BRCA1 and BRCA2 mutations. The normal lobular epithelium exhibited strong membranous immunoreactivity for E-cadherin, while LBC demonstrated absence of membranous immunoistochemical staining. Proband’s maternal grandfather died for right colon cancer, while his greatgrandmother from maternal line died for breast cancer. To our knowledge this is the first case of LBC harbouring germline, isolated CDH1 partial deletion without involvement of other tumour-related flanking genes previously reported as rearranged together with CDH1 within 16q22 in LBC. To verify possible correlation between proband’s phenotype and chromosome 16q22, genotyping excluded uniparental disomy for the region containing ZFP90. Fine molecular characterization of 16q22 deleted region demonstrated that the proband’s healthy brother inherited the same maternal chromosome 16 but the deleted region was replaced with a normal segment as a result of a double crossing-over, explaining his healthy phenotype. Studies to demonstrate maternal imprinting of 16q22 region, and in particular of ZFP90 gene, and FISH

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analysis on LBC tissue to search for the second hit, are currently underway. 12.22-P Investigation of cryptic imbalances in patients with mental retardation and/or multiple congenital abnormalities using array-CGH C. Sismani (1) V. Anastasiadou (2) P. Evangelidou (1) I. Ioannou (2) S. Hadjiloizou (3) P. Nicolaidou (3) S. Kitsiou-Tzeli (4) C. Christodoulou (1) M. Ioannides (1) P. Patsalis (1) (1) The Cyprus Institute of Neurolgy and Genetics (2) Arch Makarios III Hospital (3) Paidi center for specialized Paediatrics (4) University of Athens, Choremio Research Laboratory Chromosomal abnormalities constitute the major cause of mental retardation (MR). Conventional chromosomal analysis still remains the most important tool for identification of genomic imbalances causing MR, however subtle aberrations are missed by routine karyotyping. Array-CGH was recently introduced to clinical practice, significantly increasing the detection rate of chromosomal abnormalities. The aim of the current study was to investigate 220 patients with various degrees of mental retardation and/or congenital abnormalities for cryptic chromosomal imbalances. All patients had normal chromosomal, FISH and Fragile X results and approximately half of the patients (127/ 220–58%) also had normal subtelomeric screening results. Array-CGH was performed using BAC-based array (Cytochip -BlueGnome) with a median resolution of 565 kb for the entire genome and enhanced resolution (100–250 kb) at regions of known genetic conditions. Clinically significant submicroscopic chromosomal imbalances were detected in 15 patients (6.8%) and in another two patients (0.9%) the same imbalances were also found in the non-affected parent. All abnormal results were confirmed with additional methods. Seven deletions, eight duplications, one unbalanced translocation and one mosaic case with both a deletion and duplication were identified. Seven of the detected abnormalities were de-novo, five were familial, while the remaining five are still under investigation. Two out of the seventeen detected abnormalities would have been identified by subtelomeric screening but the majority of them (15) would have been missed. The total percentage of identified abnormalities

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in the current study (7.7%) is lower than previously reported by other studies (8.9–24%), probably due to the fact that no specific selection criteria were applied in our patients and also 58% of the patients were pre-screened for subtelomeric abnormalities. Array-CGH is a powerful tool for the identification of novel chromosomal syndromes/rearrangements and identification of new cases of known syndromes that will allow more accurate prognosis and phenotype-genotype correlations. 12.23-P Minor anomalies in a child carring a mosaic partial trisomy 8p. High resolution SNP array characterisation of the sSMC A. Tabet (1) M. Rajguru (2) N. Le¨Dû (3) M. Maurin (1) A. Delahaye (5) S. Homnes (4) J. Oury (6) S. Serero (3) B. Benzacken (1) A. Aboura (5) (1) APHP (2) APHP (3) laboratoire CBCM (4) APHP (5) APHP (6) APHP Small supernumerary marker chromosome derived from chromosome 8 are relatively common (Liehr et al, 2004 ; 2006). The phenotype ranges from almost normal to variable degree of abnormalities and up to date, genotype–phenotype correlation is not clear. Prenatal sSCM discoveries are a major problem in genetic counselling. Classical cytogenetic technique cannot provide a detailed characterization of the markers. With molecular cytogenetic techniques such as FISH using specific BAC probes, we are able to better characterize the cytogenetic content of small SMC. Nevertheless, when the SMC is very small, the morphological ultrasound echography and the cerebral foetal MRI are normal, the clinical outcome is very difficult to predict. We report on a new case of a de novo prenatal mosaic sSMC derived from chromosome 8. The rate of mosaicism was 30% in cultured cells. The anomaly was discovered in an amniotic fluid exam for advanced maternal age. Pericentromeric specific FISH probes of chromosome 8 were used during prenatal diagnosis: RP1173M19 (8p11.2) confirming that euchromatin was present in the sSCM allowing us to diagnose a partial trisomy 8p. Ultrasound morphological echography and cerebral foetal MRI were normal. After genetic counselling, parents decided to keep the pregnancy. A healthy baby girl was born at term. The clinical examination was without particularity. A

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constitutional postnatal karyotype confirmed the presence of the SMC in 40% of lymphocytes culture cells. At 1 month of life, clinical examination shows no morphological feature and a normal neurological exam. We only noted a laryngeal stridor. Because of unexpected minor anomalies, we used High-resolution Single Nucleotide Polymorphism array (HumanCNV370-Duo DNA Analysis BeadChip, Illumina, San Diego, CA, USA) to better define the partial 8p trisomy. After a brief review, we believe that this new case will contribute to a better genotype–phenotype correlation in order to precise the genetic counselling. 12.24-P Interstitial deletion 4q due to a complex rearrangement involving chromosomes 1, 2, 4, 8, 14 and 16 G. Toksoy (1) B. Röthlisberger (2) B. Türköver (1) C. Sayar (1) A. Huber (2) P. Miny (3) (1) Zeynep Kamil Women and Children Diseases Education and Research Hospital (2) Department of Biomedicine, University Children’s Hospital (3) Cantonal Hospital Complex chromosome rearrangements (CCR’s) involving multiple breaks in two or more chromosomes are rare. The precise characterization of a CCR is difficult and may be inaccurate even by using molecular cytogenetic techniques. Various new molecular techniques such as MLPA, array techniques (CGH, BAC, oligo, SNP, etc) have proved to be powerful tools for the characterization of CCR’s. We present here a patient with a de novo CCR involving chromosomes 1, 2, 4, 8, 14, 16. He was investigated cytogenetically because of multiple congenital anomalies , such as macrocephaly with a prominent forehead, epicantus, ptotic eyelids , micrognathia, low set ears, short neck, pectus exscavatum, adducted right foot, cryptorchidism, hypotonia and neurodevelopmental delay. Cytogenetic analysis revealed an abnormal karyotype (46,XY,der(1),der (2), der (4), t(8;14), der(16)), while the parents had a normal chromosome count. After FISH investigations using different commercial probes the karyotype was interpreted as ish t(1:16), ins(4;2), t(8;14). The

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rearrangements were apparently balanced at a 500– 550 band level and revealed no obvious explanation for the phenotype of the index patient. Therefore, an array-CGH analysis (NimbleGen) was initiated and a 14,7 Mb gross deletion was found in chromosome 4q (del(4)(q21.23q23)) including approximately 50 genes. The clinical findings of the patient will be presented in detail and genotype-phenotype correlations will be discussed.

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lution of 130 Kb. As suggested by the literature, GATA-4 deletion could explain our patient cardiac anomaly, while his microcephaly can’t be ascribed to MCPH1 haploinsufficiency, since this gene is located outside the deleted region. Despite TNKS deletion our patient doesn’t show any Cornelia de Lange feature. This evidence argues against considering TNKS a candidate gene for CdL syndrome. 12.26-P

12.25-P A new case of 8p23.1 deletion including a proposed candidate gene for Cornelia de Lange syndrome without the associated phenotype L. Ballarati (1) R. Caselli (1) M. Recalcati (1) A. Selicorni (2) S. Maitz (2) A. Cereda (2) C. Valtorta (1) P. Finelli (3) L. Larizza (4) D. Giardino (1) (1) IRCCS Istituto Auxologico italiano (2) IRCCS Policlinico Milano, Italia. (3) Università di Milano (4) Università di Milano, Polo H S. Paolo Deletions of 8p23.1 chromosomal region are associated with a recently described syndrome mainly characterized by developmental impairment, mild to moderate mental retardation, microcephaly, congenital heart disease, diaphragmatic hernia and a peculiar behaviour with hyperactivity and impulsiveness. Interstitial 8p23.1 deletions of about 3,4 Mb in size involve GATA-4 (GATA binding protein 4 ) and TNKS (tankyrase 1) genes. GATA-4 haploinsufficiency is considered to be responsible for the cardiac anomalies observed in 8p23.1 deleted patients. TNKS gene has been recently proposed as a further candidate for Cornelia de Lange (CdL) phenotype due to the CdL-like dysmorphic features observed in a 8p23.1 deleted patient and its role in sister chromatid cohesion, in keeping with the function of other CdL genes. Larger 8p23.1 deletions may also include MCPH 1 (microcephalin) gene, considered to be responsible for the microcephaly observed in 8p23.1 deleted patients. We report on a patient with psychomotor and developmental delay, microcephaly, minor facial dysmorphisms, interatrial defect, pulmonary stenosis and Attention-Deficit/ Hyperactivity Disorder (ADHD) carrying an interstitial 4,1 Mb deletion at 8p23.1, identified by array-CGH with a mean reso-

An International Standardised Cytogenomic Array (ISCA) Consortium approach to the design, validation, implementation and reporting of constitutional oligo array-CGH J. Barber (1) S. Huang (1) D. Ledbetter (2) C. Martin (2) S. Aradhya (3) S. Knight (4) K. Smith (5) K. Kok (6) J. Vermeesch (7) J. Crolla (1) (1) Salisbury NHS Foundation Trust (2) Emory University (3) GeneDx (4) Oxford Partnership Comprehensive Biomedical Research Centre (5) Oxford Regional Cytogenetics Laboratory (6) University Medical Center Groningen (7) University of Leuven Experience to date in ~1,000 reported cases from the National Genetics Reference Laboratory (Wessex) in the UK has shown that use of a customised 4×44k oligonucleotide array in karyotypically normal patients ascertained for developmental delay or mental retardation with or without congenital abnormalities, results in the detection of ~25% significant copy number changes (CNCs) ~15% of which are de novo. The NGRL (Wessex) is also part of the ISCA Consortium which is a collaboration comprising ~70 laboratories in the USA, Canada, South America and Europe. Two ISCA workshops held in 2008 led to agreement in two main areas, the first of which was the design, testing and implementation of oligo arrayCGH utilising multiplex formats including 8× 60k, 2×105k and 4×180k. The consensus design is evidence based and derived from extensive experience principally from five laboratories each of which independently custom designed constitutional cytogenetic arrays using Agilent’s eArray software. The ISCA oligo array combines targeting for ~500 genomic regions of known or suspected pathogenicity

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together with an even: “backbone” coverage of one oligo probe every ~25 kb. The second ISCA objective is to set up an NCBI hosted genome browser-based data sharing between Consortium Laboratories to develop data sets which will help to differentiate between benign and pathogenic copy number changes. Three ISCA laboratories are currently validating the consensus 4×180k array and these results together with a summary of the NGRL (Wessex) results to date with the 4×44k array will be presented. 12.27-P A Novel 16q24 microdeletion syndrome involving the FOX transcription factor gene cluster. P. Stankiewicz (1, 14) P. Sen (2, 14) S. Bhatt (1) M. Storer (3, 4) Z. Xia (1) B.A. Bejjani (5) Z. Ou (1) J. Wiszniewska (1) D.J. Driscoll (6) J. Bolivar (7) M. Bauer (7) E.H. Zackai (8) D.McDonald-McGinn (8) M.J. Nowaczyk (9) M. Murray (10) T.H. Shaikh (8) V. Martin (3, 4) M. Tyreman (11) I. Simonic (11) 14689 L.Willatt (11) J. Paterson (12) S. Mehta (12) S. Gribble (4) E. Prigmore (4) A. Patel (1) L.G. Shaffer (5) N. P. Carter (4) S. Wai Cheung (1) C. Langston (13) C. Shaw-Smith (3, 4) (1) Dept of Molecular & Human Genetics (2) Dept of Pediatrics—Nutrition, Baylor College of Medicine, Houston, Texas (3) Institute of Child Health, London, UK (4) Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK (5) Signature Genomic Laboratories, LLC, Spokane, Washington (6) Division of Pediatric Genetics and Metabolism, University of Florida College of Medicine, Gainesville, Florida (7) Dept of Pathology, Miami Children’s Hospital, Miami, Florida (8) Division of Human Genetics, Children’s Hospital of Philadelphia, Pennsylvania (9) Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada (10) Department of Medical Genetics, University ofWashington, Seattle, Washington (11) Regional Cytogenetics Laboratory (12) Dept of Medical Genetics, Addenbrooke’s Hospital, Cambridge, UK (13) Dept of Pathology, Texas Children’s Hospital, Baylor College of Medicine, Houston, Texas (14) Misalignment of pulmonary veins (MPV) with alveolar capillary dysplasia (ACD) is a rare developmental disorder of the lung that affects newborns.

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A characteristic of MPV/ACD is that it is typically associated with multiple congenital malformations (MCM). We report six overlapping microdeletions encompassing the FOX gene cluster at chromosome 16q24.1q24.2 in patients with ACD, MPV and MCM, using array CGH technology. Subsequent sequencing of the candidate gene FoxF1 in unrelated patients with ACD/MPV & MCM identified four different heterozygous mutations (frameshift, nonsense and no-stop). Additional MPV/ACD samples were analysed by a custom designed high resolution microarray and revealed one microdeletion within the FOXF1 gene and two microdeletions upstream of FOXF1, implicated a positional effect. This data identifies a novel microdeletion syndrome that has important implications for explaining the genetic etiology of ACD/MPV. 12.28-P Array-CGH in 400 patients with idiopathic mental retardation M. Malacarne (1) S. Cavani (1) C. Viaggi (1) M. Mogni (1) M. Piccione (2) M. Cavaliere (3) T. Mattina (4) P. Striano (5) F. Faravelli (1) M. Pierluigi (1) (1) E.O. Ospedali Galliera, Genova (2) A.O. Universitaria Policlinico (3) A.O.R.N. A. Cardarelli (4) Azienda Policlinico (5) IRCCS Gaslini Cryptic chromosome aberrations are a common cause of idiopathic mental retardation (MR), growth delay (GD) and congenital malformations (CM). A review of 16 studies show chromosome aberrations in 16,1% of this patients (range 4–34.1%). Among these aberrations there were well known syndromes as well as some rare variants. Many of these chromosomal imbalances are submicroscopic and cannot be detected by conventional cytogenetic methods. Microarray-based comparative genomic hybridization (array-CGH) is considered to be superior in the investigation of chromosomal deletions or duplications and has been demonstrated to improve the diagnostic detection rate for these small chromosomal abnormalities. Array-CGH have many potential advantage: rapid genome-wide analysis at high resolution and directely linked of the results with physical and genetic maps.

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In this study, 400 patients with idiopathic MR were analysed using array CGH Agilent 4X44K chip (Agilent Technologies, Santa Clara, CA). Alterations were validated and parents were tested to determine de novo occurrence. This study confirms the utility of molecularcytogenetic screening in patients with MR/CM and as one of the most reliable technique with a high diagnostic yield.

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It is in itself a rarity, which is inherited to the child. It is most likely a recombinant crossover at the bottom of chromosome 4q, leading to the loss of 4p terminal. Conclusion: Child with deletion of 4p terminal because of a rare maternel subtelomeric rearrangement. Array CGH analysis is a fairly good method to find quite small imbalances in the human genome, whereas FISH analysis is a unique method for analysis of complex genome rearrangement.

12.29-P 12.30-P Detectives ArrayCGH and FISH A. Nielsen (1) S. Pedersen (1) G. Hartmann (1) C. Lautrup (1) (1) Aarhus University Hospital Introduction: For 3½ years we have offered Array CGH analysis especially to patients with mental retardation. This technique detects small cryptic chromosome abnormalities. In 75 of 554 cases (14%) we have found small quantitative genomic changes. Method: Array CGH Equal parts of patient genomic DNA (CY3) and reference DNA (CY5) incubate on a BAC array with 4000 1 Mb BAC clones representing the entire genome. The Array is subsequently scanned and BlueFuse software calculates and produces graphic presentations of data from each BAC clone that has a known position in the Human Genome. Case: Girl, 13 years old, with dysmorfic features. Previously diagnosed as 46,XX in both Q-band and R-band (Prophase), with focus on chromosome 5p, due to clinical suspicion of Cri du Chat. Results: Array CGH analysis showed a small subtelomeric deletion on chromosome 4p. We verified with a subtelomer 4p FISH probe and a probe for Wolf Hirschhorn, as several of the deleted clones are located in this region. The patients diagnosis is Wolf Hirschhorn syndrome. This syndrome occurs in 1:50000 children, 85% are de novo, whereas 15% arises from a balanced translocation in one of the parents. In this case the mother had a balanced rearrangement of chromosome 4, in which 43% were mosaic, where subtelomer 4p was moved down terminally on chromosome 4q. In 57% there was a normal distribution of subtelomer 4p and 4q.

Characterization of a novel 4q21 microdeletion syndrome in 7 unrelated patients with severe mental retardation, absent speech, specific facial dysmorphism and severe growth delay C. Bonnet (1) J. Andrieux (2) B. Leheup (3) O. Boute (4) H. Copin (5) C. Le Caignec (6) V. Malan (7) V. Cormier-Daire (8) P. Jonveaux (1) D. Sanlaville (9) (1) CHU Nancy (2) , Hôpital Jeanne de Flandre, CHRU de Lille (3) CHU Nancy (4) Hôpital Jeanne de Flandre, CHRU de Lille (5) Centre de Gynécologie-Obstétrique, Hôpital Nord, Amiens (6) CHU Nantes (7) Hôpital Necker Enfants Malades, Paris (8) Hopital Necker Enfants Malades, Paris (9) Hospices Civils de Lyon, Bron Genome-wide screening of large patients cohorts with mental retardation using microarray-based comparative genomic hybridization (array-CGH) has recently lead to identify several novel microdeletion and microduplication syndromes. Here, we report on 7 unrelated patients with a de novo interstitial deletion including region 4q21 detected using CGH-array. These 7 patients present with a recognizable clinical phenotype including severe mental retardation, absent speech, specific facial dysmorphism and severe growth delay (between −6 SD and −3.5 SD). The boundaries and the sizes of the 7 deletions were different (between 3.2 and 15.1 Mb) but an overlapping region of 1.84 Mb was defined. This region included 9 genes and among these one is a strong candidate gene for mental retardation and severe growth delay observed in all our 7 patients because of its tissue expression and functions. Based on our 7 patients we propose a new 4q21 microdeletion syndrome with a distinctive clinical phenotype and a strong candidate gene.

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12.31-P 44K array-CGH in 1000 patients presenting mental retardation/multiple congenital malformations J. Andrieux (1) M. Holder-Espinasse (2) O. BouteBenejean (2) A. Dieux-Coeslier (2) M. Mathieu (3) H. Copin (3) B. Delobel (4) G. Plessis (5) C. VincentDelorme (2) S. Manouvrier-Hanu (2) (1) CHRU de Lille (2) CHRU de Lille (3) CHU d’Amiens (4) GHICL (5) CHU de Caen Since 2008, 11 array-CGH platforms have been enforced in France in order to detect constitutional cryptic genomic imbalances as a routine diagnosis. To date, 1000 DNAs from patients presenting with MR/MCA have been studied using Agilent 44K array-CGH in our centre: 914 post-natal cases and 86 prenatal cases (63 after termination of pregnancy and 13 during pregnancy). Considering post-natal cases, 160 (17.5%) genomic imbalances have been detected: 128 (14%) were considered deleterious (i.e. de novo or transmitted from a parent showing the same phenotype), 13 (1.4%) were found in a healthy parent but for the remaining 19 (2.1%) no parental samples were available. For the prenatal cases, 15 (17.4%) genomic imbalances were detected: 12 (14%) were considered deleterious and 3 (3.5%) were identified in a healthy parent. Among the 140 deleterious genomic imbalances: –

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11 (7.9%) corresponded to known microdeletion syndrome not diagnosed on clinical findings: 3 were telomeric (two 1pter and one 22qter) and 6 were typical interstitial (three del 22q11.2 (DiGeorge), one del 17p11.2 (SMS), one del 15q11.2 (Angelman syndrome), one 7q11.23 (Williams-Beuren)). Two were Xq28 duplications involving MECP2. 4 (2.9%) were imbalanced telomeric anomalies (partial monosomy associated with partial trisomy). 23 patients presented new microdeletion syndromes. Three del 17q21 (MAPT), 3 del 15q13.3 (CHRNA7), 6 del 1q21/3 dup 1q21 (microcephaly/macrocephaly), and 4 del 16p11.2/ 7 dup 16p11.2. 5 patients presented single gene deletions.

