Mutations in the MTM1 gene implicated in X-linked myotubular myopathy
Mutations in the MTM1 gene implicated in X-linked myotubular myopathy
Group 1:Jocelyn Laporte1, Christophe Guiraud-Chaumeil1, Marie-Claire Vincent1, Jean-Louis Mandel1,*, Group 2: Stephan M. Tanner2, Sabina Liechti-Gallati2, Group 3: Carina Wallgren-Pettersson3, Niklas Dahl4, Wolfram Kress5, Pieter A. Bolhuis6, Michel Fardeau7, Françoise Samson8, Enrico Bertini9 and members of the ENMC International Consortium on Myotubular Myopathy
1IGBMC, CNRS/INSERM/ULP, 1 rue Laurent Fries, B.P.163, 67404 Illkirch Cedex, France, 2Department of Clinical Research, University of Berne, 3010 Berne, Switzerland, 3Department of Medical Genetics, University of Helsinki, and the Folkhälsan Department of Medical Genetics, PO Box 211, FIN-00251 Helsinki, Finland, 4Department of Clinical Genetics and Pediatrics, University Hospital, 75185 Uppsala, Sweden, 5Institut der Humangenetik, Universitat Würzburg, 8700 Würzburg, Germany, 6Department of Neurology, AMC,1105 AZ Amsterdam zuid-oost, The Netherlands, 7INSERM U153, Institut de Myologie, Hopital Pitié-Salpêtrière, Paris, France, 8CNRS-URA 1159, Hopital Marie Lannelongue, 92350 Le Plessis Robinson, France and 9Servizio di Neurofisiologia, Ospedale Bambino Gesú, 00165 Roma, Italy
Received April 3, 1997;Revised and Accepted June 17, 1997
X-linked recessive myotubular myopathy (XLMTM) is characterized by severe hypotonia and generalized muscle weakness, with impaired maturation of muscle fibres. The gene responsible, MTM1, was identified recently by positional cloning, and encodes a protein (myotubularin) with a tyrosine phosphatase domain (PTP). Myotubularin is highly conserved through evolution and defines a new family of putative tyrosine phosphatases in man. We report the identification of MTM1 mutations in 55 of 85 independent patients screened by single-strand conformation polymorphism for all the coding sequence. Large deletions were observed in only three patients. Five point mutations were found in multiple unrelated patients, accounting for 27% of the observed mutations. The possibility of detecting mutations and determining carrier status in a disease with a high proportion of sporadic cases is of importance for genetic counselling. More than half of XLMTM mutations are expected to inactivate the putative enzymatic activity of myotubularin, either by truncation or by missense mutations affecting the predicted PTP domain. Additional mutations are missenses clustered in two regions of the protein. Most of these affect amino acids conserved in the homologous yeast and Caenorhabditis elegans proteins, thus indicating the presence of other functional domains.
X-linked recessive myotubular myopathy (XLMTM; OMIM31040) is a congenital muscle disorder characterized by severe hypotonia and generalized muscle weakness. Spontaneous movements are weak or absent. Prenatal onset can often be indicated by polyhydramnios and decreased fetal movements. Miscarriages and stillbirths are frequently observed among obligate carriers. No significant clinical symptoms or signs of myopathy occur in female carriers. The characteristic muscle histopathology consists of small rounded muscle cells with centrally located nuclei surrounded by a halo devoid of contractile elements. These resemble fetal myotubes, and it has been suggested that the disorder results from an arrest in the normal development of muscle fibres (1 ). XLMTM is usually lethal in males because of respiratory failure. Most patients die within the first months of life. A few males have survived several years with a spontaneous improvement of the respiratory function after birth. The reason for this clinical improvement is unknown. Since the first report of myotubular myopathy (2 ), ~100 cases have been documented (3 -5 ). Autosomal centronuclear/myotubular myopathies (CNM) are related disorders with similar histopathological findings, but the age of onset is later with a milder clinical course (4 ).
Exons a, b, c, d and e from Laporte et al. (13) are numbered respectively exon 8, 9, 11, 14 and 15. The stop codon is 1863-1865.
