Germline mosaicism in X-linked myotubular myopathy

X-linked myotubular myopathy (XLMTM; OMIM310400) is a congenital muscle disorder characterized by severe hypotonia and respiratory insufficiency. The disorder was mapped to Xq28 by linkage studies and the MTM1 gene was isolated by positional cloning. The gene product is a 603 amino acid protein named myotubularin. A small domain in its sequence shows high homology to a consensus active site of tyrosine phosphatases, a diverse class of proteins involved in signal transduction, control of cell growth, and differentiation. In this report, two brothers affected with XLMTM are shown to have a point mutation (G1187A) in exon 11 of the MTM1 gene. Surprisingly, their mother does not have this mutation in her lymphocytes. Therefore, she likely has a germline mosaicism. As this is the third report of germline mosaicism in XLMTM, the finding has important implications for genetic counseling.     

Characterization of mutations in fifty North American patients with X‐linked myotubular myopathy

X‐linked myotubular myopathy (MTM1) is a rare developmental disorder of skeletal muscle that is characterized by the presence of abnormal central nuclei in biopsy specimens taken from affected individuals. To date 133 different mutations have been identified in the MTM1 gene worldwide. We report here mutations detected in 50 additional U.S. families with biopsy‐proven MTM1. Forty‐one of the patients have not been described previously, including 18 with novel mutations. Eighty‐eight percent of the mothers of sporadic cases that were studied were identified as carriers, extending the previously reported high‐carrier frequency for this disorder. Clinical information collected on the majority of patients helps to further correlate genotype with phenotype, and implications of these data for genetic counseling in families are discussed. Hum Mutat 19:114–121, 2002. © 2002 Wiley‐Liss, Inc.     

Mutations in the MTM1 gene implicated in X-linked myotubular myopathy. ENMC International Consortium on Myotubular Myopathy. European Neuro- Muscular Center

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 myotubular myopathy in a family with three adult survivors

We describe a family with an extremely mild form of X-linked myotubular myopathy. Three affected males survived to adulthood with sufficient muscle strength to enable them to carry out normal daily activities. The mildness of the myopathy in this family is highlighted by the following: no neonatal or infant mortality resulting from the myopathy; one affected male who did not have neonatal asphyxia and had normal early motor milestones - this affected male was able to increase his muscle bulk and strength to normal by weightlifting; and a 55-year-old male who still lives an independent life. DNA sequencing identified a novel missense mutation - G469A (E157K) - in exon 7 of the MTM1 gene in this family. To our knowledge, this is the third X-linked myotubular myopathy family, with multiple adult survivors, to be reported in the literature.     

MTM1 mutations in X-linked myotubular myopathy

X-linked myotubular myopathy (XLMTM; MIM# 310400) is a severe congenital muscle disorder caused by mutations in the MTM1 gene. This gene encodes a dual-specificity phosphatase named myotubularin, defining a large gene family highly conserved through evolution (which includes the putative anti-phosphatase Sbf1/hMTMR5). We report 29 mutations in novel cases, including 16 mutations not described before. To date, 198 mutations have been identified in unrelated families, accounting for 133 different disease-associated mutations which are widespread throughout the gene. Most point mutations are truncating, but 26% (35/133) are missense mutations affecting residues conserved in the Drosophila ortholog and in the homologous MTMR1 gene. Three recurrent mutations affect 17% of the patients, and a total of 21 different mutations were found in several independent families. The frequency of female carriers appears higher than expected (only 17% are de novo mutations). While most truncating mutations cause the severe and early lethal phenotype, some missense mutations are associated with milder forms and prolonged survival (up to 54 years).     

Identification of novel mutations in the MTM1 gene causing severe and mild forms of X-linked myotubular myopathy.

X-linked myotubular myopathy (XLMTM) is a congenital muscular disease characterized by severe hypotonia and generalized muscle weakness, leading in most cases to early postnatal death. The gene responsible for the disease, MTM1, encodes a dual specificity phosphatase, named myotubularin, which is highly conserved throughout evolution. To date, 139 MTM1 mutations in independent patients have been reported, corresponding to 93 different mutations. In this report we describe the identification of 21 mutations (14 novel) in XLMTM patients. Seventeen mutations are associated with a severe phenotype in males, with death occurring mainly before the first year of life. However, four mutations-three missense (R241C, I225T, and novel mutation P179S) and one single-amino acid deletion (G294del)-were found in patients with a much milder phenotype. These patients, while having a severe hypotonia at birth, are still alive at the age of 4, 7, 13, and 15 years, respectively, and display mild to moderate muscle weakness.     