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All detected anomalies are available on the BACH database (https://www.genopole-lille.fr/bach/menu. php) with login/password on demand. 12.32-P A rare, prenatally detected, unbalanced complex chromosome rearrangement, studied with FISH and oligonucleotide array CGH A. Knegt (1) M. Berens (1) M. Baars (1) C. Bilardo (0) A. Polstra (1) (1) AMC Amsterdam (2) AMC Amsterdam A healthy 29-year-old woman, gravida 2 para 1 was referred for amniocentesis at 20 weeks of gestation because repeated ultrasound examinations showed multiple congenital anomalies. At the cerebral level: enlarged 3rd ventricle, abnormal fossa posterior with absent cerebellar vermis. The profile was abnormal due to a flat nose and retrognathia. Further diaphragmatic hernia and polihydramnios were noted. Conventional karyotyping was performed on amniotic fluid. Cytogenetic analysis identified a 46,XY, der(10p).ish ins (?;10) wcp10-,10pter(GS-23-B11x2) dn karyotype. The parents decided to continue the pregnancy. At 35.4 weeks of gestation a boy was born who died shortly after birth. A postmortal MRI was performed and showed pachygyria, Dandy Walker malformation, a large diaphragmatic hernia, aplastic left and hypoplastic right lung and low signal intensity of liver and long bones. FISH, array-CGH and microsatellite genotyping were performed postnatally on peripheral blood of the patient and his parents. FISH and custom Oxford 105K (Agilent) arrayCGH revealed a rare complex de novo unbalanced chromosome rearrangement (CCR) ascertained as 46, XY,ins(11;10)(q23.1q25;p11.21) .arr cgh dup(5) (q22.2q22.2),del(10)(p14p12.2),del(10) ( p11 .2 1 p 11 . 1 ) , d u p ( 11 ) ( q2 3 . 1 q 25 ) , de l ( 1 2 ) (p11.23p11.22). Microsatellite genotyping determined paternal inheritance of two of the aberrations. Conventional prenatal cytogenetics revealed a rare chromosome insertion. Array-CGH found 4 additional aberrations and was essential for detecting the complete unbalanced (complex) chromosome rearrangement. Dual-color FISH was used to make a

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reconstruction of the derivative chromosome 10. Array-CGH is more and more likely to be the first choice technique to use in clinical cytogenetics for patients with congenital abnormalities, mental retardation and in the near future in case of (multiple) congenital anomalies detected at prenatal ultrasound. 12.33-P Oligonucleotide-based array-CGH in subjects with mental retardation/developmental delay revealed unexpected findings in subjects both with normal and abnormal karyotype R. Ciccone (1) M. Fichera (2) F. Papa (3) C. Romano (2) A. Renieri (3) O. Zuffardi (1) (1) University of Pavia (2) IRCCS Maria SS (3) University of Siena Mental retardation occurs in 1–3% of the general population and its aetiology is extremely heterogeneous. Chromosomal rearrangements detectable by conventional karyotyping can be found in about 10% of mentally retarded individuals even though at least half of these subjects are carriers of trisomy 21, which is associated with a well recognizable phenotype. The implementation of conventional cytogenetics with subtelomeric screening allowed to highlight cryptic rearrangements at chromosome ends in about 2–6% of the subjects with normal karyotype, However, the large-scale application of genome-wide screening techniques allowed to identify much more cryptic imbalances causative of pathological phenotype not otherwise identifiable by other methods. We report on array-CGH investigation performed by three Italian centres (University of Pavia, IRCCS Oasi Maria SS and University of Siena) by using three different microarrays (Agilent 44B, 105A and 244A) which have an average resolution of about 100 Kb, 40 Kb and 20 Kb respectively. Array-CGH analyses have been performed on more than 1000 individuals affected by mental retardation and/or congenital anomalies and normal karyotype. Moreover, we investigated 350 individuals with known rearrangements, previously detected either by karyotype analysis or subtelomeric screening, who underwent array-CGH analysis for a more accurate investigation. We identified potentially causative copy number variations (CNVs) in 149 subjects with normal karyo-

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type, with a diagnostic yield of about 15%. Among them, 58 had imbalances mediated by segmental duplications. In addition, 107 subjects had CNVs of uncertain clinical significance generally inherited from a healthy parent. Array-CGH investigations on individuals with de novo, apparently balanced chromosome rearrangements revealed that hidden imbalances can be highlighted in about 40% of the translocations and 90% of the complex chromosome rearrangements. Additionally the analysis of subjects with unbalanced rearrangements, defined by other cytogenetic techniques, revealed that the initial interpretation was incorrect in about 16% of the cases. In conclusions, our data emphasize the importance of genome-wide investigations in clinical practice both in case with normal and abnormal karyotype. On the other hand, these investigations did reveal an unexpected number of rare CNVs inherited from healthy parents making the incomplete penetrance an emerging problem diagnosis of genetic diseases. The accumulation of array-base investigations on large sets of patients and healthy subjects will further increase our capability to interpret the biological role played by copy number variations. 12.34-P Atypical proximal and distal rearrangements generating supernumerary derivative chromosome 15 C. Largo (1) M. Gallegos (2) L. Avila (1) E. Ferrada (1) J. Castrillo (1) S. Avila (1) (1) GENETADI Biotech (2) IMMS Small supernumeray marker chromosome (SMC) are a heterogeneous group of chromosomes with an estimated frequency of 0.4-1.5/1000 newborns. SMC (15) often referred to as INV dup(15) is the most common marker chromosome in humans, representing 50% of all reported markers. We describe the case of a patient analysed for dysmorphic features. The family history is noncontributory. The propositus showed hydrops at seventh month of pregnancy and hypotonia, bilateral equinovarus and other dysmorphisms, at birth. The general report (3 years and 3 months) shows low weight, psychomotor retardation, hypertrophy of the

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palatal shelves, low-set ears, wide nasal bridge, parrot-beaked nose, hypotonia, bilateral epicanthus and microcephaly. Using cytogenetic analysis, a large dicentric bisatellited SMC chromosome 15 was identified in the propositus, Both parents showed a normal karyotype. The oligo array CGH analysis showed trisomy of a 15.6 Mb region in 15q11–q14 and also tetrasomy of 7.2 Mb region in 15q25.3–q26.2. FISH analysis were conducted in order to confirm the pattern detected by aCGH and to establish the structure of the SMC. Translocations or other possible structural rearrangements were discarded by M-FISH analysis in the three members of the family. Possibly a complex interchromosome and intrachromosomal exchange was involved in the generation of the idic(15) chromosome, suggesting that not all der(15) chromosomes arise through nonhomologous allelic recombinations mediated by LCRs present within the imprinted region of the proximal arm of chromosome 15. 12.35-P Microdeletion 21q22.11 detected with array-CGH: genotype/phenotype correlation E. Nalesso (1) S. Bigoni (2) A. Pini (3) A. Baldan (1) S. Gomirato (1) L. Cardarelli (1) (1) Department of Histology, Microbiology and Medical Biotechnologies (2) Medical Genetic Section (3) Child Neuropsychiatric Unit (4) Citotest Laboratory We describe the case of a patient analysed with array-CGH for mental retardation and dysmorphic features. Pregnancy was uneventful until the 37th week when a cesarean section was performed because of fetal cardiac arrhythmia. Normal auxological neonatal parameters. Psychomotor development was considerably delayed in the first year of life because of important axial hypotonia. At the age of 4 years an insufficient control of balance reactions, inadequate motor skills, dyspraxia, speech delay, attention deficit, scialorrea were noticed; diagnosis of moderated mental retardation was made. MRI at the age of 1 and 4 year revealed the corpus callosum hypoplasia, mild dilatation of lateral ventricles, mild frontal cortical atrophy, focal white

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matter hyperintensity mainly in the frontal regions, mega cisterna magna. Actually, at the age of 15 years old, we report normal auxological parameters, dysmorphisms, moderated mental retardation; now tremors are developing. Genetic investigations: normal high resolution karyotype and telomeric FISH probes, negative FRAXA mutation. Array-CGH has identified a deletion of about 1,7Mb in chromosome 21q22.11: the first oligomere deleted is at 32183910,bp the last one is at 33918947bp. Among the several genes mapped in this region, we draw the attention to olig1 and olig2. Olig1, Oligodendrocyte transcription factor 1, promotes formation and maturation of oligodendrocytes, especially within the brain. Cooperates with OLIG2 to establish the pMN domain of the embryonic neural tube. Olig2, Oligodendrocyte transcription factor 2, (Class B basic helix- loop-helix protein 1) (bHLHB1) (Protein kinase C-binding protein RACK17) (Protein kinase C-binding protein 2). Required for oligodendrocyte and motor neuron specification in the spinal cord, as well as for the development of somatic motor neurons in the hindbrain. Cooperates with OLIG1 to establish the pMN domain of the embryonic neural tube. Discussion of the case and review of the literature. 12.36-P FMR1 gene expansion, large deletion of Xp and skewed X-inactivation in a girl with autism and mental retardation A. Vazna (1) Z. Musova (1) M. Vlckova (1) D. Novotna (1) L. Dvorakova (2) M. Hrdlicka (3) M. Havlovicova (1) Z. Sedlacek (1) (1) Charles University in Prague—2nd Faculty of Medicine (2) Charles University 1st Faculty of Medicine and General University Hospital (3) Charles University 2nd Faculty of Medicine and University Hospital Motol We describe a girl with mild mental retardation, mild facial dysmorphy and atypical autism. The occurrence of late-onset tremor and premature ovarian failure in the maternal branch of the family pointed to a possible FMR1 defect, which was confirmed by DNA analysis. However, classical cytogenetic examination revealed

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in addition a large deletion on the short arm of one of the X chromosomes of the patient. We describe here the detailed molecular cytogenetic and molecular analysis of the genetic defects in this patient. The analysis of the FMR1 gene using PCR and Southern blotting showed a full mutation (about 460 CGG repeats) on the maternal FMR1 allele. Interestingly, both the expanded and the normal FMR1 alleles were almost completely methylated in the patient. Karyotyping and subsequent mapping using oligonucleotide array CGH showed a large, about 17 Mb long deletion of Xp22.11–p22.31. Microsatellite analysis indicated that the de novo deletion affected the paternal X chromosome. The X-inactivation status was also assessed using the analysis of the AR locus, which confirmed almost complete inactivation of the paternal X chromosome. The deletion was the most likely cause of this skewing. The breakpoints of the deletion were fine mapped using long-range PCR and sequenced. The absence of homologous repeats at the breakpoints and the presence of three bases (TTC) added at the deletion junction pointed to nonhomologous end joining as the most probable mechanism of the aberration. The phenotype of the girl resulted most likely primarily from the inactivation of both alleles of the FMR1 gene. The large deletion of Xp removed about 90 genes, but as the affected X chromosome was almost completely inactivated, the main consequence of the deletion could be the unmasking of the FMR1 defect. However, reduced dose of several genes escaping the Xinactivation could also play a role. Supported by IGA NR/9457 - 3, MZO00064203, INCORE and CHERISH.

and thus cell-to-cell variability in human cleavage stage embryos as well. As a consequence, these findings likely provide a biological basis for the recent reports that challenge the view that preimplanation genetic screening after in vitro fertilization increases the baby take home rate. DNA from 165 single blastomeres from 23 human cleavage stage embryos with good morphology from young normal fertile couples (<35 years), who had preimplantation genetic diagnosis (PGD) for sex- or microdeletion selection, were profiled cell by cell by 3K BAC and 250K SNP array. This way, genome-wide copy number variations and loss of heterozygosity were scored for the first time in single cells. Only 2 out of the 23 embryos were chromosomally normal in all blastomeres (2/23, 9%). Surprisingly, in the remaining embryos, not only mosaicism for whole chromosome aneuploidies were frequent (21/23, 91%), but in addition, a large number of segmental imbalances was observed (16/23, 75%). Furthermore, these segmental deletions, duplications and amplifications were reciprocal in sister blastomeres implying the occurrence of breakage-fusion-bridge cycles. This explains the low human fecundity and identifies post-zygotic chromosome instability as a leading cause of constitutional chromosomal disorders. Moreover, these data proof that FISH based preimplantation genetic screening of cleavage stage embryos cannot be accepted as a method to increase the baby take home rate in routine clinical patient care, since this will lead to the rejection of potential viable embryos.

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Genome wide array analysis in prenatal diagnosis: preliminary results of oligo array-CGH analysis on fetuses with normal or abnormal karyotype and echographic abnormalities

Chromosome instability is common in human cleavage stage embryos E. Vanneste (1) T. Voet (1) M. Ampe (2) P. Konings (3) C. Melotte (1) J. Fryns (1) G. Verbeke (2) T. D’Hooghe (4) Y. Moreau (3) J. Vermeesch (1) (1) K.U.Leuven (2) K.U.Leuven (3) K.U.Leuven (4) U.Z. Gasthuisberg Chromosome instability (CIN), which is characterized by an elevated rate of gains and/or losses of (parts of) chromosomes per cell cycle, is well known in tumorigenesis. Unexpectedly, this study found CIN

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A. Vetro (1) F. Lalatta (2) E. Rossi (1) J. Messa (1) M. Dioli (1) A. Pettinari (3) E. Manolakos (4) G. Croci (5) F. Franchi (5) O. Zuffardi (1) (1) University of Pavia (2) IRCSS Policlinico Mangiagalli (3) Ospedali Riuniti di Ancona (4) Bioiatriki (5) Arcispedale S. Maria Nuova Genome-wide array is having a strong impact on clinical genetics, because of its higher diagnostic rate in respect to traditional cytogenetic techniques.

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High-pitched debate is now ongoing on the appropriateness of using genome-wide array in prenatal diagnosis although conventional cytogenetics has huge limitations because of low resolution, need cell cultures and may arise unsolvable questions as it is the case of de novo apparently balanced translocations or marker chromosomes. We already studied by array-CGH analysis (Agilent oligomer platform at a resolution of about 100 Kb) 38 DNA samples (from chorion villi and/or amniocytes) from foetuses with echographic abnormalities such as IUGR, cystic hygroma and complex malformations. Conventional cytogenetic analysis has been done in all cases. In 26 cases we didn’t find any imbalance; in the remaining 12 cases we detected or re-defined genomic imbalances, obtaining information otherwise not achievable through the sole conventional cytogenetic. One case had concurrent deletion and duplication of about 20 Mb each at 5p: the similar size of the two imbalances prevented their detection by conventional cytogenetics. Two cases of de novo small supernumerary marker chromosomes (sSMC) revealed surprising results. In one case the sSMC was constituted by two non contiguous portions of chromosome 18 (short arm, proximal long arm, distal long arm), in the other case it was constituted by a derivative chromosome 15 (15p11.2;q13.1) to which the distal portion of chromosome 19p was translocated. These findings make clear that sSMCs are a category of chromosome rearrangements largely misunderstood by conventional cytogenetics. Altogether, our results show that genome-wide array in prenatal diagnosis may provide information useful to appropriate genetic counselling although present scarse knowledge of the benignity vs pathogenicity of several CNVs and the incomplete penetrance/ variable expressivity of others create important limitations. 13.2-P Prenatal diagnosis of inherited unbalanced X-Y translocation in a male fetus B.García García*; L.Varela Sanz*: MJ Álvarez Blanco*; C.Coca Martín*. E.Mansilla Aparicio**; F. García Santiago**H.U.PRÍNCIPE DE ASTURIAS. ALCALÁ HENARES.MADRID * H.U. LA PAZ.MADRID**

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Introduction. Translocations between X and Y chromosomes are rare. We present a case of prenatally ascertained (Xp;Yq) translocation characterized using conventional and molecular cytogenetic. The important genotypic defect is the deletion of the distal Xp segment, with possible loss of several genes. Clinical case. 36-year-old patient, in her second pregnancy. She refers cytogenetic abnormalities in the amniocentesis of the first pregnancy, inherited from her. That resulted in a phenotipycally normal daughter. Amniocentesis was performed at 16 weeks. Genetic study Karyotype in amniotic fluid: Cytogenetic analysis revealed an X;Y translocation: 46,Y,der(X),t(X;Y)(p22.3;q11). Karyotype in maternal peripheral blood: 46,X,der(X),t(X;Y)(p22.3;q11) C bands: Additional heterochromatic material on the distal short arm of the X chromosome. FISH in amniotic fluid: Hibridation “in situ” with probes for subtelomeric regions Xp (Cytocell), for region of Shox gene (Xp22.3/Yp11.3)(Kreatech) and for critic region of Kallmann Sd. (Xp22.3) (Vysis) Results: ish der (X) (DXYS129-; DXYS153->DXYS130-, KAL+) FISH in maternal peripheral blood: Applying the same probes as above the results are the same: ish der (X) (DXYS129-; DXYS153- >DXYS130-, KAL+) Discussion. We have a typical case of familial (Xp;Yq) translocation. The phenotype depends on the size of deletion in Xp. The mother, and her ten-year-old daughter, have a partial monosomy for the Xp segment, so they are characteristically fertile and of normal intelligence. As SHOX is deleted, the monosomic (haploinsuficient) state for this gene determines a particular form of short stature. Regarding the male fetus, partially nullisomic, his phenotype depends on the location of the

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breakpoint. We know that the critical region for Sd Kallman is present and the region for Shox gene is absent, but genes between both, ARSE (arylsulfatase E, for chondrodysplasia punctata),STS (steroid sulfatase, for ichthyosis), MRX (mental retardation) and OAI (ocular albinism) may be absent too, so, genetic counselling is very difficult in this case. 13.3-P Antenatal cytogenetic diagnostics of chromosomal anomalies of the fetus in various terms of pregnancy G. Abildinova (1) M. Bayanova (1) A. Wiebe (1) B. Kamalieva (1) (1) National reseach center mother and child For the purpose of studying of frequency and structure of chromosomal infringements of a fetus to various terms of pregnancy, prenatal cytogenetic researches have been held among 461 pregnant women from a high risk group. A material for carrying out prenatal cytogenetic researches was cells of chorion villi, placenta cells, and also lymphocytes of fetus umbilical blood. In 96, 5% a normal karyotype of a fetus has been diagnosed—46,XX; 46,XY. In 23 cases there were chromosomal infringements of a prenatal fetus that has made 5%. In 22 cases there has been revealed chromosomal anomalies aneuploidy, and a structural chromosomal pathology in 1 case. Among numerical anomalies of chromosomes five cases of a Edwards syndrome are diagnosed , a Down syndrome in four cases, anomalies on sexual chromosomes a Turner syndrome, the simple and mosaic form-45, ÕÎ in six cases, a triple Õ syndrome—47, ÕÕÕ in two cases and polyploidy—92, ÕÕÓÓ in one case. Structural chromosomal reorganization—46, XY, dup13q (q21; q22)—is revealed in one of the cases. Numerical chromosomal anomalies prevail in 95, 6% in a chromosomal pathology structure. The received results show the information of a cytogenetic research of cells of a fetus for preventive maintenance of a birth of children with a chromosomal pathology.

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13.4-P Use of FISH-method for identification of chromosomal abnormalities during prenatal diagnosis T. Zolotukhina (1) N. Shilova (0) Z. Markova (0) T. Tsvetkova (0) (1) Research Centre for Medical Genetics (2) Research Centre for Medical Genetics (3) Research Centre for Medical Genetics (4) Research Centre for Medical Genetics We report the prenatal diagnosis of three cases with different structural chromosomal abnormalities in fetuses, confirming by FISH-analysis. Case 1. The 40 year old woman was referred for cordocentesis at 21 weeks of gestation because of advanced age. The fetus do not affect phenotype by ultrasounds. Fetal karyotype was determined as mos 46,XX,fra(16)(q22)[11]/46,XX[39]. Moreover small supernumerary marker chromosome was detected in few cells. FISH analysis with wcp 16 probe confirmed fra(16)(q22), also the supernumerary marker as being of chromosome 16 origin. Karyotype of mother have the similar rearrangement. Pregnancy was continued and at term health girl was born. Case 2. The 23 year old woman was referred for cordocentesis at 21 weeks of gestation because multiple fetal abnormalities were identified by ultrasounds: goloprosencephaly, abnormal foots, smoothed profile, intrauterine growth retardation. Fetal karyotype was ascertained as 46, XY,add(7)(q35) on cord blood culture lymphocytes. Parental karyotypes were normal. mFISH analysis confirmed the additional material in chromosome 7 as being of chromosome 9 origin. FISH with subtelomeric probes showed 9pter+, in derivate chromosome. Fetal karyotype was 46,XY,der(7)t(7;9)(q35;p13) de novo. Thus fetal phenotype depended generally from monosomy 7 (q35-qter). Case 3. The 26 year old woman was referred for CVS at the 11 weeks of gestation because NT was 5 mm. Derivate chromosome was detected in direct CVS sample. In 22 weeks multiple fetal anomalies were identified by ultrasounds: choroid plexus cysts, hypoplasia vermis cerebelli, two hyperechogenic focuses in heart ventricle, intra-

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uterine growth retardation. Karyotype of fetus was ascertained as 46,XY,add(8)(p23) after cordocentesis. Parental karyotypes were normal. mFISH analysis confirmed the additional material as being of chromosome 8 origin. Thus fetal karyotype was determined as 46,XY,dup(8) (p12p23).

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13.5-P

F. Grati (1) A. Barlocco (1) B. Grimi (1) S. Milani (1) F. Dulcetti (1) S. Chinetti (1) S. De Toffol (1) M. Clementi (2) F. Maggi (1) G. Simoni (1) (1) TOMA, Advanced Biomedical Assays (2) Dipartimento di Pediatria, Università di Padova

Unbalanced translocation (14;21) in the fetus as a result of a rare mode of paternal malsegregation N. Shilova (1) T. Zolotukhina (0) (1) Research Centre for Medical Genetics of RAMS (2) Research Centre for Medical Genetics of RAMS The 21 year old woman was referred for cordocentesis at 21 weeks of gestation because multiple fetal abnormalities were identified by ultrasonography: intrauterine growth retardation, congenital heart defect.. Fetal karyotype was determined as 46,XX,der (21) in cultured cord blood lymphocytes. FISH analysis with different DNA-probes (Abbott,Vysis) in cultured cord blood lymphocytes and parental chromosomal investigation using conventional peripheral blood culture method were performed to detect origin of the derivate chromosome. FISH analysis with LSI 21 probe (21q22.13–22.2) has revealed two copies of analyzed chromosomal region. However, FISH with wcp 21 probe has shown presence of centromere-containing DAPI-positive material in the proximal part of the derivate chromosome. FISH with wcp 13, 14, 15, and 22 probes has allowed to define the abnormal chromosome as der(14). Paternal karyotype was 46,XY,t(14;21)(q21;q21). Maternal karyotype was normal. Finally fetal karyotype was determined as 46,XX,+der(14),t(14,21)(q21;q21)pat,-21. Thus fetal phenotype depended from partial trisomy 14 (14pter→q21) and partial monosomy 21 (21pter→q21). The pregnancy was terminated at 23th weeks of gestation. This paternally inherited unbalanced translocation in the fetus was result of adjacent-2 segregation in paternal meiosis as an uncommonly observed mode of malsegregation in translocation heterozygotes.

Chromosome abnormalities investigated by noninvasive prenatal screening only account for approximately 50% of fetal chromosomal abnormalities associated with a relevant clinical phenotype

During the past 20 years, non-invasive screening tests have been increasingly applied in the practice of prenatal diagnosis (PD). Considerable effort has been exerted by multicentric consortia to evaluate the performance of invasive screening tests in detecting women with an increased risk of having a pregnancy affected by trisomies 21, 18, and 13, monosomy X, and triploidies. However, an evaluation addressing how much of this group of abnormal karyotypes accounts for the total number of phenotypically-relevant fetal chromosomopathies has not been conducted. Herein we report an attempt aimed to quantify this proportion. We undertook a retrospective analysis of a homogeneous survey of 115,128 invasive PD (84,470 amniotic fluid samples and 30,658 chorionic villi samples). All cases were classified according to the indication for invasive PD. We focussed on results from 96,416 karyotype analyses from pregnant women in which the advanced maternal age (≥35y) and the gestational anxiety (<35y) were the sole indications for invasive sampling since they usually are the beneficiaries of non-invasive screening tests. The cumulative amount of fetal chromosomopathies with a significant phenotypic effect that might be detected by prenatal screenings was calculated subtracting from the observed amounts of T21, T18, T13, 45,X, and triploidies the portion that would be undetected due to the specific detection rate of the screening test. Finally, this value was related to the total chromosomopathies associated with a clinically abnormal phenotype ranging from moderate-tosevere. Our findings indicate that the chromosomal abnormalities investigated by screening tests represented <50% of the fetal chromosomopathies associated with a high-intermediate risk of an

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abnormal outcome in women <35y (45.79% and 39.61% in the 1st and 2nd trimesters, respectively), and a sensitively >50% in women ≥35y (65.10% and 61.78%, respectively). In conclusion, approximately 50% of the phenotypically-relevant abnormal karyotypes cannot be investigated by non-invasive prenatal screening tests. 13.7-P Comparison among indications to test UPD in prenatal diagnosis: study of 370 cases F. Crosti (1) N. Villa (1) E. Sala (1) S. Redaelli (2) S. Lissoni (1) L. Spaccini (3) F. Lalatta (4) B. Gentilin (4) S. Mariani (5) L. Dalpra’ (2) (1) S. Gerardo Hospital (2) University of MilanoBicocca (3) ICP (4) F.O.Maggiore Policlinico Mangiagalli Regina Elena (5) S. Gerardo Hospital Uniparental disomy (UPD) is the inheritance of homologues chromosomes from only one parent. Possible mechanisms of formation are trisomy rescue, monosomy rescue, gametic complementation, and somatic recombination. The indications to perform the test were mosaicism or rearrangement, inherited or de novo, observed during conventional cytogenetic analysis. Problems associated with UPD include trisomy mosaicism (placental and fetal mosaicism), genomic imprinting, homozygosity of autosomal recessive mutations, or even a combination of these. Phenotypic effects from UPD have been described as certain for several chromosomes (PWS, AS, SRS, BWS, 14mat). We studied 370 cases in prenatal diagnosis: 112 robertsonian translocations (30%), 83 mosaicisms (22%), 65 single clone trisomies (18%), 34 SMCs (9%), 42 reciprocal translocations (11%), 27 IUGRs (7%), 5 inversions (1%), 1 probably BWS, 1 placental pathology. No UPD positive cases in robertsonian translocation, reciprocal translocation, IUGR, inversion, BWS and placental pathology. 3 mosaicism cases (UPD15mat and two UPD16mat) (3.6%), 1 single clone trisomie (UPD15mat)(1.5%) and 5 SMCs (UPD13mat, UPD15mat, UPD16mat, UPD16mat segmental, UPD16pat segmental)(14.7%) were positive (total:2.4%). The presence of a SMC may interfere with the segregation of the structurally normal homologous

