Deletions in Xq28 in two boys with myotubular myopathy and abnormal genital development define a new contiguous gene syndrome in a 430 kb region
Deletions in Xq28 in two boys with myotubular myopathy and abnormal genital development define a new contiguous gene syndrome in a 430 kb regionLing-Jia Hu+, Jocelyn Laporte+, Wolfram Kress1, Petra Kioschis2, Renate Siebenhaar2, Annemarie Poustka2, Michel Fardeau3, Aida Metzenberg4, Emiel A. Janssen5, Nick Thomas6, Jean Louis Mandel* and Niklas Dahl7,*
Institut de Génetique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, 67404 Illkirch Cedex, Strasbourg, France,1Department of Human Genetics, University of Wurzburg Biocentre, 8700 Wurzburg, Germany, 2Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany, 3INSERM U153, 75005 Paris, France, 4Department of Biology, California State University Northridge, Northridge, CA 91330-8303, USA, 5Department of Neurology, Academical Medical Center, 1105 AZ Amsterdam, The Netherlands, 6Department of Genetics, University of Wales College of Medicine, Cardiff CF4 4XN, UK and 7Department of Clinical Genetics, University Hospital, S-751 85 Uppsala, Sweden
Received September 9, 1995; Revised and Accepted October 10, 1995
We have recently described a female patient with myotubular myopathy (MTM1) and an interstitial deletion at Xq28. Characterisation of the deletion allowed us to position the MTM1 gene to a 600 kb region between DXS304 and DXS497. In order to further restrict the region we screened for deletions in a set of 38 patients. We found two overlapping deletions in boys that in addition to MTM1 showed an unexpected abnormal genital development. As the latter phenotype is not found in the other non-deleted MTM1 patients, our observations are best explained by a contiguous gene syndrome. The deletions define a 430 kb region that contains the MTM1 gene and most likely a gene implicated in male sexual development. A high resolution physical map of this region is presented.
X-linked recessive myotubular myopathy (MTM1; MIM31040) is characterised by congenital hypotonia and muscle weakness. As a consequence, marked respiratory insufficiency is present. A muscle biopsy shows small rounded fibers resembling myotubes with nuclei in central position. Stillbirth and perinatal death is common and affected boys rarely survive the first year of life. No significant clinical manifestation has been noted in female carriers. The rare autosomal form of myotubular myopathy is distinguished from the X-linked type by a lesser severity of clinical symptoms in affected males in combination with findings in muscle biopsies (1 ).
Previous linkage studies have shown evidence for linkage between MTM1 and several polymorphic DNA markers at Xq28 (2 -6 ). A candidate region spanning 10-12 cM was delimited (7 ) but the relative position of the MTM1 gene within the interval remained unclear due to the lack of informative families and key recombinations. In recent reports we have provided evidence for a much more precise location of the MTM1 gene in the proximal part of Xq28 (8 -9 ). Linkage data and the analysis of the deletion in a female patient refined the localisation of the disease gene to a physical region of 600 kb.
The region has previously been cloned in YACs (10 -11 ). As part of a more detailed characterisation of the region we constructed a partial cosmid contig and continued the search for deletions. We describe here two microdeletions in boys affected by myotubular myopathy and abnormalities of their external genitalia. The findings allowed us to further restrict the MTM1 gene location and suggest the presence within these two deletions of a gene involved in male sexual development.
A physical map of the candidate MTM1 region is shown in Figure 1 . The MTM1 gene was localised to a 600 kb region flanked by the marker loci DXS304 and DXS497 (9 ). This localisation was based on the results from two deletions, one in a female affected by MTM1 and another in a male affected by Hunter syndrome but without MTM1. In order to characterise the candidate region in more detail we localised known polymorphic probes from DXS304 to DXS497 using DNA from the two previously described individuals with overlapping deletions. Five marker loci were mapped to the interval (DXS334, DXS462, DXS341, DXS455 and 545R). The probes were hybridised to membranes containing cosmids from the ICRF flow-sorted X chromosome library (12 ), the Lawrence Livermore X chromosome-specific library or a Xq28 specific cosmid library (13 ). A cosmid contig spanning ~300 kb was constructed (Fig. 1 ). The region has previously been covered in YACs (10 -11 ) and positive cosmids were verified by hybridisation to YACs XY316 and XY545.
