Advances in Laboratory Evaluation of Turner Syndrome and Its Variants: Beyond Cytogenetics Studies D a y n n a J. Wolff

Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charteston, SC, USA AbslmcL Turner syndrome is a clinically defined phenotype that is characterized by partial or complete X chromosome monosomy. A host of cytogenetic aberrations and mosaicism have been associated with this syndrome. Some individuals, Turner syndrome variants, have cytogenetic findings consistent with Turner syndrome, but exhibit atypical clinical phenotypes. Recently, several molecular tests have been presented to allow for the refined clinical study of Turner syndrome and its variants. [Indian J Pediatr 2000; 67(11) : 825-829]

l(eyworcl$ : Turner syndrome; X Chromosome monosomy; Cytogenetic aberrations; Mosaicism Turner syndrome, with an incidence of approximately 1 in 5000 live female births 1, is a clinically defined phenotype that is associated with partial or complete X chromosome monosomy. The syndrome is characterized by short stature, ovarian failure and the variable presence of several somatic features such as w e b b e d neck, congenital heart disease, cubitas valgus and kidney abnormalities. Just over half of the females with a classic Turner syndrome phenotype have a 45, X karyotype. The remainder of Turner syndrome individuals have a structurally a b n o r m a l sex c h r o m o s o m e a n d / o r mosaicismo The Turner syndrome phenotype is thought to result from a deficiency of specific X-linked genes. These genes must be expressed by both X chromosomes (escape from X inactivation) and must also be present on the Y chromosome, as males with one X and one Y do not exhibit the Turner syndrome phenotype2. Recent studies have identified a stature gene (SHOX) on the distal tip of the X short arm 3 that is expressed in osteogenic cells and is thought to be associated with linear growth. In addition, extensive deletion analyses have localized a locus (or loci) for short stature, ovarian failure and high-arched palate to a region of the X short arm just distal to the centromere, Xp11.2-p22.14. Continued definition of the areas that harbor genes important for the Turner syndrome

Reprint requests : Daynna J. Wolff,Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 165 Ashley Avenue, Suite 309, Charleston, SC 29425.

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phenotype will eventually aid in the counseling of patients with partial X monosomy and allow for genotypephenotype correlations to be made. A subset of patients with partial or complete monosomy X do not exhibit a typical Turner syndrome phenotype. These individuals have been termed "Turner syndrome variants". This review discusses some of the recent molecular studies that are beginning to elucidate mechanisms responsible for the atypical findings in these patients. Y CHROMOSOME MOSAICISM IN TURNER SYNDROME Monosomy X is frequent in spontaneous abortions and is seen in about 10 - 20% of all miscarriagesL It is estimated that as many as 1-2% of all conceptuses are 45,X; however, the vast majority of 45,X conceptuses (>99%) are spontaneously lost prior to 28 weeks of gestation. About half of Turner syndrome liveborn individuals have a 45,X karyotype and the remainder have a structurally abnormal sex chromosome a n d / o r are mosaic. Studies of aborted fetuses have shown that non-mosaic Turner syndrome (45,X) fetuses are more likely to be aborted than mosaic cases 1. These findings have led to the hypothesis that all liveborn Turner s y n d r o m e individuals harbor a mosaic karyotype, although the mosaicism may not be detected using a standard chromosome evaluation. Detection of mosaicism for Y chromosome-specific material in females with Turner syndrome is clinically significant. Females with dysgenetic gonads and a Y

Daynna J. Wolff

chromosome have a 15-25 % risk of developing a gonadoblastoma 6. Routine cytogenetic analysis of a patient with suspected Turner syndrome typically includes the study of 30 metaphase cells, which allows for the detection of 10% mosaicism at the 95% confidence level. Thus, low level mosaicism (<10%) may be missed with standard testing. Given the risk of neoplasm in females with Y chromosome material, investigators have turned to more sensitive molecular methods to study the incidence of mosaicism for Y chromosome DNA in females with Turner syndrome. Detection of Y chromosome sequences in Turner syndrome individuals using polymerase chain reaction (PCR) has been performed by several groups who have found a wide variation in positive results (reviewed in reference 7 groups). The best-controlled studies reveal that approximately 5% of Turner syndrome patients with a 45,X karyotype are mosaic for Y chromosome material, and thus may be at risk for gonadoblastoma. It is hypothesized that gonadoblastoma is caused by a gene, or genes, on the Y chromosome designated GBY(s) (Fig. 1). In normal males, this gene most likely plays a role in normal spermatogenesis. However, when this gene is expressed inappropriately in dysgenetic gonads of females, tumors develop and the gonads must be removed. The location of the GBY gene(s) is not known, although two groups have localized the gene to two separate areas of the Y chromosome8,9.As many genes on the Y chromosome have multiple copies dispersed throughout the chromosome, it is possible that GBY is present at several locations. Once the true GBY gene(s) is found, clinical laboratorians will be able to direct the molecular approaches and determine the population at risk for gonadoblastoma. Not only will this approach detect the persons at risk, but will determine persons