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chromosomes during meiosis and mitosis, giving rise to aneuploidy. Since aneuploidy correction is one mechanism of formation for UPD, the presence of an SMC could, in theory, increase the risk of UPD. 13.8-P Low or absent maternal serum unconjugated estriol and STS gene deletion N. Jenčíková (1) H. Peková (1) V. Nedomová (1) D. Stejskal (1) (1) GENNET s.r.o. Low or undetectable levels of unconjugated estriol (uE3) with normal AFP and hCG values are rare findings on maternal serum triple marker screening in the second trimester. These findings may i.e. serve as an indicator of steroid sulfatase (STS) deficiency in male fetus. If uE3 is under 0.25 multiple of median (MoM) we carry out the FISH assay of steroid sulphatase gene deletion (Xp22.3 locus) on amniocytes. Between 2004– 2009 we analyzed 35 amniotic fluid samples with low (<0.25 MoM) or undetectable levels of uE3. At 18/35 (51.4%) cases low uE3 was caused by STS locus deletion and all these aberrations were inherited and maternally transmitted. One male fetus at this group had a pathological sonography and poor pregnancy outcome. Clinical features vary broadly depending on the lenght of the deletion or these disorders may occur as a contiguous gene syndrome. If both fetal sonoanatomy is normal and family history negative then the postnatal manifestation of STS deficiency in males is mostly Xlinked ichthyosis. Isolated positive FISH result is not regarded as a reason for any clinical action in pregnancy. 13.9-P Redefining PGS for patients with advanced maternal age M. Milán-Sánchez (1) C. Rubio (1) E. Mateu (1) V. Peinado (1) P. Mir (1) A. Mercader (1) P. Buendía (1) A. Delgado (1) J. Remohí (1) A. Pellicer (1) (1) Instituto Valenciano de Infertilidad, Ivi Valencia Introduction: There is a direct correlation between maternal age and the incidence of aneuploidies. The aim of this study is to present the results of our

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Preimplantation Genetic Screening (PGS) program in patients with advanced maternal age (AMA), defining subsets of patients in which PGS could be especially beneficial. Material and methods: Retrospective study including 1881 patients with AMA (38–44 years) since January 2003 to October 2008. 1042 patients were subjected to PGS using fluorescence in situ hybridization (FISH) for chromosomes 13, 15, 16, 18, 21, 22, X and Y. In control group, 839 patients were included in a regular in vitro fertilization (IVF) cycle. Clinical outcome was analyzed according to women’s age in a year by year fashion. Results: The selection of euploid embryos for the analyzed chromosomes produced significantly higher ongoing implantation rates (18.2% vs. 22.6% in 38–40 years, p=0.036; 3.9% vs. 15.6% in 41–44 years, p= 0.006; control vs. PGS, respectively). In terms of ongoing pregnancy rate per cycle (OPR/C), we observed no benefit of PGS in patients with 38– 40 years when compared to the control group, whereas a clear benefit was observed in the PGS group in patients with 41–44 years of age (2.3% vs. 7.4% in 41–44 years, p=0.039; control vs. PGS, respectively). Conclusions: In the present study we show that, attending only to OPR/C, the clinical indication AMA could be redefined. Patients with 38–40 years still have good possibilities of becoming pregnant in a regular IVF cycle without a benefit of being included in a PGS program. In patients with 41–44 years, the possibilities of becoming pregnant are dramatically reduced in a regular IVF cycle and the inclusion of these patients in a PGS program results in approximately a 3 fold increase in their clinical possibilities. Poster preferred 13.10-P Indication and outcome of CVS prenatal diagnosis performed by a single operator in the years 2003–2008 L. Eggenschwiler (1) F. Bottini (1) J. Achermann (1) G. Savoldelli (2) R. Spiegel (1) (1) Genetica AG, Laboratory of Human Genetics (2) Medical practice (Poster presentation) A retrospective study is presented based on prenatal diagnosis results from more than 6,000 patients

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undergoing chorionic villus sampling (CVS) performed by a single experienced operator in Zürich, Switzerland, in the years 2003 to 2008. The statistical evaluation of patient data focuses on three main topics: 1. Predictive value of referring indications, such as advanced maternal age (MA), abnormal ultrasound (US) and first trimester test (1TT) compared to cytogenetic results. 2. Frequency and accuracy of abnormal cytogenetic findings when comparing short-term culture (ST) with long-term culture (LT), as well as follow-up investigations (amniocentesis, fetal tissue etc.) 3. Procedure-related reliability in terms of frequency of successful laboratory analysis. Among the main referring indications, US showed the highest predictive value, with 29.5% true chromosomal abnormalities detected, followed by 1TT with 4.8%. The predictive values of MA (1.9%) and maternal anxiety (1.6%) did not differ significantly. The total frequency of abnormal cytogenetic findings in either ST and/or LT was 7.07%. 4.41% were clinical significant abnormalities detected both in ST and LT (trisomy 21 1.67%, trisomy 18 0.86%, trisomy 13 0.28%, rare trisomies 0.02%, sSMC 0.06%, triploidy 0.14%, aneuploidy of sex chromosomes 0.60%, structural aberrations 0.78%). Suspected fetal mosaicism, with normal cytogenetic results in either ST or LT was found in 0.11%. In one instance a prenatally not detected submicroscopic aberration was ascertained only postnatally. Thus, overall detection rate for clinical abnormalities was 99.62%. Confined placental mosaicism (CPM) was detected in 2.55% (CPM type I 1.72%, type II 0.66% and type III 0.17%). More than 40% of total CPM cases were due to rare trisomies. In 100% of ST a cytogenetical result was obtained. LT culture failure occurred in only 0.27%. 13.11-P Confined placental mosaicism for isochromosome 11q in chorionic villus sample M. Martorell (1) M. Alegre (2) R. Batallé (1) M. Solà (1) R. Romero (1) M. Cadiñanos (3) (1) Unitat Reproducció Humana i Diagnòstic Genètic. Clínica Girona (2) Secció d’Ecografia Prenatal i Ginecològica. Clínica Girona (3) Centre Ginecològic GiDona

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Confined placental mosaicisms (CPM) in chorionic villus samples have been documented in many reports and the frequency of CPM in prenatal diagnosis is about 1%. Here we present a case of discrepancy between the karyotype in short-term culture and long-term culture from a chorionic villus sample. The methodology used was a short-term cultured villi (STC), as it provides rapid results. At the same time, we performed a long-term cultured villi (LTC) whenever possible. In our case, the karyotype was mos 47,XX,+i(11)(q10)/46,XX in cells from the STC and 46,XX from the LTC. Cytogenetic studies on amniotic fluid did not detect any abnormal cells. Molecular studies did not reveal uniparental disomy for chromosome 11 in the fetus either. The ultrasound findings were normal and the woman who, was 43 years old and had previously had three miscarriages, decided to continue the pregnancy. 13.12-P Prenatal diagnosis of Wolf-Hirschhorn Syndrome (4p-) in association with growth restriction, nasal hypoplasia, mild hydronephrosis and unilateral club foot A. Tokutake (1) (1) Citogene—Laboratório de Diagnóstico de Doenças Genéticas (2) IMMEF—Instituto da Mulher e Medicina Fetal (3) Colorado Genetics Laboratory Introduction: The Wolf-Hirschhorn syndrome, caused by partial deletion of the short arm of one chromosome 4, is characterized by severe growth restriction and mental defect, microcephaly, ‘Greek helmet’ facies, and closure defects (cleft lip or palate, coloboma of the eye, and cardiac septal defects). Case report: We report a patient, AZF, 31 year-oldwoman, G II P I, referred at 24 weeks and 2 days gestation with IUGR (intrauterine growth restriction) and suspected nasal hypoplasia. Ultrasound examination revealed nasal hypoplasia, hydronephrosis and unilateral club foot. Chromosome analysis of amniotic fluid revealed a de novo 46,XX,del(4)(p15.2) karyotype, which was confirmed by FISH. Conclusion: Ultrasonographic findings may suggest chromosomal abnormalities. This study emphasizes

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the importance of karyotyping fetuses with IUGR without vascular abnormalities and suggests the possibility of recognizing a particular phenotype of WHS in utero. 13.13-P Prenatal Detection of Variegated Aneuploidy E. Winsor (1) S. Blaser (2) B. Fitzgerald (1) K. Fong (3) S. Keating (1) N. Martin (4) P. Shannon (1) A. Toi (3) D. Chitayat (4) (1) Mount Sinai Hospital (2) Hospital for Sick Children (3) Mount Sinai Hospital (4) Mount Sinai Hospital Case 1: Amniocentesis was performed at 15 weeks on a G4,P1,SA2 woman because of increased nuchal translucency (2.6 mm, >98th percentile at 12.2 wk). Eleven of 14 metaphases from 5 culture vessels had gain or loss of different chromosomes. Premature centromeric division was not observed. Ultrasound examination at 19 weeks revealed symmetric IUGR with a head circumference <3rd percentile. There was suspicion of partial agenesis of the cerebellar vermis. Follow up cytogenetic testing of umbilical cord (3/ 10), skin (4/10), cartilage (2/20) and placenta (5/12) confirmed the mosaicism consistent with variegated aneuploidy. Molecular testing for BUB1B gene mutations is pending. Case 2: Chromosome analysis was performed on cultured umbilical cord obtained from a stillborn fetus with severe microcephaly. Six of 50 metaphases had one or two extra chromosomes consistent with variegated aneuploidy. Premature centromeric division was not observed. Amniocentesis was not performed. Fetal autopsy revealed lissencephaly, absence of rostrum of the corpus callosum and fusion of the thalami posteriorly. DNA analysis did not reveal any mutation of the BUB1B gene. Microcephaly was also detected in a previous pregnancy of this couple and no aneuploidy was detected in 25 metaphases examined from amniotic fluid. In retrospect, it seems likely that both fetuses had the same condition. Conclusions: Prenatal detection of variegated aneuploidy is very rare. Because of variation in the distribution of aneuploidy in different tissue types, it is possible that diagnosis of this condition could missed or the findings misinterpreted as culture artifact.

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13.14-P Rapid prenatal diagnosis of common aneuploidies by QF-PCR, four years experience of Hacettepe University D. Aktas (1) B. Kutukcu (2) G. Utine (3) Y. Alanay (4) O. Deren (5) K. Boduroglu (6) S. Beksac (7) M. Alikasifoglu (8) (1) Hacettepe University Medical School (2) Hacettepe University Medical School (3) Hacettepe University Medical School (4) Hacettepe University Medical School (5) Hacettepe University Medical School (6) Hacettepe University Medical School (7) Hacettepe University Medical School (8) Hacettepe University Medical School Quantitative fluorescent polymerase chain reaction (QF-PCR) is an alternative method for rapid detection of common aneuploidies, using highly polymorphic short tandem repeat (STR) markers. We applied a QF-PCR test on prenatal diagnosis. We present results of 799 samples (amniotic fluid, cord blood and chorionic villus samples) analyzed for trisomies 13, 18 and 21 and sex chromosome aneuploidies using different commercial kits (Dviser kit, ChromoQuant version 1 and version 2, Aneufast kit). Sixteen cases of trisomy 21 (2%), 6 cases of trisomy 18 (0.75%), 3 cases of trisomy 13 (0.37%), 1 case of Turner syndrome (0.12%), 1 case of Klinefelter syndrome (0.12%) and 1 case of mosaicism (0.12%) were detected. Overall 95% of clinically relevant abnormalities were readily detected and termination of affected pregnancies could be performed without waiting for the cytogenetic results. We discuss the performance of QFPCR and STR markers and suggest that QF-PCR is an appropriate choice for rapid aneuploidy testing in the Turkish population. 13.15-P Confined placental mosaicism for tetraploidy and the association with intrauterine growth restriction J. Bryan (1) M. Peters (1) R. Lourie (2) (1) Mater Health Services (2) Mater Health Services (3) Mater Health Services

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Confined placental mosaicism (CPM) for a chromosome aneuploidy has been reported in association with clinically significant intrauterine growth restriction (IUGR), reduced postnatal growth and intrauterine fetal death (IUFD) CPM occurs in around 1–2% of viable pregnancies at 10–12 weeks gestation and is usually detected when an abnormal cell line in a chorionic villus sample (CVS) is not confirmed in follow up analysis of amniotic fluid (AF) or other fetal tissues. The mechanism by which CPM affects fetal growth and may even lead to IUFD is likely multifactorial with the association difficult to correlate given the many maternal and fetal factors which impact fetal growth. As abnormal fetal growth represents a major cause of perinatal morbidity and mortality and reduced fetal growth has been linked to subsequent development of diseases such as diabetes, heart disease and hypertension, it is clearly important to investigate avenues such as CPM. While CPM for many chromosome aneuploidies have been reported, of interest to us was the reported association of mosaic tetraploidy in the placentas of growth restricted newborns and growth restricted stillbirths and miscarriages. While mosaic tetraploidy in AF is often observed as the result of cultural artefact the occurrence of high level placental tetraploidy appears to represent a real cell lineage with possible clinical consequences. We report here data collected from fifty post delivery CV cultures of IUGR fetuses correlated against fifty matched non IUGR cases. This initial analysis was used to determine if a more formal study was warranted and/or if our protocol for this population of patients required amendment. 13.16-P Prenatal detection and follow-up of a mosaic trisomy 8 case E. Lloveras Caballe (1) F. Molina (2) J. Barrionuevo (3) M. Herrero (1) L. Barranco (1) N. Palau (1) A. Canellas (1) M. Costa (1) A. Plaja (1) (1) Departament de Citogenètica. General Lab, Laboratoris d’Anàlisi. Barcelona. (2)Unidad de Ecografía. Centro Gutenberg. Málaga. (3) FEA Pediatria. Endocrino y Dismorfología. HMI. Virgen de las Nieves. Granada. (4) Unitat de Genètica. Hospital MaternoInfantil Vall d’Hebron. Barcelona.

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Mosaic aneuploidy detected in prenatal diagnosis can lead to problems in genetic counseling. The risk for an abnormal outcome varies according to the percentage of trisomic cells and the specific chromosomes and tissues involved. We present a prenatal finding of mosaic trisomy 8 true mosaicism detected in long term cultured villi (100% cells analyzed) and confirmed in Amniotic Fluid cells (4%). High-resolution ultrasound examination of the fetus was normal. After genetic counseling, parents opted to continue the pregnancy and an apparently normal male baby was delivered. Follow-up postnatal investigations confirmed the mosaicism in peripheral blood (36%). At clinical level only a microcytic anemia was detected at 10 months of life and a transfusion was required. At 1,2 years of age facial features, growth development and clinical parameters were normal. Prenatal diagnosis and genetic counseling in cases of mosaic trisomy 8 is difficult since systematic data about clinical outcome is scarce. Available date indicates that affected fetuses show a pattern of low levels or absence of trisomic cells in STC, high levels in LTC and low levels in AF. This distribution probably is due to tissue related differences in trisomic cells viability in a mosaic produced by a post-zygotic non-disjunction error. Although our patient shows a normal development by the moment, an accurate hematological follow-up is required because several reports highlight a possible association between constitutional trisomy 8 mosaicism and an increased risk of leukaemia. 13.17-P Molecular cytogenetic delineation of a cryptic subtelomeric deletion caused by insertion of an segmental inverted duplication at chromosome 18q in a fetus with near-normal phenotype during second trim C. Chen (1) M. Chen (2) (1) Changhua Christian Hospital (2) Changhua Christian Hospital Objective To delineate a cryptic subtelomeric deletion caused by insertion of an segmental inverted duplication at chromosome 18q in a fetus with near-normal pheno-

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type except choroid plexus cyst and high risk maternal serum Down syndrome screening during second trimester pregnancy Patient and Method A 31-year-old primigravida received regular antepartum examination at our hospital since early pregnancy. Ultrasound performed at 20 weeks’ gestation demonstrated an unilateral choroid plexus cyst. She received 2nd trimester maternal serum screening for Down syndrome at 16 weeks’ gestation. The risk result revealed 1/149. Amniocentesis was suggested and performed at 21 weeks’ gestation. Conventional G-banded karyotyping was performed under the resolution possible at a 550-band level (4 Mb). FISH analysis was performed by using the mixture 2 probes of ToTelVysionTM Multicolor DNA Probe Panel (Cat# 33-270000, Abbot-Vysis Inc., Downers Grove, IL, USA) to identify a possible 18qter deletion in the subtelomere region. Spectral karyotyping (SKY) was performed using multicolor-labeled painting probes, which were purchased from Applied Spectral Imaging (SkyPaint; Migdal Ha’Emek, Israel) and processed according to the manufacturer’s instructions. The mBAND FISH was carried out byXCyte 18 mBAND probe kit. Result Conventional G-banded karyotype analysis on amniocytes revealed an abnormal chromosome 18 with additional material on the q arm. Spectral karyotyping confirmed the initial dubiousness that the additional material on the chromosome 18q was a duplication involving chromosome 18 material. FISH analysis showed that subtelomeric region of 18q was deleted on the aberrant chromosome 18. mBAND FISH presented that the abnormal chromosome 18 had been formed by a invert duplication of the region 18q11.1 to 18q22. The fetus therefore has trisomy for this region and loss of one copy of 18q subtelomere. The karyotype was further characterized as 46,XY,der(18) del(18)(q22)dup(18)(q22q11.1). Because of an apparent segmental aneuploidy in this fetus, termination was applied at 24 weeks’ gestation. A dead male baby was born with body weight 735 g via vaginal delivery. Conclusion An segmental inverted duplication at chromosome 18q was evident for G-banding and confirmed by various FISH studies. Both parents were without this aberration. The 18qter change was de novo. A

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segmental aneuploidy may present a near normal phenotype. Increased serum down risk and choroid plexus cyst may have association with aneuploidy whatever severe or mild. 13.18-P Prenatal detections of Turner syndrome trough amniocentesis and early spontaneous abortions I. Tonkovic Durisevic (1) D. Muzinic (1) K. Crkvenac Gornik (1) R. Lasan (1) L. Letica (1) D. Begovic (1) (1) University Hospital Centre Zagreb Turner syndrome (TS) occurs in 1:5000 live births (1:2,500 females) and is associated with absence of one sex chromosome 45,X but also with presence of a mosaicism and/or an abnormal X or Y chromosome (deletion, isochromosome X, ring chromosome). It has been estimated that almost 98–99% of TS foetuses end in abortion. TS was detected in 6,23% (23 cases) of the 321 karyotyped first trimester spontaneous abortions. Ttwenty-two cases were 45,X and only one was 45, X/46,XX mosaicism. The mean maternal age was 30 years and gestational age ranged from 9 to 14 weeks. From 23401 amniocentesis it was found 33 cases (0,14%) of TS, using routine cytogenetic analysis of cultured amniotic fluid cells with GTG-banding. The most frequent karyotype in TS was 45,X ( 42,5%), followed by 45,X/46,XX mosaicism (30,3%), 45,X/ 47,XXX (6%), and a single cases: 45,X/46,X,r(Y); 45,X/46,X,del(Y); 45,X/46,X,r(X); 45,X/46,X,i(Xq); 46,XX,del(X)(q23-qter); 45,X,der(15;21)(q10;q10), +21/46,XY. The indications for prenatal diagnosis were abnormal sonografic findings, advanced maternal age, congenital anomalies in previous pregnancy, drugs, radiation and parental request. We found that the presence of fetal malformations on ultrasound was absolutely higher in 45,X cases (100%) than in 45,X mosaicism (0%). Mean maternal age in 45,X cases was 26 years. To the contrary, all mosaic cases 45,X/ 46,XX and 45,X 47,XXX were found in women over 35 without ultrasound markers. Our dates support that increased maternal age is not a risk factor of Turner syndrome. This study confirms that during gestation, typical forms can be diagnosed by ultrasound examination, but mild

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mosaic forms are discovered incidentally during amniocentesis for unrelated reasons (advanced maternal age). It was suggested that the monosomy X arises relatively late during embryonal development and an existence of second line could increase the chances of survival. 13.19-P Prenatal detection and characterization of 21 small supernumerary marker chromosomes out of 25,000 prenatal cases in Greece E. Manolakos (1) R. Neroutsou (1) M. Lagou (1) K. Kefalas (1) N. Trigka (1) P. Tsoplou (1) H. Kontos (4) P. Michael (3) M. Aikaterini (1) T. Liehr (2) (1) BIOIATRIKI S.A. (2) University of Jena (3) Aghia Sofia Hospital (4) Genomedica Small supernumerary marker chromosomes (sSMC) were recently defined as structurally abnormal chromosomes that cannot be identified or characterized by conventional cytogenetics alone; they are genetally equal in size or smaller than a chromosome 20. Molecular cytogenetics techniques are needed for their characterization. Supernumerary markers chromosome are relatively common in unselected prenatal cases and occur in about 0.075% per thousand fetuses. The risk for an abnormal phenotype in prenatally ascertained de novo cases with sSMC is given as ~13%. Twenty one fetuses with marker chromosomes were detected following diagnostic villocentesis and amniocentesis in our laboratories among 25,000 prenatal samples (0.84 per 1000). Using conventional cytogenetics including special staining techniques in combination with fluorescence in situ hybridization (FISH), we successfully characterized all, which assisted markers subsequent genetic counseling. Twenty of the SMCs were proven to be of autosomal origin. Of the autosomal SMCs, eight originated from chromosome 15, one from chromosome 9, three from chromosome 14, one from chromosome 16, two from chromosome 18, one from chromosome 20, one from chromosome 21, and three from chromosome 22. One marker chromosome was of sex chromosome origin. Euchromatin material was found in 8 cases. Ten out of 21 marker chromosomes were associated with abnormal outcome.

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Our study demonstrates that molecular characterization of prenatal marker chromosomes is of great significance in facilitating genotype -phenotype correlation.

13.21-P

13.20-P

B. Faas (1) I. van de Burgt (1) A. Kooper (1) R. Pfundt (1) A. Smits (1) N. de Leeuw (1) (1) University Medical Centre Nijmegen

The replacement of cytogenetic analysis of chorionic villi sampling direct preparation samples by quantitative fluorescence PCR S. Christopoulou (1) G. Christopoulou (1) A. Hatzaki (1) J. Donoghue (1) M. Karkaletsi (1) T. Tseva (1) P. Kaminopetros (2) D. Kappou (3) S. Sifakis (3) V. Velissariou (1) (1) Mitera Hospital (2) Mitera Hospital (3) University Hospital of Heraklion Aim: to assess the replacement of chromosomal analysis of chorionic villi (CV) direct preparation samples (DIR) and short-term culture (STC) by quantitative fluorescence PCR (QF-PCR) and to determine its advantages in routine prenatal diagnosis. Methods: in a total of 4964 CV samples, rapid results were obtained either by conventional cytogenetic analysis of DIR in 2770 samples, or by QF-PCR analysis in 2194 samples. The final results were given after long-term culture (LTC). Results: the frequencies of unbalanced fetal karyotypes were not significantly different, being 4.8% by the DIR-LTC and 4.2% by the QF-PCR-LTC. Four out of 134 abnormal cases (2.98%) were not detected by the DIR. Of the 99 unbalanced chromosomal abnormalities diagnosed by QF-PCRLTC, 81 (81.81%) numerical abnormalities of chromosomes 13, 18, 21, X and Y were initially detected by QF-PCR and confirmed after LTC, while 16 (18.18%) were detected after LTC only. No false-negative or -positive results were obtained with either approach. Conclusion: QF-PCR can replace chromosomal analysis of CV-DIR in most cases during routine prenatal diagnosis, requiring smaller CV samples and being more labour effective. If QF-PCR were to be applied alone 18.18% of the clinically significant chromosomal abnormalities would be undetected. However, when coupled with LTC, QF-PCR is a robust diagnostic approach with high predictive value for the most frequent fetal trisomies.

