Dominantly inherited distal nemaline/cap myopathy caused by a large deletion in the nebulin gene.

We report the first family with a dominantly inherited mutation of the nebulin gene (NEB). This ∼100 kb in-frame deletion encompasses NEB exons 14-89, causing distal nemaline/cap myopathy in a three-generation family. It is the largest deletion characterized in NEB hitherto. The mutated allele was shown to be expressed at the mRNA level and furthermore, for the first time, a deletion was shown to cause the production of a smaller mutant nebulin protein. Thus, we suggest that this novel mutant nebulin protein has a dominant-negative effect, explaining the first documented dominant inheritance of nebulin-caused myopathy. The index patient, a young man, was more severely affected than his mother and grandmother. His first symptom was foot drop at the age of three, followed by distal muscle atrophy, slight hypomimia, high-arched palate, and weakness of the neck and elbow flexors, hands, tibialis anterior and toe extensors. Muscle biopsies showed myopathic features with type 1 fibre predominance in the index patient and nemaline bodies and cap-like structures in biopsies from his mother and grandmother. The muscle biopsy findings constitute a further example of nemaline bodies and cap-like structures being part of the same spectrum of pathological changes.

Interpreting Genetic Variants in Titin in Patients With Muscle Disorders.

Importance: Mutations in the titin gene (TTN) cause a wide spectrum of genetic diseases. The interpretation of the numerous rare variants identified in TTN is a difficult challenge given its large size. Objective: To identify genetic variants in titin in a cohort of patients with muscle disorders. Design, Setting, and Participants: In this case series, 9 patients with titinopathy and 4 other patients with possibly disease-causing variants in TTN were identified. Titin mutations were detected through targeted resequencing performed on DNA from 504 patients with muscular dystrophy, congenital myopathy, or other skeletal muscle disorders. Patients were enrolled from 10 clinical centers in April 2012 to December 2013. All of them had not received a diagnosis after undergoing an extensive investigation, including Sanger sequencing of candidate genes. The data analysis was performed between September 2013 and January 2017. Sequencing data were analyzed using an internal custom bioinformatics pipeline. Main Outcomes and Measures: The identification of novel mutations in the TTN gene and novel patients with titinopathy. We performed an evaluation of putative causative variants in the TTN gene, combining genetic, clinical, and imaging data with messenger RNA and/or protein studies. Results: Of the 9 novel patients with titinopathy, 5 (55.5%) were men and the mean (SD) age at onset was 25 (15.8) years (range, 0-46 years). Of the 4 other patients (3 men and 1 woman) with possibly disease-causing TTN variants, 2 (50%) had a congenital myopathy and 2 (50%) had a slowly progressive distal myopathy with onset in the second decade. Most of the identified mutations were previously unreported. However, all the variants, even the already described mutations, require careful clinical and molecular evaluation of probands and relatives. Heterozygous truncating variants or unique missense changes are not sufficient to make a diagnosis of titinopathy. Conclusions and Relevance: The interpretation of TTN variants often requires further analyses, including a comprehensive evaluation of the clinical phenotype (deep phenotyping) as well as messenger RNA and protein studies. We propose a specific workflow for the clinical interpretation of genetic findings in titin.

TIA1 variant drives myodegeneration in multisystem proteinopathy with SQSTM1 mutations.

Multisystem proteinopathy (MSP) involves disturbances of stress granule (SG) dynamics and autophagic protein degradation that underlie the pathogenesis of a spectrum of degenerative diseases that affect muscle, brain, and bone. Specifically, identical mutations in the autophagic adaptor SQSTM1 can cause varied penetrance of 4 distinct phenotypes: amyotrophic lateral sclerosis (ALS), frontotemporal dementia, Paget's disease of the bone, and distal myopathy. It has been hypothesized that clinical pleiotropy relates to additional genetic determinants, but thus far, evidence has been lacking. Here, we provide evidence that a TIA1 (p.N357S) variant dictates a myodegenerative phenotype when inherited, along with a pathogenic SQSTM1 mutation. Experimentally, the TIA1-N357S variant significantly enhances liquid-liquid-phase separation in vitro and impairs SG dynamics in living cells. Depletion of SQSTM1 or the introduction of a mutant version of SQSTM1 similarly impairs SG dynamics. TIA1-N357S-persistent SGs have increased association with SQSTM1, accumulation of ubiquitin conjugates, and additional aggregated proteins. Synergistic expression of the TIA1-N357S variant and a SQSTM1-A390X mutation in myoblasts leads to impaired SG clearance and myotoxicity relative to control myoblasts. These findings demonstrate a pathogenic connection between SG homeostasis and ubiquitin-mediated autophagic degradation that drives the penetrance of an MSP phenotype.