who may not need prophylactic surgery for remo.val of the gonads. When considering molecular analyses for detection of Y-chromosomal material in females with a 45,X karyotype, there are several considerations. The PCR studies should include an adequate number of loci to be tested (>10), and most importantly, should include the regions that are thought to contain genes responsible for the tumor formation. In addition, for patients with a positive PCR result, it is prudent to confirm the findings with fluorescence in situ hybridization (FISH) with Y chromosome probes 7, particularly given the large risk of contamination with PCR testing. GENOTYPE/PHENOTYPE CORRELATIONS IN PATIENTS WITH SMALL MARKER X CHROMOSOMES Approximately 6-11% of Turner syndrome patients have a small marker or ring chromosome'~ the majority of which are derived from the X chromosome '2. Many patients with a 45,X/46,X, mar(X) karyotype have a classic Turner syndrome phenotype. However, a subset of patients have a more severe phenotype with mental retardation and other features uncharacteristic of Turner s y n d r o m e . M o l e c u l a r d e f i n i t i o n of the m a r k e r c h r o m o s o m e s in these i n d i v i d u a l s has led to the association of the phenotype and the X inactivation status of the X-linked material. The process of X inactivation may be separated into three steps: initiation, propagation of the inactivation signal along the length of the X chromosome, and maintenance of the inactive status. X inactivation is initiated by a gene on the X chromosome termed XIST (inactive X-

Marker X Chromosomes

Fig. 2A

Fig. 1. Schematic of the Y chromosome. Areas most often included in a PCR screen for Y chromosome sequences are shown. 826

F~g. 2B

Fig. 2. Cartoon of a normal X (left of each pair) and a ring(X) (right). XIST DNA is present on most cases with a Turner syndrome phenotype (A) while a more severe phenotype results when the XIST gene is deleted (B). Indian Journal of Pediatrics, 2000; 67 (11)

Evaluation of Turner Syndrome

Pig 3. Schematic representation (left) of androgen receptor analysis results: U = undigested DNA control lane; C = DNA digested with methylation sensitive enzyme. Results of normal male (only one X chromosome) and a patient with a r(X) chromosome and a completely non-random X inactivation pattern (right). specific transcript). This gene encodes a large heterogeneous RNA that associates with the DNA of the inactive X chromosomelL The mechanism of action of XIST, and h o w the inactivation signal is propagated along the length of the entire X chromosome and how the inactivation is maintained are not understood. It is known however, that XIST is necessary for normal development in all humans with more than one X chromosome. Upon the discovery of the XIST gene is role in the initiation of X inactivation, the presence or absence of this g e n e o n small marker X chromosomes was correlated with phenotype 1~5 (Fig. 2). These initial reports found that all marker X cases that were positive for a XIST DNA signal detected by FISH presented with Turner syndrome, w h i l e those lacking XIST had a more severe phenotype. Subsequent reports on severely affected patients with intact XIST DNA and impaired expression of the gene 16 further evidenced that the atypical Turner syndrome phenotype was caused by disomy for X-linked genes. While no standard guidelines exist for the clinical assessment of individuals with a small marker X chromosome, the clinical utility of molecular testing is evident. All females with a small marker chromosome and an atypical Turner syndrome phenotype with mental or Indian Joumal of Pediatrics, 2000; 67 (11)

severe somatic growth retardation should be assessed with FISH using an XIST probe and / or studies of XIST RNA. However, it is important to note that there are females with a typical Turner syndrome phenotype and a very tiny marker X chromosome that lacks XIST17observation). These individuals presumably have only heterochomatic material (centromeric repeats) present on the marker X chromosome. X INACTIVATION PATTERN AND ITS CLINICAL ASSOCIATIONS In chromosomaUy normal mammalian females, one of the two X chromosomes becomes randomly inactivated early in embryogenesis to allow for dosage compensation of Xlinked genes between XX females and XY males TM. In m o s t females w i t h a n o r m a l 46,XX k a r y o t y p e , X inactivation is r a n d o m with respect to which X is inactivated. However, in females with structurally abnormal X chromosomes, usually the abnormal X is inactivated d u e to selection against one cell line. Selection ensures maintenance of the most genetically "balanced" situation, and these females are usually phenotypicaUy normal or present with features of Turner 827