Application of genome wide 250k SNP array analysis in prenatal diagnosis

Objectives: In prenatal diagnosis, the use of microarray analysis is under debate, as chromosomal imbalances with unknown/uncertain clinical consequences are likely to be found. For this reason, so far most studies used targeted microarrays. We explore the possibilities for the prenatal application of the Affymetrix 250k SNP array platform. Patients/methods: 250k NspI SNP array analyses were carried out on DNA from 16 fetuses (after TOP; n=11 or IUFD; n=5) and 3 newborns. All cases were prenatally karyotyped because of ultrasound anomalies and issued as normal (n=18) except for one, in whom a de novo translocation was detected. DNA was isolated from amniotic fluid (n=10), blood cells (n=2) or from cultured amniocytes, chorionic villi or fibroblasts (n = 7). CNVs (gains>200 kb and losses>150 kb) were categorized as either benign or (possibly) clinically significant. Results: Aberrations were detected in 6 cases. Three were highly likely clinically relevant and cytogenetically unvisible: a de novo 2.9 Mb loss in 17p13 (IUFD), a 5 Mb loss in 3q26.33q27.2 (TOP; de novo t (3;18)(q26.2;q21.3)) and a maternal UPD 16 (live born child with MCA). In a fourth fetus a CNV with (yet) unknown clinical significance was detected: a 340 kb gain in 17q12 (TOP; no parental analysis so far). Furthermore, in a case of IUFD with no fetal karyotyping possible and mother being carrier of a t (4;22)(q12;q11.1), a 56.5 Mb gain of 4p16.3q12 was found. In a newborn with MCA, a 22qter deletion detected postnatally was further characterized and shown to be 6.1 Mb in size. Conclusion: In 6/19 cases, genome-wide SNP array analysis enabled the detection of imbalances that otherwise would have remained undetected. Its high resolution increases the reliability for detecting imbalances, but more knowledge is essential to improve the interpretation of CNVs. Moreover, criteria need to be established for the conscientious application of prenatal genome-wide array analysis.

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13.22-P Prenatal diagnosis: Beware of interphase FISH results. Risk of misdiagnosis! L. Kraoua (1) N. Le Dù (2) S. Dreux (3) E. Pipiras (4) A. Sartor (5) J. Oury (5) S. Serero (2) B. Benzacken (1) A. Aboura (1) A. Tabet (1) (1) AP-HP (2) LABM (3) AP-HP (4) AP-HP (5) AP-HP Discordance between interphase FISH results and karyotype of amniocytes or chorionic villus cells has been documented. A dozen of false positive results were reported since 1993. Here we present two new cases of interphase FISH false positive results. For the first patient, FISH analysis were performed on uncultured amniotic fluid using the AneuVysion EC assay kit and an alpha-satellite 12-chromosome DNA probe (D12Z1) because of diaphragmatic hernia. Results have shown three signals of D12Z1 in 60% of the analyzed nuclei. The fetus sex was XX. Surprisingly, the karyotype obtained after amniotic fluid culture was: 46,XX; no supernumerary marker chromosome was found. FISH analysis of metaphase cells from cultured amniotic fluid revealed that the third signal resulted from hybridization to the heterochromatic region of chromosome 16. Parental karyotype was performed. This same D12Z1 hybridation pattern was observed in 60% of the maternal cultured lymphocytes. For the second case, FISH analysis were performed using the AneuVysion EC assay kit because of cranial circumference < 10 th percentile. Three signals of chromosome 18 alpha-satellite DNA probe (D18Z1) were shown in 75% of the analyzed nuclei resulting in an initial cytogenetic impression of trisomy 18. Maternal serum markers including αFP, βHCG and PAPP-A, which are diminished in trisomy 18, were normal. The karyotype revealed a small supernumerary marker chromosome (sSMC) in a mosaic form. FISH analysis performed on cultured amniocytes showed the sSMC to be positive for the D18Z1 and negative for the whole chromosome 18 painting (wcp18) .This sSMC (18) was found in 75% of the maternal cultured lymphocytes. For these two cases, interphase FISH results and ultrasonographic findings could result in falsepositive errors (tetrasomy 12p for the first report

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and trisomy 18 for the second one). Thus we underscore again the importance of karyotype which remains the gold standard analysis for clinical decision. 13.23-P Reliability of 12 hours Comparative Genomic Hybridization in fibroblasts and blastomeres for a comprehensive Aneuploidy Screening M. Rius (1) A. Obradors (1) G. Daina (1) J. Cuzzi (2) L. Marquès (3) G. Calderón (4) E. Velilla (5) M. Oliver-Bonet (1) J. Benet (1) J. Navarro (1) (1) Universitat Autònoma de Barcelona (2) Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo (3) Centre de Reproducció Assistida Clínica Sagrada Família (4) IVI Barcelona (5) Institut Marquès Introduction: The aim of this study is to reduce the hybridization period of Comparative Genomic Hybridization (CGH) to 12h, in order to make this technique suitable for the PGS of Day-3 embryos, avoiding the cryopreservation of the biopsied embryos. Material & methods: To assess the reliability of the modified protocol in single cell, we attempted to characterize 30 fibroblasts from six cell lines (Coriell) containing one or two different aneuploidies by 12h-CGH. Thirty-three blastomeres from eight embryos of five patients, discarded after PGS by FISH with 9 probes, were analysed with the 12h-CGH protocol. The results obtained were validated with standard CGH (72h of hybridization). Results: In 100% of the fibroblasts, the specific aneuploidies of each cell line were confirmed by 12h-CGH. Of 33 blastomeres, six blastomeres belonging to two embryos did not present any aneuploidy and 27 were aneuploid. The chromosomes most frequently involved in aneuploidy were 22 and 16, but also aneuploidies for chromosomes 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 15, 17, 18, 19, 20, 21, X and Y were detected. Globally, we found 87 aneuploid events, 39 (45%) of them corresponding to chromosomes which are not analysed by FISH for nine chromosomes.

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The results of 17 out of 33 (51.5 %) blastomeres analysed by 12h-CGH were totally in agreement with FISH results. The standard 72h-CGH analysis and the re-hybridization with FISH probes in blastomeres of those embryos with no agreement with FISH results corroborated the 12h-CGH results. Conclusions: – –

Our preliminary results demonstrate the high reliability of the 12h-CGH, as results obtained are consistent with standard 72h-CGH. This 12h-CGH study on blastomeres shows that this technique is a valuable alternative to FISH for predetermined chromosomes because it provides a complete karyotype for aneupoidy screening. Moreover, the 12h-CGH allows for the application of this procedure in PGS of Day-3 blastomeres without the need of cryopreserving biopsied embryos.

Acknowledgements: FIS-ISCIII (PI 051395), Grup de Suport a la Recerca. Generalitat de Catalunya (2005SGR00495) and Càtedra de Recerca Euginfunded this study. The first author has a predoctoral grant from the Ministerio de Educación y Ciencia (AP2006-02211). Keywords: PGS, CGH, FISH, Aneuploidy, Blastomere 13.24-P Two-year experience of Double-Factor Preimplantation Genetic Diagnosis: preliminary results A. Obradors (1) M. Rius (1) G. Daina (1) J. Cuzzi (2) O. Martínez-Passarell (3) A. Polo (3) J. Séculi (4) M. Oliver-Bonet (1) J. Benet (1) J. Navarro (1) (1) Universitat Autonoma De Barcelona (2) Universidade de São Paulo (3) Fundació PuigVert (4) Hospital Sant Joan de Deu Background In couples affected by a monogenic disease, Double-Factor Preimplantation Genetic Diagnosis (DF-PGD) allows doubly selection of embryos, i.e. free of the monogenic disease and being potentially euploid. The intention of this manuscript is to evaluate the feasibility and possible positive effect on implantation of DF-PGD after 2 years of clinical application.

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Methods Ten couples participated in this study, four suffered AMA (mean 39.2 y.o.), but not the other six (mean 32.3 y.o.). After ICSI, the first polar bodies were biopsed and analysed using Comparative Genomic Hybridization to screen aneuploidies in the whole-chromosome complement. On day 3, a blastomere was biopsed and amplified using MDA, following by couple-specific mutation detection protocol. On day 4, both monogenic detection and 1PB-CGH results were obtained allowing for a double selection of embryos. Results During the DF-PGD, only 1PB-CGHs from developing embryos were analysed, 45.45% of them being aneuploid (35/77), only fifteen (42.8%) obtained from AMA patients. Forty-five blastomeres were non-affected by the respective disease. In 35 of them, the 1PBs-CGH was available, resulting that 15 (42.9%) were potentially euploid, thus classified as DF-PGD-transferable embryos. The remaining 10 non-affected embryos without informative 1PB-CGH were tagged as PGD-transferable. Nine of the DF-PGD-transferable embryos and eight of PGD-transferable embryos were selected for transfer, achieving pregnancy in three (33.3%) and in one (12.5%) of the transferred embryos, respectively. Conclusion Despite the reduced number of cycles performed using DF-PGD; it seems to be a good tool to increase the implantation rate in couples affected by a monogenic disease. Acknowledgements This research study has been funded by the Ministerio de Sanidad y Consumo Fondo de Investigación Sanitaria Instituto de Salud Carlos III (FIS-ISCIII; PI 051395), the Grup de Suport a la Recerca de la Generalitat de Catalunya 2005SGR00495 and by the Càtedra de Recerca EUGIN. Albert Obradors has a predoctoral grant from the Generalitat de Catalunya (2005FI00108). 13.25-P Prenatal diagnosis of double trisomy: Down and Klinefelter syndrome (48,XXY,+21) A. Vičić (1) T. Hafner (1) F. Stipoljev (1) (1) General Hospital Sveti Duh (2) General Hospital Sveti Duh (3) General Hospital Sveti Duh

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Poster presentation The occurrence of double trisomy, the existence of two chromosomes in trisomic state in the same conceptus, is rare chromosomal abnormality. Although, several cases of Down and Klinefelter syndrome have been reported among liveborns, only 15 reports of prenatally detected 48,XXY,+21 have been published. In 7 cases patients were referred due to abnormal fetal sonogram, in 7 cases prenatal diagnosis was performed because of advanced maternal age, and in one case because of abnormal maternal serum screening. We present a case of conceptus with Down and Klinefelter syndrome prenatally diagnosed at 13+5 weeks gestation. Ultrasonic evaluation demonstrated the fetus with large, septated cystic hygroma measuring 7.9 mm. The first-trimester combined screening (free β-hCG, PAPP-A,nuchal translucency thickness and maternal age) showed an increased risk for trisomy 21 (1:15) and also for trisomy 18 (>1:10). After genetic counseling, parents elected to terminate the pregnancy and cytogenetic analysis of fetal skin and chorionic villi showed a nonmosaic 48,XXY,+21 karyotype in both specimens. Parental karyotypes were normal, with no evidence of mosaicism. Since there are a small number of cases that have been discovered prenatally, parameters associated with prenatal diagnosis of Down and Klinefelter syndrome are still not clearly established. We suggest that ultrasonographic examination and biochemical screening in addition to maternal age are all important in prenatal detection of Down and Klinefelter syndrome. 13.26-P

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error (diandry) and maternal meiotic error (digyny), while digyny is, according to recent investigations, the most frequent mechanism. In contrast with some other aneuploidies, triploidy is not dependent on increased maternal age. Molecular investigations of recurrent triploidies could be helpful in determining genetic basis of triploidy. We present a case of a 33year-old woman who was referred for ultrasonographic fetal evaluation. Her previous pregnancy was terminated at 20 weeks gestation due to triploidy. Ultrasonographic examination demonstrated severe olygohydramnios, ventriculomegaly and IUGR. Chorionic villi sampling was performed at 18 weeks of gestation, and cytogenetic analysis of both shortterm and long-term culture of chorionic villi showed a full 69,XXX karyotype. Microsatellite analysis of DNA samples extracted from chorionic villi and parental blood samples showed a maternal origin of extra haploid chromosomal set. To date, only three studies of recurrent triploidy have been published, all reporting maternal origin of extra set of chromosomes. These findings suggest the existence of genetic predisposition which causes the errors in oogenesis. 13.27-P Comparison of Autosomal Aneuploidies detected in placental samples from early miscarriages and in chorionic villus samples for prenatal diagnosis P. Ng (1) E. Lau (2) W. Tam (2) S. Ding (2) W. Lam (2) S. Chan (2) L. Ng (2) M. Tang (2) (1) The University of Hong Kong (2) Tsan Yuk Hospital

Recurrent triploidy of maternal origin F. Stipoljev (1) A. Vičić (1) T. Hafner (1) I. Drmić Hoffman (2) (1) General Hospital Sveti Duh (2) General Hospital Sveti Duh (3) General Hospital Sveti Duh (4) University Hospital and Medical School Split Poster presentation Triploidy is one of the most common chromosome abnormalities in humans, occurring in approximately 1–2% of all conceptions. Mechanisms by which triploidies may arise are dispermy, paternal meiotic

From 1998 to 2008, one hundred sixty four placental samples from spontaneous miscarriages and forty nine hundred ninety six chorionic villus samples before 14 weeks gestation were received for chromosome analysis. Cytogenetic analysis was based on Giemsa banded metaphases from cultured fibroblasts. The type and frequency of non-mosaic autosomal aneuploidies detected in both groups were analysed. Autosomal aneuploidies involving chromosome 13, 18, 21 and 5, 8, 14, 16, 20 were detected in both placental samples and chorionic villus samples. Autosomal aneuploidies involving chromosome 4, 6,

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7, 10, 15 and 22 were detected only in placental samples from early spontaneous miscarriages. Autosomal aneuploidies involving chromosome 2, 9 and 11 were detected only in chorionic villus samples. Autosomal aneuploidies of chromosome 1, 3, 12, 17 and 19 were not detected in both groups. The prevalence of non-mosaic autosomal aneuploidies in placental samples from early spontaneous miscarriages and in chorionic villus samples for prenatal diagnosis were 25% (41/164) and 6% (291/4996) respectively. 13.28-P Non-Invasive Prenatal Diagnosis: Preliminary Results in Our Lab F. Fernández Martínez (1) A. Moreno Izquierdo (1) A. Galindo Izquierdo (2) A. Garcia Burguillo (2) C. Vargas Gallego (3) C. Pascual Pérez (1) E. Barreiro (1) (1) Hospital 12 de Octubre (2) Hospital 12 de Octubre (3) Hospital 12 de Octubre Objective: To evaluate the sensitivity, specificity and clinical utility of non-invasive prenatal determination (NIPD) of fetal sex and RHD using cell free fetal DNA (cffDNA), in order to incorporate this technique to our routine. Background: Current genetic prenatal diagnostics relies mainly on invasive testing by chorionic villus sampling or amniocentesis, which carry a significant risk of fetal loss (0,8–1%). The discovery of the presence of cffDNA in maternal plasma in conjunction with highly sensitive and specific techniques such as real-time PCR (RT-PCR) has led to the possibility of NIPD. Methods: cffDNA was extracted using a modified spin-protocol for the QIAamp DSP Virus Kit and compared to results of extraction with the QIAamp DNA Blood Mini Kit. DNA extract was amplified with RT-PCR specific for SRY and DYS14 together with a control RT-PCR for b-globin for fetal sex and exons 7 and 10 for fetal RHD determination. Results: In a 2 month period 75 pregnancies were tested, 41 were confirmed to be male and 34 female by QF-PCR in uncultured amniocytes. This assay detected Y chromosome sequence at very low levels with 100% specificity and 100% sensitivity. Regarding to fetal RHD results, many pregnancies are still

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ongoing and outcomes will be available in the coming months to enable accurate evaluation of test sensitivity and specificity when performed in our laboratory, together with clinical utility in terms of avoiding antenatal Rh prophylaxis. Conclusions: NIPD is the future if we talk about the possibility to know the genetic status of the fetus. Detection of Y chromosomal sequences of fetal origin is highly reliable for early fetal sex determination using this approach. On the other hand, due to RTPCR is now as sensitive as postnatal serology for the determination of the fetal’s RhD-status, antenatal Rh prophylaxis in all D-negative pregnant women can be re-evaluated. 13.29-P Results from 4 years of preimplantation genetic diagnostics using fluorescent in situ hybridization (FISH) method in Slovenia A. Veble (1) M. Volk (1) K. Writzl (1) B. Dolničar (1) Ž. Remec (1) J. Kmecl (2) B. Valentinčič-Gruden (2) I. Virant-Klun (2) T. Tomaževič (2) B. Peterlin (1) (1) Institute of Medical Genetics (2) Reproductive Unit We report the 4 years’ experience of preimplantation genetic diagnosis (PGD) at the University Medical Centre Ljubljana. We started in July 2004. Up to December 2008, 31 couples were enrolled. The most frequent indications were structural and numerical chromosomal abnormalities (74%), followed by recurrent spontaneous abortions with numerical chromosomal abnormities and repeated implantation failure (20%), and X-linked genetic disorders (6%). Fifty-four oocyte pick-up cycles were carried out. The oocytes were inseminated by in vitro fertilization (IVF) or by intracytoplasmic sperm injection (ICSI). The embryos were biopsied on the third day of development. Genetic analysis was performed using FISH method and one or two unaffected embryos were transferred on the fifth day. We transferred 1.7 embryos per cycle during the course of 44 transfer procedures and 14 ongoing pregnancies were achieved. The ongoing pregnancy rate per cycle was 25.9%, per transfer was 31.8%, and per couple was 45.2%. Seven unaffected children were born, three pregnancies are still ongoing, and four pregnancies ended in miscarriage, two of them after

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amniocentesis procedure. There was no misdiagnosis. Our series demonstrate that PGD is a reliable and successful method, with pregnancy rates comparable to those of IVF or ICSI. Parental chromosomal abnormalities are still the leading indication for PGD in our country. 13.30-P Unbalanced structural aberration in X-chromosome not detected by QF-PCR E. Solsona Moix (1) J. Cabada Juez (1) C. Fernandez Ruiz (1) A. Alvarez Colmeiro (1) M. Martin Corbella (1) A. Alcala San Martin (1) M. Caselles Criballes (1) S. Gomez Sandin (1) J. Martinez (1) M. Ortega Blanco (1) (1) Balague Center Structural aberrations are not detectable by ordinary molecular techniques (QF-PCR and FISH) excluding when they are not balanced and the implicated regions are amplified. A rare de novo structural aberration of the X chromosome was detected during routine prenatal diagnostic, but it was not diagnosed by QF-PCR. This case was derived because of an increased risk in first trimester triple screening test, maternal age was 38. The result of the cytogenetic analysis was a derivative X chromosome, with an addition of unknown material in the q extreme of X chromosome and a terminal Xq deletion. It was shown after comparative genomic hybridization (CGH) that this material came from chromosome 10, so there was a duplication of 10pter-10p13 and a deletion of Xq21.2-Xqter. In conclusion, the good practises advises the use of all possible polymorphisms in QF-PCR and confirm the result with cytogenetic analysis. 13.31-P Prenatal diagnosis of a fetus with a recombinant inversion: a case report E. Talavera (1) R. Lopez-Ortega (1) C. Garrido (2) V. Català (2) (1) Hospital Universtitari Arnau de Vilanova (2) Prenatal Genetics

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Trisomy 16 is the most frequent chromosomal abnormality at conception associated with a high probability of fetal death. We present a case of a partial trisomy 16 in a pregnancy of 17 weeks of gestation. A 33 years-old healthy pregnant woman without previous miscarriages or fertility problems. She was referred to us because the second trimester screening was 1/125, ultrasound screening was normal. Prenatal chromosome diagnosis was performed in amniotic fluid cells. QF-PCR technique was normal but in conventional cytogenetics analysis it was found an abnormal rearrangement of one chromosome 16. We required peripheral blood from both parents to determine the origin of this abnormal 16. Maternal peripheral blood revealed an inversion of one chromosome 16, (46,XX,inv(16)(p13.3q22)), the paternal karyotype was normal (46,XY) FISH analysis, in the maternal blood sample, showed a balanced inversion of one chromosome 16 (FISH: ish 16(pter,qter)x2) whereas a recombinant inversion was found on the fetus (FISH: ish der16 (pter-qter+,qter+). Cytogenetics studies from the maternal family showed the same inverted 16 in both father and brother. Inverted 16 has no phenotypic effect but the risk of having pregnancies with a recombinant unbalanced 16 is very high (45% HCForum database). Chromosome unbalanced alterations follows with clinical findings, congenital malformations and mental retardation. This family emphasizes the need for family followup when a chromosomal abnormality is detected in a prenatal diagnosis. 13.32-P A woman carrier of a t(13;21)(q32.3;q22.1) with an abnormal viable offspring A. Cisneros (1) N. Ruiz-Xivillé (1) I. Granada (1) A. Zientalska (2) D. Esteban (3) E. Santafé (1) C. Villena (1) J. Grau (1) M. Xandri (1) F. Millá (1) (1) Institut Català d’Oncologia - Badalona (2) Hospital Germans Trias i Pujol (3) Hospital Germans Trias i Pujol

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Introduction. Patients carrying balanced translocations have a risk to produce genetically unbalanced gametes. The reproductive risk of any reciprocal translocation depends upon the chromosomes involved, the breakpoints, and the number and position of chiasmata. We present a woman carrier of a t (13;21)(q32.3;q22.1) with a hight risk to produce unbalanced gametes. Case report. A 35-years-old woman with any previous spontaneous abortions was refered for amniocentesis at 30 week’s gestation due to ventriculomegaly, magna mega cisterna and intrauterin growth retardation observed by echography. Prenatal screening in maternal serum was normal. Interphase FISH analysis was performed on uncultured amniotic fluid cells using AneuVysion assay kit (Vysis). There was a normal hybridization pattern for chromosomes 18, 21, X and Y (male fetus), but there were 3 signals for chromosome 13. Conventional cytogenetic study revealed and abnormal karyotype with an apparent monosomy of chromosome 21 and trisomy of chromosome 13. Due to this contradiction, metaphases were hybridized with specific probes for chromosomes 13 and 21 (Vysis). The second signal of 21 chromosome was localized on one of three chromosomes 13. Finally, the fetal karyotype was: 46,XY,+13,der(13)t(13;21)(q32.3;q22.1),-21[20]. Parent’s analysis revealed a normal father’s karyotype and a balanced translocation between chromosomes 13 and 21 in mother’s karyotype: 46, XX,t(13;21)(q32.3;q22.1)[20]. This study suggests that the maternal gamete was genetically unbalanced due to an adjacent 2:2 segregation. The fetus was born at 31 week’s gestation; clinical examination show trigonocephaly with normal fontanels, first finger with camptodactyly and clinodactyly, normal palate and micropenis without testicles. He died 15 days later due to cardio respiratory arrest. Conclusions. FISH analysis is a rapid and useful technique but it must be complemented by conventional cytogenetics to avoid wrong interpretations. Our patient presented a translocation involving small acrocentric chromosomes and small translocated segments, so the risk to produce genetically unbalanced gametes and viable offspring was high. In this case the genetic counseling to achieve viable pregnancies is preimplantation genetic diagnosis or an oocyte donor.