A novel FLNC frameshift and an OBSCN variant in a family with distal muscular dystrophy.

A novel FLNC c.5161delG (p.Gly1722ValfsTer61) mutation was identified in two members of a French family affected by distal myopathy and in one healthy relative. This FLNC c.5161delG mutation is one nucleotide away from a previously reported FLNC mutation (c.5160delC) that was identified in patients and in asymptomatic carriers of three Bulgarian families with distal muscular dystrophy, indicating a low penetrance of the FLNC frameshift mutations. Given these similarities, we believe that the two FLNC mutations alone can be causative of distal myopathy without full penetrance. Moreover, comparative analysis of the clinical manifestations indicates that patients of the French family show an earlier onset and a complete segregation of the disease. As a possible explanation of this, the two French patients also carry a OBSCN c.13330C>T (p.Arg4444Trp) mutation. The p.Arg4444Trp variant is localized within the OBSCN Ig59 domain that, together with Ig58, binds to the ZIg9/ZIg10 domains of titin at Z-disks. Structural and functional studies indicate that this OBSCN p.Arg4444Trp mutation decreases titin binding by ~15-fold. On this line, we suggest that the combination of the OBSCN p.Arg4444Trp variant and of the FLNC c.5161delG mutation, can cooperatively affect myofibril stability and increase the penetrance of muscular dystrophy in the French family.

A 'second truncation' in TTN causes early onset recessive muscular dystrophy.

Mutations in the gene encoding the giant skeletal muscle protein titin are associated with a variety of muscle disorders, including recessive congenital myopathies ±cardiomyopathy, limb girdle muscular dystrophy (LGMD) and late onset dominant distal myopathy. Heterozygous truncating mutations have also been linked to dilated cardiomyopathy. The phenotypic spectrum of titinopathies is emerging and expanding, as next generation sequencing techniques make this large gene amenable to sequencing. We undertook whole exome sequencing in four individuals with LGMD. An essential splice site mutation, previously reported in dilated cardiomyopathy, was identified in all families in combination with a second truncating mutation. Affected individuals presented with childhood onset proximal weakness associated with joint contractures and elevated CK. Cardiac dysfunction was present in two individuals. Muscle biopsy showed increased internal nuclei and immunoblotting identified reduction or absence of calpain-3 and demonstrated a marked reduction of C-terminal titin fragments. We confirm the co-occurrence of cardiac and skeletal myopathies associated with recessive truncating titin mutations. Compound heterozygosity of a truncating mutation previously associated with dilated cardiomyopathy and a 'second truncation' in TTN was identified as causative in our skeletal myopathy patients. These findings add to the complexity of interpretation and genetic counselling for titin mutations.

Targeted Next-Generation Sequencing Reveals Novel TTN Mutations Causing Recessive Distal Titinopathy.

Tibial muscular dystrophy (TMD) is the first described human titinopathy. It is a mild adult-onset slowly progressive myopathy causing weakness and atrophy in the anterior lower leg muscles. TMD is caused by mutations in the last two exons, Mex5 and Mex6, of the titin gene (TTN). The first reported TMD mutations were dominant, but the Finnish founder mutation FINmaj, an 11-bp insertion/deletion in Mex6, in homozygosity caused a completely different severe early-onset limb-girdle muscular dystrophy 2J (LGMD2J). Later, we reported that not all TMD mutations cause LGMD when homozygous or compound heterozygous with truncating mutation, but some of them rather cause a more severe TMD-like distal disease. We have now performed targeted next-generation sequencing of myopathy-related genes on seven families from Albania, Bosnia, Iran, Tunisia, Belgium, and Spain with juvenile or early adult onset recessive distal myopathy. Novel mutations in TTN Mex5, Mex6 and A-band exon 340 were identified in homozygosity or compound heterozygosity with a frameshift or nonsense mutation in TTN I- or A-band region. Family members having only one of these TTN mutations were healthy. Our results add yet another entity to the list of distal myopathies: juvenile or early adult onset recessive distal titinopathy.

