Am. J. Hum. Genet. 51:316-322, 1992
Characterization of a Deletion at Xq27-q28 Associated with Unbalanced Inactivation of the Nonmutant X Chromosome J. T. R. Clarke, * P. J. Wilson, t C. P. Morris, t J. J. Hopwood, t R. 1. Richards, t G. R. Sutherland,t and P. N. Ray* *Division of Clinical Genetics, Departments of Pediatrics and Genetics, Hospital for Sick Children, University of Toronto, Toronto; and
tDepartment of Chemical Pathology and $Department of Cytogenetics and Molecular Genetics, Adelaide Children's Hospital, North Adelaide
Summary We report the results of studies on the characterization of the mutation associated with marked unbalanced expression of the mutant X chromosome in a karyotypically normal girl with Hunter disease (mucopolysaccharidosis type II). Southern analysis of DNA extracted from somatic cell hybrids containing only the mutant X chromosome showed deletion of the Xq27.3-q28 loci: DXS297 (VK23AC), DXS293 (VK16), FRAXA (pfxa3), DXS296 (VK21A), and the 3' end of the iduronatesulfatase (IDS) gene. The flanking loci-DXS52 (Stl4-1), DXS304 (U6.2), and DXS369 (RN1)-were intact. On the basis of these results, we concluded that the mutation was a simple deletion extending a maximum of 3-5 cM to the centromeric side of the IDS gene. Both Southern analysis of DNA from somatic cell hybrids, using short segments of IDS cDNA, and PCR of reverse-transcribed RNA from cultured skin fibroblasts indicated that the telomeric terminus of the deletion was localized to a region near the middle of the coding sequences of the gene.
Introduction
Hunter disease (mucopolysaccharidosis type II) is an X-linked mucopolysaccharide storage disease (Neufeld and Muenzer 1989) caused by deficiency of the enzyme iduronatesulfatase (IDS) (Bach et al. 1973). The gene is localized to Xq28 (Roberts et al. 1989); a full-length cDNA for the human gene has recently been isolated (Wilson et al. 1990). Hunter disease in females is exceedingly rare. We recently reported a case in which clinical and biochemical manifestations of the disease occurred in a girl as a result of a de novo mutation of the paternal IDS gene, associated with unbalanced inactivation of the maternal X chromosome (Clarke et al. 1991). We report here the partial characterization of the mutation in this patient.
Received December 12, 1991; final revision received March 23, 1992. Address for correspondence and reprints: Dr. J. T. R. Clarke, Division of Clinical Genetics, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8. X 1992 by The American Society of Human Genetics. All rights reserved. 0002-9297/92/5102-001 1$02.00
316
Material and Methods Cell Lines and Media Cultured skin fibroblast lines were maintained in a-MEM supplemented with 10% FCS, according to a method described elsewhere (Clarke et al. 1991). Rodent-human somatic cell hybrids were produced by brief treatment of mixed cultures of Hprt- Chinese
hamster (RJK88) and human fibroblasts with polyethylene glycol (Davidson and Gerald 1976), followed by selection for hybrids in HAT-ouabain medium (10-4 M hypoxanthine, 1.6 x 10 - M thymidine, 4.0 x 10-7 M aminopterin, and 1.89 x 10 -6 M ouabain) (Clarke et al. 1991). Southern Analysis
Aliquots (6 gg) of DNA were digested with restriction enzymes HindIII, PstI, or EcoRI (Boehringer Mannheim), were separated by electrophoresis on 0.4%-0.6% (EcoRI digests) or 0.8% agarose gels, and were transferred to nylon filters (GeneScreenPlus; New England Nuclear) (Southern 1975). The filters were prehybridized in 5 x SSPE (Maniatis et al. 1982), 1% SDS, 40% formamide, herring sperm DNA (100 jgg/ml), 0.5°/% skim milk powder, and 10%
317
IDS Mutation with Unbalanced X Inactivation dextran sulfate for 4-6 h at 420. Hybridization was done in the same solution by using probes radiolabeled with [32P]dCTP by random-hexamer labeling (Feinberg and Vogelstein 1983). After the probing, the filters were washed in 0.5 x SSPE, 0.1% SDS at 650C and then were exposed to Kodak X-Omat AR film for 1-14 d at -700C. Probes included a 1.7-kb insert of IDS cDNA prepared by NotIIXhoI digestions of plasmid containing a 2.3-kb full-length IDS cDNA polynucleotide (Wilson et al. 1990); 5' and 3' oligonucleotides representing the two ends of IDS cDNA (see below); and U6.2, VK21A, VK16, VK23AC, pfxa3, and pS8 (Hyland et al. 1989; Richards et al. 1991; Sutherland et al. 1991; Yu et al. 1991). Extraction and Analysis of mRNA
Total cellular RNA was extracted from cultured skin fibroblasts by the acid guanidinium thiocyanatephenol-chloroform procedure (Chomczynski and Sacchi 1987). Monolayers of cells cultured to confluency were washed three times with PBS and were flooded with 1.8 ml guanidinium thiocyanate solution (Solution D [Chomczynski and Sacchi 1987])/T75, and the extracts from seven flasks were combined for further processing. After phenol-chloroform extraction and precipitation with isopropanol, the RNA was resuspended in Solution D and was precipitated with cold ethanol. The final pellets were dissolved in DEPCtreated water and were stored at 200C. cDNA was prepared from total cellular RNA by reverse transcription. Aliquots (6 gg) of RNA from patient and control fibroblasts were added to reaction mixes containing 1 x Moloney murine leukemia virus (M-MLV) reverse-transcriptase (RT) buffer (Bethesda Research Laboratories), 80 units RNAsin (Promega), 1,000 ng of random DNA octamers, 0.5 mM deoxyribonucleotides (Boehringer Mannheim), and 400 units of M-MLV RT (Bethesda Research Laboratories), to a final volume of 100 il. The mixtures were incubated at 370C for 1 h, followed by alkaline hydrolysis of the RNA by addition of 10 pl 3 M NaOH and incubation for another 30 min at 370C. The reactions were neutralized by addition of 2.5 jl concentrated HCl, and cDNA was precipitated with ethanol and was redissolved in 100 jil H20. Aliquots of 5 td, corresponding to 300 ng of total cellular RNA, were used in typical PCR analyses.