Single copy RFLP markers, STSs and the newly isolated markers between DXS304 and DXS497 were used to screen for deletions in DNA from an initial set of 24 patients with an MTM1 diagnosis. From this analysis, two deletions were found (Fig. 2 ). Interestingly, when going back to the clinical information, we found out that both patients were described as having malformed external genitalia. In patient 474, the marker locus DXS497 failed to hybridise whereas DXS304 gave a positive signal. This indicates a deletion telomeric of DXS304. In patient 441, marker locus DXS304 failed to hybridise whereas DXS497 was present, indicating a deletion centromeric of DXS497. Further analyses showed that the 545R marker, located centromeric of DXS497, was present in 441. The single copy markers DXS334, DXS462, DXS341, DXS455, et13, etb5, 3a22 and m22t were deleted in both individuals (Fig. 3 ).
We have initially collected 24 unrelated patients with an MTM1 diagnosis. All patients fulfilled the diagnostic criterias for MTM1 (1 ). Two of the patients (cases 474 and 441) had, in addition to myotubular myopathy, abnormalities in the development of external genitalia.Case 474. The proband was born at 41 weeks of gestation after a normal delivery but with apgar scores of 4, 5 and 5 (at 1 min, 5 min and 10 min respectively). There was no family history of neuromuscular disorders, late abortions or stillbirth. A generalized hypotonia was observed and he was treated for respiratory difficulties. Muscle biopsy revealed small rounded muscle fibers with centrally placed nuclei. Staining for glycogen and oxidative enzymes was increased. Physical examination showed subnormal tendon reflexes and mild joint hyperextensibility. Genitalia were ambigous including enlarged clitoris or micropenis and bifid scrotum without palpable testes (Fig. 4 ). An introitus vagina was present and genitography showed a vaginal pouch of 2-3 cm. He was initially considered female but chromosome analysis showed a normal 46,XY karyotype.
The MTM1 gene was recently mapped to a region of 600 kb at Xq28. A map was constructed over the region and probes were tested by hybridisation and PCR on a set of 24 unrelated patients. Another set of 14 patients were more cursorily studied using PCR of STSs only. Submicroscopic deletions were found in two boys with MTM1 and genital abnormalities. The two deletions extend in opposite directions with a common overlap of no more than 430 kb. Suitable single copy markers in a 180 kb region distal to DXS304 have a potential to precisely locate the breakpoint in 474 and to further narrow down the candidate region. Two putative CpG islands were observed and correlate with the ones described previously (17 ).
Both patients had a classical MTM1 phenotype but their genital abnormalities strongly suggest that, within these deletions, lies a gene implicated in external genital development in males. There have been no previous reports about MTM1 and associated genital abnormalities and none of the other MTM1 patients we studied shared such symptoms. In a recent survey describing clinical findings associated with MTM in 37 independent cases (1 ) a high proportion showed cryptorchidism as the only feature affecting external genitalia. Whether this is due to a primary or secondary disturbance of genital development is unclear.
Although the MTM phenotype was classical in both cases, the genital abnormalities differed in the two boys. Patient 474 showed severe genital malformations including severe hypospadias, micropenis and a bifid scrotum. Hormone levels were found normal. The patient 441 showed less pronounced genital abnormalities with hypospadias but a normal scrotum. An abnormal androgen binding was found in cultured fibroblasts from this patient, suggesting a qualitative and quantitative binding defect. The action of androgen is of importance in male genital development and mutant androgen receptors are associated with a spectrum of genital abnormalities in males including hypospadias (18 ). As a consequence, two different mutations on a single X chromosome might possibly explain the phenotype in case 441. Deficiency of 5-alpha-reductase has been associated with hypospadias and pseudohermaphroditism but this was excluded in the two deleted patients. However, the fact that genital abnormalities were seen only in the two boys with deletions and not in the 36 other patients speaks in favour of a contigous gene syndrome involving a gene for male sexual development. Such cases greatly favor the mapping and isolation of disease genes (19 ).