Homozygosity of the Dominant Myotilin c.179C>T (p.Ser60Phe) Mutation Causes a More Severe and Proximal Muscular Dystrophy.

Most myotilinopathy patients present with a dominant late onset distal phenotype and myofibrillar pathology, although the first MYOT mutation in a family reported to have LGMD phenotype. We report here a French family affected with a late onset proximal and distal muscle weakness and myofibrillar myopathy on muscle pathology, in which the siblings known to be clinically affected were homozygous for the c.179C>T (p.Ser60Phe) myotilin gene mutation. One subjectively asymptomatic member of the family was heterozygous for this mutation. This is the first report of a family with patients being homozygous for a known dominant MYOT mutation. Dominant negative mutations are generally considered not to cause a more severe disease in homozygosity, but our data clearly demonstrate the existence of dominant MYOT mutations with a possible dose effect causing a more severe disease phenotype in homozygosity in the spectrum of myofibrillar myopathies (MFM).

The genetic basis of undiagnosed muscular dystrophies and myopathies: Results from 504 patients.

OBJECTIVE: To apply next-generation sequencing (NGS) for the investigation of the genetic basis of undiagnosed muscular dystrophies and myopathies in a very large cohort of patients. METHODS: We applied an NGS-based platform named MotorPlex to our diagnostic workflow to test muscle disease genes with a high sensitivity and specificity for small DNA variants. We analyzed 504 undiagnosed patients mostly referred as being affected by limb-girdle muscular dystrophy or congenital myopathy. RESULTS: MotorPlex provided a complete molecular diagnosis in 218 cases (43.3%). A further 160 patients (31.7%) showed as yet unproven candidate variants. Pathogenic variants were found in 47 of 93 genes, and in more than 30% of cases, the phenotype was nonconventional, broadening the spectrum of disease presentation in at least 10 genes. CONCLUSIONS: Our large DNA study of patients with undiagnosed myopathy is an example of the ongoing revolution in molecular diagnostics, highlighting the advantages in using NGS as a first-tier approach for heterogeneous genetic conditions.

Mutations in HSPB8 causing a new phenotype of distal myopathy and motor neuropathy.

OBJECTIVE: To report novel disease and pathology due to HSPB8 mutations in 2 families with autosomal dominant distal neuromuscular disease showing both myofibrillar and rimmed vacuolar myopathy together with neurogenic changes. METHODS: We performed whole-exome sequencing (WES) in tandem with linkage analysis and candidate gene approach as well as targeted next-generation sequencing (tNGS) to identify causative mutations in 2 families with dominant rimmed vacuolar myopathy and a motor neuropathy. Pathogenic variants and familial segregation were confirmed using Sanger sequencing. RESULTS: WES and tNGS identified a heterozygous change in HSPB8 in both families: c.421A > G p.K141E in family 1 and c.151insC p.P173SfsX43 in family 2. Affected patients had a distal myopathy that showed myofibrillar aggregates and rimmed vacuoles combined with a clear neurogenic component both on biopsy and neurophysiologic studies. MRI of lower limb muscles demonstrated diffuse tissue changes early in the disease stage progressing later to fatty replacement typical of a myopathy. CONCLUSION: We expand the understanding of disease mechanisms, tissue involvement, and phenotypic outcome of HSPB8 mutations. HSPB8 is part of the chaperone-assisted selective autophagy (CASA) complex previously only associated with Charcot-Marie-Tooth type 2L (OMIM 60673) and distal hereditary motor neuronopathy type IIa. However, we now demonstrate that patients can develop a myopathy with histologic features of myofibrillar myopathy with aggregates and rimmed vacuoles, similar to the pathology in myopathies due to gene defects in other compounds of the CASA complex such as BAG3 and DNAJB6 after developing the early neurogenic effects.

