Characterization of mutations in the myotubularin gene in twenty six patients with X-linked myotubular myopathy
Characterization of mutations in the myotubularin gene in twenty six patients with X-linked myotubular myopathyBeatrice M. de Gouyon, Wei Zhao, Jocelyn Laporte1, Jean-Louis Mandel1, Aida Metzenberg2 and Gail E. Herman+,*
Department of Molecular and Human Genetics and Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA, 1Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM/ULP, Illkirch, CU de Strasbourg, France and 2Department of Biology, California State University, Northridge, CA 91330, USA
Received March 26, 1997;Revised and Accepted June 17, 1997
A candidate gene, myotubularin, involved in the pathogenesis of X-linked myotubular myopathy (MTM1) was isolated recently. Mutations originally were identified in 12% of patients examined for 40% of the coding sequence, raising the possibility that additional genes could be responsible for a proportion of X-linked cases. We report here the identification of mutations in 26 of 41 independent male patients with muscle biopsy-proven MTM, by direct genomic sequencing of 92% of the known coding sequence of the myotubularin gene. Eighteen patients had point mutations, including one A/G transition found in four patients which alters a splice acceptor site in exon 12 and leads to a three amino acid insertion. Six patients had small deletions involving <6 bp, while two larger deletions encompassed two or six exons, respectively. No differences were noted among the types of mutations between familial and sporadic cases. However, all of the five patients with a mild phenotype had missense mutations. While 50% of the mutations were found in exons 4 and 12, and three distinct mutations were found in more than one patient, no single mutation accounted for more than 10% of the cases. The low frequency of large deletions and the varied mutations identified suggest that direct mutation screening for molecular diagnosis may require gene sequencing.
X-linked myotubular myopathy (MTM1; McKusick #31040) is a congenital myopathy in which affected boys present at birth with severe hypotonia and respiratory insufficiency, leading to a high neonatal mortality. Surviving patients have prolonged ventilator dependence and grossly delayed motor milestones, but they usually have intact intelligence (1 -3 ). Autosomal recessive and autosomal dominant forms of MTM with milder phenotypes have also been described (1 ,3 ). Until recently, the diagnosis of MTM1 rested on finding the characteristic pathology of central nuclei in muscle biopsies from affected males and on a family history of similarly affected male relatives in the maternal lineage. In sporadic cases, characteristic pathology and a severe clinical presentation suggest the X-linked form of the disease. No specific clinical signs are detectable in obligate female carriers, and abnormal muscle biopsies are found in only 50-70% (3 ,4 ).
Since 1990, several groups have reported linkage of MTM1 to Xq28 (5 ,6 ). Further linkage studies and the analysis of a female with mental retardation and a mild form of myotubular myopathy helped to refine the minimum critical region to ~600 kb (7 ,8 ). Several complementary positional cloning approaches (9 -11 ) eventually led to the isolation of a candidate MTM1 gene in 1996, and mutations were found in several patients (12 ). The gene codes for a protein named myotubularin and appears to be ubiquitously expressed, with a 3.9 kb transcript detected in all adult and fetal tissues studied. A second 2.4 kb transcript is found only in skeletal muscle and testis (12 ). The originally reported cDNA may lack some sequences at the 5' end, and exon-intron boundaries were described for only five exons. The myotubularin protein is predicted to contain 603 amino acids (Laporte et al., in preparation), and a small domain in its protein sequence shows high homology to a consensus active site of tyrosine phosphatases. In addition to MTM1, three highly related human genes, designated MTM-related one, two and three (MTMR1, 2 and 3) were also identified (11 ,12 ). MTMR1, corresponding to the cDNA contig XAP86 (11 ), lies ~100 kb telomeric to MTM1. This gene could also be a candidate for MTM1 since in the original report of the gene isolation only seven mutations were identified in the 60 independent MTM1 patients studied (12 ). However, only five exons were screened for mutations and, for some of these exons, PCR primers were designed to sequences within the exon itself. Fifteen exons are now known to comprise the MTM1 locus (Laporte et al., in preparation), and much of the genomic structure has been elucidated, enabling a more systematic search for mutations.
