Hum Genet (1998) 103 : 51–56
© Springer-Verlag 1998
ORIGINAL INVESTIGATION
Tsutomu Ogata · Keiko Wakui · Koji Muroya Hirofumi Ohashi · Nobutake Matsuo Donna M. Brown · Takashi Ishii Yoshimitsu Fukushima
Microphthalmia with linear skin defects syndrome in a mosaic female infant with monosomy for the Xp22 region: molecular analysis of the Xp22 breakpoint and the X-inactivation pattern Received: 16 December 1997 / Accepted: 25 February 1998
Abstract This paper describes a female infant with microphthalmia with linear skin defects syndrome (MLS) and monosomy for the Xp22 region. Her clinical features included right microphthalmia and sclerocornea, left corneal opacity, linear red rash and scar-like skin lesion on the nose and cheeks, and absence of the corpus callosum. Cytogenetic studies revealed a 45,X[18]/46,X,r(X)(p22q21) [24]/46,X,del(X)(p22)[58] karyotype. Fluorescence in situ hybridization analysis showed that the ring X chromosome was positive for DXZ1 and XIST and negative for the Xp and Xq telomeric regions, whereas the deleted X chromosome was positive for DXZ1, XIST, and the Xq telomeric region and negative for the Xp telomeric region. Microsatellite analysis for 19 loci at the X-differential region of Xp22 disclosed monosomy for Xp22 involving the critical region for the MLS gene, with the breakpoint between DXS1053 and DXS418. X-inactivation analysis for the methylation status of the PGK gene indicated the presence of inactive normal X chromosomes. The Xp22 deletion of our patient is the largest in MLS patients with molecularly defined Xp22 monosomy. Nevertheless, the result
T. Ogata (✉) · K. Muroya · N. Matsuo Department of Pediatrics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-0016, Japan e-mail:
[email protected], Tel.: +81-3-3353-1211, Fax: +81-3-5379-1978 K. Wakui · Y. Fukushima Department of Hygiene and Medical Genetics, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto-shi, Nagano 390, Japan H. Ohashi Division of Medical Genetics, Saitama Children’s Medical Center, 2100 Magome, Saitama 339, Japan D. M. Brown Research Genetics, 2700 Memorial Pky S., Huntsville, AL 35801, USA T. Ishii Mitsubishi Kagaku Bioclinical Laboratories, 3-30-1 Shimura, Itabashi-ku, Tokyo 174, Japan
of X-inactivation analysis implies that the normal X chromosomes in the 46,X,del(X)(p22) cell lineage were more or less subject to X-inactivation, because normal X chromosomes in the 45,X and 46,X,r(X)(p22q21) cell lineages are unlikely to undergo X-inactivation. This supports the notion that functional absence of the MLS gene caused by inactivation of the normal X chromosome plays a pivotal role in the development of MLS in patients with Xp22 monosomy.
Introduction Microphthalmia with linear skin defects syndrome (MLS; MIM 309801) is a rare congenital developmental disorder characterized by microphthalmia, sclerocornea, and irregular linear areas of skin hypoplasia involving the face and neck. In addition, this disorder is often associated with other features such as retinal lacunae, agenesis of corpus callosum, mental retardation, seizures, and costovertebral anomalies. Since all patients with MLS are female, with the exception of a 46,XX male (reviewed in Ballabio and Andria 1992), MLS has been regarded as a male-lethal Xlinked dominant disorder. MLS is predominantly observed in patients with monosomy for the Xp22 region (reviewed in Ballabio and Andria 1992). This suggests that the MLS gene resides in Xp22, and deletion mapping in patients with various types of Xp22 monosomy has localized the MLS gene to a 570-kb region at Xp22 (Wapenaar et al. 1994), although the MLS gene has not been cloned to date. Furthermore, the manifestation of MLS in patients monosomic for Xp22 implies that the MLS gene is subject to X-inactivation, and that the normal X and the abnormal X undergo random X-inactivation at least in a specific tissue at a particular developmental stage (Ballabio and Andria 1992). If the MLS gene escapes X-inactivation, as has been demonstrated for the genes for CDPX1, STS, and KAL1, assigned to Xp22.3 distal to the MLS critical region (Ropers et al. 1981; Franco et al. 1991, 1995), the MLS phenotype, as well as the features of
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CDPX1, STS, and KAL1, would not be exhibited by patients with Xp22 monosomy. Similarly, if the abnormal X chromosome undergoes selective X-inactivation, patients with Xp22 monosomy, as well as those with large Xp deletions and monosomy X, would not manifest the MLS phenotype. Thus, functional absence of the MLS gene caused by inactivation of the normal X chromosome is believed to play a critical role in the development of MLS in patients with Xp22 monosomy (Ballabio and Andria 1992). In addition, it has been suggested that a variable X-inactivation pattern in different tissues may account for the clinical divergence in such patients (Ballabio and Andria 1992). In this report, we describe a girl with MLS and mosaic Xp22 monosomy in whom the Xp22 breakpoint and the Xinactivation pattern were molecularly determined.