AAV-mediated intramuscular delivery of myotubularin corrects the myotubular myopathy phenotype in targeted murine muscle and suggests a function in plasma membrane homeostasis

Myotubular myopathy (XLMTM, OMIM 310400) is a severe congenital muscular disease due to mutations in the myotubularin gene (MTM1) and characterized by the presence of small myofibers with frequent occurrence of central nuclei. Myotubularin is a ubiquitously expressed phosphoinositide phosphatase with a muscle-specific role in man and mouse that is poorly understood. No specific treatment exists to date for patients with myotubular myopathy. We have constructed an adeno-associated virus (AAV) vector expressing myotubularin in order to test its therapeutic potential in a XLMTM mouse model. We show that a single intramuscular injection of this vector in symptomatic Mtm1-deficient mice ameliorates the pathological phenotype in the targeted muscle. Myotubularin replacement in mice largely corrects nuclei and mitochondria positioning in myofibers and leads to a strong increase in muscle volume and recovery of the contractile force. In addition, we used this AAV vector to overexpress myotubularin in wild-type skeletal muscle and get insight into its localization and function. We show that a substantial proportion of myotubularin associates with the sarcolemma and I band, including triads. Myotubularin overexpression in muscle induces the accumulation of packed membrane saccules and presence of vacuoles that contain markers of sarcolemma and T-tubules, suggesting that myotubularin is involved in plasma membrane homeostasis of myofibers. This study provides a proof-of-principle that local delivery of an AAV vector expressing myotubularin can improve the motor capacities of XLMTM muscle and represents a novel approach to study myotubularin function in skeletal muscle.     

Extensive germinal mosaicism in a family with X linked myotubular myopathy simulates genetic heterogeneity.

A family with two male cousins affected with myotubular myopathy (MTM) was referred to us for genetic counselling. Linkage analysis appeared to exclude the Xq28 region. As a gene for X linked MTM was recently identified in Xq28, we screened the obligatory carrier mothers for mutation. We found a 4 bp deletion in exon 4 of the MTM1 gene, which originated from the grandfather of the affected children and which was transmitted to three daughters. This illustrates the importance of mutation detection to avoid pitfalls in linkage analysis that may be caused by such cases of germinal mosaicism.     

Phenotypic heterogeneity in British patients with a founder mutation in the FHL1 gene

Mutations in the four-and-a-half LIM domain 1 (FHL1) gene, which encodes a 280-amino-acid protein containing four LIM domains and a single zinc-finger domain in the N-terminal region, have been associated with a broad clinical spectrum of X-linked muscle diseases encompassing a variety of different phenotypes. Patients might present with a scapuloperoneal myopathy, a myopathy with postural muscle atrophy and generalized hypertrophy, an Emery-Dreifuss muscular dystrophy, or an early onset myopathy with reducing bodies. It has been proposed that the phenotypic variability is related to the position of the mutation within the FHL1 gene. Here, we report on three British families with a heterogeneous clinical presentation segregating a single FHL1 gene mutation and haplotype, suggesting that this represents a founder mutation. The underlying FHL1 gene mutation was detected by direct sequencing and the founder effect was verified by haplotype analysis of the FHL1 gene locus. A 3-bp insertion mutation (p.Phe127_Thr128insIle) within the second LIM domain of the FHL1 gene was identified in all available affected family members of the three families. Haplotype analysis of the FHL1 region on Xq26 revealed that the families shared a common haplotype. The p.Phe127_Thr128insIle mutation in the FHL1 gene therefore appears to be a British founder mutation and FHL1 gene screening, in particular of exon 6, should therefore be indicated in British patients with a broad phenotypic spectrum of X-linked muscle diseases.     

