X-linked Recessive Myotubular Myopathy Research Articles

Characterization and genetic diagnosis of centronuclear myopathies in seven Chinese patients.

Centronuclear myopathies (CNMs) are a group of clinically and genetically heterogeneous muscle disorders. Here, we report a cohort of seven CNM patients with their clinical, histological, and morphological features. In addition, using the next-generation sequencing (NGS) technique (5/7 patients), we identified small indels: intronic, exonic, and missense mutations in MTM1, DNM2, and RYR1 genes. Further genetic studies revealed skewed X-chromosome inactivation in two female patients carrying MTM1 mutations. Based on the results of genetic analysis, these seven patients were classified as (1) X-linked recessive myotubular myopathy (patients 1-3) with MTM1 mutations and mild phenotype, (2) the autosomal dominant CNM (patients 4-6) with DNM2 mutations, and (3) the autosomal recessive CNM (patient 7) with RYR1 mutations. In all patients, histological findings featured a high proportion of fibers with central nuclei. Radial arrangement of the sarcoplasmic strands was observed in DNM2-CNM and RYR1-CNM patients. Muscle magnetic resonance imaging (MRI) revealed a proximal pattern of involvement presented in both MTM1-CNM and RYR1-CNM patients. A distal pattern of involvement was present in DNM2-CNM patients. Our findings thereby identified a number of novel features that expand the reported clinicopathological phenotype of CNMs in China.

P.254 - Severe X-linked myotubular myopathy with unexpected inheritance from the grandfather and identification of necklace fibers in an asymptomatic male

X-linked recessive myotubular myopathy (XLMTM) usually affects boys and is associated with mutations in the myotubularin gene (MTM1). In this study we describe a family with a severely affected infant boy with a centronuclear myopathy due to unexpected grand paternal transmission of a novel mutation in MTM1 (c.646_648dupGTT; p.Val216dup). The mother of the affected boy was heterozygous carrier of the variant whereas the maternal grandmother was homozygous for the wild-type allele. The maternal grandfather, who was reported to be healthy, was identified with both the mutated MTM1 gene together with wild-type MTM1. A muscle biopsy showed occasional necklace fibers, a hallmark of MTM1-associated myopathies, especially in manifesting heterozygous carriers. The grandfather, who had a normal karyotype (46,XY), demonstrated equal amounts of DNA with the wild-type sequence and DNA with the c.646_648dupGTT variant in five different tissues that were analyzed. These results were interpreted as postzygotic mosaicism and the mutation had probably occurred at the first mitosis of the zygote. This family demonstrate an unusual pattern of inheritance for a severe X-linked disorder, where the grandfather was transmitting the mutation. Muscle biopsy in the grandfather with presence of necklace fibers was important to further establish the pathogenicity of the novel MTM1 mutation. The results also have important implications for genetic counseling in this family.

G.P.42: Mild clinical phenotype in two siblings carrying myotubular myopathy

Among congenital myopathies, X-linked recessive myotubular myopathy is one of the most severe forms. The majority of patients present with severe hypotonia at birth and respiratory insufficiency and most of them die in the first year of life. However, there is phenotypic variability. Here, we report two patients carrying a missense mutation in MTM1 gene and presenting with a mild phenotype. The first boy was born on time with respiratory insufficiency and generalised hypotonia. He required ventilator support for 3 weeks, and then he went home without specific care. He walked at 11 month. He is now 24 and can run, practices canoe and works as a salesperson. At the exam, he presents mild proximal weakness and restrictive syndrome with no ventilator support (VC = 57%). His brother was born on time with respiratory insufficiency and hypotonia. He stayed in intensive care unit for 6 weeks, then went home without support. He walked at 18 months. Respiratory involvement was more severe than his brother with a severe restrictive syndrome (VC = 33%) but the young man refused non-invasive night-time ventilation proposed at the age of 16. Being now 20, he can walk without any difficulty but cannot run. Clinical exam reveals a mild proximal weakness. Both patients underwent muscle biopsy showing the typical centronuclear myopathy pattern. Molecular analysis of MTM1gene found a hemizygous mutation c.575A>G in exon 8 leading to a missense p.Tyr192Cys. A decreased level of the MTM1 protein was observed by western blot analysis on lymphoblasts. To date, about 300 different mutations in around 540 families have been identified in MTM1 gene, including point mutations, insertions and small and large deletions and duplication. Around 70% of patients presenting with a mild or moderate phenotype carries a missense mutation, which is also the most frequent type of mutation. Another patient has been described with the same mutation (c.575A>G) and presented also with a mild phenotype.