Daynna J. Wolff syndrome19,20. In a minor percentage of females with an a b n o r m a l X c h r o m o s o m e , the a b n o r m a l X is not inactivated in all of the cells, resulting in excess or decreased expression of X chromosome genes 21. These individuals may present with an atypical abnormal appearance and mental retardation. Historically, late replication banding has been used to assess the X inactivation pattern in females with structurally abnormal X chromosomes 22. This technically challenging and subjectively interpreted testing modality is not routine for most clinical cytogenetic laboratories. Recently, several more sensitive, quantitative molecular methods have been described to assess directly X inactivation patterns23,24,2s. The single most useful analysis for the study of X inactivation is a methylation analysis at the androgen receptor (AR) locus. For AR analysis, methylation of the CpG island adjacent to the polymorphic CAG repeat in exon I of the gene is evaluated 23 and, based on the banding pattern, X inactivation may be classified as completely nonrandom, skewed or random (Figure 3). While most carriers of a structurally abnormal X chromosome exhibit a completely nonrandom X inactivation pattern and have a normal or Turner syndrome phenotype, carriers with skewed or random X inactivation have functional disomy or monosomy of X chromosomal sequences. Thus, molecular assessment of the X inactivation pattern may be correlated with phenotype. The X inactivation status of females with structurally abnormal X chromosomes and an abnormal phenotype should be assayed as part of a routine clinical work-up 19,2i. This should aid in the understanding of abnormal phenotypes in livebom individuals with abnormal X chromosomes, including some patients with a chromosome analysis consistent with Turner syndrome and a variant phenotype.

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13. CONCLUSION Molecular testing is an important adjunct to cytogenetic studies for patients with Turner syndrome and variant Turner syndrome phenotypes. As the special properties of the sex chromosomes, such as X inactivation and the p r e v a l e n c e of viable m o s a i c i s m , b e c o m e b e t t e r understood, additional studies will move from the research to clinical setting. Thus, correlative molecular testing will likely become a routine part of genetic evaluation of patients with Turner syndrome in the future.

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REFERENCES 17. 1. Hook EB, Warburton D. The distribution of chromosomal genotypes associated with Turner syndrome : Livebirth prevalence and evidence for