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13.33-P Morbidity and mortality among carriers of de novo balanced reciprocal translocations and inversions—a status after 38 years of prenatal examination Christina Halgren1, Nete M. Nielsen2, Susanne Kjaergaard3, Karen Brøndum-Nielsen4, Peter K.A. Jensen5, Dorthe G. Crüger6, Kirsten Rasmussen7, Jan Hansen5, Thue Bryndorf8, Morten Frisch2, Niels Tommerup1 and Iben Bache1. 1 Wilhelm Johannsen Centre for Functional Genome Research, Department of Cellular and Molecular Medicine, University of Copenhagen, Denmark. 2 Department of Epidemiology Research, Statens Serum Institut, Denmark. 3 Department of Clinical Genetics, UniversityHospital of Copenhagen: Rigshospitalet, Denmark. 4 Kennedy Center, Medical Genetics Laboratory, Glostrup, Denmark. 5 Department of Clinical Genetics, Århus University Hospital, Denmark. 6 Department of Clinical Genetics, Vejle Hospital, Denmark. 7 Department of Biochemistry, Pharmacology and Genetics, Odense University Hospital, Denmark. 8 Department of Gynecology and Obstetrics, HvidovreUniversityHospital, Copenhagen, Denmark. Abstract Genetic counseling in prenatally diagnosed de novo balanced chromosomal rearrangements represents a diagnostic dilemma, where the risk figures of 6–9% derives from a systematic follow-up on the cumulative morbidity and mortality in the first two years of life (1). However, many disorders may be difficult to diagnose in the first years of life, especially cognitive deficits. We identified all persons recorded with a prenatally diagnosed de novo balanced chromosomal rearrangement in the Danish Cytogenetic Central Registry for the period 1970–2003 and linked this database to nation-wide health registries to identify congenital malformations (CM) and diseases that required hospital contact among carriers. We identified a total of 109 de novo balanced reciprocal translocations or inversions. More than half of these pregnancies were terminated. The mean follow up of the 48 liveborn carriers (42 translocations, 6 inversions) was 13.2 years, and diagnoses were confirmed by

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scrutiny of the original medical records. Three carriers (6.3%) had severe CM diagnosed at birth, complying with the 6–9% risk reported previously. However, a range of cognitive, behavioral and neurological disorders were diagnosed after the age of two: A total of six carriers were mentally retarded (12.5%), including two of those with CM. Three carriers had psychiatric disorders (one with severe, debilitating depression and two with severe behavioral disorders). All in all, a total of 20.8% had either a severe malformation, were mentally retarded or had a psychiatric disorder, resulting in a 2–3 fold higher morbidity risk than previously shown for de novo rearrangements (1), and a 3 fold higher general morbidity risk than in 240 age, laboratory and gender matched controls with a prenatally diagnosed normal karyotype. We are now reexamining the cohort clinically and assessing whether genome-wide array CGH can improve genetic counseling. 1 Warburton D. Am J Hum Genet. 1991;49:995-1013. We would like to make an oral presentation.

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Chip; Operon, Köln) and fluorescence in situhybridisation with BACs revealed a de novo rearranged 5p chromosome with subtelomeric deletion del(5)(pterp15.1), duplication and inversion inv dup(5)(p14.3p12). To date, only two patients of inv dup del 5p have been reported (Sreekantaiah et al. 1999, American Journal of Medical Genetics; Wang et al. 2008, American Journal of Medical Genetics). Both cases were identified postnatally using FISH analysis or Molecular Karyotyping. The first patient, described by Sreekantaiah et al., had a catlike cry from birth but no other findings of cridu-chat syndrome. The patient described by Wang et al. had no catlike cry or other characteristics of cri-du-chat syndrome. In both patients the cri-du-chat critical region on 5p15.2 was not deleted. To our knowledge our case is the first case with inv dup del 5p including a deletion of the cri-du-chat critical region. The presented case shows the power of modern cytogenetic methods, allowing a more detailed diagnosis in affected individuals, and therefore, facilitating a more reliable prenatal diagnosis.

13.34-P 13.35-P Prenatal Molecular Karyotyping revealed an inverted duplication with deletion of 5p spanning the cri-du-chat critical region

Clinical outcome in patients with chromosomal rearrangements included in a PGD program

B. Klink (1) A. Stadler (1) M. Entezami (2) M. Stumm (2) E. Schrock (1) R. Wegner (3) (1) Institut für Klinische Genetik (2) 2Zentrum für Pränataldiagnostik (3) Institut für Humangenetik

P. Buendía (1) C. Rubio (1) M. Martínez (2) L. Rodrigo (1) E. Mateu (1) M. Milán-Sánchez (1) A. Mercader (1) A. Delgado (1) L. Escrich (1) C. Simón (1) (1) IVI Valencia (2) IVI Murcia

Although very rare, inverted duplications with terminal deletion (inv dup del) have been reported at different chromosomal ends. We report here on the first case of an inv dup del 5p detected by prenatal diagnosis. Chorionic villi sampling was performed in the 13th week of gestation because of fetal abnormalities detected by ultrasound examination. Ultrasound findings included generalized skin oedema, compressed choroid plexus as early sign of a hydrocephalus, tricuspid regurgitation, club feet, a hyperechogenic cystic structure at the right side of the fetal neck (differential diagnosis: teratoma, encephalocele). Conventional cytogenetic and molecular cytogenetic techniques were applied to determine the correct karyotype of the affected fetus. Molecular Karyotyping by Array-CGH analysis using a whole genome oligonucleotide chip (35k OpArray V4 70mer Oligonucleotid

Introduction: The aim of this study was to evaluate the efficacy and clinical outcome of preimplantation genetic diagnosis (PGD) using fluorescence in situ hybridization (FISH) in couples with structural chromosome abnormalities. Material & methods: Retrospective study of 856 embryos, from 165 cycles of our PGD program (1997– 2008). Inclusion criteria: Couples with Robertsonian translocations and couples with other chromosomal rearrangements, including reciprocal translocations and inversions. Embryo biopsy was performed on day 3 and one or two cells were removed from each embryo. Interphase nuclei were analysed by FISH with different combinations of centromeric, locus specific and subtelomeric probes for each of the cases evaluated (Vysis Inc., Downers Grove,Il., USA). Embryos were cocultured with epithelial endometrial cells during the

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analysis and normal/balanced embryos were transferred on day 5. Results: Blastocyst rates were significantly higher (p<0.05) in normal/balanced embryos compared to unbalanced ones in Robertsonian translocations, reciprocal translocations and inversions (61.3% vs. 36.8%; 64.8% vs. 47.5%; 91.3% vs. 50.0% respectively). Conclusions:Successful pregnancy and implantation rates can be achieved with PGD in carriers of structural rearrangements in both, female and male carrier of translocations, despite significantly higher percentage of unbalanced embryos was observed in female carriers of Robertsonian translocations. 13.36-P Accuracy of cytogenetic findings on chorionic villus sampling (CVS): D. Carbonell (1) L. Cusido (0) M. Rigola (0) C. Hernando (0) R. Fernandez (0) S. Escorihuela (0) N. Pujol (0) C. Preciado (0) J. Hernandez (0) M. Grao (0) (1) Cerba.Sae. Laboratorios de Analisis Clinicos Accuracy of cytogenetic findings on chorionic villus sampling (CVS): Laboratory experience of 2200 consecutive cases Carbonell D, Cusidó L, Rigola MA, Hernando C, Fernández R, Escorihuela S, Pujol N, Muñoz C, Comadran L, Preciado C, Martos J, Leal S, Casablancas O, Rodríguez E, García B, Cuartero M, Pleguezuelos M, Hernández J, Grao MP. Departamento de Citogenética. Laboratorio CERBA Internacional SAE. Sabadell. Barcelona. Spain.

Objective: To assess the predictive value of cytogenetic diagnosis after CVS. Methodology: We report our experience in 2200 consecutive CVS over a 7-year period (2002–2009). Cytogenetic analysis was performed by short-term culture (STC) and long-term culture (LTC) when the sample was enough quantity. Results: An aberrant karyotype was found in 8.5% (187/2200) of which 7.27% were numerical abnormalities, 0.60% structural rearrangements and 0.63% mosaic chromosomal aberrations. A discrepancy between the fetal and the villi karyotype was observed in 0.9% (20/2200) as a result of confined placental mosaicism (CPM). There were no reports of false-negative findings or maternal cell contamination. The overall rate of false-positive aberration was 0.9% (20/2200) of which 0.55% were false-positive mosaic and 0.35% false-positive nonmosaic aberrations. Mosaic aberrations found in CVS were most of them CPM. Non-mosaic aberrations involved monosomy X, trisomy 13 and trisomy 18 encountered after STC without ultrasound abnormalities could have been suspected of being confined to the placenta. These figures determined a sensitivity of CVS for prenatal detection of chromosome aberrations of 99.1%. Conclusions: Our data confirm the high diagnostic accuracy of CVS and the utility of combinated use of STC and LTC in minimizing diagnostic errors. When a CVS mosaicism or a non-mosaic abnormal karyotype is encountered without ultrasound abnormalities, the definitive prenatal cytogenetic diagnosis should be obtained through subsequent amniocentesis.

Robertsonian translocations

Reciprocal translocations

Inversions

♂

♀

♂

♀

♂

♀

No. of cycles

50

34

40

30

4

7

Previous miscarriages ± SD

0.5±1.0

1.9±1.4

1.4±1.5

2.1±1.4

0.7±0.8

0.3±0.4

Mean age ± SD

33.8±3.5

33.9±3.6

35.1.±4.7

32.4±2.9

37.2±3.4

35.0±2.8

% Abnormal embryos

59.9

83.2

82.4

62.1

54.8

Pregnancy rate/transfer

64.1

41.7

55.0

54.5

50.0

60.0

Miscarriages

0

0

1 (N/A)

0

0

1 (tris 7)

Implantation rate

49.3

35.1

37.9

33.3

33.3

50.0

ab

p=0.0029

a

73.9

b

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13.37-P Evaluation of technologies for detection and quantification of foetal DNA in maternal plasma in a clinical setting

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foetal DNA comparable to that obtained by Qiamp MinElute, but most importantly produced a lower amount of total DNA (maternal plus foetal), thereby achieving enrichment in foetal DNA by a factor of 1.6. 13.38-P

K. Stergianou (1) K. Carpenter (2) K. Ocraft (1) G. Cross (2) M. Hultén (3) T. Parkin (1) (1) Nottingham University Hospitals NHS Trust (2) Nottingham University Hospitals NHS Trust (3) Nottingham University Hospitals NHS Trust (4) Nottingham University Hospitals NHS Trust (5) University of Warwick (6) Nottingham University Hospitals NHS Trust The discovery of cell free foetal DNA in maternal plasma is leading to new horizons of non-invasive prenatal diagnosis. This study is aiming to: (i) validate foetal sexing from maternal blood using a TaqMan real-time PCR assay (ii) assess claims that formaldehyde treatment increases the yield of foetal DNA (iii) ascertain the optimum DNA extraction method for foetal sequences, and (iv) establish a collection of maternal plasma samples with known karyotypes. Following ethical approval and informed consent, 10–15 mls of venous blood was obtained from pregnant women before chorionic villus sampling/ amniocentesis, one aliquot of which was treated with formaldehyde. The validation of foetal sexing from maternal plasma was achieved by a blind comparison of results obtained by real-time PCR with DYS14 with the karyotype outcome from these pregnancies. Foetal sex determination was successful in 117 out of 118 pregnancies with one female pregnancy remaining unresolved. In order to evaluate the effect of the formaldehyde treatment, the amount of cell free foetal DNA recovered from treated and non-treated maternal plasma samples was statistically compared in 48 male pregnancies, using the paired t-test. The differences in the quantity of cell free foetal DNA and in the ratio of foetal to total DNA (maternal and foetal) were not statistically significant. Three DNA extraction systems were compared: the manual protocol Nucleospin Plasma XS (MacheryNagel), the Qiamp MinElute Virus Spin manual extraction kit (Qiagen) and the automated EZ1 (Qiagen). The Qiamp MinElute virus kit yielded the highest amount of foetal DNA, whereas our modified protocol for Nucleospin Plasma XS not only produced yields of

Parents’ preference in prenatal testing: rapid aneuploidy detection versus traditional karyotyping, a one-year experience in clinical service A. Kooper (1) B. Faas (1) E. Boormans (2) A. Eggink (3) H. Zondervan (4) F. Boekkooi (5) R. Quartero (6) R. Rijnders (7) I. van der Burgt (1) A. Smits (1) (1) Radboud University Nijmegen Medical Centre (2) Academic Medical Centre Amsterdam (3) Radboud University Nijmegen Medical Centre (4) Rijnstate Hospital Arnhem (5) St Elisabeth Hospital / TweeSteden Hospital Tilburg (6) Medical Spectrum Twente (7) Jeroen Bosch Hospital ‘s-Hertogenbosch In 2008, as final part of the national Dutch study M.A.K.E. (MLPA And Karyotyping, an Evaluation) in five prenatal clinics of the Network Prenatal Diagnostics Nijmegen, pregnant women with advanced maternal age or at risk for Down syndrome after first trimester screening (NT<3.5 mm) were offered a choice between either (i) rapid DNA testing by MLPA as a stand-alone test or (ii) traditional karyotyping before undergoing amniocentesis. Over a 1-year period, we both evaluated the performance of the MLPA test and traditional karyotyping in ~625 amniotic fluid samples and examined the parents’ preferences for testing. In 93% the MLPA test results, measured in duplicate, were available within 3 working days, the remaining 7% within one week. There were no test failures and in ~4% an aneuploidy for either one of the chromosomes 21, 18, 13, X or Y was detected. An average of 57% of the parents opted for the rapid MLPA test. This choice, however, appeared to range from ~40% to 90% between the different prenatal clinics. This unexpected observation prompted us to search for factors affecting the decision-making process. Communication of information to parents may play a key role in its final outcome. Whereas in all prenatal clinics the parents received identical brochures with information on both the DNA test and karyotyping, differences were noted in transferring this information to the parents. In

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some of the prenatal clinics, in addition to the brochure, oral consults were held by genetic advisors and/or gynaecologists, in other clinics the parents’ decision was based on the information in the brochure. We will present our laboratory workflow of rapid aneuploidy detection and discuss the information content of the brochure and the differences in communication with parents in the context of the ultimate test choices that were made. 13.39-P Familial 18 centromere variant resulting in difficulties in interpreting prenatal interphase FISH C. Yardin (1) S. Bourthoumieu (1) F. Esclaire (1) F. Terro (1) P. Brosset (2) M. Fiorenza (3) V. Aubard (3) M. Beguet (1) (1) CHU Dupuytren (2) CHU Dupuytren (3) CHU Dupuytren We report here on a familial case of centromeric heteromorphism of chromosome 18 detected by prenatal interphase FISH analysis transmitted by the mother to her fetus, and resulting in complete loss of one 18 signal. A 36-years-old woman was referred for an amniocentesis because of the presence of an isolated fetal cardiac abnormality (common arterial trunk). Direct preparations from amniocytes were labelled with the AneuVysion probe set (Vysis, Downer’s Grove, IL, USA). Two signals were seen for both chromosomes 13 and 21, whereas only one centromeric 18 signal was detectable on interphase nuclei, along with one signal for the Y and one for the X chromosomes. Further studies were performed using 18 telomeric probes (tel18p: locus D18S552, 18q: locus D18S1390, Vysis, Downer’s Grove, IL, USA). Two signals for each probe were present on all examined nuclei. Furthermore the FISH study with the LSI DiGeorge 22q11.2 kit (Vysis, Downer’s Grove, IL, USA) showed the absence of one TUPLE 1 signal on all examined nuclei leading to the diagnosis of a 22q11.2 microdeletion. The results of the interphasic FISH on fetal nuclei being difficult to interpret (complex rearrangement involving both chromosomes 18 and 22?), buccal smears from each parents were labelled with the same protocol as the fetal cells. Similar results concerning the chromosome 18 to the fetal ones were obtained for the maternal nuclei, that is only one signal for the 18 centromeric probe. The interphase FISH for chromosome 22 was normal for both

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the mother and the father. The fetal R-banded karyotype displayed the presence of a difference in the length of chromosomes 22 with normal chromosomes 18. In particular the centromeric regions of the chromosomes 18 were unremarkable. The FISH analysis performed on fetal metaphases confirmed the results of interphase FISH, with the absence of one TUPLE 1 signal for chromosome 22 and the absence of one centromeric signal for chromosomes 18. Same results were obtained for the mother concerning chromosomes 18. This case illustrates once again that the locus specific (LSI) probes are more effective than the alpha centromeric probes for interphase analysis. The development of high-quality LSI probes for chromosomes 18, X and Y could avoid the misinterpretation of prenatal interphase FISH leading to numerous additional and expensive investigations. 13.40-P Prenatal diagnosis of de novo terminal deletion 4p in 5 cases B. Karaman (1) H. Kayserili (1) I. Kalelioglu (2) R. Has (2) S. Basaran (1) (1) Istanbul University, Istanbul Medical Faculty Wolf Hirschorn Syndrome (WHS) is a clinically well described contiguous gene syndrome caused by a deletion of the short arm of chromosome 4 including critical region 4p16.3. Phenotype of the syndrome varies depending on the size and breakpoints of the deletion. Here we describe 5 WHS cases diagnosed at antenatal period. De novo pure terminal deletion of 4p with proximal breakpoints varying from 4p15.2 to 4p16.3 was observed in four of them. A mosaic ring chromosome 4 with the breakpoints p15.3 and q31.3 was found in one case. All cases were investigated cytogenetically due to the pathological ultrasonographic findings in second and third trimester of the pregnancies. Intrauterine growth retardation was observed as the common finding although renal, cardiac anomalies and cleft lip and palate was found in two of them. After genetic counseling session, one of the families opted to continue, while 4 of them decided for medical abortion. In this study the ultrasonographic, cytogenetic, molecular cytogenetic and clinical findings of these cases will be presented.

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13.41-P Prenatal diagnosis on fetal cells of uncommon origin R. Wegner (1) M. Stumm (1) M. Entezami (1) K. Karman (2) R. Becker (1) (1) Zentrum für Pränataldiagnostik (2) Lund University Hospital Conventional prenatal cytogenetic analysis relies either on amniocytes or on chorionic villi. In cases of chromosomal mosaicism found in these tissues the question might arise if the pathological cell line derives from the extraefetal tissue or if it represents in fact the fetal karyotype. In such cases, analysis of fetal lymphocytes is considered to be of help to resolve this problem. However, case reports pointing out the limited reliability of lymphocytes in unravelling fetal tissue specific mosaicism are well known. Thus parents might ask for complementing approaches to affirm a suspected fetal chromosomal anomaly. Here we report on our experience in the cytogenetic analysis of cells obtained by sampling fetal urine, fluid of ovarian cysts or of pleural effusion. Data on 14 cases demonstrate the growth potential of the collected cells. Moreover, there are cases showing that these tissues have some advantages over fetal lymphocytes in the detection of true fetal chromosomal aberrations. We could disclose two cases of false negative results in fetal lymphocytes. The results will be discussed with respect to the advantages and disadvantages as compared to common invasive prenatal diagnosis. 13.42-P Alternative testing strategy for first trimester prenatal diagnosis in CVS V. Cirigliano (1) G. Voglino (3) E. Ordoñez (1) L. Rueda (1) E. Lloveras (4) C. Mediano (5) C. Fuster (2) (1) General Lab (2) Universitat Autonoma de Barcelona (3) Promea spa (4) General Lab (5) Hospital Vall d’Hebron Prenatal diagnosis in CVSs usually implies sequential karyotyping cytotrophoblastic cells (STC) and long term cultures (LTC). Main drawbacks of this approach are possible discordance between results obtained on different cell populations, confined placental mosaicism (CPM) and maternal contamination. The great

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majority of chromosome abnormalities can be detected by QF-PCR; in CVSs it may also provide information about the presence of different cell lines in mosaic or contaminated samples, or the origin of the extra chromosome in trisomic cases. We analysed 3696 consecutive CVSs; following an initial evaluation on 500 samples, STC was fully replaced by QF-PCR. QF-PCR was performed on a single villus within 24 hours from sampling; abnormal results were confirmed on a second frond. In alternative, DNA extraction was performed on the cellular pool obtained after digestion prior to cell culture. A total of 201 cytogenetic abnormalities were observed, 98% were detected by QF-PCR with 100% PPV and 98.7% NPV. Five CPM cases were identified by QF-PCR because of trisomic diallelic patterns observed for all informative markers in single tissue fronds. Six true mosaic fetuses were detected with STR patterns clearly confirming the meiotic origin of the extra chromosome. Maternal cell overgrowth in 2 LTC could be revealed by the discrepancy with the QF-PCR result of normal male. LTC failed in 3% of cases because of poor sample quality or amount, QF-PCR analysis was always possible avoiding a second invasive procedure in several cases. The QF-PCR test coupled with LTC is a robust diagnostic approach. This allowed discriminating true fetal abnormalities from CPM in several cases thus avoiding further investigations by amniotic fluid sampling. This testing strategy proved to be efficient and reliable also if applied on samples of limited size and quality and its routine application resulted in a significant reduction of hands on and reporting time 13.43-P Non invasive prenatal diagnosis of chromosome abnormalities of paternal origin by STR analysis of fetal DNA in maternal plasma L. Rueda (1) E. Ordóñez (1) M. Cañadas (1) C. Mediano (2) C. Fuster (3) V. Cirigliano (1) (1) General Lab (2) Hospital Universitari Vall d’Hebrón (3) Universitat Autònoma de Barcelona Since its discovery in 1997, free fetal DNA has been used for non invasive prenatal diagnosis of different genetic disorders by detecting paternally inherited

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genomic sequences. The low proportion of fetal compared to the large amount of maternal DNA still pose a technical challenge for the detection of fetal aneuploidies. We explored the possibility of using targeted assays developed to confirm or exclude the presence of fetal chromosome imbalance in cases where the father is carrier of a balanced chromosome rearrangement. Two families were selected with paternal balanced translocations (6;21) and (13;14) undergoing prenatal diagnosis by CVS. A maternal blood sample was collected in both cases before the invasive procedure together with a paternal buccal swab. Targeted QF-PCR assays were developed to amplify highly polymorphic STR markers in the chromosomal regions involved in paternal rearrangements. In the first case karyotype analysis of CVS showed the presence of the same paternal balanced rearrangement. Single paternal alleles at 2 informative markers were detected by STR analysis in maternal plasma confirming the normal fetal chromosome complement for the examined regions. The second case was diagnosed as trisomy 13 by rapid QF-PCR analysis, paternal and maternal STRs on chromosome 13 confirmed the paternal origin of the extra chromosome; the presence of the paternal derived chromosome was later confirmed by cytogenetic analysis. The inheritance of both paternal chromosomes 13 was further confirmed by detecting both paternal alleles for 2 different markers in maternal plasma. The detection of paternal STR alleles in maternal plasma has shown to be efficient and reliable; if properly selected in the chromosomal region of interest, STR markers allow non invasive prenatal diagnosis in families where the father is carrier of a chromosomal rearrangement. This approach may benefit couples undergoing PGD to confirm single cell diagnosis at no risk for the fetus.

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Until recent years, prenatal diagnosis was based only on cytogenetic analysis, which has limitations in time and chromosome resolution. New technologies allow us a rapid aneuploidy diagnosis with QF-PCR or the detection of minor genomic variations by means of MLPA or microarray CGH (aCGH), last approach being especially useful when ultrasound pathological findings cannot be fully ascertained by traditional studies. In the course of a cost-efficiency study comparing standard and novel technologies, we have already studied more than 100 consecutive prenatal samples. In addition to confirm all alterations detectable by QFPCR and/or karyotyping (including, 47, +13; 45,X; and 69,XXY), aCGH with an in-house developed array containing 2106 quatriplicated BAC probes (qChip Pre), identified a smaller rearrangement in an additional case. Amniocentesis was performed in a 20-weeks gestation due to the ultrasound diagnosis of Dandy-Walker variant with abnormal corpus callosum (absent posterior tail), confirmed by MRI. aCGH was carried out in DNA extracted from 5 mL. of amniotic fluid, while 15 mL. were cultured and used for routine cytogenetic studies. A deletion spanning the telomeric portion (6p25.1–6p25.3) was identified, affecting all clones (n=22) covering this zone and spanning at least 6.2 Mb of the euchromatic telomeric region. Reevaluation of the karyotype with this information in hand led us suspect the previously undetected structural anomaly. Gestation was terminated due to the ultrasound malformation, and genetic counselling informing of a low recurrence risk was provided to the couple. The technical success to date along with the reported case detected illustrate the efficiency and utility of a high resolution targeted aCGH in prenatal diagnosis. 13.45-P

13.44-P The third era of cytogenetics: prenatal diagnosis of a 6p25.1–6p25.3 deletion in a malformed fetus using array CGH C. Mediano (1) A. Plaja (1) L. Espi (1) M. Jallas (1) A. Fernandez (1) C. Preciado (2) T. Higueras (1) M. Garcia (3) L. Armengol (3) L. Perez-Jurado (1) (1) Hospital Vall d’Hebron (2) Universitat Pompeu Fabra (3) QGenomics S.L.

