Nasim Vasli

Recessive truncating titin gene, TTN, mutations presenting as centronuclear myopathy.

Objective: To identify causative genes for centronuclear myopathies (CNM), a heterogeneous group of rare inherited muscle disorders that often present in infancy or early life with weakness and hypotonia, using next-generation sequencing of whole exomes and genomes. Methods: Whole-exome or -genome sequencing was performed in a cohort of 29 unrelated patients with clinicopathologic diagnoses of CNM or related myopathy depleted for cases with mutations of MTM1, DNM2, and BIN1. Immunofluorescence analyses on muscle biopsies, splicing assays, and gel electrophoresis of patient muscle proteins were performed to determine the molecular consequences of mutations of interest. Results: Autosomal recessive compound heterozygous truncating mutations of the titin gene, TTN, were identified in 5 individuals. Biochemical analyses demonstrated increased titin degradation and truncated titin proteins in patient muscles, establishing the impact of the mutations. Conclusions: Our study identifies truncating TTN mutations as a cause of congenital myopathy that is reported as CNM. Unlike the classic CNM genes that are all involved in excitation-contraction coupling at the triad, TTN encodes the giant sarcomeric protein titin, which forms a myofibrillar backbone for the components of the contractile machinery. This study expands the phenotypic spectrum associated with TTN mutations and indicates that TTN mutation analysis should be considered in cases of possible CNM without mutations in the classic CNM genes.

Next generation sequencing for molecular diagnosis of neuromuscular diseases

Inherited neuromuscular disorders (NMD) are chronic genetic diseases posing a significant burden on patients and the health care system. Despite tremendous research and clinical efforts, the molecular causes remain unknown for nearly half of the patients, due to genetic heterogeneity and conventional molecular diagnosis based on a gene-by-gene approach. We aimed to test next generation sequencing (NGS) as an efficient and cost-effective strategy to accelerate patient diagnosis. We designed a capture library to target the coding and splice site sequences of all known NMD genes and used NGS and DNA multiplexing to retrieve the pathogenic mutations in patients with heterogeneous NMD with or without known mutations. We retrieved all known mutations, including point mutations and small indels, intronic and exonic mutations, and a large deletion in a patient with Duchenne muscular dystrophy, validating the sensitivity and reproducibility of this strategy on a heterogeneous subset of NMD with different genetic inheritance. Most pathogenic mutations were ranked on top in our blind bioinformatic pipeline. Following the same strategy, we characterized probable TTN, RYR1 and COL6A3 mutations in several patients without previous molecular diagnosis. The cost was less than conventional testing for a single large gene. With appropriate adaptations, this strategy could be implemented into a routine genetic diagnosis set-up as a first screening approach to detect most kind of mutations, potentially before the need of more invasive and specific clinical investigations. An earlier genetic diagnosis should provide improved disease management and higher quality genetic counseling, and ease access to therapy or inclusion into therapeutic trials.

Adult-onset autosomal dominant centronuclear myopathy due to BIN1 mutations

Centronuclear myopathies are congenital muscle disorders characterized by type I myofibre predominance and an increased number of muscle fibres with nuclear centralization. The severe neonatal X-linked form is due to mutations in MTM1, autosomal recessive centronuclear myopathy with neonatal or childhood onset results from mutations in BIN1 (amphiphysin 2), and dominant cases were previously associated to mutations in DNM2 (dynamin 2). Our aim was to determine the genetic basis and physiopathology of patients with mild dominant centronuclear myopathy without mutations in DNM2. We hence established and characterized a homogeneous cohort of nine patients from five families with a progressive adult-onset centronuclear myopathy without facial weakness, including three sporadic cases and two families with dominant disease inheritance. All patients had similar histological and ultrastructural features involving type I fibre predominance and hypotrophy, as well as prominent nuclear centralization and clustering. We identified heterozygous BIN1 mutations in all patients and the molecular diagnosis was complemented by functional analyses. Two mutations in the N-terminal amphipathic helix strongly decreased the membrane-deforming properties of amphiphysin 2 and three stop-loss mutations resulted in a stable protein containing 52 supernumerary amino acids. Immunolabelling experiments revealed abnormal central accumulation of dynamin 2, caveolin-3, and the autophagic marker p62, and general membrane alterations of the triad, the sarcolemma, and the basal lamina as potential pathological mechanisms. In conclusion, we identified BIN1 as the second gene for dominant centronuclear myopathy. Our data provide the evidence that specific BIN1 mutations can cause either recessive or dominant centronuclear myopathy and that both disorders involve different pathomechanisms.

VaRank: a simple and powerful tool for ranking genetic variants.