deoxynucleotides modified to 400 gM and with the addition of dimethyl sulfoxide (10%) in some reactions. Thirty-five cycles of denaturation at 940C for 45 s, annealing at 571C for 45 s, and elongation at 720C for 2 min were carried out. PCR products were typically analyzed by electrophoresis on 2% agarose minigels stained with ethidium bromide and were photographed by transmitted UV light. Preparation of IDS cDNA Oligonucleotide Probes Probes for the 5' and 3' ends of IDS cDNA were prepared by PCR, with the use of primers constructed on the basis of the nucleotide sequence of IDS cDNA (Wilson et al. 1990). The primers were as follows: J1,
5'-TAACTGCGCCACCTGCTGCA-3' and 5'-GGAAGAAAGGACTGGCTGAC-3'; J2, 5'-GAACTCCATGCCAACCTG-3' and 5'-GAGCACATCACATTTGCC-3'; J2.1, 5'-GAACTCCATGCCAACCTG-3' and 5'-AAGAGACACAAGTTCCACAA-3'; and J2.2, 5'-ACAGTTGATGGAGCCAGG-3' and 5'-GAGCACATCACATTTGCC-3'. The template was Xgt2S2.3 (Wilson et al. 1990), the construct containing the fulllength IDS cDNA. The relationship of J1, J2, J2.1, and J2.2 to IDS cDNA is shown in the top panel of figure 1. Results
-
PCR PCR conditions were the same as those described by Saiki et al. (1988), with the final concentration of
Southern Analysis
The Southern pattern of the patient's DNA extracted from lymphoblasts was normal when digested with HindIII. However, PstI and EcoRI digests probed with the cDNA probe, IDS1.7 (fig. 2), revealed structural alterations in the IDS locus of one of her two chromosomes. Both her parents had patterns indistinguishable from those of the controls. The sizes of the abnormal fragments could not be explained by the simple gain or loss of a single restriction-endonuclease recognition site or by a small deletion or insertion that would increase fragment sizes by a constant amount. These results were consistent with localization of the abnormality in the 3' end of IDS, which previous reports have shown to be oriented toward the centromere (Wilson et al. 1991). The approximate location of the deletion breakpoint within the IDS gene was determined by Southern analysis using fragments of IDS cDNA as probes (fig. 1, top). The Southern patterns obtained using EcoRI digestion are presented in the bottom panel of figure 1. When Ji (nucleotides 64-786 [Wilson et al. 1990]) was used to probe DNA
318
Clarke et al.
0
I
1a
0.4 mI
0.8 IE
l
0
Im
1.2 I0
1.6 I
I
m
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I
II IDS1.7
I
I"~~~~ A1 %ff I
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I
I I J2
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l.. J
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Figure I Top, Diagram of IDS cDNA probes used in Southern analyses. The size and location of various probes used in the studies reported here are shown in relation to the full-length IDS cDNA (Xgt2S2.3 [Wilson et al. 1990]) shown diagrammatically at the top of the figure. The two-headed arrow indicates the approximate location of the telomeric terminus of the deletion. Bottom, Autoradiograms showing Southern analyses of patient DNA probed with J1, J2. 1, and J2.2. The panels show the results of Southern analyses done on DNA digested with EcoRI and probed with J1 (left), J2. 1 (middle), and J2.2 (right). The sources of the DNA are shown in the same left-to-right sequence as in fig. 2: RJK88 hamster fibroblasts (lane 1), hamster/patient somatic cell hybrid (lane 2), patient lymphoblasts (lane 3), paternal lymphoblasts (lane 4), maternal lymphoblasts (lane 5), and healthy human control fibroblasts (lane 6). The positions of molecular-size markers are shown along the left side of each panel and correspond to 9.4- and 5.0-kb fragments of X phage digested with EcoRI and HindIII.
digested with EcoRI, HindIII, or PstI, the patterns were indistinguishable from the normal one. In contrast, the patterns obtained using J2 (nucleotides 6531813) as probe showed the same abnormal fragments seen when the complete IDS1.7 was used as probe. The abnormal fragments were also present in Southern patterns probed with J2.1, a 5' fragment of J2; probing with J2.2 yielded no signal at all. These results indicate that the mutation in the patient was a deletion of the 3' end of the IDS gene, with the telomeric endpoint of the deletion lying between nucleotides 785 and 1288 of the cDNA (fig 1, top). Southern analysis of somatic cell hybrid DNA by using probes for loci adjacent to IDS showed that DXS304 (probed with U6.2) was present. However,
the following probes produced no signal, indicating that the respective loci were deleted: VK16 (DXS293), VK21A (DXS296), pfxa3 (FRAXA), pS8 (DXS296), and VK23AC (DXS297). A positive signal was obtained by probing with RN1 (DXS369), setting the approximate centromeric limit of the deletion. The results of these analyses are summarized in figure 3. Analysis of mRNA
The results of previous studies indicated that the nonmutant X chromosome in this patient was inactivated (Clarke et al. 1991). In order to determine whether there was measurable expression of the nonmutant X in cultured skin fibroblasts, the production of mRNA was determined by RT-PCR analyses. The
IDS Mutation with Unbalanced X Inactivation
319
m~~~~~~~~~~~~~~~Astai
m
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-
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Pstl
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Figure 2 Autoradiograms showing Southern analyses of patient DNA probed with IDS1.7. The conditions of the analyses are described in Material and Methods. The sources of the DNA are shown in the same sequence in each panel (from left to right): RJK88 hamster fibroblasts, hamster/patient somatic cell hybrids, patient lymphoblasts, paternal lymphoblasts, maternal lymphoblasts, and healthy human control fibroblasts. The positions of molecular-size markers are shown along the left side of each panel and correspond (from top to bottom) to 9.4-, 5.0-, 2.3-, and 1.0-kb fragments of X phage digested with EcoRI and HindIll.
results of analyses carried out with the use of the same primers that were used to construct probes for use in Southern analysis showed that primer pairs amplifying 5' sequences (Jl) produced product, indicating that a stable message was produced. Under the same conditions, no product from cDNA coding for 3' sequences of IDS (J2) could be demonstrated (fig. 4). The results were interpreted as indicating that mutant fibroblasts produced at least some stable but truncated IDS message and that expression of IDS by the nonmutant X was negligible. Discussion
Previous studies on this female with Hunter syndrome suggested that the phenotype in her case was due to a single de novo mutation which disrupted the
IDS locus of her paternal X chromosome and also resulted in marked unbalanced inactivation of her maternal (nonmutant) X chromosome (Clarke et al. 1991). The present study was undertaken to characterize the mutation, anticipating that DNA sequences involved may play some role in directing normal X-chromosome inactivation. Conventional and highresolution chromosome studies had previously shown no visible disturbance of X-chromosome structure (Clarke et al. 1990). Mapping studies of the Xq distal end, around IDS and FRAXA, have provided detailed physical and linkage maps of the region (Oberle et al. 1986, 1987; Arveiler et al. 1988; Hyland et al. 1989; Mandel et al. 1989; Oostra et al. 1990; Suthers et al. 1990, 1991; Rousseau et al. 1991). Southern analysis of lymphocyte DNA from the patient herself and of DNA ex-
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Clarke et al.