De novo apparently balanced reciprocal (x;10) translocation from prenatal diagnosis of IVF achived pregnancy M. Vasilevska (1) A. Anevska Mitrevska (1) A. Stefanovska (1) E. Sekovska (1) S. Adamoska Klisaroska (1) G. Dimeska (2) (1) Special Hospital for Obstetrics and gynecology “Mala Bogorodica-Sistina”, Skopje, R. Macedonia (2) Faculty of Natural Sciences and Mathematics

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De novo balanced translocations are observed in very low frequency during cytogenetic prenatal diagnosis. We report on a couple which underwent IVF treatment due to polycistic ovaries syndrome, tubal obstruction and increased testosterone concentration in the woman. Since the pregnancy after the fresh embryo transfer did not result with delivery, after 8 months transfer with 3 thawed embryos was carried out. Single pregnancy was achieved and amniocentesis in 17 gestational week was performed. Standard cytogenetic analysis revealed de novo reciprocal (X;10) balanced translocation involving p arm of the X chromosome and q arm from 10 chromosome later confirmed with FISH analysis. Karyotypes of the parents were normal and possible implications from this condition were explained to them. All ultrasound parameters were in normal range and the parents decided to go on with the pregnancy. Healthy baby girl was born and so far no health complications are observed. 13.46-P Classical-karyotype and molecular-karyotype characterization in prenatally detected ring chromosome 18. Each approach becomes important E. Triviño (1) M. Jimenez (1) E. Barcelo (1) E. Lopez Quesada (2) A. Cueto (3) (1) CatLab (2) Hospital Universitari Mutua de Terrassa (3) Hospital Universitari Mutua de Terrassa Chromosome 18 abnormalities, including ring(18), are among the most frequently occurring autosomal anomalies, together with 18p- and 18q- being found in approximately 1/40,000 live births. There’s no genotype-phenotype well stablished map, and so, not only one approach to the diagnosis, mainly in prenatal diagnosis. We present the case of a ring(18) detected in a 11+5 week of gestation, characterized by classical karyotype, QF-PCR, FISH, and molecular karyotype Chorionic villus sampling was performed in a 38 years old woman with the only indication of maternal age. According to our standard procedures, we used quantitative fluorescent PCR (QF-PCR) and long-term-culture (LTC ). QF-PCR result was normal, in contrast with karyotype finding of 46,XX,r(18) (p11.3q23), confirmed by amniocentesis. FISH study

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in amniotic fluid cells with subtelomeric probes revealed an absence of both p and q signals. QFPCR , using additional markers was still normal. Molecular-karyotype, using qChip Pre(v2) from qgenomics revealed a 2.5 Mb deletion at 18p11.32, and 0.6 Mb deletion at 18q23. No fetal abnormalities were observed by ultrasound. Even though the chromosomal abnormality was well characterized, the clinical consequences could not be determined. Patient was informed about phenotype variability, often associated with mental retardation. She elected pregnancy termination. Classical and molecular karyotype of abortion samples confirmed diagnosis. Autopsy revealed an abnormal left lung, with two lobes, as a sole anomaly. Nowadays there is a debate about the possibility of replacing classical karyotype with QF-PCR in most of the prenatal diagnoses. In our case, each technique separately was not useful at all. Furthermore, while QF-PCR and FISH are available in most of prenatal diagnosis laboratories, and an easy complement to cytogenetics, this is not the case of molecular karyotypying. In any case, we need to value routine prenatal protocols by cost-benefit , taking advantage of the new opportunities offered by molecular biology techniques. 13.47-P Application of Touch FISH as an additional tool in the study of spontaneous abortions S. Dória (1) F. Carvalho (1) C. Ramalho (2) V. Lima (1) L. Moreira (1) A. Duarte (1) M. Sousa (3) A. Matias (2) A. Barros (1) (1) Faculty of Medicine (2) Hospital de São João (3) Biomedical Sciences Abel Salazar Introduction: Many human conceptions are genetically abnormal and end up in miscarriage. Conventional cytogenetics is well established but is associated to culture failure, external contamination and selective overgrowth of maternally derived cells. Comparative genomic hybridization (CGH) provides a genome-wide screening for unbalanced aberrations although it is unable to differentiate the ploidy status. Interphase FISH using touch preparations (Touch FISH) is an efficient approach to screen for

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ploidy changes and also to detect maternal cell contamination. Material and methods: Touch FISH was performed in 80 cases, for ploidy screening, to confirm an abnormal CGH profile, to evaluate maternal contamination and to exclude a tetraploidy due to an artefact arising during cell culture. Touch FISH was performed in small pieces of tissues that were cut and touched onto a Polysine slide, followed by a fixative process. Three probes were used (CEP probe for one autosome, CEP X and CEP Y) according to manufacturer instructions using an adapted protocol. Results and discussion: From the 80 cases, 69 were previously studied by CGH without conventional karyotype. Touch FISH confirmed the CGH profile in all cases, including 14 with abnormal profile. In 8 cases, Touch FISH indicated maternal contamination and in 4, triploidy. Touch FISH was also performed in 11 cases with karyotype, confirming maternal cell contamination in 3 cases. In the other 8 cases, tetraploid cells were seen in distinct cultures and were present in more than 20% of the cells analyzed. Touch FISH confirmed the genuine tetraploid complement in only one case. This means that a tetraploid karyotype should be carefully interpreted and must be confirmed. In conclusion, Touch FISH protocol is an efficient approach to study ploidy allowing the identification of maternal cell contamination. It can also distinguish genuine mosaicism from cases with tissue culture artifact. 13.48-P Unbalanced structural rearrangements detected in amniotic fluid samples during one year N. Clusellas (1) C. Morales (1) I. Mademont (1) M. Milà (1) A. Soler (1) A. Sánchez (3) (1) Hospital Clínic de Barcelona During 2008 our laboratory performed 2,226 prenatal diagnoses in amniotic fluid samples and 60 unbalanced chromosomal abnormalities (2.7%) were detected. Most of them (50/60) were aneuploidies of chromosomes 21, 18, 13, and sexual chromosomes. All cases but two, (+9 and +20) could be detected by QF-PCR screening. Furthermore, we detected 10 cases of unbalanced structural rearrangements, repre-

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senting the 16.6% of all unbalanced abnormalities. The karyotypes of these abnormalities are: 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

47,XY,+mar[18]/46,XY[42]dn 47,XY,+mar[3]/46,XY[47] 47,XX,+mar dn 46,X ,del(14)(q32.1) 46,XY,del(18)(q12.2q21.1) 46,XY,del(4)(q31.3) 46,XX,del(1)(q42)dn 46,XX,1p+ dn 46,XX,der(12)t(Y;12)(q12;q24.33)pat 46,X,i(Y)(p10)

The chromosome abnormalities were first detected by standard cytogenetics studies, and in most of then molecular techniques were applied to specify the diagnosis (FISH, MLPA and QF-PCR). In five cases the prenatal diagnosis was performed for detected ultrasound findings, in four cases due to a high risk of chromosomal abnormality in maternal serum screening (>1/100), and in the case of i(Y) because of parental anxiety. These results show that cytogenetic analysis with GTG banding is necessary at least in gestations with ultrasound abnormalities or high biochemical risk, and molecular techniques can be used complementarily in order to achieve the correct diagnosis and improve the genetic counselling. 13.49-P Introduction of a new protocol for first-trimester prenatal diagnosis B. Minuti (1) S. Bernabini (1) E. Pelo (1) S. Frusconi (1) I. Giotti (1) V. Formica (1) M. Trafeli (1) S. Carniani (1) F. Cutinelli (1) F. Torricelli (1) (1) Azienda Ospedaliero-Universitaria Careggi In the Italian Region Tuscany, it has been registered a great increase of transabdominal Chorionic Villus Sampling (CVS) for prenatal diagnosis of genetic disorders, but without a consistent decrease of amniocentesis. The great increase of request of first-trimester testing on CVS has lead the structure SOD Diagnostica Genetica of the Careggi Hospital of Florence (Italy) to the proposal of a new protocol of analysis. Quantitative Fluorescent Polymerase Chain Reaction (QF-PCR) will be applied in substitution of the chromosomal analysis of spontane-

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ous metaphases of CVS in order to identify the chromosome 13, 18, 21, X and Y aneuploidies. The analysis will be integrated by full karyotype analysis performed on CVS colture. Although full karyotype is the gold standard of prenatal diagnosis, it has been proposed to introduce QF-PCR in the field of prenatal diagnosis to allow rapid diagnosis of some selected chromosomal anomalies. The use of this new protocol is supported by the data present in literature, in which is reported that the risk of chromosomal anomalies, after the exclusion of the more frequent aneuploidies, is 0,4%. In this work the data collected in 2008 and in the first trimester of 2009 are presented; during this period we have studied 1200 samples of CVS evaluating molecular and citogenetic aspects, pathological samples percentage and implication of unexpected results on genetic counselling. 13.50-P Identification of protein biomarkers for Turner Syndrome using Mass Spectrometry based proteomics

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glycoprotein precursor (A1BG) and Ig lambda chain C regions (LAC) were increased in plasma samples obtained from women known to carry TS while 5 proteins Kininogen-1 precursor (KNG), Clusterin precursor (CLUS), Ig alpha-1 chain C region (IGHA1), Immunoglobulin J chain (IGJ) and Fibrinogen gamma chain precursor (FIBG) were decreased. Conclusions: The majority of the above proteins are acute phase proteins and with the exception of SHBG they are not associated with TS phenotype. All differentially expressed proteins are candidate biomarkers for TS, providing opportunities for the development of non-invasive prenatal diagnosis. As these are preliminary findings, follow-up experiments are needed for their evaluation. Acknowledgments: Funding from the European Commission for the Special Non-Invasive Advances in Fetal and Neonatal Evaluation (SAFE) Network of Excellence (LSHB-CT-2004-503243) for which this study was funded is gratefully acknowlwdged. 13.51-P

A. Kolialexi (1) G. Tsangaris (2) A. Anagnostopoulos (2) N. Papantoniou (3) A. Antsaklis (3) A. Mavrou (1) (1) Athens University

Screening and diagnosis of fetal aneuploidy: maternal age, serum testing and ultrasound screening

Background: Despite the large impact of ultrasonographic and biochemical markers on prenatal screening, the ability to accurately diagnose Turner syndrome (TS) is still limited and better diagnostic testing is needed. Materials & Methods: Plasma from 7 women carrying a TS fetus and 12 with non-TS fetuses matched for gestational age, maternal age and ethnicity, in the 2nd trimester of pregnancy were analysed using a combined approach, based on 2-DE, MALDI-TOF-MS, and Western blotting in order to identify biomarkers for TS. Plasma from women carrying TS fetuses were provided from the SAFE Biobank (London). Results: Gel comparison revealed sixteen proteins differentially expressed in maternal plasma in women with TS fetuses: Complement C3 precursor (CO3), Haptoglobin precursor (HPT), Sex hormone-binding globulin precursor (SHBG), CD5 antigen-like precursor (CD5L), Hemoglobin subunit gamma-2 (HABP2), Hemoglobin subunit beta (HBB), Ig kappa chain C region (KAC), Afamin precursor (AFAM), Complement C1s subcomponent precursor (C1S), Alpha-1B-

A. Moreno-Izquierdo (1) F. Fernández-Martínez (1) M. Gómez-Rodríguez (1) A. Galindo-Izquierdo (1) M. Fernández-Guijarro (1) E. Barreiro-Miranda (1) (1) Hospital 12 de Octubre Prenatal diagnosis of aneuploidy is widely available, but the screening of which patients should undergo prenatal diagnosis is changing. Woman aged 38 years or older, maternal serum testing and ultrasound screening are now accepting screening protocols for Down syndrome and other aneuploidies for invasive testing (amniocentesis) in our center. We prospectively assessed and compared the efficiency of three different screening strategies for fetal chromosomal abnormalities from 2007 to 2008. Of 843 woman included, 388 were over 38 years (detection rate 2%), 347 had a biochemical risk more than 1:270 (detection rate 3’4%) and 108 had ultrasound markers (detection rate 23%). New developments in maternal serum and ultrasound screening improved the ability to identify pregnancies at increased risk of Down syndrome, trisomy 18, and other chromosomal abnormalities.

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13.52-P Cystic hygroma in two fetuses with unbalanced translocations of parental origin S. Sifakis (1) E. Papadopoulou (2) D. Kappou (1) N. Mantas (1) A. Konstantinidou (3) L. Florentin (4) V. Velissariou (5) I. Matalliotakis (1) (1) University of Crete (2) University of Crete (3) University of Athens (4) Institute of Molecular Biology & Cytogenetics (5) Mitera Hospital Introduction: The estimated frequency of balanced and unbalanced translocation in newborns is about 1:500 and 1:2000, respectively. A carrier of a balanced translocation is at risk of producing offspring with unbalanced chromosomal rearrangements. Fetuses with unbalanced translocations are spontaneously aborted or present severe malformations, which may be detected by sonogram even in early pregnancy. We report two cases of fetuses with an unbalanced translocation of parental origin diagnosed at the first gestational trimester after the sonographic detection of cystic hygroma. Case reports: large cystic hygromas were detected in two pregnancies during a routine sonographic scanning of nuchal translucency measurement at 12 weeks’ gestation. The pregnant women were 28 and 24 years old respectively, both of them second gravida, with uneventful previous pregnancies. Chorioning villus sampling was performed in both cases. Two different trophoblasts culture with the use of GTG banding revealed unbalanced translocations, [46,XY,der(21),t(11;21)(p11.1;p11.1)] and [46,XX,der(4)t(4;8)(p14;p21)], respectively. The analysis of the parental karyotypes revealed that, in the first case, the mother was a carrier of a balanced translocation [46XX,t(11;21)(p11.1;p11.1)], and, in the second case, the father was a carrier of a [46, XX,t(4;8)(p14;p21)] balanced translocation. In the second case, the sonographic findings at 16 weeks’ gestation were IUGR, situs inversus, and renal hypoplasia. After genetic counseling both pregnancies were terminated by a parents’ request at 14 and 17 weeks’ gestation, respectively. The perinatal autopsy in the second case confirmed the existence of the large cystic hygroma and the other sonographically detected findings, accompanied by multiple additional abnormalities (including facial abnormalities, ventriculoseptal heart defect, asplenia,

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spina bifida, elbow arthrogryposis, clitoridomegaly, and immaturity of the chorionic villi). In the first case no further investigation was carried out. Discussion: Fetuses affected by unbalanced chromosomal rearrangements originated by a parental balanced translocation may present abnormal sonographic findings; the majority of the phenotypic abnormalities, however, will be present at the second or the third gestational trimester. Cystic hygroma may be a marker of underlying chromosomal abnormality of the fetus, and is easily detected by ultrasonogram. It will enable an early diagnosis as well as a pregnancy termination before the full sonographic appearance of the associated abnormalities. In such cases, however, a perinatal autopsy is strongly recommended; it will provide useful information for the genetic counseling and the management of the subsequent pregnancies of the couples where the existence of a balanced translocation is already known. 13.53-P A de novo ESAC marker chromosome derived from chromosome 15 prenatally diagnosed in a fetus with macroephaly and atrium aplasia D. Kappou (1) S. Sifakis (1) E. Papadopoulou (2) A. Konstantinidou (3) P. Fragkiadaki (1) P. Patsalis (4) I. Matalliotakis (1) V. Velissariou (5) (1) University of Crete (2) University of Crete (3) University of Athens (4) Institute of Neurogenetics (5) Mitera Hospital Introduction: the incidence of de novo extra structurally abnormal chromosomes (ESAC) at prenatal diagnosis is 1 in 2500 pregnancies and studies have demonstrated that approximately 13% may be associated with congenital abnormalities. The majority of ESACs have been shown to be derived from chromosomes 15 and 22. It has been shown that, when band 15q12 is included in the der(15) ESACs, the phenotype is abnormal). Here, we report on a case of a de novo ESAC detected at CVS, which was shown with FISH studies to have derived from chromosome 15. Major congenital abnormalities of the fetus, such as macrocephaly and atrium aplasia, were detected by ultrasound early in the second trimester of the pregnancy. Case report: CVS was performed in a second gravida 38 year-old woman at 13 weeks because of advanced

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maternal age. The karyotype of the fetus was 47,XX, +ESAC in all analyzed metaphases. The ESAC appeared the size of a G chromosome, monocentric with satellites on the short arm. Cytogenetic analysis of the parents revealed normal karyotypes. FISH studies showed that the ESAC had derived from chromosome 15 and therefore the karyotype of the fetus was 47,XX, +ESAC.ish der(15)(D15Z1) de novo. The parents were counseled accordingly and decided to continue with the pregnancy. A detailed sonogram at 16 weeks of gestation revealed macrocephaly (BPD and HC>95 centile), aplasia of the right atrium of the heart, and increased nuchal fold). The parents decided to terminate the pregnancy. A postnatal pathology examination of the fetus confirmed the sonographic findings, plus hypertelorism, a flat occiput, and thymus dysplasia. Conclusion: the detection of de novo ESACs during prenatal diagnosis warrants detailed cytogenetic, FISH and sonographic studies, because it has been shown that the majority are associated with abnormal outcomes. As we have presented in this case a panel of specific fetal anomalies were associated with the der(15) ESAC, but more data is needed to support this evidence. 13.54-P Chromosomal rearrangements and minor structural variations in prenatal diagnosis N. Mantas (1) S. Sifakis (1) D. Kappou (1) E. Papadopoulou (2) D. Koutroulakis (1) P. Fragkiadaki (1) L. Florentin (3) K. Pangalos (4) V. Velissariou (5) I. Matalliotakis (1) (1) University of Crete (2) University of Crete (3) Institute of Molecular Biology & Cytogenetics (4) Laboratory of Cytogenetics (5) Mitera Hospital Aim: to evaluate the incidence and the type of chromosomal variants and rearrangements in fetal karyotype detected after invasive prenatal diagnosis. Materials-Methods: We registered retrospectively chromosome rearrangements and minor structural variations (also called variants or polymorphisms) in a sample of 1901 fetal karyotypes detected after invasive antenatal procedures, within the years 1996– 2004. The main indications for invasive prenatal diagnosis (1864 amniocentesis and 37 CVS) were advanced maternal age (49%), and pathological biochemical markers of aneuploidy (13%).

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Results:Variants, chromosome rearrangements, and marker chromosomes were present in 30 out of 1901 cases of fetal karyotypes (1.57%), with the majority of them (90%) to be associated with normal phenotype. There were 18 cases of variants (0.94%), that specifically included: 13 cases of inv(9)p11;q13; 4 cases of increased satellite material of the p arm; and 1 case of increased heterochromatin. All these variants accompanied a normal phenotype. There were 10 cases (0.52%) of chromosome rearrangements, 8 balanced and 2 unbalanced. In particular: 5 derivatives originated from maternal or paternal translocations, with 2 of them to be robertsonians; 2 inversions; 1 dicentric chromosome. All the above were considered balanced rearrangements associated with normal phenotype. On the contrary, in 2 cases, there was an unbalanced derivative of a maternal translocation, in both cases involved the chromosome 21, that was followed by pregnancy termination. Finally, there were 2 cases (0.1%) of extra structural chromosome arrangement. This presence of a marker chromosome was of maternal origin in one case, and was associated with normal phenotype. The other case was a de novo extra marker associated with sonographic abnormalities, and the pregnancy was terminated. Conclusion: More commonly the fetal minor structural variations and the chromosomal rearrangements do not confer phenotypical abnormalities. However, genetic counseling and management of such cases is of high importance due to the risk resulting from unbalanced or de novo rearrangements. 13.55-P Preimplantation Genetic Diagnosis (PGD) for dir ins(7;17)(q34;q23.1q25.3) carrier L. Carey (1) P. Barahona (2) D. Wright (1) (1) Sydney IVF (2) Sydney IVF Introduction: We present a case of PGD for a 46,XY, dir ins(7;17)(q34;q23.1q25.3)mat carrier, where commercial probes were not available for the inserted region at 17q23.1–q25.3. Transmission of the insertion has been reported through four generations, with 2 abnormal individuals resulting from recombination within the inserted region, which measures 0.58% haploid autosomal length (HAL). Segregation at meiosis may result in a theoretical 30 outcomes. Aim: FISH probes were generated from BAC clones for the chromosome 17

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insertion region and a validated FISH assay was designed for PGD to identify all possible meiotic segregants among embryos. Methods: Probes were prepared from 28 BAC clones using degenerative oligonucleotide primed PCR (DOP-PCR) which were then labelled using a secondary DOP-PCR labelling reaction with fluorophore incorporation. All 28 BAC probes were screened initially for localization and signal intensity. Nine selected BAC probes were assessed for probe specificity and hybridisation efficiency. A single BAC clone (304E2) was labelled using the ULS™ arrayCGH chemical labeling kit. The final selected clone and PGD probe mixes were validated by establishing the measurement of uncertainty (MU). Results: A single BAC clone (304E2) was chosen and DOP-PCR and ULSTM arrayCGH chemical labelling were compared visually by direct fluorescent microscopy and by determining probe hybridisation characteristics (probe specificity and hybridisation efficiency) The ULS™ arrayCGH chemical labelled probe showed excellent signal intensity and hybridisation efficiency and was selected for the PGD assay. Conclusion: A PGD FISH assay has been developed using the BAC clone 304E2 for the inserted region on chromosome 17 which involves a total of four probes to identify all possible meiotic segregants using two sequential FISH hybridisations.

tion in CVS was 50/3754(1.3%) and in AF was 4/ 4697(0.09%). Mosaicism involved +2, +4, +7 +8, +9,+10, +13, +15, +16, +18, +21, +22, 4n, X0, XX/ XY, +mar and a double aneuploidy. For CVS, follow-up AF was received in 45 cases and FISH was performed for 42 cases not involving an unidentified marker chromosome. FISH showed no abnormalities in 33/42 (79%) cases. In 9/42 (21%) cases FISH detected low level mosaicism, however it was confirmed in only 5 long-term cultured karyotypes. For the four AF cases, one patient had a repeat AF, one patient a repeat AF and FBS, and 2 patients had FBS only. FISH was performed in 3 cases with an abnormality in only 1/3 (33%) of cases. In summary, mosaicism was observed in a small proportion of cases and FISH studies were beneficial in 45/54 (83%) patients indicating good utilisation. At the time of counselling for further investigation and follow-up studies, the data shown here of CVS (79%) and AF (66%) with normal results, suggests that counselling can be relatively reassuring. However, the likelihood of confirming mosaicism should not be underestimated. The data here is limited and caution is advised on using this data during counselling with further study necessary.