X-linked recessive myotubular myopathy withMTM1mutations

X-linked recessive myotubular myopathy (XLMTM) is a severe congenital muscle disorder caused by mutations in the MTM1 gene and characterized by severe hypotonia and generalized muscle weakness in affected males. It is generally a fatal disorder during the neonatal period and early infancy. The diagnosis is based on typical histopathological findings on muscle biopsy, combined with suggestive clinical features. We experienced a case of a newborn who required intubation and ventilator care because of profound hypotonia and respiratory difficulty. The preliminary diagnosis at the time of request for retrieval was hypoxic ischemic encephalopathy, but the infant was clinically reevaluated for generalized weakness and muscle atrophy. Muscle biopsies showed variability in fiber size and centrally located nuclei in nearly all the fibers. We detected an MTM1 gene mutation of c.1261-1C>A in the intron 10 region, and diagnosed the neonate with myotubular myopathy. The same mutation was detected in his mother.

Myotubular myopathy caused by multiple abnormal splicing variants in the MTM1 RNA in a patient with a mild phenotype

Mutations impacting on the splicing of pre-mRNA are one important cause of genetically inherited diseases. However, detection of splice mutations, that are mainly due to intronic variations, and characterization of their effects are usually not performed as a first approach during genetic diagnosis. X-linked recessive myotubular myopathy is a severe congenital myopathy due to mutations in the MTM1 gene encoding myotubularin. Here, we screened a male patient showing an unusually mild phenotype without respiratory distress by western blot with specific myotubularin antibodies and detected a strong reduction of the protein level.The disease was subsequently linked to a hemizygous point mutation affecting the acceptor splice site of exon 8 of MTM1, proven by protein, transcript and genomic DNA analysis. Detailed analysis of the MTM1 mRNA by RT-PCR, sequencing and quantitative PCR revealed multiple abnormal transcripts with retention of a truncated exon 8, and neighboring exons 7 and 9 but exclusion of several other exons, suggesting a complex effect of this mutation on the splicing of non-adjacent exons. We conclude that the analysis of RNA by RT-PCR and sequencing is an important step to characterize the precise impact of detected splice variants. It is likely that complex splice aberrations due to a single mutation also account for unsolved cases in other diseases.

P3.40 Myopathy in a woman and her daughter associated with a novel MTM1 mutation

X-linked recessive myotubular myopathy (XLMTM) is due to mutations in MTM1, which encodes a phosphatase called myotubularin. The disease usually affects boys and frequently presents with severe neonatal onset of hypotonia and muscle weakness. Female heterozygous carriers may manifest muscle weakness possibly due skewed X-inactivation. We have investigated a woman and her daughter with an early onset, slowly progressive myopathy. The proband had mild leg weakness since childhood and onset of upper extremity weakness at age 20.

X-linked inheritance and its implication in the diagnosis and management of female patients in Fabry disease

X-linked recessive myotubular myopathy (XLMTM) is a disorder associated with mutations in the myotubularin gene (MTM1) that usually affects boys, with transmission of the mutated allele from the mother. Here we describe a family with unexpected grand paternal transmission of a novel mutation in MTM1 (c.646_648dupGTT; p.Val216dup) identified in a severely affected infant boy with a centronuclear myopathy. We confirmed the carrier status of the mother, but surprisingly we found that her father was a carrier of the mutated MTM1 gene together with wild-type MTM1. A muscle biopsy from the grandfather revealed occasional typical necklace fibers with internalized nucleus, which is typically found in MTM1-associated myopathies. Further analysis of the grandfather revealed equal amounts of DNA with the wild-type sequence and DNA with the c.646_648dupGTT variant in five different tissues examined. In the presence of a normal karyotype (46,XY) in the grandfather and no evidence of intragenic duplication of MTM1, the result was interpreted as postzygotic mosaicism and the mutation had probably occurred at the first mitosis of the zygote. This study demonstrates the importance of considering the possibility of paternal transmission in families with severe X-linked disorders. The muscle biopsy with the finding of typical necklace fibers was important to further establish the pathogenicity of the novel MTM1 mutation.