Many genes have been identified as playing a role in male sexual development but none have been mapped to the Xq28 region. The two cases presented were sporadic and did not allow for any linkage studies in the families. In principle, in contiguous gene syndromes, one expects to find instances where phenotype is single. In our example one would expect either MTM1 only or abnormal genitalia only. The latter has not been observed as being linked to Xq28 because of various possible reasons. It is a sex-limited phenotype which is difficult to distinguish from sex-linked. Secondly, it is heterogeneous both in terms of symptoms and genes involved. However, our findings show that such a gene may indeed exist. Our findings also agree with previous locations for the MTM1 locus and have further restricted the candidate region. Several CpG islands have been identified and the cosmid contig of 300 kb is being expanded in the common deletion overlap, in the search for corresponding coding sequences.
Genomic DNA was prepared from cultured fibroblasts, cultured lymphoblastoid cell lines or venous EDTA blood (20 ) and analysed as described (21 ). The probes DXS304 (22 ), DXS334 and DXS341 (23 ), DXS462 (10 ), DXS455 (24 ), DXS497 (25 ), 3a22 (16 ; Siebenhaar, unpublished), m22t and 545R were labeled by random priming and used for Southern blot analyses of DNA as described elsewhere (21 ). M22t is a 300 bp single copy probe derived from cosmid 22 using primers defined in Table 1 . The probe 545R was generated from YAC XY545 by PCR amplification (10 ) followed by size separation on a 2% agarose gel.
The two YACs XY545 and XY316 are derived from the St Louis library (26 -27 ) and were provided by Dr Michele d'Urso, Naples, Italy. DNA from the YACs were prepared in low melting point agarose blocks using standard protocols and were directly used for PCR amplification or pulsed-field gel electrophoresis. Cosmid libraries were screened by hybridisation using high density gridded filters constructed at ICRF (12 ), LLNL (Lawrence Livermore National Laboratories, CA) or filters from a Xq28 specific cosmid library (13 ).
Exon trapping were performed using the pSPL3 vector as described previously (14 ,15 ). Spliced products (et13 and etb5) were subcloned in pBluescript (Stratagene) or pAMP1 (Boehringer) and sequenced on an ABI automated sequencer and analysed with the computer program BLAST. PCR assays for genomic DNA were developed for these correctly spliced products (Table 1 and Laporte et al., in preparation).
This study was supported by grants from the Association Francaise contre les Myopathies (AFM), Groupement de Recherche et d'Etudes sur les Génomes, INSERM, CNRS, the Swedish Medical Research Council, Markus Borgströms foundation, the Sven Jerring Foundation, The Swedish Medical Society, Barncancerfonden, Torsten and Ragnar Söderbergs fund, the Deutsche Forschungsgemeinschaft and the European community genome analysis program. We wish to thank Christine Krez for excellent technical assistance, Dr Carina Wallgren-Pettersson, Dr Gail E. Herman and members of the European MTM consortium sponsored by funds from the ENMC. We wish to thank Drs S. Warren, D. Barker and K. Davies for gift of probes and M. d'Urso for YACs. The X chromosome specific gene libraries used in this work were constructed at and distributed by the Imperial Cancer Research Fund, London, UK and Lawrence Livermore National Library, Livermore, CA 94550, USA under the auspices of the National Laboratory Gene Library Project sponsored by US DOE.
1 Wallgren-Pettersson, C. and Thomas, N. (1994) Report on the 20th ENMC sponsored international workshop: myotubular/centronuclear myopathy. Neuromusc. Disord., 4, 71-74.