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1
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_
q 2
DXS255
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_DXS369 (RN- ) -DXS297 (VK23AC) DXS293 (VK 16) -FRAXA (pfxa3) -DXS296 (VK21 A) -IDS DXS304 (U6.2)
3 5
6
Figure 3
-+
-7
3-5 cM
+
Diagram showing the extent of the deletion of Xq
in the patient.
tracted from patient-hamster fibroblast somatic cell hybrids has shown that part of the IDS locus and several unique loci centromeric of IDS were deleted. Since the loci deleted were contiguous with each other and continuous from VK23AC to the 3' end of IDS, the abnormality would appear to be a simple deletion, rather than a complex rearrangement associated with deletion of noncontiguous loci. On the basis of the location of the loci deleted, the deletion was estimated to span approximately 3-5 cM (Oberle et al. 1986, 1
2
3 4
5 6 7
8
1987; Arveiler et al. 1988; Hyland et al. 1989; Mandel et al. 1989; Oostra et al. 1990; Suthers et al. 1990, 1991; Rousseau et al. 1991), which may encompass as little as 1.5 Mb of DNA (Poustka et al. 1991). This is beyond the resolution of conventional karyotyping and is at the limit of detectability by high-resolution banding techniques. This would explain the inability to detect structural abnormalities in the X chromosome by these techniques. Our earlier results with methylation-sensitive RFLP analysis and somatic cell hybrid selection indicated unambiguously that inactivation of the X chromosomes in the patient was unbalanced (Clarke et al. 1991). They did not, however, indicate the extent or the mechanism of preferential expression of the nonmutant X chromosome. The results of the studies reported here show that the mutation in IDS involves a portion of the gene, not including the putative activesite sequence of the enzyme (J. J. Hopwood, unpublished observations). Mutant fibroblasts produce truncated mRNA from the mutant allele and little or no mRNA from the normal allele. This is consistent with both the level ofresidual enzyme activity in the patient, which is less than 0. 1 % of normal (J. Bielicki, personal communication), and the clinical and biochemical features of classical Hunter disease. We conclude from this that expression of normal enzyme activity from the nonmutant X is negligible and that inactivation
9 10 11121314
151617 181920
Results of RT-PCR analyses of fibroblast RNA. Total cellular RNA was extracted from cultured skin fibroblasts (see Figure 4 Methods) and was reverse-transcribed, and the first strand of cDNA was amplified by PCR, using the same primers as were used to synthesize probes J1, J2, and J2.2 shown in fig. 2 and using primers for human N-acetylgalactosamine-4-sulfatase (Litjens et al. 1991) as controls. Lanes 1, 9, and 15, Molecular-size standards derived from the digestion of SPP-1 with EcoRI. Lanes 14 and 20, Standards derived from pUC19 digested with HpaII. Lanes 2-5, Results obtained with the use of J1 primers and either no template (lane 2) or control female cDNA as template (lane 3), patient cDNA as template (lane 4), or Xgt2S2.3 (Wilson et al. 1990) (lane 5) as template. Lanes 6-8 and 10, Results obtained with the use of J2 primers and either no template (lane 6) or control female cDNA as template (lane 7), patient cDNA as template (lane 8), or Xgt2S2.3 (lane 10) as template. Lanes 11-13, Results obtained with the use of human N-acetylgalactosamine-4-sulfatase primers (Litjens et al. 1991) and either no template (lane 11) or control female cDNA as template (lane 12) or patient cDNA (lane 13) as template. Lanes 16-19, Results obtained with the use of J2.2 primers and either no template (lane 16) or control female cDNA as template (lane 17), patient cDNA as template (lane 18), or Xgt2S2.3 as template (lane 19).
321
IDS Mutation with Unbalanced X Inactivation
of or selection against the nonmutant X is virtually complete. To date, the only identified X-chromosome locus with properties that would make it a candidate for a major role in X inactivation is the XIST locus described by Brown et al. (1991), which is localized to Xq13. The deletion in our patient suggests that sequences in the Xq27.3-q28 region may also be important in the process. Deletion of sequences in this region might make inactivation of the deleted chromosome impossible, forcing inactivation of the nonmutant X in every cell. The nature of such a locus is still not clear; it might be the receptor for a trans-acting ligand, the origin of a cis-acting inactivation factor, or a locus that is important in the maintenance of inactivation of the chromosome. Alternatively, cells in which the deleted X has been inactivated may have been strongly selected against, by some unknown mechanism, early in embryogenesis. Schmidt et al. (1990; also see Schmidt 1992) described a deletion patient with interesting genetic features in common with our patient. The karyotype of their patient showed a relatively large deletion of the Xq27-q28 region, involving a large number of loci between DXS105 at the centromeric end and DXS304 toward the telomere, including all the loci deleted in our patient. Like our patient, she showed preferential expression of the mutant X chromosome. However, on the basis of methylation-sensitive RFLP analysis and cytogenetic studies, Schmidt et al. estimated that in 10%-15% of cultured skin fibroblasts and peripheral blood lymphocytes the nonmutant X was active. Although the abnormality included complete deletion of IDS, the patient exhibited no clinical or biochemical features of Hunter disease, presumably because production of enzyme by the IDS allele on the nonmutant X was adequate to prevent clinical manifestations of the disease. The unbalanced expression of the deleted X in both our patient and the patient reported by Schmidt et al. supports the suggestion that there exists in the Xq27.3-q28 region some locus which when deleted disrupts the normal X-inactivation process. The nature or precise location of such a locus is still unknown.