13.56-P

Evaluation of Non-Invasive Prenatal Diagnosis from Fetal Nucleated Red Blood Cells (NRBCs) isolated from Maternal Circulation

Four year retrospective review on mosaic prenatal samples S. Polihronis (1) D. Wright (1) (1) Sydney IVF A retrospective review from Jan2005 to Dec2008 was performed on prenatal samples (n=8451) encompassing the Sydney metropolitan area, including chorion (CVS) (n=3754) and amniotic fluid (AF)(n=4697) samples. Our aim was to determine the proportion of mosaic cases, the utilisation of interphase fluorescent in-situ hybridisation (FISH) follow-up studies and impact of findings on counselling and patient management. Follow-up studies involved an amniocentesis or fetal blood sampling (FBS) with both karyotyping and FISH being performed to clarify the fetal karyotype. Overall, mosaic karyotypes were identified in 54/8451 (0.64%) samples. The propor-

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K. Elisavet (1) K. Aggelki (1) P. Nikolas (2) A. Aris (2) K. Emmanuel (1) M. Ariadni (1) (1) Athens University (2) Athens University AIM: To evaluate the use of fetal NRBCs isolated from maternal blood for non-invasive prenatal diagnosis of fetal aneuploidies. Materials and methods: Twenty ml of peripheral blood were collected from 414 women, non-carriers of β-thalassaemia trait, in the 2nd trimester of pregnancy: 388 carried chromosomally normal and 26 chromosomally abnormal fetuses (Down Syndrome: n=18, trisomy 18: n=2, trisomy 13: n=1, mosaic for trisomy 12: n=1, 45, XO: n=3, and 47, XXX: n=1). NRBCs were positively selected by MACS with anti -CD71 monoclonal antibody, stained

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with May-Grunwald/Giemsa and enumerated. FISH was performed in 75 cases using X, Y and 21 chromosome specific probes. Results: NRBCs were identified in 357 samples (86%). Among 327 women with chromosomally normal fetuses, 7 NRBCs (range 1–18) were identified. In another 4 women carrying normal fetuses an increased number of NRBCs were isolated, possibly of maternal origin. In 26 cases with aneuploid fetuses the mean number of NRBCs was 33.5 (range 5–113): 66 (range 22–113) in DS fetuses, 6 (range 5–7) in trisomy 18, 37 in trisomy 13 and 8 in mosaic trisomy 12. The mean numbers of NRBCs in pregnancies carrying a 45, XO and a 47, XXX fetus were 51 (range 43–58) and 12 respectively. Fetal sex was correctly determined in 46/75 cases (61%). In all cases with DS the aneuploidy was confirmed. Conclusion: The present results confirm our previous reports indicating an increased number of NRBCs in maternal circulation in pregnancies with chromosomally abnormal fetuses, possibly due to abnormal placentation leading to increased transfer of fetal cells into maternal circulation. FISH analysis of NRBCs facilitates the distinction between fetal and maternal NRBCs when the fetus is male, but hybridization efficiency is very low due to enrichment procedures and the apoptotic status of the isolated NRBCs. 13.58-P Novel approach for Preimplantation Genetic Diagnosis in Translocation carriers J. Shamash (1) (1) the Sheba Medical Center Novel approach for Preimplantation Genetic Diagnosis in Translocation carriers Shamash J1, Rienstein S1,Wolf-Reznik H1, Pras E1 Dekel M1, Brengauz M2, Goldman B1, Litmanovitch T1, Levron J2, and Aviram-Goldring A1 1.Danek-Gertner institute of human genetics, 2.In Vitro Fertilization unit, department of obstetrics and gynecology, Chaim Sheba medical center, Tel Hashomer and SacklerSchool of medicine, Tel- Aviv University, Israel Introduction: Chromosomal translocations carriers (TCs) are at risk of having unbalanced offspring,

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resulting in severe malformations and mental retardation. TCs often suffer from infertility problems and repeated miscarriages. Preimplantation genetic diagnosis (PGD) using fluorescence in-situ hybridization (FISH) provides a reproductive solution. However, FISH does not differentiate between balanced translocated and normal embryos. This technical limitation might perpetuate infertility and other abnormalities to the next generation. Aims: To develop an improved PCR based method for PGD that is capable to identify and differentiate between balanced TCs and chromosomally normal embryos. Methods: Two translocation carriers’ families (15:21 and 13:14) were analyzed. FISH-PGD was performed using commercial probes (Abbott Inc.) according to standard manufacture’s instructions. Unbalanced embryos were haplotyped following whole genome DNA amplification of blastomere/blastocyste, using isothermal Phi 29 reaction. Results In each family, we set up segregation mode of fully informative polymorphic markers (PM) located close to the translocation breakpoint, in the parents and in a third informative related individual. By doing so, we could haplotype those alleles that correlate with the translocated and the normal chromosomes. Two embryos (E1,E2) from TC 15/21 and two embryos (E3,E4) from TC 13/14 were analyzed. E1 showed three alleles of chromosome 21 including the one that correlates with the maternal translocated chromosome indicating unbalanced translocation (trisomy 21) assesing previous FISH results. E2 was found to carry the maternal translocated chromosome alleles in a balanced form. E3 showed three alleles of chromosome 13 (two paternal including the translocated chromosome and one maternal) resulting in unbalanced translocationtrisomy 13, confirmed by FISH. E4 exhibited alleles linked to the paternal normal chromosomes 13 and 14. This embryo was translocation free. Conclusions: Our preliminary results indicate that the PM method is capable of distinguishing between normal, balanced and unbalanced TC embryos. The use of this method will improve PGD and will allow balanced TC`s to avoid transferring their fertility problems to the next generation.

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13.59-P Use of Quantitative Fluorescent Polymerase Chain Reaction (QF-PCR) in spontaneous miscarriages D. Parrini (1) B. Minuti (1) R. Casano (1) A. Lari (1) R. Abdul Samad (1) S. Cascella (1) F. Sbernini (1) R. Ciampi (1) S. Fibbi (1) F. Torricelli (1) (1) Aziena Ospedaliero-Universitaria Careggi During 2008, SOD Diagnostica Genetica of Careggi Hospital of Florence (Italy) carried out a study to assess whether the QF-PCR technique could be used as a complementary method in cytogenetic studies of spontaneous miscarriages in order to detect chromosomal anomalies. This technique was already used in the laboratory for first-trimester testing on Chorionic Villus Samples (CVS) in substitution of the chromosomal analysis of spontaneous metaphases. QF-PCR was carried out on 80 spontaneous abortion samples: 14 embryonic miscarriages, 63 foetal miscarriages (46 of the first trimester and 17 of the second one) and 3 MIF.The protocol proposed conventional tissue culturing and karyotyping beside DNA extraction for QF-PCR. Conventional tissue culturing presented some limitations such as culture failure, external contamination and selective growth of maternal cells while molecular analysis was informative in 95% of the miscarriages. Aneuploidies were detected in 32% of our samples. QF-PCR represents a useful and reliable tool to diagnose aneuploidies in spontaneous miscarriages; this technique is highly accurate, low cost, and a rapid diagnostic method able to overcome the classical cytogenetic disadvantages as a long laboratory cycle, intensive labor, and culture failure, especially when karyotyping necrotic tissues.

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copy number changes. For prenatal samples, DNA amounts are often too low for array CGH and require cell culturing. To reduce turn-around time, cell culturing could be replaced by whole genome amplification (WGA). This would enable molecular karyotyping of samples having only few nanograms of DNA or even single cells. Although WGA has been shown to be promising, further information is needed about the putative bias WGA introduces to the genomic profiles. We performed WGA on DNA samples and analyzed the resulting genomic profiles by means of array CGH. The amplification was performed with GenomePlex® (SigmaAldrich) products using few nanograms of DNA or few cells as a starting material. To assess possible bias introduced by WGA, the amplified test samples were hybridized both with the respective native and with an amplified reference DNA. PerkinElmer’s Constitutional Chip® platform and SpectralWare® analysis software were used in the array CGH analysis. Our results indicate that the Constitutional Chip® platform works effectively for generating array CGH data from amplified test and reference samples. Using this platform we compared amplified DNA with the respective native DNA and observed that specific chromosome regions continuously fail to amplify in WGA. Nevertheless, the results show that detection of aneuploidies is not hampered by the sequence dropout, although micro-level changes may be missed owing to the biased amplification. WGA has potential for expanding the use of array CGH in the analysis of limited amounts of DNA. A larger set of samples will be needed to determine the feasibility of the technology for prenatal samples. 13.61-P

13.60-P Molecular karyotyping of whole genome amplified samples H. Raussi (1) M. Mäkinen (2) S. Ruosaari (1) A. Godenhjelm (1) P. Ollikka (1) (1) Wallac Oy (2) Turku University Array comparative genomic hybridization (array CGH) has been widely used for detection of DNA

Prenatal diagnosis of a fetus carrying two balanced reciprocal translocations C. Villalon (1) M. Ferro (1) M. Talavera (1) E. Garcia-Galloway (1) J. Garcia-Sagredo (1) (1) University Hospital Ramon y Cajal We report a 30 years old pregnant woman asking for prenatal diagnosis because of she is carrier of a Robertsonian translocation 13;14.

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After amniocentesis at 16 weeks of pregnancy, fetus karyotype shows two different translocations. One is the Robertsonian translocation of the mother t (13;14) and the second one is an apparently balanced reciprocal translocation between chromosomes 4 and 15: t(4;15)(q21;q15). With this karyotype finding, 45,XX,t(4;15)(q21; q15),t(13;14), the father was analyzed and his karyotypes was 46,XY,t(4;15)(q21;q15). Delivery was at 38 weeks of pregnancy, and a normal female was born. 13.62-P Use of subtelomeric MLPA in cytogenetic prenatal diagnosis. Our experience A. Soler (1) I. Mademont (1) C. Morales (1) N. Clusellas (1) E. Margarit (1) I. Madrigal (2) A. Sánchez (1) (1) Hospital Clínic de Barcelona (2) CIBER de Enfermedades Raras It is known from postnatal studies that imbalances of subtelomeric regions are responsible for a significant part of idiopathic mental retardation and/or dysmorphic features. Among the present methods for subtelomere screening, MLPA has been developed as a reliable and inexpensive molecular technique to detect submicroscopic imbalances and has been successfully applied to individuals with unexplained mental retardation and normal karyotype. In a prenatal setting, MLPA can be used to screen for cryptic subtelomeric imbalances in fetuses with ultrasound abnormalities and normal karyotype, but also as a complementary technique to conventional cytogenetics in cases of structural chromosome rearrangements. We have applied MLPA to detect cryptic subtelomeric imbalances in 216 pregnancies presenting fetal malformations with normal karyotype, and in a selected group of pregnancies showing a structural rearrangement in order to contribute to their characterization. DNA was obtained from chorion villi samples or cultured amniocytes following standard procedures. MLPA was performed using the p036 subtelomere kit and p070 kit for confirmation (MRC-Holland). To confirm deletion findings, FISH was performed according to standard procedures.

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In the first group of pregnancies, only 3 imbalances were detected using both MLPA kits, one of them of unclear significance, so the detection rate of relevant submicroscopic imbalances was unexpectedly low: 0.9%. This finding seems to indicate that fetal malformations are not predictors of postnatal mild dysmorphic features or mental retardation. In the other group of pregnancies, MLPA analysis disclosed a second imbalance in three cases of unbalanced chromosomal rearrangements; a cryptic subtelomeric deletion in a case of a chromosome abnormality confined to a CV sample; and allowed to confirm the suspected state of balance / imbalance of subtle chromosome rearrangements diagnosed in short-cultured CVS, preventing the need to wait for long-term cultures and permitting to achieve a final diagnosis in a short period of time. 13.63-P Cytogenetic and molecular characterization of a Small Supernumerary Marker Chromosome (sSMC) found at prenatal diagnosis R. Murru (1) M. Angiolucci (2) L. Martorana (1) A. Azzena (1) S. Deidda (1) V. Licheri (1) C. Vivanet (1) G. Serra (2) S. Orru’ (1) C. Carcassi (1) (1) University of Cagliari (2) University of Cagliari Human small supernumerary marker chromosomes (sSMC) are present in 0.043% of newborn infants. They can be defined as additional centric chromosome fragments and generally are equal in size or smaller than a chromosome 20 of the same metaphase spread. Prenatal diagnosis of supernumerary marker chromosomes (SMC) is problematic because of the difficulty of predicting the phenotype. The assessment of phenotypic risk is based on the size, morphology and origin of the SMC. We found a sSMC in long term culture of chorionic villi performed for increased nuchal translucency in a 38 year old woman at 12 +4 weeks gestation. Amniocentesis was performed at 15+5 weeks: GTG-banding analysis of amniotic cells confirmed the presence of the additional marker chromosome (karyotype:47,XX,+mar). Both parents had normal karyotypes. Fluorescence in situ hybridization (FISH) analysis using chromosomal specific alphoid satellite DNA probes and whole chromosome paint probes showed that the extra sSMC was derived

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from chromosome 14. Further characterization of SMC performed by centromere-near-specific multicolor FISH (subcenM-FISH) showed it was an inv dup(14)(q11.1). This region consist of heterochromatic material: SMC without euchromatic content are more likely to result in normal phenotypes. Maternal uniparental disomy for chromosome 14 (associated with precocious puberty, short stature and highly variable developmental delay) was excluded by microsatellite analysis. Pregnancy was continued. No morphological abnormalities were observed on any ultrasound examinations performed during pregnancy. The baby was delivered by caesarean section at 38 weeks gestation. No dysmorphic features were noticed at birth. At 1 year follow-up, the child was growing and developing normally. This case illustrates how a combination of family studies, FISH characterization and molecular analyses may enhance the accuracy of the information given (diagnosis and prognosis, risk estimates) during prenatal counseling.

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After genetic counselling, parents elected to terminate the pregnancy. Fetal autopsy showed, in a female fetus normal for gestational age, micrognathia, lowset normal ears, feminine external genitalia with clitoridomegaly, marked dilatation of lateral ventricles, hypertrophic left adrenal gland partially fused with pancreas, large interatrial and interventricular defects of the heart, hypertrophy of deltoid and femoral quadriceps muscles. This case report shows a mosaic trisomy 9 fetus with most typical features of the syndrome (cardiovascular defects, urogenital abnormalities, craniofacial and central nervous system malformations), suggesting that even mosaics can present a severe phenotype. This aspect can be relevant in genetic counselling. Moreover the fetus presented muscle hypertrophy, a feature never reported before to our knowledge, that probably will deserve an evaluation in other cases of this trisomy. 13.65-P

13.64-P Case report: Prenatal diagnosis of mosaic trisomy 9

Prenatal diagnosis of trisomy 18: time of evalutation, ultrasound markers and genetic counselling. A review

V. Licheri (1) C. Vivanet (1) R. Murru (1) A. Azzena (1) L. Martorana (1) S. Deidda (1) F. Spina (1) M. Virdis (1) S. Orrù (1) C. Carcassi (1) (1) University of Cagliari

S. Deidda (1) R. Murru (1) A. Azzena (1) L. Martorana (1) V. Licheri (1) M. Virdis (1) G. Serra (2) M. Angiolucci (2) S. Orrù (1) C. Carcassi (1) (1) University of Cagliari (2) University of Cagliari

Trisomy 9 is an uncommon chromosome abnormality which can occur in a mosaic or non-mosaic state and presents with a distinct clinical picture. This is a relatively rare chromosomal abnormality, constituting only 2.7% of all trisomic cases (Roshanfekr et al. 1998). Complete trisomy 9 ends in spontaneous abortion in the first trimester. We detected a mosaic trisomy 9 in an amniotic fluid cell culture from a 40-year-old woman at 16+4 weeks’ gestation, evaluated because of advanced maternal age and anomalous First Trimester Serum Screening (background risk: 1:93; adjusted trisomy 18 risk: >1:50). Nuchal Translucency scan was normal. We analyzed 26 clones from three different cultures, and the karyotype resulted 46,XX[18]/47, XX,+9[8]. Fetal ultrasound was negative up to 18th weeks’ pregnancy, when revealed dilated lateral ventricles and micrognathia.

Trisomy 18 is a devastating genetic disorder characterized by multiple congenital anomalies. The most common fetal anomalies noted are: persistent abnormal position of fingers, abnormally shaped fetal head, heart defects, two-vessel umbilical cord, intra-uterine growth retardation, omphalocele, neural tube defects and cystic hygroma or lymphangiectasia, abnormalities of amniotic fluid volume and renal defects. Considering 2300 karyotypes were diagnosed eleven fetuses with trisomy 18 (0,48%). Seven out of then were female. The mean age of the mothers was 38,9±4,33 years (range, 31 to 45 years). The mean gestational age at the time of evalutation was 14±5.0 SD weeks (range, 11+3 to 30 weeks). All fetuses identified with trisomy 18 had sonographic abnormalities. Nine cases showed ultrasound anomalies in first-trimester: five cases showed increased nuchal thickness, four cases showed fetal hydrops.

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One patient did ultrasound assessment only at the end of gestation, that showed multiple fetal anomalies typical of trisomy 18. Only in one case, the first-trimester assessment was normal and the woman refused invasive testing for maternal age. Routine ultrasound examination at 30 weeks’ gestation, however, revealed multiple anomalies in the fetus and the prenatal diagnosis at 31 weeks identified the trisomy 18. Our data shows that most of fetuses with trisomy 18 were identified by sonographic examination in first-trimester, and that the more sensitive ultrasound marker is the nuchal translucency. Moreover, considering 167 prenatal diagnosis performed for increased nuchal translucency, eighteen cases showed trisomy 21 (10,77%) and five cases trisomy 18 (3%). Even if most of fetuses with trisomy 18 are going to be aborted early spontaneously, our previous data show that, in case of increased nuchal translucency, it is important to let the mother aware about trisomy 18 consequences in order to enable her taking a conscious decision about invasive prenatal diagnosis. 13.66-P FISH on interphasic uncultured amniocytes: experience in 328 foetuses with US anomalies E. Sala (1) N. Villa (1) F. Crosti (1) E. Gautiero (1) R. Solano (1) S. Lissoni (1) E. Martinoli (2) M. Volontè (2) S. Mariani (3) L. Dalprà (4) (1) san gerardo hospital (2) university milan (3) san gerardo hospital (4) university milan-bicocca Ultrasound malformations in pregnancy is often associated with chromosomal abnormalities. Cytogenetic results take 10–14 days due to culturing amniotic cells. This lengthy wait causes considerable parental anxiety, specially when echographic abnormalities were observed. In our clinic, we decide to perform rapid FISH, using AneuVysion probes, on 328 selected amniocentesis with foetal malformations over several years, and we identified 84 cases with aneuploidy (25.6%). In cases with a sole abnormal US finding (n=92) 22 aneuploidies were detected (23.9%—17 tris 21, 1 tris 13, 1 tris 18, 2 aneuploidies of sexual chromosomes). In the group with two or more malformations (n=236) there were 62 aneuploidies (26.3%—21 tris 21, 19 tris 18 one of them with

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mosaicism, 9 tris 13, 6 triploidy, 5 monosomy X, 1 karyotype: 48,XXY and one mosaicismo: 45,X/47, XXX). In this group conventional cytogenetics revealed five chromosomal anomalies not detectable by FISH: a trisomy 16 mosaicism, a del (4p), a derivate chromosome 18 from a t(4;18)mat, an interstitial del(5q), and add6p. Moreover in the group with a single US anomaly three karyotypes showed a structural alteration: a r(13), a r(14) and a del(13)(q31q34). In two cases FISH analysis failed (one normal karyotype and one tris 18) while in 5 cases conventional cytogenetics do not performed any result. All these cases had a normal FISH analysis. There were no false positive or false negative results by FISH. The complete concordance between FISH data and karyotype study enable us to consider the interphase FISH as a useful tool in pregnancies with high risk of chromosomal aneuploidies. When disomic status is known to be normal, the overall risk of chromosomal abnormalities is significantly reduced (residual risk: 3.3%). Detection of 8 structural chromosomal alterations only by conventional cytogenetics suggest that interphase FISH must still be considered as a complement to standard karyotyping. 13.67-P Constitutive heterochromatin traits as revealed by Acridine Orange in human embryos I. Trofimova (1) T. Kuznetzova (2) V. Baranov (2) (1) Saint-Petersburg State University (2) Ott’s Institute of obstetrics & gynecology RAMS (3) Ott’s Institute of obstetrics & gynecology RAMS Constitutive heterochromatin regions (CHR) in chromosomes of different tissues are characterized by tight condensation, late replication and methylation. Meanwhile decondensation, hypomethylation CHR in human trophoblast and tumor cells were registered. Stress-induced transcription of satellite III in some cell lines was shown. We studied definite peculiarities of CHR in direct and semi-direct slides from chorionic villi and embryonic tissue samples (8 and 3 accordingly) after artificial abortions at 5–12 week of pregnancy. Typical for single-stranded DNA and RNA bright red fluorescence of 1qh, 9qh, 16qh, Yqh, 15cenh,

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22cenh and typical for double-stranded DNA yellowgreen fluorescence along arms of chromosome set were registered after standard AO staining of untreated slides. Separate and combined pretreatment of slides with different nucleases (RNase A and DNase I) resulted in decrease of red fluorescence of CHR in most metaphase plates. Chromosome arms were relatively uniform stained or banded similar to RFA in some metaphases. Subjection to DNA ligase T4 resulted in total absence of fluorescence ("black holes") in CHR and uniform green fluorescence along chromosome arms. Staining pattern corresponded to untreated slides was observed in all control slides treated with corresponding buffer solutions. Metaphase chromosomes from peripherical and umbilical cord PHA-stimulated lymphocytes demonstrated uniform green colour in all experiments and untreated slides. Our results advocate for unusual conformation packaging of CHR which could be attributed to the feasible transcriptional activity of pericentromeric satellite DNA in embryonic and cytotrophoblast cells during embryogenesis. 13.68-P Pitfall: False-negative 46,XX finding in CVS: chromosome mosaicism involving a de novo autosomal structural abnormality and unexpected diagnosis of a third cell line in the fetus U. Mau-Holzmann (1) S. Singer (1) H. Enders (1) S. Haen (2) I. Tekesin (3) T. Liehr (4) (1) University of Tuebingen (2) University of Tuebingen (3) University of Tuebingen (4) Jena University Hospital We report on a unique case of discordant chorionic villus sampling (CVS) and fetal karyotype results. A CVS and fetal cord blood sampling were performed in a 26 years old woman because of oligohydramnios, heart defect with arrhythmia and hypertrophic placenta. Direct culture of the chorionic villi showed a normal female karyotype. Analysis of the fetal cord blood revealed a mosaic: a de novo autosomal structural abnormality [46,XX,t(8;9)] and a few cells with a normal female karyotype. CVS long term culture confirmed the normal female karyotype.

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In view of the sonographic abnormalities the woman opted for induced abortion in the 22+3 week of gestation. Chromosome analysis of different fetal tissues and the placenta confirmed the former results. Additionally, an unexpected isolated 45,X-karyotype was found in fibroblasts of the fetal cord. The presumed origin and development of this complex mosaicism will be discussed. 13.69-P Human Constitutive Heterochromatin: a New Intrigue T. Kuznetzova (1) I. Trofimova (2) I. Martynkevich (3) V. Baranov (1) (1) Ott’s Institute of Obstetrics & Gynecology (2) Saint-Petersburg State University (3) Russian Research Institute of Hematology and Transfusiology (4) Ott’s Institute of Obstetrics & Gynecology Acridine orange (AO) usually results in uniform green staining along the length of chromosome arms on untreatment slides. It is also routinely used for the standard RFA and RBA banding. As fluorescent dye AO is discriminating single-stranded from double stranded nuclei acids as orange-red and yellow-green fluorescence respectively. In our chromosome replication studies carried out on chorionic biopsies samples incubated for 24–72 h without BrDU as well as on direct chromosome preparations heterochromatin blocks of chromosomes 1, 9, 16 and Y showed red fluorescence whereas chromosome arms were uniformly yellow-green. The same staining pattern was typical for chromosomes of different embryonic tissues on the 5–10 weeks of gestation. This puzzle stimulated more thorough studies in chromosome preparations from PHAstimulated lymphocytes of both fetal & adult origin, from amniotic fluid (slides kindly provided by Y. Loginova) and adult bone marrow cells. Standard cytogenetic techniques for both pre- and postnatal karyotyping including this one applied for oncohematological practice were used throughout these studies. Regions 1qh, 9qh, 16qh & Yqh revealed red fluorescence in metaphases of all undifferentiated cells with high mitotic activity in vivo (cytotrophoblast, embryonic cells, bone marrow cells from patients with lymphoblastic leukemia).