Background. Most genetic disorders are caused by single nucleotide variations (SNVs) or small insertion/deletions (indels). High throughput sequencing has broadened the catalogue of human variation, including common polymorphisms, rare variations or disease causing mutations. However, identifying one variation among hundreds or thousands of others is still a complex task for biologists, geneticists and clinicians. Results. We have developed VaRank, a command-line tool for the ranking of genetic variants detected by high-throughput sequencing. VaRank scores and prioritizes variants annotated either by Alamut Batch or SnpEff. A barcode allows users to quickly view the presence/absence of variants (with homozygote/heterozygote status) in analyzed samples. VaRank supports the commonly used VCF input format for variants analysis thus allowing it to be easily integrated into NGS bioinformatics analysis pipelines. VaRank has been successfully applied to disease-gene identification as well as to molecular diagnostics setup for several hundred patients. Conclusions. VaRank is implemented in Tcl/Tk, a scripting language which is platform-independent but has been tested only on Unix environment. The source code is available under the GNU GPL, and together with sample data and detailed documentation can be downloaded from http://www.lbgi.fr/VaRank/.

An integrated diagnosis strategy for congenital myopathies.

Congenital myopathies are severe muscle disorders affecting adults as well as children in all populations. The diagnosis of congenital myopathies is constrained by strong clinical and genetic heterogeneity. Moreover, the majority of patients present with unspecific histological features, precluding purposive molecular diagnosis and demonstrating the need for an alternative and more efficient diagnostic approach. We used exome sequencing complemented by histological and ultrastructural analysis of muscle biopsies to identify the causative mutations in eight patients with clinically different skeletal muscle pathologies, ranging from a fatal neonatal myopathy to a mild and slowly progressive myopathy with adult onset. We identified RYR1 (ryanodine receptor) mutations in six patients and NEB (nebulin) mutations in two patients. We found novel missense and nonsense mutations, unraveled small insertions/deletions and confirmed their impact on splicing and mRNA/protein stability. Histological and ultrastructural findings of the muscle biopsies of the patients validated the exome sequencing results. We provide the evidence that an integrated strategy combining exome sequencing with clinical and histopathological investigations overcomes the limitations of the individual approaches to allow a fast and efficient diagnosis, accelerating the patient’s access to a better healthcare and disease management. This is of particular interest for the diagnosis of congenital myopathies, which involve very large genes like RYR1 and NEB as well as genetic and phenotypic heterogeneity.

Impacts of massively parallel sequencing for genetic diagnosis of neuromuscular disorders

Neuromuscular disorders (NMD) such as neuropathy or myopathy are rare and often severe inherited disorders, affecting muscle and/or nerves with neonatal, childhood or adulthood onset, with considerable burden for the patients, their families and public health systems. Genetic and clinical heterogeneity, unspecific clinical features, unidentified genes and the implication of large and/or several genes requiring complementary methods are the main drawbacks in routine molecular diagnosis, leading to increased turnaround time and delay in the molecular validation of the diagnosis. The application of massively parallel sequencing, also called next generation sequencing, as a routine diagnostic strategy could lead to a rapid screening and fast identification of mutations in rare genetic disorders like NMD. This review aims to summarize and to discuss recent advances in the genetic diagnosis of neuromuscular disorders, and more generally monogenic diseases, fostered by massively parallel sequencing. We remind the challenges and benefit of obtaining an accurate genetic diagnosis, introduce the massively parallel sequencing technology and its novel applications in diagnosis of patients, prenatal diagnosis and carrier detection, and discuss the limitations and necessary improvements. Massively parallel sequencing synergizes with clinical and pathological investigations into an integrated diagnosis approach. Clinicians and pathologists are crucial in patient selection and interpretation of data, and persons trained in data management and analysis need to be integrated to the diagnosis pipeline. Massively parallel sequencing for mutation identification is expected to greatly improve diagnosis, genetic counseling and patient management.

Altered Splicing of the BIN1 Muscle-Specific Exon in Humans and Dogs with Highly Progressive Centronuclear Myopathy

Amphiphysin 2, encoded by BIN1, is a key factor for membrane sensing and remodelling in different cell types. Homozygous BIN1 mutations in ubiquitously expressed exons are associated with autosomal recessive centronuclear myopathy (CNM), a mildly progressive muscle disorder typically showing abnormal nuclear centralization on biopsies. In addition, misregulation of BIN1 splicing partially accounts for the muscle defects in myotonic dystrophy (DM). However, the muscle-specific function of amphiphysin 2 and its pathogenicity in both muscle disorders are not well understood. In this study we identified and characterized the first mutation affecting the splicing of the muscle-specific BIN1 exon 11 in a consanguineous family with rapidly progressive and ultimately fatal centronuclear myopathy. In parallel, we discovered a mutation in the same BIN1 exon 11 acceptor splice site as the genetic cause of the canine Inherited Myopathy of Great Danes (IMGD). Analysis of RNA from patient muscle demonstrated complete skipping of exon 11 and BIN1 constructs without exon 11 were unable to promote membrane tubulation in differentiated myotubes. Comparative immunofluorescence and ultrastructural analyses of patient and canine biopsies revealed common structural defects, emphasizing the importance of amphiphysin 2 in membrane remodelling and maintenance of the skeletal muscle triad. Our data demonstrate that the alteration of the muscle-specific function of amphiphysin 2 is a common pathomechanism for centronuclear myopathy, myotonic dystrophy, and IMGD. The IMGD dog is the first faithful model for human BIN1-related CNM and represents a mammalian model available for preclinical trials of potential therapies.