The locus responsible for the disease (MTM1) was mapped to Xq28 by linkage analysis (6 ,7 ). By combining linkage analysis with the study of patients carrying deletions, we restricted the candidate region to 280 kb (8 -12 ), and the MTM1 gene was isolated recently by a positional cloning strategy (13 ). The MTM1 gene is ubiquitously expressed and shows a muscle-specific alternative transcript due to the use of a different polyadenylation signal. It encodes a protein which contains the consensus sequence for the active site of tyrosine phosphatases, a wide class of proteins involved in signal transduction, control of cell growth and differentiation. The MTM1 gene product (myotubularin) is highly conserved in Saccharomyces cerevisiae and Caenorhabditis elegans, which is surprising for a gene implicated in a muscle-specific disorder. Three new homologous human genes were found, and, together with MTM1, they define a new family of putative tyrosine phosphatases in man (13 ).
The cloning of the MTM1 gene has greatly improved the prospects for diagnosis and genetic counselling of the disease. Moreover, identification of the mutations responsible for XLMTM should give some insights into its molecular and physiological basis. We have determined the exon-intron structure of the MTM1 gene (Laporte et al., in preparation), and performed mutation screening in 85 independent patients. Mutations were found in 55 of them, and this allows accurate counselling in the relevant families. The identification of 19 missense mutations and small insertions or deletions of amino acids should be useful for functional studies of myotubularin.
The MTM1 gene, located in the proximal part of Xq28, consists of 15 exons, for which flanking intronic sequences have been determined (Laporte et al., in preparation). In the original description of the cDNA sequence, the location of the translation initiation site had not been determined. More recent data using antibodies directed against myotubularin indicate that the protein is translated starting from the methionine codon at position 19 in the putative amino acid sequence previously reported [(13 ), GenBank U46024]. The endogenous protein immunoprecipitated from myoblast extracts has the same size as that synthesized in COS cells transfected with the full-length cDNA, where the first ATG codon is at position 19 (unpublished results). We have thus renumbered the amino acid positions.
The MTM1 gene was screened for mutations using single-strand conformation polymorphism (SSCP) followed by sequencing of abnormal bands, based on the knowledge of intronic sequences flanking all of the 15 exons (see primers and conditions in Table 1 ). Among 85 patients screened for mutations for all the coding sequence, we found 52 point mutations (Table 2 ). Three patients had large deletions. Seven of the point mutations and two large deletions were reported previously (11 ,13 ). The 52 point mutations consist of 33 base changes and 19 small insertions/deletions. Sixteen of the base substitutions affect CpG dinucleotides. Twenty seven mutations are frameshift or nonsense mutations. Alteration of donor or acceptor splice sites account for six of the mutations. Including the large deletions, 65% of the mutations would result in the absence or truncation of myotubularin.
An A(1314-10)G change in intron 11 was found in four unrelated patients and creates a new functional acceptor site that leads to the insertion of three amino acids in a conserved region of the protein as indicated by RT-PCR experiments (14 ,28 ).
Missense mutations were found concentrated in three areas: exons 4 and 5, exons 8 and 9 and exons 11 and 12 (Fig. 1 ). At least 12 of the 16 different missenses or single amino acid deletions affect residues that are completely conserved in the homologous proteins predicted from yeast and C.elegans sequences (13 ), as indicated in Table 2 . The N-terminal domain of myotubularin (from exons 2 to 7) is only weakly homologous to its counterparts in yeast and C.elegans, making the alignment less reliable. The four other missense mutations are in any case non-conservative (del K47, R69C, L70F and S229P). We tested all exons on at least 40 normal chromosomes under the same SSCP conditions, and did not find these mutations. This further supports the conclusion that these alterations are indeed disease causing.
aEach patient represents one family. For DA1 and CNM5, CNM10, CNM14 and CNM19, maternal DNA was analysed, as no DNA was available from the affected boys. bSequence changes are presented according to the nomenclature proposed by the Ad Hoc Committee on Mutation Nomenclature (27). Nucleotide numbers refer to their positions in the sequence of the MTM1 transcript (GenBank U46024). Starred nucleotide changes affect a CpG dinucleotide. cThe amino acid numbering starts at the first methionine of the open reading frame [codon 19 in (13)]. The numbering has therefore been modified, for the mutations previously reported (13). dIn regions where the homology between myotubularin and yeast or C.elegans homologues is low (N-terminal domain of the protein), a question mark indicates that the alignment is ambiguous. eIn a few cases, two brothers were affected, and their age at death is indicated. fIn the mutation origin column, mc means that the mother carries the mutation, other mentions (maternal, grand paternal etc.) indicate the origin of a new mutation. gNHGMCR stands for Nigms Human Genetic Mutant Cell Repository.