Targeted next-generation sequencing assay for detection of mutations in primary myopathies.

Mutations in more than 100 different genes are known to cause hereditary primary myopathies. In patients with less distinct phenotypes several genes may have to be sequenced in order to make the correct diagnosis. The large number of possible candidate genes and overlapping phenotypes, as well as an enormous size of some of the genes such as DMD, TTN and NEB, constitute difficult challenges for molecular genetic diagnostics using conventional sequencing. Molecular characterization is nevertheless important for the final diagnosis and accurate management of the diseases. Targeted next-generation sequencing is a rapid and cost-effective method to sequence large numbers of genes simultaneously. We developed a targeted next-generation sequencing assay, MyoCap, for the coding exons and UTRs of 180 myopathy related genes including 42 novel genes that have not yet been associated with myopathies. DNA samples of four controls with known mutations and 61 patients negative for previous candidate gene approaches were sequenced. The genetic defect was totally or partly clarified in 21 patients with nine of them having potential disease-causing mutations in TTN. MyoCap provides higher read depth and coverage with lower price in the myopathy related genes compared to the whole exome sequencing and is thus very suitable for diagnostic use.

Novel mutations in DNAJB6 gene cause a very severe early-onset limb-girdle muscular dystrophy 1D disease.

DNAJB6 is the causative gene for limb-girdle muscular dystrophy 1D (LGMD1D). Four different coding missense mutations, p.F89I, p.F93I, p.F93L, and p.P96R, have been reported in families from Europe, North America and Asia. The previously known mutations cause mainly adult-onset proximal muscle weakness with moderate progression and without respiratory involvement. A Finnish family and a British patient have been studied extensively due to a severe muscular dystrophy. The patients had childhood-onset LGMD, loss of ambulation in early adulthood and respiratory involvement; one patient died of respiratory failure aged 32. Two novel mutations, c.271T > A (p.F91I) and c.271T > C (p.F91L), in DNAJB6 were identified by whole exome sequencing as a cause of this severe form of LGMD1D. The results were confirmed by Sanger sequencing. The anti-aggregation effect of the mutant DNAJB6 was investigated in a filter-trap based system using transient transfection of mammalian cell lines and polyQ-huntingtin as a model for an aggregation-prone protein. Both novel mutant proteins show a significant loss of ability to prevent aggregation.

SQSTM1 splice site mutation in distal myopathy with rimmed vacuoles.

OBJECTIVE: To identify the genetic etiology and characterize the clinicopathologic features of a novel distal myopathy. METHODS: We performed whole-exome sequencing on a family with an autosomal dominant distal myopathy and targeted exome sequencing in 1 patient with sporadic distal myopathy, both with rimmed vacuolar pathology. We also evaluated the pathogenicity of identified mutations using immunohistochemistry, Western blot analysis, and expression studies. RESULTS: Sequencing identified a likely pathogenic c.1165+1 G>A splice donor variant in SQSTM1 in the affected members of 1 family and in an unrelated patient with sporadic distal myopathy. Affected patients had late-onset distal lower extremity weakness, myopathic features on EMG, and muscle pathology demonstrating rimmed vacuoles with both TAR DNA-binding protein 43 and SQSTM1 inclusions. The c.1165+1 G>A SQSTM1 variant results in the expression of 2 alternatively spliced SQSTM1 proteins: 1 lacking the C-terminal PEST2 domain and another lacking the C-terminal ubiquitin-associated (UBA) domain, both of which have distinct patterns of cellular and skeletal muscle localization. CONCLUSIONS: SQSTM1 is an autophagic adaptor that shuttles aggregated and ubiquitinated proteins to the autophagosome for degradation via its C-terminal UBA domain. Similar to mutations in VCP, dominantly inherited mutations in SQSTM1 are now associated with rimmed vacuolar myopathy, Paget disease of bone, amyotrophic lateral sclerosis, and frontotemporal dementia. Our data further suggest a pathogenic connection between the disparate phenotypes.

Centronuclear myopathies: genotype-phenotype correlation and frequency of defined genetic forms in an Italian cohort.