aA schematic representation of the genomic structure of the MTM1 locus is shown in Figure 1. bThe numbering of nucleotides in the cDNA has been reported (12), and the abbreviations used to designate mutations are taken from (21). cAmino acids listed in bold type are conserved in the presumed C.elegans and/or S.cerevisiae MTM1 homologs. The numbering scheme for the amino acids is from (15), with translation beginning at the first methionine in the cDNA sequence (12) at bp 55. dThe phenotypic classification of patients is described in Materials and Methods. eA (-) indicates there is a negative family history. One male sibling or one or more maternal uncles were affected in those families where indicated. fA (+) indicates that the mother was analyzed and determined to be heterozygous for the mutation. A (-) indicates that the mother was analyzed and determined not to be a carrier. gWe could not distinguish between the deletion [Delta]AAAG 193-196 and [Delta]AGAA 195-198, between [Delta]ACTT 643-646 and [Delta]TTAC 645-648, or between [Delta]GTAAA 392-396 and [Delta]396A/GTAA because of the sequences adjacent to each deletion. The slash (`/') in [Delta]396A/GTAA indicates the exon-intron boundary at the end of exon 5. hNo clinical information is available on this patient, and he was not included in the totals regarding the severity of the phenotype or the presence or absence of a positive family history. iThe exact extent of the deletion is not known because of our inability to analyze exon 7.
We report here the identification of mutations in the myotubularin gene in 63% of 41 unrelated North American MTM1 patients using direct genomic sequencing of 12 exons from the MTM1 gene. While several of these mutations were detected more than once, no single mutation accounts for more than 10% of those identified. The implications of these findings for molecular diagnosis of MTM1 and for establishing whether a single X-linked gene is responsible for all X-linked cases are discussed.
As a first step toward understanding the pathogenesis of X-linked MTM, we have performed mutation studies on a collection of 41 independent males with biopsy-proven myotubular/centronuclear myopathy (see Materials and Methods). Genomic DNA from patients was screened initially for mutations using single strand conformational polymorphism (SSCP) and heteroduplex analysis (HA). However, a low number of aberrant fragments representing potential mutations were identified, and we switched our approach to utilize direct automated genomic sequencing as the structure of the MTM1 gene and intronic PCR primers became available. To date, we have found 26 mutations in the 41 patients (63%) by determining the sequence of 12 exons representing 92% of the known MTM1 coding sequence (see Materials and Methods). A single polymorphism has been detected in an intron 3' to exon 11 (IVS11+3A/G): 34% of the X chromosomes sequenced have an `A' at this position and 66% have a `G' at this position (data not shown). Additional coding sequence alterations were identified in MTM1 patients with either base at this site, suggesting that it does not constitute a mutation or significantly influence the phenotype.
A summary of the mutations that we have identified is presented in Table 1 and Figure 1 . Thirteen distinct point mutations were found, of which 10 are single amino acid missense mutations and two are nonsense mutations. One of the point mutations alters a splicing consensus site before exon 12 (see Fig. 2 A and B) and would be predicted to lead to an in-frame insertion of three amino acids. This mutation, as well as a missense mutation in exon 4, was observed in multiple unrelated patients. Two of the patients demonstrated relatively large intragenic deletions involving at least two and six exons, respectively. These deletions were detected initially as a lack of PCR amplification using intronic primers specific for the involved exons, and both were confirmed by Southern hybridization using the MTM1 cDNA (12 ) as a probe (not shown). Six patients were found to have small deletions: two distinct 4 bp deletions result in frameshift mutations (patients 1-4 and patient 13) and distal termination codons after amino acid 70 in exon 4 or after amino acid 247 in exon 9, respectively. The first deletion [Delta]193-196, was found in four independent cases. Two of the cases with this mutation are familial, and the other two have a negative family history. Finally, a third deletion event removes five nucleotides at the end of exon 5 ([Delta]392-396) in patient 9 and results in a truncated protein after amino acid 112.