Normal innervation and differentiation of X-linked myotubular myopathy muscle cells in a nerve-muscle coculture system.

To study the pathogenesis of X-linked recessive myotubular myopathy (XLMTM), we used a nerve-muscle coculture system which allows the reconstitution of functional motor units in vitro after coupling of human skeletal muscle cells with embryonic rat spinal cord explants. We used three skeletal muscle cell lines derived from subjects with known mutations in the MTM1 gene (two from embryonic tissues, associated with mutations predicted to give a severe phenotype, and one from a neonate still alive at 3 years 6 months and exhibiting a mild phenotype). We compared these three XLMTM muscle cell cultures with control cultures giving special attention to behaviour of living cocultures (formation of the myofibres, contractile activity, survival), expression of muscular markers (desmin, dystrophin, alpha-actinin, troponin-T, myosin heavy chain isoforms), and nerve-muscle interactions (expression and aggregation of the nicotinic acetylcholine receptors). We were unable to reproduce any 'myotubular' phenotype since XLMTM muscle cells behaved like normal cells with regard to all the investigated parameters. Our results suggest that XLMTM muscle might be intrinsically normal and emphasize the possible involvement of the myotubularin-deficient motor neurons in the development of the disease.

Diagnosis of X-linked myotubular myopathy by detection of myotubularin.

Mutations in the MTM1 gene cause X-linked recessive myotubular myopathy (XLMTM; MIM310400). Myotubularin, the implicated protein, is a phosphoinositide phosphatase that belongs to a large protein family conserved through evolution that also includes the antiphosphatase Sbfl and the protein hMTMR2 mutated in Charcot-Marie-Tooth type 4B. Myotubularin is detectable in a variety of cell lines by immunoprecipitation followed by Western blotting. We screened 29 independant patients with XLMTM phenotype and four with centronuclear myopathy. 87% (21/24) of patients with known MTM1 mutations showed abnormal myotubularin levels, including some with missense mutations. Moreover, myotubularin was also undetectable in a patient for whom no mutation could be identified by SSCP screening. The centronuclear cases investigated have a normal level of protein, suggesting that the centronuclear form is not the result of a decrease in myotubularin level. Thus, immunoprecipitation of myotubularin from cultured cells represents a rapid and helpful method for classifying those cases where no mutation was found. On the other hand, the amount of expression may be of diagnostic value for disease course in patients with a mutation.

Genomic Organization of a 225-kb Region in Xq28 Containing the Gene for X-Linked Myotubular Myopathy (MTM1) and a Related Gene (MTMR1)

MTM1is responsible for X-linked recessive myotubular myopathy, which is a congenital muscle disorder linked to Xq28.MTM1is highly conserved from yeast to humans. A number of related genes also exist. TheMTM1gene family contains a consensus sequence consisting of the active enzyme site of protein tyrosine phosphatases (PTPs), suggesting that they belong to a new family of PTPs. Database searches revealed homology of myotubularin and all related peptides to the cisplatin resistance-associated α protein, which implicates an as yet unknown function. In addition, homology to the Sbf1 protein (SET binding factor 1), involved in the oncogenic transformation of fibroblasts and differentiation of myoblasts, was also evident. We describe 225 kb of genomic sequence containingMTM1and the related gene,MTMR1,which lies 20 kb distal toMTM1.Although there is only moderate conservation of the exons, the striking similarity in the gene structures indicates that these two genes arose by duplication. Calculations suggest that this event occurred early in evolution long before separation of the human and mouse lineages. So far, mutations have been identified in the coding sequence of only 65% of the patients analyzed, indicating that the remaining mutations may lie in noncoding regions ofMTM1or possibly inMTMR1.Knowledge of the genomic sequence will facilitate mutation analyses of the coding and noncoding sequences ofMTM1andMTMR1.