Rare variants were found in either normal individuals or in patients carrying a characterized mutation, except for 1755C/T, found in a patient for whom no mutation has been detected yet. The first base indicated corresponds to the common allele and the frequency is calculated for the rare allele. aSee Laporte et al. (13).
Although no point mutations were found in exons 1, 10, 14 and 15, our results are consistent with a widespread occurrence of mutations along the gene. The C-terminal end of the protein may be of lesser functional importance as it is not well conserved in yeast and C.elegans, and as one would expect ~10 mutations in exons 14 + 15, based on the length of their coding sequences. Exons 4, 8, 9, 11 and 12 showed a higher tendency to mutate in this panel of patient samples.
Ten polymorphisms or rare variants have been characterized to date (Table 3 ). The T -> C transition at position 615 affects a wobble base and was found in only one patient (CU32) who carries a deleterious mutation (R224X). The (1314+17)A/G was found in G93-401, who already has the same R224X mutation, and in two normal chromosomes (15 ). The frequent (1314+3)A/G polymorphism is located in the donor splice site of intron 11. It is unlikely that it affects the splicing efficiency in the normal population as G or A are equally accepted at the +3 position of the consensus donor site. The 1755C/T variant that does not affect the amino acid sequence was found in a patient for whom no other mutation has yet been observed, but this variant is unlikely to be pathological.
The nonsense mutations R37X, R224X and R474X, corresponding to C -> T transitions at CpG sites, were found in two, three and three independent families, respectively. A 4 bp frameshift deletion in exon 4 was found in three patients. This recurrent deletion does not correspond to a replication slippage affecting a tandem repeat, a frequently observed cause of small deletions. The splice mutation in intron 11 (A 1314-10G) which was found four times, shows no obvious feature that could account for its high incidence.
Patients showing the same mutation were analysed with microsatellites in the MTM1 region [DXS8377 and 7423 (17 )] which confirmed that they were from different families (not shown), except for patients CNM15 and EA5 who carry the splice mutation in intron 11 on the same haplotype. The mutation in EA5 is a maternal new mutation, indicating that the mutations in EA5 and CNM15 are independent.
Although in many cases we did not have DNA from the mother or other family members to trace the inheritance of the mutation, in the cases where such analyses could be performed, we found a high proportion of maternal or grandparental new mutations. In particular, seven of 28 mothers tested did not carry their son's mutation, which is close to the expected one-third (Table 2 ).
The MTM1 gene, which encodes a 603 amino acid protein, was screened for mutations in 85 patients by SSCP analysis, over all the coding sequence. Fifty five mutations and 10 polymorphisms and rare variants were identified. As expected for a lethal X-linked disease, we found a high heterogeneity of mutations, widespread through the coding sequence, of which a high proportion were new mutations. Truncated proteins are predicted to result from ~65% of the mutations.
Five recurrent mutations account for nearly one-third of all mutations, and single nucleotide changes affect preferentially CpG dinucleotides. The only exception is the splice mutation in intron 11 resulting in the insertion of three amino acids (FIQ) at position 420 and observed in four independent patients. There are no obvious sequence features that may account for the high incidence of this mutation (also observed by de Gouyon et al., 28 ), and for the recurring 4 bp deletion in exon 4 (195delAGAA). The parental origin of these mutations is under investigation.
The number of mutations found is lower than expected, considering that we screened all the coding sequence. This might be accounted for by preferential localization of mutations in regions not yet analysed (regulatory sequences, introns) or by the fact that we did not detect all mutations by SSCP analysis. As sporadic cases account for a high proportion of the patients, some patients may not have XLMTM. We cannot exclude the possibility, at least in a small proportion of patients, of another gene defect causing a similar phenotype. We reported recently the finding of an MTM1 mutation in a family previously thought to indicate genetic heterogeneity in XLMTM (18 ). There is thus no more evidence of genetic heterogeneity in proven X-linked cases (15 ).