Acknowledgements This research was supported in part by grants from the Medical Research Council of Canada and from the National Health and Medical Research Council of Australia.
References Arveiler B. Oberle I, Vincent A, Hofker MH, Pearson PL, Mandel JL (1988) Genetic mapping of the Xq27-q28 region: new RFLP markers useful for diagnostic applications in fragile-X and hemophilia-B families. Am J Hum Genet 42:380-389 Bach G, Eisenberg F Jr, Cantz M, Neufeld EF (1973) The defect in the Hunter syndrome: deficiency of sulfoiduronate sulfatase. Proc Natl Acad Sci USA 70:2134-2138 Brown CJ, Lafreniere RG, Powers VE, Sebastio G, Ballabio A, Pettigrew AL, Ledbetter DH, et al (1991) Localization of the X inactivation centre on the human X chromosome in Xql3. Nature 349:82-84 Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal Biochem 162:156-159 Clarke JTR, Greer WL, Strasberg PM, Pearce RD, Skomorowski MA, Ray PN (1991) Hunter disease (mucopolysaccharidosis type II) associated with unbalanced inactivation of the X chromosomes in a karyotypically normal girl. Am J Hum Genet 49:289-297 Clarke JTR, Willard HF, Teshima I, Chang PL, Skomorowski MA (1990) Hunter disease (mucopolysaccharidosis type II) in a karyotypically normal girl. Clin Genet 37: 355-362 Davidson RG, Gerald PS (1976) Improved techniques for the induction of mammalian cell hybridization by polyethylene glycol. Somatic Cell Genet 2:165-176 Feinberg AP, Vogelstein B (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6-13 Hyland VJ, Fernandez KEW, Callen DF, MacKinnon RN, Baker E, Friend K, Sutherland GR (1989) Assignment of anonymous DNA probes to specific intervals of human chromosomes 16 and X. Hum Genet 83:61-66 Litjens T, Morris CP, Gibson GJ, Beckmann KR, Hopwood JJ (1991) Human N-acetylgalactosamine-4-sulphatase: protein maturation and isolation of genomic clones. Biochem Int 24:209-215 Mandel JL, Willard HF, Nussbaum RL, Romeo G, Puck JM, Davies KE (1989) Report of the Committee on the Genetic Constitution of the X Chromosome. Human Gene Mapping 10. Cytogenet Cell Genet 51:384-437 Neufeld EFMuenzerJ (1989) The mucopolysaccharidoses. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds) The metabolic basis of inherited disease, 6th ed. McGrawHill, New York, pp 1565-1588 Oberle I, Camerino G, Wrogemann K, Arveiler B, Hanauer A, Raimondi E, Mandel JL (1987) Multipoint genetic mapping of the Xq26-q28 region in families with fragile X mental retardation and in normal families reveals tight linkage of markers in q26-q27. Hum Genet 77:60-65 Oberle I, Heilig R, Moisan JP, Kloepfer C, Mattei MG, Mattei JF, Boue J, et al (1986) Genetic analysis of the fragile-X mental retardation syndrome with two flanking
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322 polymorphic DNA markers. Proc Natl Acad Sci USA 83: 1016-1020 Oostra BA, Hupkes PE, Perdon LF, Van Bennekom CA, Bakker E, Halley DJJ, Schmidt M, et al (1990) New polymorphic DNA marker close to the fragile site FRAXA. Genomics 6:129-132 Poustka A, Dietrich A, Langenstein G, Toniolo D, Warren ST, Lehrach H (1991) Physical map of human Xq27-qter: localizing the region of the fragile X mutation. Proc Natl Acad Sci USA 88:8302-8306 Richards RI, Shen Y, Holman K, Kozman H, Hyland VJ, Mulley JC, Sutherland GR (1991) Fragile X syndrome: diagnosis using highly polymorphic microsatellite markers. Am J Hum Genet 48:1051-1057 Roberts SH, Upadhyaya M, Sarfarazi M, Harper PS (1989) Further evidence localising the gene for Hunter's syndrome to the distal region of the X chromosome long arm. J Med Genet 26:309-313 Rousseau F, Vincent A, Rivella S, Heitz D, Triboli C, Maestrini E, Warren ST, et al (1991) Four chromosomal breakpoints and four new probes mark out a 10-cM region encompassing the fragile-X locus (FRAXA). Am J Hum Genet 48:108-116 Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, et al (1988) Primer directed amplification of DNA with a thermostable DNA polymerase. Science 239:487-491 Schmidt M (1992) Do sequences in Xq27.3 play a role in X inactivation? Am J Med Genet 43:279-281 Schmidt M, Certoma A, Du Sart D, Kalitsis P, Leversha M, Fowler K, Sheffield L, et al (1990) Unusual X chromosome
inactivation in a mentally retarded girl with an interstitial deletion Xq27: implications for the fragile X syndrome. Hum Genet 84:347-352 Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503-517 Sutherland GR, Gedeon A, Kornman L, Donnelly A, Byard RW, MulleyJC, Kremer E, et al (1991) Prenatal diagnosis of fragile X syndrome by direct detection of the unstable DNA sequence. N Engl J Med 325:1720-1722 Suthers GK, Hyland VJ, Callen DF, Oberle I, Rocchi M, Thomas NS, Morris CP, et al (1990) Physical mapping of new DNA probes near the fragile X mutation (FRAXA) by using a panel of cell lines. Am J Hum Genet 47:187195
Suthers GK, Oberle I, NancarrowJ, MulleyJC, Hyland VJ, Wilson PJ, McCure J, et al (1991) Genetic mapping of new RFLPs at Xq27-q28. Genomics 9:37-43 Wilson PJ, Morris CP, Anson DS, Occhiodoro T, Bielicki J, Clements PR, Hopwood JJ (1990) Hunter syndrome: isolation of an iduronate-2-sulphatase cDNA clone and analysis of patient DNA. Proc Natl Acad Sci USA 87: 8531-8535 Wilson PJ, Suthers GK, Callen DF, Baker E, Nelson PV, Cooper A, Wraith JE, et al (1991) Frequent deletions at Xq28 indicate genetic heterogeneity in Hunter syndrome. Hum Genet 86:505-508 Yu S, Pritchard M, Kremer E, Lynch M, NancarrowJ, Baker E, Holman K, et al (1991) Fragile X genotype characterized by an unstable region of DNA. Science 252:11791181
Characterization of a Deletion at Xq27-q28 Associated with Unbalanced Inactivation of the Nonmutant X Chromosome J. T. R. Clarke, * P. J. Wilson, t C. P. Morris, t J. J. Hopwood, t R. 1. Richards, t G. R. Sutherland,t and P. N. Ray* *Division of Clinical Genetics, Departments of Pediatrics and Genetics, Hospital for Sick Children, University of Toronto, Toronto; and
tDepartment of Chemical Pathology and $Department of Cytogenetics and Molecular Genetics, Adelaide Children's Hospital, North Adelaide
Summary We report the results of studies on the characterization of the mutation associated with marked unbalanced expression of the mutant X chromosome in a karyotypically normal girl with Hunter disease (mucopolysaccharidosis type II). Southern analysis of DNA extracted from somatic cell hybrids containing only the mutant X chromosome showed deletion of the Xq27.3-q28 loci: DXS297 (VK23AC), DXS293 (VK16), FRAXA (pfxa3), DXS296 (VK21A), and the 3' end of the iduronatesulfatase (IDS) gene. The flanking loci-DXS52 (Stl4-1), DXS304 (U6.2), and DXS369 (RN1)-were intact. On the basis of these results, we concluded that the mutation was a simple deletion extending a maximum of 3-5 cM to the centromeric side of the IDS gene. Both Southern analysis of DNA from somatic cell hybrids, using short segments of IDS cDNA, and PCR of reverse-transcribed RNA from cultured skin fibroblasts indicated that the telomeric terminus of the deletion was localized to a region near the middle of the coding sequences of the gene.