Mutation spectrum of the MTM1 gene in XLMTM patients: 10 years of experience in prenatal and postnatal diagnosis

X‐linked myotubular myopathy (XLMTM) is a congenital neuromuscular disorder defined by severe hypotonia, respiratory failure and histopathologic changes in muscle biopsy. The objective of this report is to inform about our experience of genetic analysis on a group of 25 unrelated XLMTM patients, clinically diagnosed by several Italian and European Medical Institutes from 2006 to 2015. The molecular strategy used for genotyping involved Sanger sequencing of coding and intron/exon regions and the Multiplex Ligation Probe Amplification method. A total of 13 different point variants (6 nonsense, 5 missense, 1 splicing and 1 small deletion) were found in 15 patients (60%). Three were new missense variants: c.185G>T p.(Arg62Ile), c.719T>A p.(Val240Glu), and c.1262G>T p.(Arg421Leu). No large duplications/deletions have been identified. We performed carrier testing of at‐risk female relatives. Only one mutation was de novo. Successively, we offered XLMTM prenatal testing for seven pregnancies in five unrelated families. In this context, the aim to propose an effective molecular diagnostic service is to confirm clinical XLMTM diagnosis, to monitor the cause‐disease mutation segregation in the family and to offer genetic counseling to have correct information regarding offspring risks and the prenatal testing.     

Inhibition of activin receptor type IIB increases strength and lifespan in myotubularin-deficient mice

X-linked myotubular myopathy (XLMTM) is a congenital disorder caused by deficiency of the lipid phosphatase, myotubularin. Patients with XLMTM often have severe perinatal weakness that requires mechanical ventilation to prevent death from respiratory failure. Muscle biopsy specimens from patients with XLMTM exhibit small myofibers with central nuclei and central aggregations of organelles in many cells. It was postulated that therapeutically increasing muscle fiber size would cause symptomatic improvement in myotubularin deficiency. Recent studies have elucidated an important role for the activin-receptor type IIB (ActRIIB) in regulation of muscle growth and have demonstrated that ActRIIB inhibition results in significant muscle hypertrophy. To evaluate whether promoting muscle hypertrophy can attenuate symptoms resulting from myotubularin deficiency, the effect of ActRIIB-mFC treatment was determined in myotubularin-deficient (Mtm1δ4) mice. Compared with wild-type mice, untreated Mtm1δ4 mice have decreased body weight, skeletal muscle hypotrophy, and reduced survival. Treatment of Mtm1δ4 mice with ActRIIB-mFC produced a 17% extension of lifespan, with transient increases in weight, forelimb grip strength, and myofiber size. Pathologic analysis of Mtm1δ4 mice during treatment revealed that ActRIIB-mFC produced marked hypertrophy restricted to type 2b myofibers, which suggests that oxidative fibers in Mtm1δ4 animals are incapable of a hypertrophic response in this setting. These results support ActRIIB-mFC as an effective treatment for the weakness observed in myotubularin deficiency.     

Sporadic cardiac and skeletal myopathy caused by a de novo desmin mutation

Desmin myopathy is a familial or sporadic disorder characterized by intracytoplasmic accumulation of desmin in the muscle cells. We and others have previously identified desmin gene mutations in patients with familial myopathy, but close to 45% of the patients do not report previous family history of the disease. The present study was conducted to determine the cause of desmin myopathy in a sporadic patient presenting with symmetrical muscle weakness and atrophy combined with atrioventricular conduction block requiring a permanent pacemaker. A novel heterozygous R406W mutation in the desmin gene was identified by sequencing cDNA and genomic DNA. Expression of a construct containing the patient's mutant desmin cDNA in SW13 (vim-) cells demonstrated a high pathogenic potential of the R406W mutation. This mutation was not found in the patient's father, mother or sister by sequencing and restriction analysis. Testing with five microsatellite markers and four intragenic single nucleotide polymorphisms excluded alternative paternity. Haplotype analysis indicates that the patient's father was germ-line mosaic for the desmin mutation. We conclude that de novo mutations in the desmin gene may be the cause of sporadic forms of desmin-related cardiac and skeletal myopathy.     

Autosomal dominant Brody disease cosegregates with a chromosomal (2;7)(p11.2;p12.1) translocation in an Italian family

Brody disease is a rare muscle disorder characterized by exercise-induced impairment in muscle relaxation, due to a markedly reduced influx of calcium ions in the sarcoplasmic reticulum. A subset of autosomal recessive families harbour mutations in the ATP2A1 gene, encoding the fast-twitch skeletal muscle sarcoplasmic reticulum Ca(2+) ATPase (SERCA1). Rare autosomal dominant families have been described, in which ATP2A1 was excluded as the causative gene, further supporting genetic heterogeneity. We report four individuals from a three-generation Italian family with a clinical phenotype of Brody disease, in which linkage analysis excluded ATP2A1 as the responsible gene. The disease cosegregates in an autosomal dominant fashion with an apparently balanced constitutional chromosome translocation (2;7)(p11.2;p12.1), suggesting a causal relationship between the rearrangement and the phenotype. FISH analysis using YAC and PAC clones as probes refined the breakpoint regions to genomic segments of about 164 and 120 kb, respectively, providing a possible clue to pinpoint the location of a novel gene responsible for this rare muscle disorder.     