The proportion of sporadic cases in an X-linked lethal disease like XLMTM is >50%. Genetic counselling is especially difficult in such cases, as there is no reliable way of assessing carrier status in females (4 ,15 ). Direct mutation detection thus becomes the most reliable method for prenatal diagnosis and carrier detection (13 ,14 ). Exons 4, 8, 9, 11 and 12 showed a higher tendency to mutate and may be tested first. Together, these five exons accounted for 73% of all the mutations found. Three of the recurrent mutations are included in these five exons. RT-PCR can be an alternative method in order to avoid analysis of individual exons, but this requires adequately stored cells.
In the future, the assessment of the presence of the protein in muscle biopsies or in leukocytes by Western blot or immunohistochemistry (19 ) may become an alternative to mutation screening, if the level of expression is sufficient. Sixty five percent of the mutations are predicted to truncate the protein, thus probably leading to its degradation. Furthermore, some missense mutations may also lead to selective degradation of myotubularin by affecting its stability or its appropriate targeting to subcellular compartments.
The MTM1 gene is probably involved in a signal transduction pathway necessary for late myogenesis, although its ubiquitous expression suggests a wider function. Almost all changes, insertion or deletion of amino acids, observed to date in patients affect regions and residues that are conserved in the homologous yeast and C.elegans proteins, indicating their functional importance.A tyrosine phosphatase active site is predicted in myotubularin. Although phosphatase activity has not yet been demonstrated, two of the disease-causing mutations are missenses within the PTP signature (amino acids 373-385) and eight involve neighbouring amino acids (encoded in exons 11 and 12) that may belong to the broader PTP domain which usually encompasses ~250 amino acids around the active site (20 ). The A(1314-10)G intronic mutation causes an insertion of three amino acids (420insFIQ) in a very conserved region close to the PTP site, where we also found the R421Q missense.
We observed two other preferential regions for missenses. Four missense mutations were found in exons 8 and 9, corresponding to a region conserved throughout evolution (13 ). The other region corresponds to exons 4 and 5, but here the conservation in the yeast or C.elegans homologous genes is low. These two domains of the protein may be involved in substrate recognition, appropriate targeting to cellular compartments or interaction with other proteins. The missense mutations we observed will be useful when studying putative functions or protein interactions of myotubularin. Although no missense mutations were found in exons 14 and 15, the recurring R474X nonsense mutation truncates this part of the protein, indicating that the C-terminal domain is important for the structure/stability of myotubularin.
Milder autosomal forms of CNM have been described (4 ). Candidate genes for such forms are the MTM-related genes, and genes coding for putative proteins that interact with myotubularin or belong to the same signal transduction pathway. Identification of genes implicated in autosomal CNM may thus shed light on the function of myotubularin.
Given the ubiquitous expression of the gene and the existence of a well-conserved gene in yeast, it is surprising that no tissues other than muscle have been clearly found to be affected in XLMTM patients (21 ). Other associated phenotypes might have been missed, as most patients die very early. Alternatively, tissue-specific pathology could result from interaction with muscle-specific proteins, or from a different balance of proteins with overlapping functions encoded by the MTM-related genes.
Additional clinical features were only described for patients with large deletions, and suggest a contiguous gene syndrome (11 ). A gene involved in male sexual development must be centromeric to MTM1 in the region deleted in G89-441 and G89-474, but present in CM73. The F18 gene and XAP80-XAP87 transcripts represent candidates for such function (22 ,23 ).
From our data, there is no obvious correlation between the type of mutation and the severity of the disease. Some patients with nonsense or frameshift mutations have survived 7-14 years (mutations 451delAT, C718T and 890delC), while other patients with missense mutations died in the neonatal period. Moreover, patient 4711 (451delAT) is still alive at 7 years but a brother died neonatally. The missense R241C led to a mild phenotype in two cases [patient N2-9 in this study, and (24 )], while a R241L mutation caused a severe phenotype (28 ). Because of the existence of such milder phenotypes, it may be important to check for mutations in cases diagnosed as autosomal recessive centronuclear/myotubular myopathy if the autosomal inheritance is not well established.