Introduction
Hunter disease (mucopolysaccharidosis type II) is an X-linked mucopolysaccharide storage disease (Neufeld and Muenzer 1989) caused by deficiency of the enzyme iduronatesulfatase (IDS) (Bach et al. 1973). The gene is localized to Xq28 (Roberts et al. 1989); a full-length cDNA for the human gene has recently been isolated (Wilson et al. 1990). Hunter disease in females is exceedingly rare. We recently reported a case in which clinical and biochemical manifestations of the disease occurred in a girl as a result of a de novo mutation of the paternal IDS gene, associated with unbalanced inactivation of the maternal X chromosome (Clarke et al. 1991). We report here the partial characterization of the mutation in this patient.
Received December 12, 1991; final revision received March 23, 1992. Address for correspondence and reprints: Dr. J. T. R. Clarke, Division of Clinical Genetics, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8. X 1992 by The American Society of Human Genetics. All rights reserved. 0002-9297/92/5102-001 1$02.00
316
Material and Methods Cell Lines and Media Cultured skin fibroblast lines were maintained in a-MEM supplemented with 10% FCS, according to a method described elsewhere (Clarke et al. 1991). Rodent-human somatic cell hybrids were produced by brief treatment of mixed cultures of Hprt- Chinese
hamster (RJK88) and human fibroblasts with polyethylene glycol (Davidson and Gerald 1976), followed by selection for hybrids in HAT-ouabain medium (10-4 M hypoxanthine, 1.6 x 10 - M thymidine, 4.0 x 10-7 M aminopterin, and 1.89 x 10 -6 M ouabain) (Clarke et al. 1991). Southern Analysis
Aliquots (6 gg) of DNA were digested with restriction enzymes HindIII, PstI, or EcoRI (Boehringer Mannheim), were separated by electrophoresis on 0.4%-0.6% (EcoRI digests) or 0.8% agarose gels, and were transferred to nylon filters (GeneScreenPlus; New England Nuclear) (Southern 1975). The filters were prehybridized in 5 x SSPE (Maniatis et al. 1982), 1% SDS, 40% formamide, herring sperm DNA (100 jgg/ml), 0.5°/% skim milk powder, and 10%
317
IDS Mutation with Unbalanced X Inactivation dextran sulfate for 4-6 h at 420. Hybridization was done in the same solution by using probes radiolabeled with [32P]dCTP by random-hexamer labeling (Feinberg and Vogelstein 1983). After the probing, the filters were washed in 0.5 x SSPE, 0.1% SDS at 650C and then were exposed to Kodak X-Omat AR film for 1-14 d at -700C. Probes included a 1.7-kb insert of IDS cDNA prepared by NotIIXhoI digestions of plasmid containing a 2.3-kb full-length IDS cDNA polynucleotide (Wilson et al. 1990); 5' and 3' oligonucleotides representing the two ends of IDS cDNA (see below); and U6.2, VK21A, VK16, VK23AC, pfxa3, and pS8 (Hyland et al. 1989; Richards et al. 1991; Sutherland et al. 1991; Yu et al. 1991). Extraction and Analysis of mRNA
Total cellular RNA was extracted from cultured skin fibroblasts by the acid guanidinium thiocyanatephenol-chloroform procedure (Chomczynski and Sacchi 1987). Monolayers of cells cultured to confluency were washed three times with PBS and were flooded with 1.8 ml guanidinium thiocyanate solution (Solution D [Chomczynski and Sacchi 1987])/T75, and the extracts from seven flasks were combined for further processing. After phenol-chloroform extraction and precipitation with isopropanol, the RNA was resuspended in Solution D and was precipitated with cold ethanol. The final pellets were dissolved in DEPCtreated water and were stored at 200C. cDNA was prepared from total cellular RNA by reverse transcription. Aliquots (6 gg) of RNA from patient and control fibroblasts were added to reaction mixes containing 1 x Moloney murine leukemia virus (M-MLV) reverse-transcriptase (RT) buffer (Bethesda Research Laboratories), 80 units RNAsin (Promega), 1,000 ng of random DNA octamers, 0.5 mM deoxyribonucleotides (Boehringer Mannheim), and 400 units of M-MLV RT (Bethesda Research Laboratories), to a final volume of 100 il. The mixtures were incubated at 370C for 1 h, followed by alkaline hydrolysis of the RNA by addition of 10 pl 3 M NaOH and incubation for another 30 min at 370C. The reactions were neutralized by addition of 2.5 jl concentrated HCl, and cDNA was precipitated with ethanol and was redissolved in 100 jil H20. Aliquots of 5 td, corresponding to 300 ng of total cellular RNA, were used in typical PCR analyses.