Novel molecular diagnostic approaches for X-linked centronuclear (myotubular) myopathy reveal intronic mutations.

X-linked centronuclear myopathy (XLMTM), also called myotubular myopathy, is a severe congenital myopathy characterized by generalized hypotonia and weakness at birth and the typical histological finding of centralization of myo-nuclei. It is caused by mutations in the MTM1 gene encoding the 3-phosphoinositides phosphatase myotubularin. Mutations in dynamin 2 and amphiphysin 2 genes lead to autosomal forms of centronuclear myopathy (CNM). While XLMTM is the most frequent and severe form of CNM, no mutations are found in about 30% of patients by sequencing all MTM1 exons. Moreover, the impact of MTM1 sequence variants is sometimes difficult to assess. It is thus important to devise a complete molecular diagnostic strategy that includes analysis of the myotubularin transcript and protein expression. We therefore developed novel antibodies against human myotubularin and showed that they are able to detect the endogenous protein by direct Western blot from muscle samples and from cultured cells. In conjunction with RT-PCR analysis we validated the consequences of missense and splice mutations on transcript integrity and protein level. We also detected and characterized a novel deep intronic mutation consisting of a single nucleotide change that induces exonisation of a conserved intronic sequence. Patients with centronuclear myopathy and no molecular diagnosis should be investigated for MTM1 defects at the cDNA and protein level.

Affected female carriers of MTM1 mutations display a wide spectrum of clinical and pathological involvement: delineating diagnostic clues

X-linked myotubular myopathy (XLMTM), a severe congenital myopathy, is caused by mutations in the MTM1 gene located on the X chromosome. A majority of affected males die in the early postnatal period, whereas female carriers are believed to be usually asymptomatic. Nevertheless, several affected females have been reported. To assess the phenotypic and pathological spectra of carrier females and to delineate diagnostic clues, we characterized 17 new unrelated affected females and performed a detailed comparison with previously reported cases at the clinical, muscle imaging, histological, ultrastructural and molecular levels. Taken together, the analysis of this large cohort of 43 cases highlights a wide spectrum of clinical severity ranging from severe neonatal and generalized weakness, similar to XLMTM male, to milder adult forms. Several females show a decline in respiratory function. Asymmetric weakness is a noteworthy frequent specific feature potentially correlated to an increased prevalence of highly skewed X inactivation. Asymmetry of growth was also noted. Other diagnostic clues include facial weakness, ptosis and ophthalmoplegia, skeletal and joint abnormalities, and histopathological signs that are hallmarks of centronuclear myopathy such as centralized nuclei and necklace fibers. The histopathological findings also demonstrate a general disorganization of muscle structure in addition to these specific hallmarks. Thus, MTM1 mutations in carrier females define a specific myopathy, which may be independent of the presence of an XLMTM male in the family. As several of the reported affected females carry large heterozygous MTM1 deletions not detectable by Sanger sequencing, and as milder phenotypes present as adult-onset limb-girdle myopathy, the prevalence of this myopathy is likely to be greatly underestimated. This report should aid diagnosis and thus the clinical management and genetic counseling of MTM1 carrier females. Furthermore, the clinical and pathological history of this cohort may be useful for therapeutic projects in males with XLMTM, as it illustrates the spectrum of possible evolution of the disease in patients surviving long term.

Recessive mutations in the kinase ZAK cause a congenital myopathy with fibre type disproportion

Congenital myopathies define a heterogeneous group of neuromuscular diseases with neonatal or childhood hypotonia and muscle weakness. The genetic cause is still unknown in many patients, precluding genetic counselling and better understanding of the physiopathology. To identify novel genetic causes of congenital myopathies, exome sequencing was performed in three consanguineous families. We identified two homozygous frameshift mutations and a homozygous nonsense mutation in the mitogen-activated protein triple kinase ZAK. In total, six affected patients carry these mutations. Reverse transcription polymerase chain reaction and transcriptome analyses suggested nonsense mRNA decay as a main impact of mutations. The patients demonstrated a generalized slowly progressive muscle weakness accompanied by decreased vital capacities. A combination of proximal contractures with distal joint hyperlaxity is a distinct feature in one family. The low endurance and compound muscle action potential amplitude were strongly ameliorated on treatment with anticholinesterase inhibitor in another patient. Common histopathological features encompassed fibre size variation, predominance of type 1 fibre and centralized nuclei. A peculiar subsarcolemmal accumulation of mitochondria pointing towards the centre of the fibre was a novel histological hallmark in one family. These findings will improve the molecular diagnosis of congenital myopathies and implicate the mitogen-activated protein kinase (MAPK) signalling as a novel pathway altered in these rare myopathies.