Samples were available to analyze eight of the 17 mothers of sporadic cases for which a mutation was identified (see Table 1 ). For the mother of T.D., one of two sporadic cases with the [Delta]AAAG deletion in exon 4, we were able to detect the heterozygous mutation by SSCP (not shown). We subsequently sequenced multiple clones obtained from her PCR product and confirmed the SSCP result. No mutation was detected after sequencing 11 clones from the mother of the second sporadic case (W.C.) or upon direct genomic sequencing of her PCR product. This mother is very unlikely to be a carrier (<10% risk), although we cannot exclude the possibility of gonadal mosaicism. All of the other sporadic cases for which a maternal sample was available involved point mutations, and the SSCP and HA results were inconclusive. Direct genomic sequencing and sequencing of cloned PCR products were employed to establish the maternal carrier status (see Table 1 ). In total, seven of the eight mothers studied (88%) were found to be carriers. Additional families will need to be studied to extend these results and to determine whether they deviate significantly from the 67% predicted for a lethal X-linked recessive disorder where mutation rates are equal in male and female gametes (13 ).
The data presented here confirm that the majority, if not all, patients with X-linked myotubular myopathy have mutations in the recently identified myotubularin gene (12 ). In the initial report, seven mutations were detected in 60 patients by screening five exons using SSCP; the mutations were then confirmed by DNA sequencing. We have analyzed 41 unrelated, male MTM1 patients and detected mutations in 26 of them (63.4%). The diagnosis of MTM1 was confirmed by finding the characteristic pathology on a muscle biopsy, and a diagnosis of congenital myotonic dystrophy was excluded in all 41 patients. Three of the patients for whom no mutation was found have had an extremely mild course, with their initial presentations between 5 and 8 months of age, and they may represent autosomal forms of MTM (G. Herman, in preparation). We screened only 92% of the known coding sequence because exon-intron boundaries and intronic primer sequences to study exons 1, 2 and 7 were not available. We detected 10 different missense, two nonsense and one splicing mutation, as well as five distinct intragenic deletions. We also established the carrier status of eight mothers of sporadic cases, determining that 88% are carriers.
It is likely that alterations associated with a truncated protein are null alleles; however, it may be more difficult to correlate a severe clinical phenotype with some of the missense mutations. For each unique mutation, the entire available coding sequence was analyzed, and no additional amino acid substitutions were detected. Eight of the 10 missense mutations listed in Table 1 alter an amino acid which is conserved in the homologous gene from Saccharomyces cerevisiae and/or Caenorhabditis elegans (12 ). In addition, five of the mutations alter the charge of the predicted protein. The two missense mutations which are not conserved in lower eukaryotes are among those which alter the charge of the protein (patients 5-7 and patient 16). Both of these mutations are associated with a mild phenotype.
Three of the mutations were present in more than a single patient. The C to T transition at base pair 259, found in three of our patients (J.F., S.S. and A.V.) and in one additional patient (15 ), is associated with a CpG dinucleotide; however, there are no obvious predisposing features in the sequence surrounding the recurrent splicing site mutation in exon 12. The three small deletions in exons 4, 5 and 8 are associated with direct repeats of 2-4 bp adjacent to or included within the deletion. Such repeats frequently are associated with deletions of <20 bp, the presumed mechanism being slippage during DNA replication (14 ). The exon 12 splicing mutation and deletion in exon 4 were seen in four and three distinct patients, respectively, studied by Laporte et al. (15 ).
Despite the extensive evolutionary conservation of MTM1, its function remains completely unknown. The only homology found to other known proteins is with the active site of the tyrosine phosphatases. This finding suggests that myotubularin could be involved in a signal transduction pathway. In our studies, no mutations were detected within the 39 bp of this conserved phosphatase domain in exon 11, although the entire exon is deleted in patient 11 (J.L.) and two missense mutations within the conserved phosphatase domain were among those reported by Laporte et al. (12 ).