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diminished fetal mortality and severity in genotypes associated with structural X abnormalitiesor mosaicism. Hum Genet 64 : 1983; 24-27. Zinn AR, Page DC, Fisher EMC.Turner syndrome - the case of the missing sex chromosome. Trends Genet 9 : 1993; 90-93. Rao E, Weiss B, Fukami M et al. Pseudoautosomal deletions encompassing a novel homeobox gene cause growth failure in idiopathic short stature and turnersyndrome. Nat Genet 16 : 1997; 54-63. Zinn AR, Tonk VS, Chen Z et al. Evidence for a Turner syndrome locus or loci at Xp11.2-p22.1. Am JHum Genet 63 : 1998; 1757-1766. Bou6A, Bou6 J, Gropp A. Cytogenetics of pregnancy wastage. Ann Rev Genet 14 : 1985; 1-571. Verp MS and Simpson JL. Abnormal sexual differentiation and neoplasia. Cancer Genet Cytogenet 25 : 1987; 191-218. Quilter CR, Taylor K, Conway GS, Nahwani N, Delhanty JDA. Cytogeneticand molecularinvestigations of Y chromosome sequences and their role in Turner syndrome. Ann Hum Genet 62 : 1998; 99-106. Tsuchiya K, Reijo R, Page D, Disteche MC. Gonadoblastoma molecular definition of the susceptibility region of the Y chromosome. Am J Hum Genet 57 : 1995; 1400-1407. Salo P, K/i/iri/iinen H, Petrovic V, Peltom/iki P, Page DC, De la Chapelle A. Molecular mapping of the putative gonadoblastoma locus on the Y chromosome. Genes Chromosomes Canc 14 : 1995; 210-214. Schmid W, Naef E, Murset G, Prader A. Cytogenetic finding in 89 cases of Turner's syndrome with abnormal karyotypes. Hum Genet 24 : 1974; 93-104. CocoR, Bergada C. Cytogeneticfindings in 125 patients with Turner's syndrome and abnormal karyotypes. J Genet Hum 25 : 1977; 95-107. Schwartz S, Depinet TW, Leana-Cox Jet al. Sex chromosome markers: characterization using fluorescence in situ hybridization and review of the literature. Am J Med Genet 71 : 1997; 1-7. BrockdoffN and Duthie SM. X chromosomeinactivation and the Xist gene. Cell Mol Life Sci 54 : 1998; 104-112. WolffDJ, Brown CJ, Schwartz S, Duncan AMV, Surti U, Wfllard HF. Small marker X chromosomes lack the X inactivation center: Implications for karyotype/phenotype correlations. Am J Hum Genet 55 : 1994; 87-95. Cole H, Huang B, Salbert BA et al. Mental retardation and Ullrich-Turner syndrome in cases with 45,X/ 46,X,+mar : Additional support for the loss of the X inactivation center hypothesis. Am ] Med Genet 52 : 1994; 136-145. Migeon BR, Luo S, Jani M, Jeppesen P. The severe phenotype of females with tiny ring X chromosomes is associated with inability of these chromosomes to undergo X inactivation. Am J Hum Genet 55 : 1994; 497504. Turner C, Dennis NR, Skuse DH, Jacobs. PA. Jacobs.PA. Seven ring (x) chromosomes lacking the XISTlocvs, six with an unexpectedly mild phenotype. Hum Genet 106 : 2000; 93-100.

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Evaluation of Turner Syndrome 18. Lyon MF. Gi~ne action in the X-chromosome of the mouse (Mus musculus L). Nature 190 : 1961; 372-373. 19. Schmidt M, Du Sart D. Functional disomies of the X chromosome influence the cell selection and hence the X inactivation pattern in females with balanced Xautosome translocations: a review of 122 cases. Am ] Med Genet 42 : 1992; 161-169. 20. Shapiro LJ, Mohandas T. DNA methylation and the control of gene expression on the human X chromosome. Cold Spring Harb Syrup Quant Biol 47 : 1983; 631-637. 21. WolffD, Schwartz S, Carrel L. X inactivation pattern, as measured with molecular techniques, correlates with phenotype in females with a structurally abnormal X chromosome. Genet in Med, 2 : 2000; 136-141. 22. Zuffardi O (1983) Cytogenetics of human X/autosome

translocations. In : A. A Sandberg Cytogenetics of the Mammalian X Chromosome, Part B: BasicMechanisms of X chromosome Behaviour, ed (New York : Alan R Liss, Inc) 193-210. 23. Allen RC, Zoghbi HY, Moseley AB, Rosenblatt HM, Belmont JW. Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgenreceptor gene correlates with X chromosome inactivation. Am J Hum Genet 51 : 1992; 1229-1239. 24. CarrelL, Willard HF. An assay for X inactivation based on differential methylation at the FragileX locus, FMR1. Am J Med Genet 64 : 1996; 27-30. 25. Rupert JL, Brown CJ, Willard HF. Direct detection of non-random X chromosome inactivation by use of transcribed polymorphism in the XISTgene. Eur J Hum Genet 3 : 1995; 333-343.

DOES D I E T I N F L U E N C E H Y P E R A C T I V I T Y ? A lengthy list of dietary substances has been proposed as triggers for hyperactivity in children, including sugar, additives, dyes, preservatives and natural salicylates found in vitamin C-rich foods. After their literature review, Dr. Levy and Susan Hyman, MDs, FAAP, Assistant Professors of Pediatrics at University of Rochester School of Medicine and Children Hospital at Strong, concluded there may be a small, poorly defined subgroup o f children who show behavioral changes in response to food additives or other dietary eon~aonents-with the exception of sugar, for which no convincing link has been shown. "The Scientific literature has largely laid the matter to rest of sugar and and hyperactivity. But it has taken a life of its own outside of the medical community. It is not at all uncommon for parents to say, 'I don't know why he's still hyperactive. I took sugar out of his diet', commented Dr. Hyman. Abstracted from : AAP News October 2000; 17

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