Centronuclear myopathies (CNMs) are a group of clinically and genetically heterogeneous muscle disorders. To date, mutation in 7 different genes has been reported to cause CNMs but 30 % of cases still remain genetically undefined. Genetic investigations are often expensive and time consuming. Clinical and morphological clues are needed to facilitate genetic tests and to choose the best approach for genetic screening. We aimed to describe genotype-phenotype correlation in an Italian cohort of patients affected by CNMs, to define the relative frequencies of its defined genetic forms and to draw a diagnostic algorithm to address genetic investigations. We recruited patients with CNMs from all the Italian tertiary neuromuscular centers following clinical and histological criteria. All selected patients were screened for the four 'canonical' genes related to CNMs: MTM1, DNM2, RYR1 and BIN1. Pathogenetic mutations were found in 38 of the 54 screened patients (70 %), mostly in patients with congenital onset (25 of 30 patients, 83 %): 15 in MTM1, 6 in DNM2, 3 in RYR1 and one in TTN. Among the 13 patients with a childhood-adolescence onset, mutations were found in 6 patients (46 %), all in DNM2. In the group of the 11 patients with adult onset, mutations were identified in 7 patients (63 %), again in DNM2, confirming that variants in this gene are relatively more common in late-onset phenotypes. The present study provides the relative molecular frequency of centronuclear myopathy and of its genetically defined forms in Italy and also proposes a diagnostic algorithm to be used in clinical practice to address genetic investigations.

Atypical phenotypes in titinopathies explained by second titin mutations.

OBJECTIVE: Several patients with previously reported titin gene (TTN) mutations causing tibial muscular dystrophy (TMD) have more complex, severe, or unusual phenotypes. This study aimed to clarify the molecular cause of the variant phenotypes in 8 patients of 7 European families. METHODS: Clinical, histopathological, and muscle imaging data of patients and family members were reanalyzed. The titin protein was analyzed by Western blotting and TTN gene by reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing. RESULTS: Western blotting showed more pronounced C-terminal titin abnormality than expected for heterozygous probands, suggesting the existence of additional TTN mutations. RT-PCR indicated unequal mRNA expression of the TTN alleles in biopsies of 6 patients, 3 with an limb-girdle muscular dystrophy type 2J (LGMD2J) phenotype. Novel frameshift mutations were identified in 5 patients. A novel A-band titin mutation, c.92167C>T (p.P30723S), was found in 1 patient, and 1 Portuguese patient with a severe TMD phenotype proved to be homozygous for the previously reported Iberian TMD mutation. INTERPRETATION: The unequal expression levels of TTN transcripts in 5 probands suggested severely reduced expression of the frameshift mutated allele, probably through nonsense-mediated decay, explaining the more severe phenotypes. The Iberian TMD mutation may cause a more severe TMD rather than LGMD2J when homozygous. The Finnish patient compound heterozygous for the FINmaj TMD mutation and the novel A-band titin missense mutation showed a phenotype completely different from previously described titinopathies. Our results further expand the complexity of muscular dystrophies caused by TTN mutations and suggest that the coexistence of second mutations may constitute a more common general mechanism explaining phenotype variability.

Hereditary myopathy with early respiratory failure: occurrence in various populations.

OBJECTIVE: Several families with characteristic features of hereditary myopathy with early respiratory failure (HMERF) have remained without genetic cause. This international study was initiated to clarify epidemiology and the genetic underlying cause in these families, and to characterise the phenotype in our large cohort. METHODS: DNA samples of all currently known families with HMERF without molecular genetic cause were obtained from 12 families in seven different countries. Clinical, histopathological and muscle imaging data were collected and five biopsy samples made available for further immunohistochemical studies. Genotyping, exome sequencing and Sanger sequencing were used to identify and confirm sequence variations. RESULTS: All patients with clinical diagnosis of HMERF were genetically solved by five different titin mutations identified. One mutation has been reported while four are novel, all located exclusively in the FN3 119 domain (A150) of A-band titin. One of the new mutations showed semirecessive inheritance pattern with subclinical myopathy in the heterozygous parents. Typical clinical features were respiratory failure at mid-adulthood in an ambulant patient with very variable degree of muscle weakness. Cytoplasmic bodies were retrospectively observed in all muscle biopsy samples and these were reactive for myofibrillar proteins but not for titin. CONCLUSIONS: We report an extensive collection of families with HMERF with five different mutations in exon 343 of TTN, which establishes this exon as the primary target for molecular diagnosis of HMERF. Our relatively large number of new families and mutations directly implies that HMERF is not extremely rare, not restricted to Northern Europe and should be considered in undetermined myogenic respiratory failure.