It is possible that in most cases survival depends more on clinical treatment and other parameters (prematurity, putative modifying genes) than on the nature of the mutation. It has indeed been suggested that, once a patient survives beyond a certain point, he may have improvement in his condition (4 ).
We conclude that the MTM1 gene is indeed the principal and probably the only gene implicated in XLMTM. Mutation detection has already proven to be very useful for genetic counselling, carrier detection and prenatal diagnosis in families.
Genomic DNA was prepared from cultured fibroblasts, cultured lymphoblastoid cell lines or venous EDTA blood (25 ). Primer sets were designed from intronic and exonic sequences flanking the 15 exons of the MTM1 gene. Primer sequences and PCR conditions are summarized in Table 1 . Briefly, 100 ng of patient DNA were amplified in 10 mM Tris-HCl pH 8.3, 50 mM KCl, 200 [mu]M dNTP, the defined MgCl2 concentrations, with 10 pmol of each primers, 1 nmol (3[mu]Ci) of [32P]dCTP and 1 U of Taq polymerase in a total volume of 25 [mu]l. Thermocycling was carried out in the PTC-100 Programmable Thermal Controller (MJ Research, Inc.) at 94oC for 5 min followed by 30 cycles at 94oC for 10 s, annealing temperature for 10 s, 72oC for 10 s, and a final elongation step at 72oC for 5 min. For exon 1, the PCR was performed with 3 min elongation at each cycle and with 10% dimethylsulfoxide (DMSO) and 3.75 nmol of C7deaza guanine; then the product was digested by SalI into two fragments (176 and 279 nucleotides).
After PCR, the products were denaturated at 94oC for 10 min with 6 [mu]l of SSCP loading buffer (0.2 M NaOH, 0.25% w/v each bromophenol blue and xylene cyanol in formamide) and separated on a 6% native polyacrylamide gel. For each primer set, gels were run at 4oC under two conditions: overnight at 6 W and 5 h at 30 W. Shifted bands were excised from the gel and reamplified in 200 [mu]l, then purified using the Geneclean Kit (Bio 101, Inc.) and sequenced on an Applied Biosystems (ABI) automated sequencer with fluorescent dideoxynucleotides using the same primers as for PCR.
For patients identified as CNMx, the same PCR amplification was carried out in a Thermal Cycler 2400 (Perkin Elmer). Initial denaturation was at 95oC for 2 min, followed by 28 cycles of 95oC for 15 s, annealing for 15 s (at 52oC: exons 5, 10, 12, 13, 14; at 56oC: exons 3, 4, 6, 8, 9, 11, 15), and 72oC for 45 s. Final extension was at 72oC for 7 min. PCR products were analysed after denaturation on 12% acrylamide gels containing 7.25% glycerol using a two-buffer system (Liechti-Gallati et al., submitted). DNA was visualized by silver staining (26 ). Sequence changes were detected either by double or single strand band shifts or by the generation of heteroduplex on the same gel. SSCP variants were purified directly using QIAquick PCR purification columns (QIAGEN) according to the supplier and sequenced.
Large deletions were confirmed by PCR amplification of the different exons using the primers in Table 1 , and by Southern blot from patient genomic DNA (11 ) digested with BstYI or MspI and hybridized with exon-specific PCR products.
In order to confirm that patients with recurrent mutations were from different families, they were all genotyped using polymorphic markers DXS8377 and DXS7423 closely flanking the MTM1 gene (17 ).
We wish to thank Christine Kretz, Serge Vicaire and Vreni Schneider for excellent technical assistance in SSCP analysis and the sequencing work. We would also like to thank all clinicians listed in Table 2 and the Genethon for tissues and blood samples of XLMTM families.Collaboration within the ENMC International MTM Consortium was sponsored by the EAMDA and the European Neuro-Muscular Center (Baarn, The Netherlands). This study was supported by the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique, the Centre Hopitalier Universitaire Régional, and by grants from the Association Française contre les Myopathies (AFM), the Swiss National Research Foundation (No. 32-043631.95) and Ronald McDonalds fund.
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*To whom correspondence should be addressed. Tel: +33 388 653244; Fax: +33 388 653246; Email: mandeljl@igbmc.u-strasbg.fr
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