deoxynucleotides modified to 400 gM and with the addition of dimethyl sulfoxide (10%) in some reactions. Thirty-five cycles of denaturation at 940C for 45 s, annealing at 571C for 45 s, and elongation at 720C for 2 min were carried out. PCR products were typically analyzed by electrophoresis on 2% agarose minigels stained with ethidium bromide and were photographed by transmitted UV light. Preparation of IDS cDNA Oligonucleotide Probes Probes for the 5' and 3' ends of IDS cDNA were prepared by PCR, with the use of primers constructed on the basis of the nucleotide sequence of IDS cDNA (Wilson et al. 1990). The primers were as follows: J1,
5'-TAACTGCGCCACCTGCTGCA-3' and 5'-GGAAGAAAGGACTGGCTGAC-3'; J2, 5'-GAACTCCATGCCAACCTG-3' and 5'-GAGCACATCACATTTGCC-3'; J2.1, 5'-GAACTCCATGCCAACCTG-3' and 5'-AAGAGACACAAGTTCCACAA-3'; and J2.2, 5'-ACAGTTGATGGAGCCAGG-3' and 5'-GAGCACATCACATTTGCC-3'. The template was Xgt2S2.3 (Wilson et al. 1990), the construct containing the fulllength IDS cDNA. The relationship of J1, J2, J2.1, and J2.2 to IDS cDNA is shown in the top panel of figure 1. Results
-
PCR PCR conditions were the same as those described by Saiki et al. (1988), with the final concentration of
Southern Analysis
The Southern pattern of the patient's DNA extracted from lymphoblasts was normal when digested with HindIII. However, PstI and EcoRI digests probed with the cDNA probe, IDS1.7 (fig. 2), revealed structural alterations in the IDS locus of one of her two chromosomes. Both her parents had patterns indistinguishable from those of the controls. The sizes of the abnormal fragments could not be explained by the simple gain or loss of a single restriction-endonuclease recognition site or by a small deletion or insertion that would increase fragment sizes by a constant amount. These results were consistent with localization of the abnormality in the 3' end of IDS, which previous reports have shown to be oriented toward the centromere (Wilson et al. 1991). The approximate location of the deletion breakpoint within the IDS gene was determined by Southern analysis using fragments of IDS cDNA as probes (fig. 1, top). The Southern patterns obtained using EcoRI digestion are presented in the bottom panel of figure 1. When Ji (nucleotides 64-786 [Wilson et al. 1990]) was used to probe DNA
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0
I
1a
0.4 mI
0.8 IE
l
0
Im
1.2 I0
1.6 I
I
m
2.0 kb I0
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I
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I
I"~~~~ A1 %ff I
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I
I I J2
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Figure I Top, Diagram of IDS cDNA probes used in Southern analyses. The size and location of various probes used in the studies reported here are shown in relation to the full-length IDS cDNA (Xgt2S2.3 [Wilson et al. 1990]) shown diagrammatically at the top of the figure. The two-headed arrow indicates the approximate location of the telomeric terminus of the deletion. Bottom, Autoradiograms showing Southern analyses of patient DNA probed with J1, J2. 1, and J2.2. The panels show the results of Southern analyses done on DNA digested with EcoRI and probed with J1 (left), J2. 1 (middle), and J2.2 (right). The sources of the DNA are shown in the same left-to-right sequence as in fig. 2: RJK88 hamster fibroblasts (lane 1), hamster/patient somatic cell hybrid (lane 2), patient lymphoblasts (lane 3), paternal lymphoblasts (lane 4), maternal lymphoblasts (lane 5), and healthy human control fibroblasts (lane 6). The positions of molecular-size markers are shown along the left side of each panel and correspond to 9.4- and 5.0-kb fragments of X phage digested with EcoRI and HindIII.
digested with EcoRI, HindIII, or PstI, the patterns were indistinguishable from the normal one. In contrast, the patterns obtained using J2 (nucleotides 6531813) as probe showed the same abnormal fragments seen when the complete IDS1.7 was used as probe. The abnormal fragments were also present in Southern patterns probed with J2.1, a 5' fragment of J2; probing with J2.2 yielded no signal at all. These results indicate that the mutation in the patient was a deletion of the 3' end of the IDS gene, with the telomeric endpoint of the deletion lying between nucleotides 785 and 1288 of the cDNA (fig 1, top). Southern analysis of somatic cell hybrid DNA by using probes for loci adjacent to IDS showed that DXS304 (probed with U6.2) was present. However,
the following probes produced no signal, indicating that the respective loci were deleted: VK16 (DXS293), VK21A (DXS296), pfxa3 (FRAXA), pS8 (DXS296), and VK23AC (DXS297). A positive signal was obtained by probing with RN1 (DXS369), setting the approximate centromeric limit of the deletion. The results of these analyses are summarized in figure 3. Analysis of mRNA
The results of previous studies indicated that the nonmutant X chromosome in this patient was inactivated (Clarke et al. 1991). In order to determine whether there was measurable expression of the nonmutant X in cultured skin fibroblasts, the production of mRNA was determined by RT-PCR analyses. The
IDS Mutation with Unbalanced X Inactivation
319
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m
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-
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Pstl
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Figure 2 Autoradiograms showing Southern analyses of patient DNA probed with IDS1.7. The conditions of the analyses are described in Material and Methods. The sources of the DNA are shown in the same sequence in each panel (from left to right): RJK88 hamster fibroblasts, hamster/patient somatic cell hybrids, patient lymphoblasts, paternal lymphoblasts, maternal lymphoblasts, and healthy human control fibroblasts. The positions of molecular-size markers are shown along the left side of each panel and correspond (from top to bottom) to 9.4-, 5.0-, 2.3-, and 1.0-kb fragments of X phage digested with EcoRI and HindIll.