Welander distal myopathy is caused by a mutation in the RNA-binding protein TIA1.

OBJECTIVE: A study was undertaken to identify the molecular cause of Welander distal myopathy (WDM), a classic autosomal dominant distal myopathy. METHODS: The genetic linkage was confirmed and defined by microsatellite and single nucleotide polymorphism haplotyping. The whole linked genomic region was sequenced with targeted high-throughput and Sanger sequencing, and coding transcripts were sequenced on the cDNA level. WDM muscle biopsies were studied by Western blotting and immunofluorescence microscopy. Splicing of TIA1 and its target genes in muscle and myoblast cultures was analyzed by reverse transcriptase polymerase chain reaction. Mutant TIA1 was characterized by cell biological studies on HeLa cells, including quantification of stress granules by high content analysis and fluorescence recovery after photobleaching (FRAP) experiments. RESULTS: The linked haplotype at 2p13 was narrowed down to <806 kb. Sequencing by multiple methods revealed only 1 segregating coding mutation, c.1362 G>A (p.E384K) in the RNA-binding protein TIA1, a key component of stress granules. Immunofluorescence microscopy of WDM biopsies showed a focal increase of TIA1 in atrophic and vacuolated fibers. In HeLa cells, mutant TIA1 constructs caused a mild increase in stress granule abundance compared to wild type, and showed slower average fluorescence recovery in FRAP. INTERPRETATION: WDM is caused by mutated TIA1 through a dominant pathomechanism probably involving altered stress granule dynamics.

Titin mutation segregates with hereditary myopathy with early respiratory failure.

In 2001, we described an autosomal dominant myopathy characterized by neuromuscular ventilatory failure in ambulant patients. Here we describe the underlying genetic basis for the disorder, and we define the neuromuscular, respiratory and radiological phenotype in a study of 31 mutation carriers followed for up to 31 years. A combination of genome-wide linkage and whole exome sequencing revealed the likely causal genetic variant in the titin (TTN) gene (g.274375T>C; p.Cys30071Arg) within a shared haplotype of 2.93 Mbp on chromosome 2. This segregated with the phenotype in 21 individuals from the original family, nine subjects in a second family with the same highly selective pattern of muscle involvement on magnetic resonance imaging and a third familial case with a similar phenotype. Comparing the mutation carriers revealed novel features not apparent in our original report. The clinical presentation included predominant distal, proximal or respiratory muscle weakness. The age of onset was highly variable, from early adulthood, and including a mild phenotype in advanced age. Muscle weakness was earlier onset and more severe in the lower extremities in nearly all patients. Seven patients also had axial muscle weakness. Respiratory function studies demonstrated a gradual deterioration over time, reflecting the progressive nature of this condition. Cardiomyopathy was not present in any of our patients despite up to 31 years of follow-up. Magnetic resonance muscle imaging was performed in 21 affected patients and revealed characteristic abnormalities with semitendinosus involvement in 20 of 21 patients studied, including 3 patients who were presymptomatic. Diagnostic muscle histopathology most frequently revealed eosinophilic inclusions (inclusion bodies) and rimmed vacuoles, but was non-specific in a minority of patients. These findings have important clinical implications. This disease should be considered in patients with adult-onset proximal or distal myopathy and early respiratory failure, even in the presence of non-specific muscle pathology. Muscle magnetic resonance imaging findings are characteristic and should be considered as an initial investigation, and if positive should prompt screening for mutations in TTN. With 363 exons, screening TTN presented a major challenge until recently. However, whole exome sequencing provides a reliable cost-effective approach, providing the gene of interest is adequately captured.