action (PCR) for 19 loci assigned to the X-differential region of Xp22 (Table 1). Amplification was performed in a reaction volume of 10 µl containing 40 ng template genomic DNA, 6 pmol fluorescently labeled forward primer, 6 pmol unlabeled reverse primer, 15 nmol dNTPs, and 0.25 U Taq polymerase. The primer sequences and the PCR conditions have been reported in Gyapay et al. (1994) and in the Genome Database. Multiple PCR products based on color and size separation were mixed with a size standard and loading buffer, and the mixure was denatured for 5 min at 95°C, rapidly cooled on ice, and loaded onto an autosequencer (ABI PRISM 377). The sizes of the PCR products were determined by Genotyper. Analysis of X-inactivation pattern The X-inactivation pattern was assessed for the methylation status of the PGK gene according to the method of Gilliland et al. (1991) with minor modifications. In brief, the region encompassing a polymorphic BstXI site and a methylation-sensitive HpaII site was amplified by PCR, and the PCR products were subjected to BstXI digestion and
Subjects and methods Case report This female infant was born as the first child to healthy nonconsanguineous parents at 40 weeks of gestation. In the third trimester of the pregnancy, fetal ultrasound examinations delineated a hydrocephalic appearance. There was no prenatal history of drug exposure or intrauterine infection. At birth, the length was 48 cm (mean–0.9 SD), the weight 3.0 kg (mean–0.5 SD), and the head circumference 34.5 cm (mean+0.9 SD). At 1 month of age, the girl was referred to Shinshu University Hospital because of ocular and cutaneous abnormalities. Physical examination revealed right microphthalmia and sclerocornea, left corneal opacity, anteverted nostril, linear red rash and scar-like skin lesion on the nose and cheeks, hemangioma on the right wrist, and nail hypoplasia. The retina was not visualized because of the corneal clouding. Turner somatic stigmata such as lymphedema were apparently absent. Computed tomography of the brain showed absence of the corpus callosum and colpocephaly. Chest and spine radiographs were normal with no costovertebral defects or scoliosis. On the basis of the above findings, she was diagnosed as having the MLS phenotype. Subsequent development was relatively well preserved with no seizures. She controlled the head at 3 months old, and walked without support and spoke single words at 14 months old. At 18 months of age, her developmental quotient was estimated to be 95 by the Enjoji method. At present, she is 23 months old, measures 77.0 cm (mean–2.5 SD), and weighs 10.7 kg (mean–1.0 SD). Visual acuity appears to be almost lost for the right eye and relatively well preserved for the left eye. Conventional and molecular cytogenetic studies Chromosomal preparations were made from 3-day cultures of peripheral lymphocytes of the patient and the parents. G-banding analysis was performed on 100 metaphases of the patient and 20 metaphases of the parents. For the patient, high-resolution G-banding was also carried out with ethidium bromide. Fluorescence in situ hybridization (FISH) analysis was performed on metaphases of the patient, using the probes for DXZ1, XIST, the Xp/Yp telomeric region (CY29), and the Xq/Yq telomeric region (c8.2/1). The methods used were as described in the manufacturer’s protocol for DXZ1 and XIST (Oncor) and in Ning et al. (1996) for the telomeric regions. Microsatellite analysis of Xp22 Genomic DNA was extracted from peripheral blood leukocytes of the patient and the parents, and was amplified by the polymerase chain re-
Fig. 1A,B X-inactivation analysis using the methylation status of the PGK gene. A Map of the PGK gene around polymorphic BstXI and methylation-sensitive HpaII sites. Arrows show the positions of primers used in the present study. Polymerase chain reaction (PCR) amplification followed by BstXI digestion results in a 530-bp product for an allele negative for the BstXI site and in 433-bp plus 97-bp products for an allele positive for the BstXI site. After HpaII digestion, the same region on the inactive X chromosome alone is amplified by PCR, because of the methylation of the HpaII site. B Assessment of X-inactivation pattern in the family (the father, the patient, and the mother) and the control females (control 1, a normal 46,XX female; control 2, a 46,XX female known to have skewed X-inactivation; control 3, a nonmosaic 46,X,del(X)(p21) female; and control 4, a nonmosaic 46,X,del(X)(p11) female). Examination of BstXI heterozygosity before HpaII digestion indicates that the patient is heterozygous for the BstXI site, with the 433-bp band derived from the hemizygous father and the 530-bp band from the homozygous mother. The four control females are heterozygous for the BstXI site. After HpaII digestion, a faint 433-bp band and a more distinct 530-bp band are detected in the patient. No band is seen in the father. Two bands with similar intensity are detected in control 1, two bands with distorted intensity are detected in control 2, and only a single band is detected in controls 3 and 4
53 Fig. 2 The X-chromosomal constitution of the 45,X cell lineage (left), the 46,X,del(X)(p22) cell lineage (middle), and the 46,X,r(X)(p22q21) cell lineage (right). Solid arrows show the breakpoint of the Xp22 deletion chromosome, and dotted arrows indicate the breakpoints of the ring X chromosome
loaded onto a 2% agarose gel, to examine the BstXI heterozygosity (Fig. 1A). This procedure was also carried out after HpaII digestion, to amplify the same region on the inactive X chromosome alone. Amplification was performed for 35 cycles of 1 min at 94°C, 1 min at 58°C, and 2 min at 72°C, in a 50-µl reaction mixture containing 1.0 µg genomic DNA, 20 pmol forward primer (5′-AGCTGGACGTTAAAGGGAAGCGGGTCGTTA-3′), 20 pmol reverse primer (5′-TACTCCTGAAGTTAAATCAACATCCTCTTG-3′), 0.2 mM dNTPs, and 2.5 U Taq polymerase. For controls, four females heterozygous for the BstXI site, i.e., a normal 46,XX female (control 1), a 46,XX female with skewed X-inactivation described by Okamoto et al. (1996) (control 2), and non-mosaic 46,X,del(X)(p21) and 46,X,del(X)(p11) females (controls 3 and 4), were similarly analyzed.
Results Conventional and molecular cytogenetic studies The karyotype of the patient was initially interpreted as 45,X[18]/46,X,r(X)[24]/46,XX[58]. However, high-resolution G-banding analysis revealed a small Xp-terminal deletion with the breakpoint at the border of Xp22.3 and Xp22.2, in one of the two seemingly normal X chromosomes (Fig. 2). The breakpoints of the ring X chromosome appeared to reside in the middle part of Xp22 and in the distal part of Xq21. FISH analysis showed that the ring X chromosome was positive for DXZ1 and XIST and negative for the Xp and Xq telomeric regions, whereas the deleted X chromosome was positive for DXZ1, XIST, and the Xq telomeric region and negative for the Xp telomeric region (data not shown). The parental karyotypes were normal. Microsatellite analysis of Xp22 The results are summarized in Table 1. For DXS1060, DXS996, DXS7103, DXS7104, DXS987, and DXS1053, maternal alleles were not inherited by the patient and paternal alleles alone were transmitted to the patient. For DXS418, DXS1195, DXS999, and DXS365, both parental alleles were inherited by the patient. The results of the re-
Table 1 Microsatellite analysis of the Xp22 region. The locus order is based on the report of Ferrero et al. (1995) and the report of the Sixth International Workshop on Human X Chromosome Mapping 1995 (Nelson et al. 1995). The MLS critical region resides between DXS7108 and DXS1043 (Nelson et al. 1995). (N.I. not informative) Locus
DXS1060 DXS996 DXS1223 DXS7103 DXS7108 DXS1043 DXS7104 DXS7109 DXS1224 DXS987 DXS207 DXS1053 DXS43 DXS418 DXS1195 DXS999 DXS7161 DXS443 DXS365
Product size (bp)
Copy number of patient
Father
Mother
Patient
148 165 160 127 254 154 177 138 159 218 110 200 86 145 233 268 248 208 215
134, 136 157 160 133, 135 254, 258 154 181 138 159 212, 214 110 196, 198 86 143, 147 237, 239 276 248, 250 208, 210 205, 211
148 165 160 127 254 154 177 138 159 218 110 200 86 145, 147 233, 237 268, 276 248 208 205, 215
1 1 N.I. 1 N.I. N.I. 1 N.I. N.I. 1 N.I. 1 N.I. 2 2 2 N.I. N.I. 2
maining nine loci were not informative for copy number in the patient. Analysis of X-inactivation pattern The results are shown in Fig. 1B. Examination of the BstXI polymorphism showed that the patient was heterozygous for the BstXI site, with a strong 433-bp band derived from