results of analyses carried out with the use of the same primers that were used to construct probes for use in Southern analysis showed that primer pairs amplifying 5' sequences (Jl) produced product, indicating that a stable message was produced. Under the same conditions, no product from cDNA coding for 3' sequences of IDS (J2) could be demonstrated (fig. 4). The results were interpreted as indicating that mutant fibroblasts produced at least some stable but truncated IDS message and that expression of IDS by the nonmutant X was negligible. Discussion
Previous studies on this female with Hunter syndrome suggested that the phenotype in her case was due to a single de novo mutation which disrupted the
IDS locus of her paternal X chromosome and also resulted in marked unbalanced inactivation of her maternal (nonmutant) X chromosome (Clarke et al. 1991). The present study was undertaken to characterize the mutation, anticipating that DNA sequences involved may play some role in directing normal X-chromosome inactivation. Conventional and highresolution chromosome studies had previously shown no visible disturbance of X-chromosome structure (Clarke et al. 1990). Mapping studies of the Xq distal end, around IDS and FRAXA, have provided detailed physical and linkage maps of the region (Oberle et al. 1986, 1987; Arveiler et al. 1988; Hyland et al. 1989; Mandel et al. 1989; Oostra et al. 1990; Suthers et al. 1990, 1991; Rousseau et al. 1991). Southern analysis of lymphocyte DNA from the patient herself and of DNA ex-
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P -
(M270)
2 3
1
-F9
_
q 2
DXS255
2
_DXS369 (RN- ) -DXS297 (VK23AC) DXS293 (VK 16) -FRAXA (pfxa3) -DXS296 (VK21 A) -IDS DXS304 (U6.2)
3 5
6
Figure 3
-+
-7
3-5 cM
+
Diagram showing the extent of the deletion of Xq
in the patient.
tracted from patient-hamster fibroblast somatic cell hybrids has shown that part of the IDS locus and several unique loci centromeric of IDS were deleted. Since the loci deleted were contiguous with each other and continuous from VK23AC to the 3' end of IDS, the abnormality would appear to be a simple deletion, rather than a complex rearrangement associated with deletion of noncontiguous loci. On the basis of the location of the loci deleted, the deletion was estimated to span approximately 3-5 cM (Oberle et al. 1986, 1
2
3 4
5 6 7
8
1987; Arveiler et al. 1988; Hyland et al. 1989; Mandel et al. 1989; Oostra et al. 1990; Suthers et al. 1990, 1991; Rousseau et al. 1991), which may encompass as little as 1.5 Mb of DNA (Poustka et al. 1991). This is beyond the resolution of conventional karyotyping and is at the limit of detectability by high-resolution banding techniques. This would explain the inability to detect structural abnormalities in the X chromosome by these techniques. Our earlier results with methylation-sensitive RFLP analysis and somatic cell hybrid selection indicated unambiguously that inactivation of the X chromosomes in the patient was unbalanced (Clarke et al. 1991). They did not, however, indicate the extent or the mechanism of preferential expression of the nonmutant X chromosome. The results of the studies reported here show that the mutation in IDS involves a portion of the gene, not including the putative activesite sequence of the enzyme (J. J. Hopwood, unpublished observations). Mutant fibroblasts produce truncated mRNA from the mutant allele and little or no mRNA from the normal allele. This is consistent with both the level ofresidual enzyme activity in the patient, which is less than 0. 1 % of normal (J. Bielicki, personal communication), and the clinical and biochemical features of classical Hunter disease. We conclude from this that expression of normal enzyme activity from the nonmutant X is negligible and that inactivation
9 10 11121314
151617 181920
Results of RT-PCR analyses of fibroblast RNA. Total cellular RNA was extracted from cultured skin fibroblasts (see Figure 4 Methods) and was reverse-transcribed, and the first strand of cDNA was amplified by PCR, using the same primers as were used to synthesize probes J1, J2, and J2.2 shown in fig. 2 and using primers for human N-acetylgalactosamine-4-sulfatase (Litjens et al. 1991) as controls. Lanes 1, 9, and 15, Molecular-size standards derived from the digestion of SPP-1 with EcoRI. Lanes 14 and 20, Standards derived from pUC19 digested with HpaII. Lanes 2-5, Results obtained with the use of J1 primers and either no template (lane 2) or control female cDNA as template (lane 3), patient cDNA as template (lane 4), or Xgt2S2.3 (Wilson et al. 1990) (lane 5) as template. Lanes 6-8 and 10, Results obtained with the use of J2 primers and either no template (lane 6) or control female cDNA as template (lane 7), patient cDNA as template (lane 8), or Xgt2S2.3 (lane 10) as template. Lanes 11-13, Results obtained with the use of human N-acetylgalactosamine-4-sulfatase primers (Litjens et al. 1991) and either no template (lane 11) or control female cDNA as template (lane 12) or patient cDNA (lane 13) as template. Lanes 16-19, Results obtained with the use of J2.2 primers and either no template (lane 16) or control female cDNA as template (lane 17), patient cDNA as template (lane 18), or Xgt2S2.3 as template (lane 19).
321
IDS Mutation with Unbalanced X Inactivation
of or selection against the nonmutant X is virtually complete. To date, the only identified X-chromosome locus with properties that would make it a candidate for a major role in X inactivation is the XIST locus described by Brown et al. (1991), which is localized to Xq13. The deletion in our patient suggests that sequences in the Xq27.3-q28 region may also be important in the process. Deletion of sequences in this region might make inactivation of the deleted chromosome impossible, forcing inactivation of the nonmutant X in every cell. The nature of such a locus is still not clear; it might be the receptor for a trans-acting ligand, the origin of a cis-acting inactivation factor, or a locus that is important in the maintenance of inactivation of the chromosome. Alternatively, cells in which the deleted X has been inactivated may have been strongly selected against, by some unknown mechanism, early in embryogenesis. Schmidt et al. (1990; also see Schmidt 1992) described a deletion patient with interesting genetic features in common with our patient. The karyotype of their patient showed a relatively large deletion of the Xq27-q28 region, involving a large number of loci between DXS105 at the centromeric end and DXS304 toward the telomere, including all the loci deleted in our patient. Like our patient, she showed preferential expression of the mutant X chromosome. However, on the basis of methylation-sensitive RFLP analysis and cytogenetic studies, Schmidt et al. estimated that in 10%-15% of cultured skin fibroblasts and peripheral blood lymphocytes the nonmutant X was active. Although the abnormality included complete deletion of IDS, the patient exhibited no clinical or biochemical features of Hunter disease, presumably because production of enzyme by the IDS allele on the nonmutant X was adequate to prevent clinical manifestations of the disease. The unbalanced expression of the deleted X in both our patient and the patient reported by Schmidt et al. supports the suggestion that there exists in the Xq27.3-q28 region some locus which when deleted disrupts the normal X-inactivation process. The nature or precise location of such a locus is still unknown.