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But they stained in green in terminally differentiated cultured lymphocytes and amniocytes. These results advocate in favor of the principal cytochemical and conformational differences in heterochromatin regions in different cells which could be attributed to the variability of their DNA and hystone epigenetic modifications and probably to the transcription activity of the satDNAs. Further studies of embryonic and stem cells as well as in malignant cells are in progress. The option to use AO for more detailed analysis of heterochromatin participation in normal and abnormal development is discussed.

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For genetic service purposes and compared to data in the literature we conclude that our success rates were satisfactory, however the approach is labour-intensive. Placental tissue has a superior growth rate, however possesses a risk of MCC. Cytogenetic diagnostics of IUFD should not be restricted to fetuses with visible phenotypic abnormalities. The relatively low frequency of unbalanced IUFD karyotypes is most likely due to the use of prenatal diagnosis and screening in our population. It would be relevant to investigate the feasibility of molecular cytogenetic techniques for IUFD genetic diagnostics. 13.71-P

13.70-P Quality of cytogenetic diagnostics on solid tissue in 394 second and third trimester intrauterine fetal deaths J. Hahnemann (1) G. Bennich (2) (1) Kennedy Center (2) Roskilde Hospital We evaluated the quality in our laboratory of cytogenetics on solid tissue obtained after intrauterine fetal death (IUFD) ≥ 14 weeks of gestation, to provide the basis for optimising this genetics service. Laboratory records on samples received for karyotyping during 1999–2006 were reviewed, karyotype and growth success rates were calculated, the frequency of maternal cell contamination (MCC) was estimated, and details of pregnancies with unbalanced karyotypes were reviewed. An overall proportion of 72% (285/394) of the IUFD conceptuses were successfully karyotyped, increasing from 58% to 79% during 2000–2006. An average of 2.0 tissues per conceptus were received and set up for culturing. Growth success rates were 33%, 40%, and 88 % for achilles tendon, skin, and placental biopsies respectively. Evidence of MCC was found in six cases with an XX karyotype and discordance between karyotypic and phenotypic sex of the fetus; all six were karyotyped on cells from placenta or extrafetal tissue. Karyotyping resulted in the detection of 4.2% (12/285) chromosomal imbalances. Except from cystic hygroma in four fetuses, dysmorphism or malformations were observed at delivery/abortion or autopsy in only three out of the 12 conceptuses with a chromosomal imbalance.

Validation of QF-PCR for aneuploidy detection in an indian population S. Agarwal (1) A. Kooper (2) I. Gomes (2) J. DerksWillemen (2) I. Panigrahi (3) A. Smits (2) (1) SGPGIMS, Lucknow,India (2) Radboud University Nijmegen Medical Centre (3) PGI, Chandigarh, India Quantitative fluorescence polymerase chain reaction (QF-PCR) is a rapid, reliable and cost-effective method for screening of common aneuploidies, but it is not in routine use in India. Our objective was to investigate three different commercially available QF-PCR kits to select the most suitable kit for the application of rapid aneuploidy detection in prenatal samples in the Indian population. There are differences in heterozygosity and in frequency of triallelic patterns in markers in different populations and therefore evaluation of the STR markers in the different kits were compared for the Indian population. Three different kits for the detection aneuploidies of 13,18, 21, X and Y were tested from Aneufast (Genomed, UK), Elucigene QST*R plus (Tepnel, UK) and the Compact kit (Devyser, Sweden), respectively, on 18 blood samples from parents and 29 children affected with Down syndrome. Informativeness, differences in labor intensiveness and costeffectiveness of the kits were examined in order to select the most suitable kit. Test results will be presented for discussion. For prenatal aneuploidy screening, QF-PCR needs to be efficient and reliable. Considering the frequency triallelic patterns of one STR marker needs careful

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evaluation in each marker when it is applied to a different population. 13.72-P FISH identification of aneuploid circulating fetal cells in maternal bood between 14 and 17 weeks of gestation G. Palka (1) G. Calabrese (1) G. Sitar (2) D. Fantasia (1) A. Colombo (3) E. Morizio (1) M. Alfonsi (1) P. Guanciali Franchi (1) (1) University of Chieti/D’Annunzio Foundation (2) Policlinico S. Matteo (3) University of Milan We analyzed 15 peripheral blood specimens obtained from women having a trisomy 21 (9 patients), trisomy 18 (5 patients), and trisomy 15 (1 patient) following invasive prenatal diagnosis. Blood samples were enriched in fetal cells content with a modification of our previously described ‘non phisiological’ system (Sitar et al 2005). Fixed cells were subsequently spread on slides by cytospin (50–100×103 cells/slide). FISH experiments using two commercial probes, labelled in different colors, mapping at different loci of the same chromosome were carried out. Cells showing a two-green/two-red signal FISH pattern were classified as normal, and cells with a three-green/three red signal pattern as trisomic. At least 900 cells per sample were scored. In 11 cases 4–7 trisomic cells /1000 scored cells per slide were found (range 0.36–0.76% trisomic cells/ sample). In one case bearing a +15 at chrorionic villous sampling, only 2/1700 abnormal cells (0.12%) were observed. In the remaining 3 cases some technical artifacts possibly due to insufficient cell storage conditions before cell manipulation prevented further analysis. The same procedure applied on slides from normal pregnancies showed in 5 cases a maximum of 2 apparent trisomic abnormal cells in 2000 scored cells by FISH. Present preliminar results: a) confirmed previous results obtained from normal and trisomy cases using X and Y chromosome probes by scoring a minimum of 7000 cells; b) demonstrated that modification in enrichment techniques decrease work load for aneuploid cell search (1000 cells/case) when blood samples are correctly stored before enrichment and FISH protocols.

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13.73-P A translocation t(11;22) patient with recurrent spontaneous abortions and preimplantation genetic diagnosis (PGD) F. Celep (1) M. Yırmıbes Karaoguz (2) Y. Saglam (3) S. Basaran (4) A. Karaguzel (1) (1) Karadeniz Technical University Medical Faculty (2) Gazi University, Faculty of Medicine, (3) Goztepe Medical Park Hospital (4) İstanbul Universıtyİstanbul Medıcal Faculty (5) Karadeniz Technical University, Medical Faculty Reciprocal translocations are of great clinical importance and the carriers are phenotypically normal, except for rise to reproductive problems and usually recurrent pregnancy loss, chromosomally abnormal offspring, or, in some cases, infertility. Here we report a patient who presented with balanced translocation of both chromosomes 11 and 22 and. The patient, 28 year-old, was referred to our laboratory with a reproductive history. She was a phenotypically normal female and the obstetric history revealed missed abortions at 8, 7 and 8 weeks of gestation. The patient’s karyotype was identified as 46,XX,t (11;22)(q23;q11.2) according to the standard cytogenetic methods using G-banding technique. This result was confirmed by FISH (Fluorescence in situ hybridization) on fixed metaphases. The patient’s husband and her parent’s chromosomes were normal. Genetic counseling and prenatal testing is offered to family. In first and second abortions, no cytogenetic analyses had been performed but in the last abortion, amniocentesis was performed and revealed 46,XX,der(13;14)(q10;q10),+13 karyotype. PGD (Preimplantation genetic diagnosis) is nowadays widely applied which diagnose genetically or chromosomally abnormal embryos of couples at risky group. Therefore, the family was directed to PGD. We present the results of PGD applications in our patient. 13.74-P 45, X karyotype and abnormal ultrasound findings in early pregnancy loss M. Angiolucci (1) R. Murru (2) V. Mais (1) A. Azzena (2) L. Martorana (2) S. Deidda (2) V. Licheri (2) A. Sammarco (1) S. Orru’ (2) C. Carcassi (2) (1) University of Cagliari (2) University of Cagliari

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The aim of this study was to verify if a specific chromosomal abnormality could determine abnormal biometric ultrasound (US) measurements in early pregnancy. Transvaginal (TV) US findings obtained in 9 cases of early pregnancy loss (EPL) with 45, X karyotype were compared with those obtained in 58 cases of EPL with normal karyotype. In all cases, the TV US scan performed at the moment of the diagnosis of EPL to measure the size of both the fetus and the gestational sac (GS) was not the first one performed in that pregnancy. Cytogenetical analysis was performed on abortion material both directly and with cultured cells using GTG banding. For each sample, no less than 8 clones or 15 metaphases were observed. When a normal 46, XX karyotype was found, maternal and fetal DNA were compared by molecular analysis in order to exclude maternal contamination. Fisher’s exact test was used for statistical analysis. In 7 out of 9 cases of EPL with 45, X karyotype the TV US scan performed at the time of diagnosis found a condition of “small fetus”(SF), meaning that the crownrump length (CRL) of the fetus was below the 5th percentile in the presence of a GS of normal size for the gestational age. The difference between the mean diameter of the GS and the CRL ranged between 25 and 35 mm. A SF was observed only in one out of 58 cases of EPL with normal karyotype. Therefore, the 45, X karyotype seems to be significantly associated with a TV scan revealing precocious fetal arrested growth (P<0.0001). Our results show that an USstudy of the size of both the fetus and the GS in early pregnancy makes it possible to identify pregnancies at risk for chromosomal abnormalities at a very early stage; this will undoubtfully have a significant impact on prenatal genetic counselling. 13.75-P Problems in interphase investigations by mutations in the centromeric regions D. Hansmann (1) G. Schwanitz (2) U. Paetzold (2) U. Gamerdinger (3) T. Eggermann (4) (1) Institute of Prenatal Medicine and Genetics (2) University of Bonn (3) University Medical Center of Giessen and Marburg (4) RWTH Aachen We present the case of a familial mutation of 21q10 detected after CVS, AC and subsequent chromosome analysis of the mother.

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Prenatal chromosome analysis was performed because of increased maternal age at pregnancy (41 years). Two previous pregnancies lead to the birth of healthy children. At this time, the first trimester was complicated by spontaneous bleedings. An increased NT of 2.9 mm was diagnosed by ultrasound investigation. CVS was performed in pregnancy week 12+5. A level I mosaic with 3/46 (6.5%) of mitoses revealing different derivates of chromosome 13 was detected after long term cell culture. Therefore amniocentesis at week 15+5 was performed. Interphase FISH with DNA probes 18, X, Y cen and LSI 13, 21 revealed normal results in 208 cells, 1 cell (0,48%) showed 3 signals for chromosome 13 (LSI 13q14). Additional hybridizations with the probe 13, 21 cen showed only 1 signal in all cells analyzed (n= 159). The missing centromere was identified as 21q10 after cell cultivation. Besides this peculiarity, chromosome 21 had a normal morphology and by additional chromosome investigations it could be delineated as maternal in origin. After cultivation of amniocytes the chromosomes were analyzed from two cultures. Four of 92 mitoses showed different derivates of chromosome 13 in culture 1. In culture 2, all mitoses (n=35) had a normal karyotype (total amount of pathologic cells 3%). The ultrasound findings at week 15+5 were normal. This investigation was repeated at week 21+5, again with normal results. The parents decided to continue the pregnancy after genetic counselling. A healthy child was born at term and showed a normal development in its first six months of life. Additional investigations were started to analyze the centromere mutation in mother and child. 13.76-P Evaluation of a QF-PCR kit for Rapid Aneuploidy Detection F. Lonardo (1) G. Cantalupo (1) M. Ciavarella (1) M. Della Monica (1) C. Lombardi (1) M. Maioli (1) L. Masella (1) G. Scarano (1) (1) A.O.R.N. Gaetano Rummo Prenatal diagnosis for chromosome abnormalities is routinely undertaken by full karyotype analysis of

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chromosomes from cultured cells, so pregnant women must wait on average 15 days for a result. However, trisomies 13, 18 and 21, as well as sex chromosome aneuploidies and triploidies, which account for more than 80% of significant chromosomal aberrations, can be detected by other techniques within 24–48 h. We recently introduced in our Prenatal Diagnosis Service a test for Rapid Aneuploidy Detection (RAD). Among the commercial kits validated for In Vitro Diagnostics and CE labelled we selected Devyser® Complete Kit (DevyserAB, Stockholm, Sweden). The test is based on Quantitative Fluorescence PCR (QFPCR) of seven markers each for chromosomes 13, 18, 21 and XY. The high number of markers minimises the need for re-runs of samples due to homozygous or uninformative results. A unique marker for true detection of Turner syndrome is also included. The aim of the current study was to evaluate the RAD markers chosen and to estimate their informativeness in our population (coming mainly from South Italy). We prospectively analysed about 400 amniotic fluid samples. The data obtained from the Devyser kit were compared to corresponding data from karyotyping to verify the diagnostic power of the tests. For each marker we evaluated number and size of alleles, character of the repeats and heterozygosity. RAD by QF-PCR has proven to be a sensitive, efficient and reliable method for detecting major aneuploidies, but it requires an accurate selection of markers, as ambiguous results are likely to increase parental anxiety. The use of markers with a high coefficient of informativeness decreases the probability of homozygosity and therefore that of uninformative QF-PCR profiles. In our clinical setting the markers amplified by Devyser Complete Kit showed a high heterozygosity rate, and allowed a diagnostic result in all cases except those with significant maternal cell contamination. 13.77-P Familiar inversion of chromosome 4 in a Tunisian family resulting in trisomy 4p l. ben jemaa (1) m. chaabouni (1) m. kessentini (1) r. meddeb (1) l. euchi (1) f. maazoul (1) r. mrad (1) s. gaigi (2) h. chaabouni (1) (1) EPS Charles nicolle (2) maternité de la rabta

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A 26 year old women was referred to our center at 12 weeks gestation for abnormal nucale translucency at 4,3 mm. Paternal age was 34 years. Amniocentesis was performed at 15 weeks, the cytogenetic analysis of the cultured amniotic fluid cells revealed a male karyotype with trisomy 4p who is inherited from a double anomaly of chromosome 4 in his father consisting in 46,XY,rec(4) (dup)(4p)inv(4)(p12q35), rec(4)(del)(4p) inv(4) (p12q35). The maternal karyotype is normal. FISH analysis was performed in the foetus with WHS chromosome 4 probe (LSf WHS VYSIS) located to 4p16.3 and showed a double signal in the abnormal chromosome 4 indicated a trisomy 4p. The pregnancy was terminated at 19 weeks . the autopsy showed a macrocephaly, facial dysmorphy , microrethrognathy, short neck , overlapping of 2nd and 5th finger, increased inter-nipple distance. We realised karyotype in all the family: the parents of the father who are consanguineous and his two healthy sisters has the same pericentric inversion of chromosome 4: inversion (4)(p12q35), another sister has the some anomaly of his brother. In the familiar history the father has a brother with mental retardation, facial dysmorphy micropenis, overlapping of 2nd and 5th finger, his karyotype was normal, it will be interesting to search for a parental disomy in this case. To date about 12 cases of recombinant of chromosome 4 pericentic inversion have been described , we report familiar cases with the same pericentric inversion in a consanguineous couple. 13.78-P Prenatal diagnosis today—what we need to change E. Ribeiro (1) M. Martins (1) C. Santana (1) O. Carmo (1) I. Pinto (1) M. Souto (1) P. Botelho (1) O. Moutinho (1) R. Pinto Leite (1) (1) Centro Hospitalar de Trás-os-Montes e Alto Douro The main objective to perform an invasive technique in prenatal diagnosis (DPN) still is the diagnosis of trisomy 21. Despite the knowledge of ultrasound, the discovered of new ecographics markers and the effectiveness of the biochemical screening, the advanced maternal age still remains, in spite of the low

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sensitivity, the most frequent motive for the request to perform the amniocentesis. The author presents the cytogenetic results obtained in prenatal diagnosis performed from 1998 to February 2009 in the Centro Hospitalar de Trásos-Montes e Alto Douro and makes some reflection about those results. In the 4454 amniocentesis performed at the Prenatal Center, 96% of them had a normal cytogenetic result. The trisomy 21 was the most frequent chromosomal anomaly. The indication that found a higher incidence of chromosomal aneuploidies was echographic abnormalities (12% of cases) followed by ultrasound markers (6% of cases) and positive biochemistry screening (4% of the cases. When the indication for the achievement of a DPN was advanced maternal age (72% of the total indications), 98% of them had a normal cytogenetic result. It will be described in detail the cytogenetic abnormalities found and his relationship with the indication to perform prenatal diagnosis. The scientific and technological development occurring in recent decades, particularly in DPN, requires a different behavior to perform DPN as well as an urgent necessity to change the DPN Portuguese law.

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inversions inv(9) 6 cases, inv(10) 1 case; ring chromosome r(6) 1 case; deletions:del(7)(q21-ter) 1 case, del(1)(q32.1-ter) 2 cases, structural anomalies of X chromosome del(X)(q26-ter) 1 case. A karyotype using classical banding methods is only fully informative method able to detect all chromosomal abnormalities. 13.80-P Molecular diagnosis of fetal chromosomal defects in Costa Rica I. Castro-Volio (1) (1) University of Costa Rica Molecular diagnosis of fetal chromosomal defects in Costa Rica. Wendy Malespín-Bendaña1, Fernando Ortiz-Morales1 & Isabel Castro-Volio1 1 Instituto de Investigaciones en Salud (INISA), Universidad de Costa Rica Isabel Castro-Volio, INISA. Ciudad de la Investigación, Universidad de Costa Rica, P.O. Box 2060 San Pedro, San José, Costa Rica, Central America. Tel: (506) 2511 3293. Fax (506) 2511 5130. isabel. [email protected]

13.79-P Cytogenetic analysis in prenatal diagnosis— A report of 550 pregnancy C. Gug (1) (1) University of Medicine and Pharmacy “Victor Babes”, Timisoara Prenatal cytogenetic diagnosis is a part of prenatal care. In this paper we report results of 550 fetal karyotypes performed by cultured amniocytes analysis. The cytogenetic analysis with GTG banding of amniotic fluid cells revealed 14 aneuploidies (3.9%) and 20 structural abnormalities (2.9%). We found omogen trisomy 21 (8 cases), mosaic trisomy 21 (3 cases), omogen trisomy 18 (1 case), omogen trisomy 13 (1 case), triplidy 69,XXX (1 case). The structural abnormalities includes: 2 cases with robertsonian translocations trob(13;22), trob(21;21); balanced reciprocal translocations t(7;10)(p22;p12.1) 2 cases and t(17;20)(q12;q11) 1 case; pericentric

Key words: QF-PCR, prenatal diagnosis, Costa Rica Justification and aims: In Costa Rica, the diagnosis of chromosomal fetal anomalies is performed only by conventional cytogenetic analysis of chromosomes obtained from cellular cultures. The waiting for the results can be long. Moreover, with some frequency culture fails due to bad quality of the sample or the chromosomes cannot be analyzed. This makes it necessary to have a simple and cheap methodology to obtain an accurate and rapid fetal diagnosis of trisomy 21, 18 or 13, in pregnancies of high genetic risk. Materials and methods: Three multiplex PCRs were designed to amplify four different STRs of each of the chromosomes 21, 18 and 13. We collected 93 samples (88 amniotic fluids and 5 fetal bloods). The results of the QF-PCR were compared with the karyotypes obtained of the same samples to establish the accuracy and reliability of the assay. Results: Accuracy of the assay was 100% and it was possible to obtain results within 48 h.

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Conclusion: QF-PCR turned out to be a simple, accurate and rapid methodology, and a useful complementary tool of chromosomal conventional analysis. The securing of rapid results in cases of antenatal diagnosis might diminish the period of parental anxiety in waiting for the results, as well as to allow a better therapeutic management of the affected fetuses. In search of cancer genes in solid tumors of Bloom deficient mice Last minute poster presentation abstracts R Banerjee, N. Conte, B. L. Ng, F. Yang, N. P. Carter and A. Bradley Molecular Cytogenetics, The Wellcome Trust Sanger Institute, The Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA The aim of our study is to find recurrent rearrangements involving specific chromosomes in solid tumour cell lines of irradiated Bloom deficient mice. Loss of DNA helicase Bloom function leads to higher rate of mitotic recombination resulting in increased genomic instability and cancer predisposition. Forty two solid tumor cell lines, derived from primary tumors of different cell types have been characterized using high resolution array CGH (aCGH) and Multicolour FISH (M FISH). A 21 colour pool was developed in our laboratory for mouse M-FISH. The study has shown how complementary the two techniques are in characterizing complex karyotypes. In particular, M-FISH has revealed balanced and unbalanced translocations, marker chromosomes and clonal evolution within tumours. In one particular tumour cell line, a copy number breakpoint in chromosome 6, as revealed in aCGH, was shown to be the result of an unbalanced translocation between chromosomes 5 and 6, coinciding with the BRAF oncogene. The underlying mechanism of this rearrangement could only be determined from the M –FISH data. On the other hand, aCGH has revealed hidden deletions (CDKN2A/CDKN2B) and amplifications (cMyc and MET) in apparently normal karyotypes. In conclusion, the two methods are proving to be a very powerful approach to gene discovery and in unravelling underlying mechanisms in cancer.

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Family with two Down syndrome children: genotype to phenotype Panigrahi I, Sharda S, Kulkarni S, Khullar M*, Kaur S, Kalra J* Genetic-Metabolic Unit, Dept. of Pediatrics, Advanced Pediatric Center, Department of *Obstetrics and Gynecology, PGIMER Dept. of **Experimental Medicine; PGIMER, Chandigarh-12, India Down syndrome (DS) is a common cause of mental retardation, mostly due to trisomy 21. Translocation trisomy accounts for only 4% of the cases. Folate polymorphisms in mothers have been found to increase risk of DS in offsprings in certain situations. In present report, we describe a family from North India with two DS children without cardiac defect. In one pregnancy, the mother had an abortion. In fourth pregnancy, the mother had come to fetal medicine clinic for genetic counseling. Karyotype done in two of the affected children showed translocation between chromosomes 14 and 21. Polymorphisms in MTHFR, RFC and CBS genes are also being studied in the family. The implications of translocation DS and the other results will be discussed. De novo double 1;13 and 5;11 translocation in a patient with short stature, mental retardation and mild facial dysmorphism Valerica Belengeanu, Simona Farcas, Cristina Popa, Monica Stoian, Nicoleta Andreescu Department of Medical Genetics, University of Medicine and Pharmacy “Victor Babes” Timisoara, Romania The vast majority of apparently balanced structural chromosome abnormalities are not associated with abnormal phenotypes and may be transmitted through several generations without detection, but the cytogenetic identification of an unbalanced constitutional de novo chromosome abnormality in patients is usually accepted as the underlying cause of their abnormal phenotype. Rarely, individuals might be carriers of double translocations; a few cases are described in the literature. We present an unusual additional case in which an individual carries a double translocation. The patient is a 7 years old boy evaluated for mental retardation and growth retardation. On clinical exam-

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ination of the patient the following were noticed: microcephaly (cranial circumference of 45.5 cm), downwards slanting palpebral fissures, retrognathia, dental malposition, clinodactyly 2nd and 5th finger both hands, umbilical hernia, suggesting a chromosomal disorder. Conventional cytogenetic analysis performed from peripheral blood lymphocytes with GTG banding surprisingly revealed a double translocation, one involving chromosomes 1 and 13, and the other involving chromosomes 5 and 11. For establishing whether the translocations were inherited, chromosomal analysis was performed for both parents; their karyotypes were normal. Analyzing the banding pattern of patient’s chromosomes implicated in translocations, we established that between chromosomes

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1 and 13, the chromosomal fragments implicated in translocation do not present a modified banding pattern, and we can sustain that the translocation is balanced [t(1;13)(p31.2;q32)]. But for the other translocation the banding pattern is modified, and we couldn’t establish the exact breakpoints, but the following rearrangement might be involved in translocation t(5;11)(q14;q25). Only aCGH analysis could allow us to identify the exact breakpoints and to evaluate the genomic imbalance responsible for the phenotype anomalies. As aCHG is not available in Romania so far, we present this case hoping that it will raise interest for a laboratory where aCGH is available and support us in solving it. Key words: double chromosomal translocation