54 Table 2 X-inactivation pattern in MLS patients with partial monosomy for the Xp22 region. The patient described by Johnston et al. (1987) with random X inactivation has currently been interpreted as being free from MLS (Shaefer et al. 1993; Wapenaar et al. 1994) Case
Cytogenetic abnormality
Method of X inactivation
Analyzed cells (number)
Inactive X
Inactivation pattern
Reference
1
der(X)t(X;3)a
Cytogeneticb
Lymphocytes and skin fibroblasts (65)
der(X) only
Nonrandom
Ropers et al. (1982)
2
der(X)t(X;Y)
Cytogeneticb
Lymphocytes (50)
der(X) only
Nonrandom
Al-Gazali et al. (1990), patient 1
3
del(X)
Cytogeneticb
Lymphocytes (43)
del(X) ~42/43, normal X ~1/43
Almost nonrandom
Thies et al. (1991)
4
del(X)
Cytogeneticb
Lymphocytes (47)
del(X) only
Nonrandom
Naritomi et al. (1992), patient 1
5
del(X)
Cytogeneticb
Lymphocytes (35)
del(X) ~18/35 normal X ~17/35
Random
Naritomi et al. (1992), patient 2
6
der(X)t(X;?)
Cytogeneticb
Lymphocytes (30)
der(X) only
Nonrandom
Lindor et al. (1992)
7
del(X)
Cytogeneticb
Lymphocytes (100)
del(X) only
Nonrandom
Lindsay et al. (1994), patient 1
8
del(X) (mosaic)
Molecularc
Leukocytes
del(X) and normal X
Uncertainc
Present case
a b c
This patient has a complex chromosomal rearrangement Replication analysis See text for details
the father and a weak 530-bp band from the mother. After HpaII digestion, both the 433-bp and 530-bp bands were still detected in the patient, although the 433-bp band had become faint. The results of the control females indicated random X-inactivation in control 1, skewed X-inactivation in control 2, and nonrandom X-inactivation in controls 3 and 4. In addition, the 433-bp band of the father, with an active single X chromosome, disappeared after HpaII digestion.
Discussion Our patient had a 45,X/46,X,r(X)(p22q21)/46,X,del(X) (p22) karyotype and MLS features [here, the deleted X chromosome, although it should retain a functioning telomere, is referred to as having a terminal rather than interstitial deletion because of the absence of the subtelomeric region demonstrated by the Xp/Yp telomere FISH analysis, as has been suggested by Ballabio and Andria (1992)]. Since functional nullisomy for the MLS gene is postulated for the development of MLS (Ballabio and Andria 1992), it is inferred that 46,X,del(X)(p22) cells with an inactive normal X chromosome alone are responsible for the development of MLS. The 46,X,del(X)(p22) cells with an active normal X, as well as 45,X and 46,X,r(X)(p22q21) cells, in which
the normal X chromosome should be active, would not cause the MLS phenotype. Although the karyotype of lymphocytes was examined, mosaicism would also be present in tissues/organs that are targets for the MLS gene, such as the eyes and the skin. Thus, the relatively small cell number relevant to the development of MLS would account for the mildness of the MLS phenotype in our patient. In addition, the mosaicism would also explain the apparent lack of Turner stigmata, except for short stature, which is primarily ascribed to haploinsufficiency of the pseudoautosomal SHOX gene (Rao et al. 1997), in all three cell lineages. Microsatellite analysis showed that our patient had monosomy for the Xp22 region with the breakpoint between DXS1053 and DXS418, and that the rearranged X chromosomes were of maternal origin whereas the normal X chromosome was of paternal origin in the three cell lineages. The result is consistent with loss of the MLS gene from the del(X)(p22) chromosome, because the MLS gene has been located between DXS7108 and DXS1043 (Table 1) (Nelson et al. 1995). It might be possible that the breakpoint defined by the microsatellite analysis represents that of the ring X chromosome rather than the del(X)(p22) chromosome. In this case, the del(X)(p22) chromosome is assumed to have a larger deletion including the MLS gene. According to the Report of the Sixth International Workshop on Human X Chromosome Mapping (Nelson et al. 1995), the Xp22 breakpoint in our patient resides in a posi-
55