Myotubularin, a phosphatase deficient in myotubular myopathy, acts on phosphatidylinositol 3-kinase and phosphatidylinositol 3-phosphate pathway

Myotubular myopathy (MTM1) is an X-linked disease, characterized by severe neonatal hypotonia and generalized muscle weakness, with pathological features suggesting an impairment in maturation of muscle fibres. The MTM1 gene encodes a protein (myotubularin) with a phosphotyrosine phosphatase consensus. It defines a family of at least nine genes in man, including the antiphosphatase hMTMR5/Sbf1 and hMTMR2, recently found mutated in a recessive form of Charcot-Marie-Tooth disease. Myotubularin shows a dual specificity protein phosphatase activity in vitro. We have performed an in vivo test of tyrosine phosphatase activity in Schizosaccharomyces pombe, indicating that myotubularin does not have a broad specificity tyrosine phosphatase activity. Expression of active human myotubularin inhibited growth of S.pombe and induced a vacuolar phenotype similar to that of mutants of the vacuolar protein sorting (VPS) pathway and notably of mutants of VPS34, a phosphatidylinositol 3-kinase (PI3K). In S.pombe cells deleted for the endogenous MTM homologous gene, expression of human myotubularin decreased the level of phosphatidylinositol 3-phosphate (PI3P). We have created a substrate trap mutant which shows relocalization to plasma membrane projections (spikes) in HeLa cells and was inactive in the S.pombe assay. This mutant, but not the wild-type or a phosphatase site mutant, was able to immunoprecipitate a VPS34 kinase activity. Wild-type myotubularin was also able to directly dephosphorylate PI3P and PI4P in vitro. Myotubularin may thus decrease PI3P levels by down-regulating PI3K activity and by directly degrading PI3P.     

Two related Dutch families with a clinically variable presentation of cardioskeletal myopathy caused by a novel S13F mutation in the desmin gene

Desmin-related myopathy is characterised by skeletal muscle weakness often combined with cardiac involvement. Mutations in the desmin gene have been described as a cause of desmin-related myopathy (OMIM 601419). We report here on two distantly related Dutch families with autosomal dominant inheritance of desmin-related myopathy affecting 15 family members. A highly heterogeneous clinical picture is apparent, varying from isolated dilated cardiomyopathy to a more generalised skeletal myopathy and mild respiratory problems. Morphological analysis of muscle biopsies revealed intracytoplasmic desmin aggregates (desmin and p62 staining). In both families we identified an identical novel pathogenic heterozygous missense mutation, S13F, in the 'head' domain of the desmin gene which cosegregates with the disease phenotype. This is the 5th reported missense mutation located at the 'head' domain of the desmin gene and the first reported Dutch family with desmin-related myopathy. This article illustrates the importance of analysing the desmin gene in patients with (familial) cardiac conduction disease, dilated cardiomyopathy and/or a progressive skeletal myopathy resembling limb-girdle muscular dystrophy.     

Novel skeletal muscle ryanodine receptor mutation in a large Brazilian family with malignant hyperthermia

Malignant hyperthermia (MH) is an autosomal dominant disorder that predisposes susceptible individuals to a potentially life-threatening crisis when exposed to commonly used anesthetics. Mutations in the skeletal muscle calcium release channel, ryanodine receptor (RYR1) are associated with MH in over 50% of affected families. Linkage analysis of the RYR1 gene region at 19q13 was performed in a large Brazilian family and a distinct disease co-segregating haplotype was revealed in the majority of members with diagnosis of MH. Subsequent sequencing of RYR1 mutational hot spots revealed a nucleotide substitution of C to T at position 7062, causing a novel amino acid change from Arg2355 to Cys associated with MH in the family. Haplotype analysis of the RYR1 gene area at 19q13 in the family with multiple MH members is an important tool in identification of genetic cause underlying this disease.