Although extremely preliminary, we can begin to correlate the mutant genotypes with clinical phenotypes for MTM1 patients. All of the patients with a mild phenotype had missense mutations, and the single missense mutation R69C in exon 4 was associated with a mild clinical course in both patients for whom clinical information was available. Specifically, patients J.F. and A.V. were intubated at birth for only 2 days and 2 weeks, respectively; they are now ~24 and 18 months old and have not required further ventilatory support. They have vocabularies of several words and could sit unassisted by 1 year of age (G. Herman, unpublished results). Similarly, all of the patients with nonsense mutations, large or small deletions or splicing mutations have had severe phenotypes, suggesting that the myotubularin protein is exquisitely sensitive to more than very minimal alterations.
An early goal after the isolation of a disease gene is often establishing or improving pre- and post-natal molecular diagnosis of the disorder. Linkage analysis using closely flanking polymorphic markers is well established in familial cases (7 ,10 ); however, verifying the carrier status of mothers in sporadic cases or determining whether mildly affected males have the X-linked form of the disorder can be difficult. Our results clearly demonstrate the ability to detect carrier females by direct sequencing of genomic PCR products or cloned fragments once the mutation in the proband is known. The results also suggest that no single mutation is likely to account for more than 10-20% of cases, although 50% of the mutations identified were detected in exons 4 and 12, and rare deletions detectable on Southern hybridization are encountered. Molecular diagnosis of MTM1 is likely to require DNA sequence analysis of each proband, with subsequent diagnoses relying on direct sequencing or linkage studies depending on the exact circumstances and available resources for that family.
Patients with MTM1 were referred to A.M. or G.E.H. by their physician or by the X-linked myotubular myopathy resource group, a family support group. For all of the patients studied, the diagnosis was confirmed by muscle biopsy. Medical records or the medical history were reviewed for all patients by a clinician (G.E.H.), except for one patient (S.S.) for whom no records or clinical information were available. The families were ethnically diverse (36 Caucasian, two Hispanic, one Vietnamese, one Japanese/Caucasian) and distributed throughout the United States. We have verified that the patients and their extended families are distinct from those reported by Laporte et al. (12 ,15 ). Clinical data concerning patients J.S. and M.M. have been reported (2 ). Detailed clinical descriptions of the remaining patients will be published elsewhere (G. Herman et al., in preparation). For the purposes of the mutation studies, the patients have been grouped based on the presence or absence of a positive family history and their clinical severity. Thirteen patients had a positive family history of one or more affected male siblings or one or more affected maternal uncles, or both. Twenty nine patients demonstrated the classical severe phenotype, presenting at birth and requiring prolonged ventilatory support. Eight patients were classified as mild based on a need for mechanical ventilation in the newborn period, but rapid weaning from the ventilator and a milder course with fewer hospitalizations and more rapid attainment of motor milestones than typically reported for MTM1 (1 -3 ). Finally, three of the 41 males did not exhibit symptoms until 5-8 months of age and may not represent the X-linked form of the disease. None of the patients had ambiguous genitalia which has been observed in two cases with large deletions (>300 kb) encompassing the entire MTM1 locus (9 ).
DNA was prepared from freshly obtained peripheral blood, cultured lymphoblasts or cultured skin fibroblasts, using an Applied Biosystem nucleic acid extractor. For all patients, a diagnosis of congenital myotonic dystrophy was excluded by Southern hybridization of NcoI-digested genomic DNA with a genomic probe (MDY1) from the 3' end of human myotonin kinase gene which contains the expandable triplet repeat (16 ). Confirmation of the large intragenic deletions observed in patients S.C. and J.L. was performed using Southern hybridization of EcoRI-, TaqI- or BamHI-digested genomic DNA with the human MTM1 cDNA (Ig38) (12 ). Genomic DNA was digested as recommended by the enzyme manufacturer (Boehringer-Mannheim) and separated on a 1% agarose gel in 1* TAE buffer (0.04 M Tris-acetate/1 mM EDTA) by migration overnight at 55 V. After electrophoresis, DNA was transferred to Sure blot hybridization membranes (Oncor) and hybridized with [[alpha]-32P]dCTP-labeled probe prepared using the random hexamer method (17 ). Hybridizations and washes were performed at 65oC as described (18 ). Membranes were exposed overnight at -80oCwith X-OMAT film (Kodak).