tion roughly 17.5 Mb from the Xp telomere or 15 Mb from the pseudoautosomal boundary. To our knowledge, the molecularly defined Xp22 breakpoints in previously reported MLS patients lie in the region distal to DXS16 at a position approximately 12 Mb from the pseudoautosomal boundary, with the most proximal breakpoint defined by the patient reported by Ropers et al. (1982) (Shaefer et al. 1993; Lindsay et al. 1994; Wapenaar et al. 1994; Ferrero et al. 1995). Thus, our patient appears to have the largest Xp22 deletion in molecularly defined MLS patients. In this context, since our patient had no features other than MLS and short stature, it is suggested that a dominant disease gene, except for the MLS gene, is absent from the approximately 15-Mb Xdifferential region. The PCR analysis for the PGK gene detected a faint but definite 433-bp band after HpaII digestion in the patient, together with a more distinct 530-bp band (the intensity difference between the two bands before HpaII digestion would be related to mosaicism of the patient). This implies that normal X chromosomes were more or less subject to Xinactivation in our patient. Although variable methylation at the HpaII site might be possible, the results in the control females and the father suggest that our method well reflects the X-inactivation status. Since normal X chromosomes in the 45,X and 46,X,r(X)(p22q21) cell lineages are unlikely to undergo X-inactivation, the 433-bp band found after HpaII digestion is considered to represent inactive normal X chromosomes in the 46,X,del(X)(p22) cell lineage. Thus, although it is uncertain from the PCR-based data in the mosaic patient whether the normal X chromosome and the del(X)(p22) chromosome underwent skewed or random Xinactivation, the results support the idea that functional absence of the MLS gene caused by inactivation of the normal X chromosome plays a pivotal role in the development of MLS in patients with monosomy for the Xp22 region (Ballabio and Andria 1992). The X-inactivation pattern has also been studied in seven MLS patients with monosomy for the Xp22 region (Table 2, cases 1–7). The results are not straightforward, and most patients show nonrandom X-inactivation, although a certain degree of random X-inactivation is assumed, at least in a specific tissue at a particular developmental stage. There are three possible explanations for the nonrandom Xinactivation. First, cell selection due to the chromosomal abnormalities may result in nonrandom X-inactivation, at least in the examined cells by the time of investigation. In this regard, cases 1, 4, and 7 are associated with nonrandom X-inactivation, despite the molecularly defined size of the Xp22 monosomic region being less than that in our patient (Shaefer et al. 1993; Lindsay et al. 1994; Ferrero et al. 1995). Such a difference in cell-selection effect could occur if the effect of Xp22 deletion (and non-X-chromosomal material translocated onto Xp22) is variable among patients, because of other genetic and environmental factors. Second, nonchromosomal factors such as a stochastic event and a coexisting mutation leading to positive or negative selection may be involved in the different X-inactivation pattern, at least in the examined cells. Such factors have been indicated for skewed X-inactivation in 46,XX females (Bel-
mont 1996). Third, cytogenetic replication analysis of a limited number of cells may be insufficient to detect cryptic levels of inactive normal X chromosomes. In this context, it is likely that the application of the PCR method may have served to detect an inactive normal X chromosome in our patient. If so, it is expected that a PCR-based X-inactivation analysis would improve the identification of inactive normal X chromosomes in MLS patients monosomic for the Xp22 region. In summary, molecular studies on our patient revealed the largest Xp22 deletion in molecularly defined MLS patients, and indicated the presence of inactive normal X chromosomes. Further analysis of similarly affected patients will allow clarification of the role of X-inactivation in the development of MLS. Acknowledgements We would like to thank Dr. Katsuyuki Arai for referring the patient, Dr. Kenji Ohyama for providing us with a blood sample from the female with skewed X-inactivation, and Prof. David H. Ledbetter for providing us with the Xp/Yp and Xq/Yq telomeric probes. This work was supported in part by a grant-in-aid from the Ministry of Education, Science, Sports and Culture, a grant for Pediatric Research from the Ministry of Health and Welfare, and a grant for Medical Research from Keio University. The experiments comply with current Japanese laws.
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