Acknowledgements This research was supported in part by grants from the Medical Research Council of Canada and from the National Health and Medical Research Council of Australia.
References Arveiler B. Oberle I, Vincent A, Hofker MH, Pearson PL, Mandel JL (1988) Genetic mapping of the Xq27-q28 region: new RFLP markers useful for diagnostic applications in fragile-X and hemophilia-B families. Am J Hum Genet 42:380-389 Bach G, Eisenberg F Jr, Cantz M, Neufeld EF (1973) The defect in the Hunter syndrome: deficiency of sulfoiduronate sulfatase. Proc Natl Acad Sci USA 70:2134-2138 Brown CJ, Lafreniere RG, Powers VE, Sebastio G, Ballabio A, Pettigrew AL, Ledbetter DH, et al (1991) Localization of the X inactivation centre on the human X chromosome in Xql3. Nature 349:82-84 Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal Biochem 162:156-159 Clarke JTR, Greer WL, Strasberg PM, Pearce RD, Skomorowski MA, Ray PN (1991) Hunter disease (mucopolysaccharidosis type II) associated with unbalanced inactivation of the X chromosomes in a karyotypically normal girl. Am J Hum Genet 49:289-297 Clarke JTR, Willard HF, Teshima I, Chang PL, Skomorowski MA (1990) Hunter disease (mucopolysaccharidosis type II) in a karyotypically normal girl. Clin Genet 37: 355-362 Davidson RG, Gerald PS (1976) Improved techniques for the induction of mammalian cell hybridization by polyethylene glycol. Somatic Cell Genet 2:165-176 Feinberg AP, Vogelstein B (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6-13 Hyland VJ, Fernandez KEW, Callen DF, MacKinnon RN, Baker E, Friend K, Sutherland GR (1989) Assignment of anonymous DNA probes to specific intervals of human chromosomes 16 and X. Hum Genet 83:61-66 Litjens T, Morris CP, Gibson GJ, Beckmann KR, Hopwood JJ (1991) Human N-acetylgalactosamine-4-sulphatase: protein maturation and isolation of genomic clones. Biochem Int 24:209-215 Mandel JL, Willard HF, Nussbaum RL, Romeo G, Puck JM, Davies KE (1989) Report of the Committee on the Genetic Constitution of the X Chromosome. Human Gene Mapping 10. Cytogenet Cell Genet 51:384-437 Neufeld EFMuenzerJ (1989) The mucopolysaccharidoses. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds) The metabolic basis of inherited disease, 6th ed. McGrawHill, New York, pp 1565-1588 Oberle I, Camerino G, Wrogemann K, Arveiler B, Hanauer A, Raimondi E, Mandel JL (1987) Multipoint genetic mapping of the Xq26-q28 region in families with fragile X mental retardation and in normal families reveals tight linkage of markers in q26-q27. Hum Genet 77:60-65 Oberle I, Heilig R, Moisan JP, Kloepfer C, Mattei MG, Mattei JF, Boue J, et al (1986) Genetic analysis of the fragile-X mental retardation syndrome with two flanking
Clarke et al.
322 polymorphic DNA markers. Proc Natl Acad Sci USA 83: 1016-1020 Oostra BA, Hupkes PE, Perdon LF, Van Bennekom CA, Bakker E, Halley DJJ, Schmidt M, et al (1990) New polymorphic DNA marker close to the fragile site FRAXA. Genomics 6:129-132 Poustka A, Dietrich A, Langenstein G, Toniolo D, Warren ST, Lehrach H (1991) Physical map of human Xq27-qter: localizing the region of the fragile X mutation. Proc Natl Acad Sci USA 88:8302-8306 Richards RI, Shen Y, Holman K, Kozman H, Hyland VJ, Mulley JC, Sutherland GR (1991) Fragile X syndrome: diagnosis using highly polymorphic microsatellite markers. Am J Hum Genet 48:1051-1057 Roberts SH, Upadhyaya M, Sarfarazi M, Harper PS (1989) Further evidence localising the gene for Hunter's syndrome to the distal region of the X chromosome long arm. J Med Genet 26:309-313 Rousseau F, Vincent A, Rivella S, Heitz D, Triboli C, Maestrini E, Warren ST, et al (1991) Four chromosomal breakpoints and four new probes mark out a 10-cM region encompassing the fragile-X locus (FRAXA). Am J Hum Genet 48:108-116 Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, et al (1988) Primer directed amplification of DNA with a thermostable DNA polymerase. Science 239:487-491 Schmidt M (1992) Do sequences in Xq27.3 play a role in X inactivation? Am J Med Genet 43:279-281 Schmidt M, Certoma A, Du Sart D, Kalitsis P, Leversha M, Fowler K, Sheffield L, et al (1990) Unusual X chromosome
inactivation in a mentally retarded girl with an interstitial deletion Xq27: implications for the fragile X syndrome. Hum Genet 84:347-352 Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503-517 Sutherland GR, Gedeon A, Kornman L, Donnelly A, Byard RW, MulleyJC, Kremer E, et al (1991) Prenatal diagnosis of fragile X syndrome by direct detection of the unstable DNA sequence. N Engl J Med 325:1720-1722 Suthers GK, Hyland VJ, Callen DF, Oberle I, Rocchi M, Thomas NS, Morris CP, et al (1990) Physical mapping of new DNA probes near the fragile X mutation (FRAXA) by using a panel of cell lines. Am J Hum Genet 47:187195
Suthers GK, Oberle I, NancarrowJ, MulleyJC, Hyland VJ, Wilson PJ, McCure J, et al (1991) Genetic mapping of new RFLPs at Xq27-q28. Genomics 9:37-43 Wilson PJ, Morris CP, Anson DS, Occhiodoro T, Bielicki J, Clements PR, Hopwood JJ (1990) Hunter syndrome: isolation of an iduronate-2-sulphatase cDNA clone and analysis of patient DNA. Proc Natl Acad Sci USA 87: 8531-8535 Wilson PJ, Suthers GK, Callen DF, Baker E, Nelson PV, Cooper A, Wraith JE, et al (1991) Frequent deletions at Xq28 indicate genetic heterogeneity in Hunter syndrome. Hum Genet 86:505-508 Yu S, Pritchard M, Kremer E, Lynch M, NancarrowJ, Baker E, Holman K, et al (1991) Fragile X genotype characterized by an unstable region of DNA. Science 252:11791181