2 Thomas, N., Williams, H., Cole, G., Roberts, K., Clarke, A., Leichti-Gallati, S., Gerber, A., Meier, C., Moser, H. and Harper, P.S. (1990) X-linked centronuclear/myotubular myopathy: evidence for linkage to Xq28 DNA marker loci. J. Med. Genet., 27, 284-287. MEDLINE Abstract
3 Darnfors, C., Larsson, B., Oldfors, A., Kyllerman, M., Gustavson, KH., Bjursell, G. and Wahlström, J. (1990) X-linked myotubular myopathy: a linkage study. Clin. Genet., 37, 335-340.MEDLINE Abstract
4 Lehesjoki, A.E., Sankila, E., Miao, J., Somer, M., Salonen, R., Rapola, J. and de la Chapelle, A. (1990) X-linked neonatal myotubular myopathy: one recombination detected with four polymorphic DNA markers from Xq28. J. Med. Genet., 27, 288-291.
5 Starr, J., Lamont, M., Iselius, L., Harvey, J. and Heckmatt, J. (1990) A linkage study of a large pedigree with X-linked centronuclear myopathy. J. Med. Genet., 27, 281-283.MEDLINE Abstract
6 Liechti-Gallati, S., Müller, B., Grimm, T., Kress, W., Müller, C., Boltshauser, E., Moser, H. and Braga, S. (1991) X-linked centronuclear myopathy: mapping the gene to Xq28. Neuromusc. Disord., 4, 239-245.
7 Janssen, E.A., Hensels, G., W., van Oost, B., A., Hamel, B., Kemp, S, Baas, F., Weber, J., Barth, P., G. and Bolhuis, P., A. (1994) The gene for X-linked myotubular myopathy is located in a 8 Mb region at the border of Xq27.3 and Xq28. Neuromusc. Disord., 4, 455-461.
8 Dahl, N., Hu, L., Chery, M., Fardeau, M., Gilgenkrantz, S., Nivelon-Chevallier, A., Sidaner-Noisette, I., Mugneret, F., Gouyon, J., Gal, A., Kioschis, P., d'Urso, M. and Mandel, JL. (1995) Myotubular myopathy in a girl with a deletion at Xq27-q28 and unbalanced X inactivation assigns the MTM1 gene to a 600 kb region. Am. J. Hum. Genet., 56, 1108-1116.MEDLINE Abstract
9 Dahl, N., Samson, F., Thomas, N., Hu, L., Gong, W., Herman, G., Laporte, J., Kioschis, P., Poutska, A. and Mandel, JL. (1994) X linked myotubular myopathy (MTM1) maps between DXS304 and DXS305, closely linked to the DXS455 VNTR and a new, highly informative microsatellite marker (DXS1684) J. Med. Genet., 31, 922-924.MEDLINE Abstract
10 Palmieri, G., Romano, G., Casamassimi, A., D'Urso, M., Little, R., Abidi, F., Schlessinger, D., Lagerstöm, M., Malmgren, H., Steen-Bondeson, ML., Pettersson, U. and Landegren, U. (1993) 1.5 mb YAC contig in Xq28 formatted with sequence-tagged sites and including a region unstable in the clones. Genomics, 16, 586-592.MEDLINE Abstract
11 Rogner, U., Kioschis, P, Wilke, K., Gong, W., Pick, E., Dietrich, A., Zechner, U., Hameister, H., Pragliola, A., Herman, G., Yates, J., Lehrach, H. and Poustka, A. (1994) A YAC clone map spanning 7.5 megabases of human chromosome band Xq28. Hum. Mol. Genet., 3, 2137-2146.MEDLINE Abstract
12 Nizetic, D., Zehetner, G., Monaco, A.P., Gellen, L., Young, B.D. and Lehrach H (1992) Construction, arraying and high-density screening of large insert libraries of human chromosomes X and 21: their potential use as reference libraries. Proc. Natl. Acad. Sci. USA, 88, 3233-3237. MEDLINE Abstract
13 Kioschis, P., Gross, B., Nizetic, D., Zehetner, G., Lehrach, H. and Poustka, A. (1991) Molecular analysis of the Xq28 region. Cytogenet. Cell Genet., 58, 2070.