SSCP and HA were performed using standard methods (19 ). PCR amplification of genomic DNA was performed as described (15 ) except that the Mg concentration was 1.5 mM for all exons tested and no glycerol was added to any of the reactions. Primer sequences for the forward intronic primer to amplify exon 8, and for the intronic primers for exons 11, 14 and 15, have been reported previously (12 ). They were designated as exons a, c, d and e, respectively, in that publication (see Fig. 1 ). The remaining intronic primer sequences used to amplify the 12 exons analyzed are provided in the accompanying manuscript by Laporte et al. (15 ).
Direct genomic cycle sequencing of PCR products was performed on an ABI373 sequencer using a Perkin-Elmer ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit including Taq FS DNA polymerase. One of the intronic primers used in the PCR amplification was also employed for the cycle sequencing. DNA sequences were aligned and screened for mutations using GCG software. For each PCR product, both strands were sequenced, and each mutation reported was detected on each strand. Each distinct mutation was confirmed by direct sequencing of a second independent PCR product from the patient. To ascertain the carrier status of mothers of sporadic cases, SSCP, HA, and direct genomic sequencing using dye terminator primers was performed as above. The carrier status was confirmed by sequencing of up to 11 cloned PCR fragments isolated with the pGEM-T vector system (Promega).
TotalRNA was isolated from human lymphoblastoid cell lines using RNAzol B (Tel Test). RT product was prepared from 5 [mu]g of total RNA using MMLV reverse transcriptase (Pharmacia) as described (20 ). To analyze the splicing mutation near exon 12, a nested PCR reaction was performed using outer primers 12-1 forward (5'-TGGATAGCTTCTATAGGAGC-3') and reverse (5'-GGCAACTATACAGATGATCC-3'), and inner nested primers 12-2 forward (5'-TGAAGGGTTCGAAATACTGG-3') and reverse (5'-TAGGAGAACGGTCAGCATCG-3'). The two forward primers contain sequences from exon 11, while the outer and nested reverse primers contain sequences from exons 13 and 12, respectively. One [mu]l of RT product was first amplified for 40 cycles at an annealing temperature of 55oC with primers 12-1. One [mu]l of 1:100 dilution of the first product subsequently was amplified for 35 cycles using an annealing temperature of 60oC and the 12-2 primers.
The authors wish to thank Cheri Walker for technical assistance and advice concerning automated DNA sequencing; Stella Madu and John Bargerstock for help in establishing and maintaining the human lymphoblastoid cell lines; Pam Watson and Wendy Gray for preparation of human DNA samples; Nelly Rivera for help preparing the manuscript; and the numerous clinicians who have referred MTM1 families to A.M. and G.E.H. We are especially grateful to the MTM resource group and to Pam, Gary and John Scoggin whose courage in living daily with this disorder provided the impetus to perform these studies. This research was supported by grants from the Muscular Dystrophy Association to G.E.H. and to B.de G. and by the Baylor College of Medicine Mental Retardation and Child Health Research Centers. J.L. and J.-L. M. were supported by grants from the Association Francaise Contre les Myopathies and the Ministere de l'Education Nationale.
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*To whom correspondence should be addressed. Tel: +1 614 722 2848; Fax: +1 614 722 2716; Email: gherman@chi.osu.edu
+Present address: Children's Hospital Research Foundation, Ohio State University, 700 Children's Drive, Columbus, OH 43205, USA
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