Genomic organization of the MTM1 gene implicated in X-linked myotubular myopathy.

X-linked recessive myotubular myopathy (XLMTM) is a very severe congenital muscular disease characterised by an impaired maturation of muscle fibres, and caused by defects in the MTM1 gene. This gene defines a new family of putative tyrosine phosphatases conserved through evolution. We have determined intronic flanking sequences for all the 15 exons to facilitate the detection of mutations in patients and genetic counselling. We characterised a new polymorphic marker in the immediate vicinity of the gene, which might prove useful for linkage analysis. Sequencing of the TATA-less predicted promoter provides the basis for transcriptional regulatory studies.

Confirmation of prenatal diagnosis results of X-linked recessive myotubular myopathy by mutational screening, and description of three new mutations in the MTM1 gene.

X-linked recessive myotubular myopathy (XLMTM; MTM1) is a severe neonatal disorder often causing perinatal death of the affected males. The responsible gene, designated MTM1, was localized to proximal Xq28 and recently isolated. The characterization of MTM1 allowed us to screen for causing mutations in three families, previously investigated by linkage analysis. Using exon amplification, single strand conformation polymorphism, and subsequent sequencing analysis, three new mutations and their mutational origin were characterized by analyzing 10 exons. An acceptor splice site and a frameshift mutation were correlated with the concurrent appearance of XLMTM in two families. A third intronic mutation was also analyzed by reverse transcription PCR and revealed a cryptic splice site mutation cosegregating with the presumed XLMTM haplotype in the third family. These results further support the implication of the MTM1 gene in XLMTM and allow efficient and reliable carrier and prenatal diagnosis in these families. Direct mutational diagnosis of families at risk in combination with haplotype analysis avoid the drawbacks using only linkage analysis, make genetic counselling far more reliable, and early clinical management of this disease more appropriate. Moreover, pedigree analyses provide first information on de novo mutation frequency in this newly identified human disease gene.

X-linked Recessive Myotubular Myopathy with a Splice-Site Mutation in the Myotubularin Gene

X-linked Recessive Myotubular Myopathy with a Splice-Site Mutation in the Myotubularin Gene

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.

Cousins with X-linked Recessive Myotubular Myopathy

Cousins with X-linked Recessive Myotubular Myopathy

X-linked myotubular myopathy: Refinement of the critical gene region

X-linked recessive myotubular myopathy (XLMTM) is a severe neonatal neuro-muscular disease characterized by muscle weakness, hypotonia, and respiratory problems. The locus for the XLMTM gene (MTM1) has previously been mapped to Xq28 between the markers DXS304 and DXS497 by linkage analyses and by determining the breakpoints of delection patients. We report linkage analysis data of 20 XLMTM families who were tested using the DNA markers DXS1113, DXS304, DXS455, DXS1684, DXS305 and DXS52 and present two families showing recombination between MTM1 and either DXS304, DXS334 or DXS305. We found each of the families to be informative for at least three markers. Based on these findings we excluded 30 women from being carriers, the carrier status of 17 obligate carrier mothers could be confirmed and eight mothers and sisters were identified as to be at high risk of carrying a MTM1 mutation. By combining recently published data with the results of our recombinant families, we suggest that the MTM1 locus maps between DXS334 and DXS497 narrowing the region of interest from 600 kb to an estimated <500 kb interval. This additional refinement in the localization of MTM1 means a further step towards the isolation of the gene in the near future, and allows more reliable and efficient carrier detection and prenatal diagnosis.

A gene mutated in X-linked myotubular myopathy defines a new putative tyrosine phosphatase family conserved in yeast.