14 Buckler, A.J., Chang, D.D., Graw, S.L., Brook, J.D., Haber, D.A., Sharp, P.A. and Housman, D.E. (1991) Exon amplification: A strategy to isolate mammalian genes based on RNA splicing. Proc. Natl. Acad. Sci. USA, 88, 4005-4009. MEDLINE Abstract
15 Church, D.M., Stotler, C.J., Rutter J.L., Murrel, J.R., Trofatter, J.A. and Buckler, A.J. (1994) Isolation of genes from complex sources of mammalian genomic DNA using exon amplification. Nature Genet, 6, 98-105.MEDLINE Abstract
16 Korn, B., Sedlacek, Z., Nizetic, D., Zehetner, G., Lerach, H. and Poustka, A. (1992) A strategy for the selection of transcribed sequences in the Xq28 region. Hum. Mol. Genet., 1, 235-242.MEDLINE Abstract
17 Palmieri, G., Romano, G., Ciccodicola, A., Casamassimi, A., Campanile, C., Esposito, T., Cappa, V., Lania, A., Johnson, S., Reinbold, R., Poutska, A., Schlessinger, D. and D'Urso, M. (1994) YAC contig organization and CpG island analysis in Xq28. Genomics, 24, 149-158.MEDLINE Abstract
18 Batch, J.A., Evans, B., Hughes, I. and Patterson, M. (1993) Mutations of the androgen receptor gene identified in perineal hypospadias. J. Med. Genet., 3, 198-201.MEDLINE Abstract
19 Tommerup, N. (1993) Mendelian cytogenetics: Chromosome rearrangements associated with Mendelian disorders. J. Med. Genet., 30, 7132-7727.
20 Miller, S., Dykes, D. and Polesky, H. (1988) A simple salting out method for extracting DNA from human nucleated cells. Nucleic Acids Res., 16, 1215.
21 Oberlé, I., Camerino, G., Kloepfer, C., et al. (1986) Characterisation of a set of X-linked sequences and of a panel of somatic cell hybrids useful for the regional mapping of the human X chromosome. Hum. Genet., 72, 43-49.MEDLINE Abstract
22 Dahl, N., Goonewardena, P., Malmgren, H., Gustavson, K-H., Flod, A., Holmgren, G., Annerén, G., Pettersson, U. (1989) Linkage analysis of families with Fragile-X mental retardation using a novel RFLP marker (DXS304) Am. J. Hum. Genet., 45, 304-309.MEDLINE Abstract
23 Barker, D., Dietz-Band, J.N., Donaldson, et al. (1989) Further localisation, characterisation and physical localisation of X chromosome RFLP markers, comparing VNTR directed and random isolation strategies. Cytogenet. Cell Genet., 51, 958.
24 Consalez, G., Stayton, C., Freimer, N., Goonewardena, P., Brown, T., Gilliam, T. and Warren, S. (1992) Isolation and characterisation of a highly polymorphic human locus (DXS455) in proximal Xq28. Genomics, 12, 710-714.MEDLINE Abstract
25 Hirst, M., Roche, A., Flint, T.J., et al. (1991) Linear order of new and establishe DNA markers around the fragile site at Xq27.3. Genomics, 10, 243-249. MEDLINE Abstract
26 Little, R.D., Porta, G., Carle G.F., Schlessinger, D. and d'Urso, M. (1989) Yeast artificial chromosomes with 200- to 800-kilobase inserts of human DNA containing HLA, Vk, 5S and Xq24-Xq28 sequences. Proc. Natl. Acad. Sci. USA, 86, 1598-1602.MEDLINE Abstract
27 Abidi, F.E., Wada, M., Little, R.D. and Schlessinger, D. (1990) Yeast artificial chromosomes containing human Xq24-Xq28 DNA: Library construction and representation of probe sequences. Genomics, 7, 363-376.MEDLINE Abstract
*To whom correspondence should be addressed+Both authors have contributed equally to this study
This page is maintained by OUP admin. Last updated Thu Oct 31 15:08:29 GMT 1996. Part of the OUP Journals World Wide Web service.Copyright Oxford University Press, 1996