X-linked recessive myotubular myopathy (MTM1) is characterized by severe hypotonia and generalized muscle weakness, with impaired maturation of muscle fibres. We have restricted the candidate region to 280 kb and characterized two candidate genes using positional cloning strategies. The presence of frameshift or missense mutations (of which two are new mutations) in seven patients proved that one of these genes is indeed implicated in MTM1. The protein encoded by the MTM1 gene is highly conserved in yeast, which is surprising for a muscle specific disease. The protein contains the consensus sequence for the active site of tyrosine phosphatases, a wide class of proteins involved in signal transduction. At least three other genes, one located within 100 kb distal from the MTM1 gene, encode proteins with very high sequence similarities and define, together with the MTM1 gene, a new family of putative tyrosine phosphatases in man.

The gene for X-linked myotubular myopathy is located in an 8 Mb region at the border of Xq27.3 and Xq28

X-linked recessive myotubular myopathy (XLMTM) is a rare and severe neonatal neuromuscular disease characterized by muscle weakness, hypotonia, and respiratory problems. Here we report an extensive linkage analysis in two families with XLMTM. Using 18 markers in the Xq27-Xqter region we found a maximum two-point lod score of Z = 4.00 at Θ = 0.00 for the marker II-10 (DXS466). Three recombinations were detected between markers and the disease locus. At the distal side of Xq27.3 a recombination was present in between RNI (DXS369) and VK23b (DXS297), another in between VK23b (DXS297) and II-10 (DXS466), and at the proximal side of Xq28 a recombination in between U6.2 (DXS304) and Cpx67 (DXS134). Combining the results of both families we conclude that XLMTM is located in the 8 Mb (11 cM) region between VK23b (DXS297) and Cpx67 (DXS134).

New Insights Into the Pathogenesis of Congenital Myopathies

Congenital myopathies are developmental disorders of muscle that are best understood in the context of ontogenesis. Segmental amyoplasia results from a defective somite, usually because of lack of induction by the notochord and neural tube; the connective tissue matrix of the muscle is derived from lateral mesoderm and is present, but the myocytes are derived from somitic mesoderm and are replaced by adipose cells. Generalized amyoplasia is due to defective myogenic regulatory genes. X-linked recessive myotubular myopathy is associated with overexpression of vimentin and desmin, fetal intermediate filaments that attach to nuclear, mitochondrial, and inner sarcolemmal membranes and Z-bands of sarcomeres to preserve the morphologic organization of the myotube. Neonatal myotonic dystrophy is a true maturational delay in muscle development. Congenital muscle fiber-type disproportion is a syndrome of multiple etiologies but in some cases is associated with cerebellar hypoplasia and may be the result of abnormal suprasegmental stimulation of the developing motor unit at 20 to 28 weeks' gestation, mediated through bulbospinal pathways but not the corticospinal tract. Maturational delay of muscle in late developmental stages is less specific than in stages before midgestation. The Proteus syndrome is a muscular dysgenesis; abnormal paracrine growth factors and perhaps altered genes that regulate muscle differentiation and growth, such as myoD and myogenin, are the suspected cause. Focal proliferative myositis may be another example of a "paracrine myopathy."

Vimentin and desmin in maturing skeletal muscle and developmental myopathies.

I studied vimentin and desmin immunoreactivities in the skeletal muscle of 30 human fetuses and children ranging from 8 weeks' gestation to 2 years of age, and in 45 infants and children and five adults with developmental neuromuscular diseases. Acridine orange-RNA fluorescence also identified regenerating myofibers in Duchenne muscular dystrophy and dermatomyositis for comparison with congenital myopathies. Vimentin and desmin are both strongly expressed in fetal myotubes and their immunohistochemical demonstration persists until 36 weeks' gestation. These cytoskeletal proteins are uniformly expressed in myofibers of neonates with X-linked recessive myotubular myopathy. Desmin but not vimentin is diffusely increased in infantile cases of myotonic dystrophy, in some cases of congenital muscle fiber-type disproportion, and in cerebrohepatorenal disease. In nemaline rod myopathy, desmin is focally increased in perinuclear zones and in regions of aggregated rods. The small myofibers in infantile spinal muscular atrophy show increased vimentin and desmin in the subsarcolemmal region. The demonstration of these intermediate filament proteins provides markers to enhance diagnostic precision in the interpretation of the infant muscle biopsy. Furthermore, persistently high fetal concentrations of vimentin/desmin may play a role in the pathogenesis of some developmental myopathies.