Anoctamin-5 related muscle disease: clinical and genetic findings in a large European cohort.

Anoctamin-5 related muscle disease is caused by biallelic pathogenic variants in the anoctamin-5 gene (ANO5) and shows variable clinical phenotypes: limb-girdle muscular dystrophy type 12 (LGMD-R12), distal muscular dystrophy type 3 (MMD3), pseudometabolic myopathy or asymptomatic hyperCKaemia. In this retrospective, observational, multicentre study we gathered a large European cohort of patients with ANO5-related muscle disease to study the clinical and genetic spectrum and genotype-phenotype correlations. We included 234 patients from 212 different families, contributed by 15 centres from 11 European countries. The largest subgroup was LGMD-R12 (52.6%), followed by pseudometabolic myopathy (20.5%), asymptomatic hyperCKaemia (13.7%) and MMD3 (13.2%). In all subgroups, there was a male predominance, except for pseudometabolic myopathy. Median age at symptom onset of all patients was 33 years (range 23-45 years). The most frequent symptoms at onset were myalgia (35.3%) and exercise intolerance (34.1%), while at last clinical evaluation most frequent symptoms and signs were proximal lower limb weakness (56.9%) and atrophy (38.1%), myalgia (45.1%) and atrophy of the medial gastrocnemius muscle (38.4%). Most patients remained ambulatory (79.4%). At last evaluation, 45.9% of patients with LGMD-R12 additionally had distal weakness in the lower limbs and 48.4% of patients with MMD3 also showed proximal lower limb weakness. Age at symptom onset did not differ significantly between males and females. However, males had a higher risk of using walking aids earlier (P = 0.035). No significant association was identified between sportive versus non-sportive lifestyle before symptom onset and age at symptom onset nor any of the motor outcomes. Cardiac and respiratory involvement that would require treatment occurred very rarely. Ninety-nine different pathogenic variants were identified in ANO5 of which 25 were novel. The most frequent variants were c.191dupA (p.Asn64Lysfs*15) (57.7%) and c.2272C>T (p.Arg758Cys) (11.1%). Patients with two loss-of function variants used walking aids at a significantly earlier age (P = 0.037). Patients homozygous for the c.2272C>T variant showed a later use of walking aids compared to patients with other variants (P = 0.043). We conclude that there was no correlation of the clinical phenotype with the specific genetic variants, and that LGMD-R12 and MMD3 predominantly affect males who have a significantly worse motor outcome. Our study provides useful information for clinical follow up of the patients and for the design of clinical trials with novel therapeutic agents.

Targeted Next-Generation Sequencing Reveals Mutations in Non-coding Regions and Potential Regulatory Sequences of Calpain-3 Gene in Polish Limb-Girdle Muscular Dystrophy Patients.

Limb-girdle muscular dystrophy type R1 (LGMDR1) is caused by mutations in CAPN3 and is the most common type of recessive LGMD. Even with the use of whole-exome sequencing (WES), only one mutant allele of CAPN3 is found in a significant number of LGMDR patients. This points to a role of non-coding, intronic or regulatory, sequence variants in the disease pathogenesis. Targeted sequencing of the whole CAPN3 gene including not only intronic, 3' and 5' UTRs but also potential regulatory regions was performed in 27 patients suspected with LGMDR1. This group included 13 patients with only one mutated CAPN3 allele detected previously with exome sequencing. A second rare variant in the non-coding part of CAPN3 was found in 11 of 13 patients with previously identified single mutation. Intronic mutations were found in 10 cases, with c.1746-20C>G variant present in seven patients. In addition, a large deletion of exons 2-8 was found in one patient. In the patients with no causative mutation previously found, we detected rare CAPN3 variants in 5 out of 10 patients and in two of them in a compound heterozygous state. Rare variants within putative regulatory sequences distant from the CAPN3 gene were found in 15 patients, although in 11 of these cases, other variants are deemed causative. The results indicate that intronic mutations are common in Polish LGMDR patients, and testing for non-coding mutations in CAPN3 should be performed in apparently single heterozygous patients.

ANO5 mutations in the Polish limb girdle muscular dystrophy patients: Effects on the protein structure.

LGMD2L is a subtype of limb-girdle muscular dystrophy (LGMD), caused by recessive mutations in ANO5, encoding anoctamin-5 (ANO5). We present the analysis of five patients with skeletal muscle weakness for whom heterozygous mutations within ANO5 were identified by whole exome sequencing (WES). Patients varied in the age of the disease onset (from 22 to 38 years) and severity of the morphological and clinical phenotypes. Out of the nine detected mutations one was novel (missense p.Lys132Met, accompanied by p.His841Asp) and one was not yet characterized in the literature (nonsense, p.Trp401Ter, accompanied by p.Asp81Gly). The p.Asp81Gly mutation was also identified in another patient carrying a p.Arg758Cys mutation as well. Also, a c.191dupA frameshift (p.Asn64LysfsTer15), the first described and common mutation was identified. Mutations were predicted by in silico tools to have damaging effects and are likely pathogenic according to criteria of the American College of Medical Genetics and Genomics (ACMG). Indeed, molecular modeling of mutations revealed substantial changes in ANO5 conformation that could affect the protein structure and function. In addition, variants in other genes associated with muscle pathology were identified, possibly affecting the disease progress. The presented data indicate that the identified ANO5 mutations contribute to the observed muscle pathology and broaden the genetic spectrum of LGMD myopathies.

Whole-exome sequencing identifies novel pathogenic mutations and putative phenotype-influencing variants in Polish limb-girdle muscular dystrophy patients.

BACKGROUND: Limb girdle muscular dystrophies (LGMD) are a group of heterogeneous hereditary myopathies with similar clinical symptoms. Disease onset and progression are highly variable, with an elusive genetic background, and around 50% cases lacking molecular diagnosis. METHODS: Whole exome sequencing (WES) was performed in 73 patients with clinically diagnosed LGMD. A filtering strategy aimed at identification of variants related to the disease included integrative analysis of WES data and human phenotype ontology (HPO) terms, analysis of genes expressed in muscle, analysis of the disease-associated interactome and copy number variants analysis. RESULTS: Genetic diagnosis was possible in 68.5% of cases. On average, 36.3 rare variants in genes associated with various muscle diseases per patient were found that could relate to the clinical phenotype. The putative causative mutations were mostly in LGMD-associated genes, but also in genes not included in the current LGMD classification (DMD, COL6A2, and COL6A3). In three patients, mutations in two genes were suggested as the joint cause of the disease (CAPN3+MYH7, COL6A3+CACNA1S, DYSF+MYH7). Moreover, a variety of phenotype-influencing variants were postulated, including in patients with an identified already known primary pathogenic mutation. CONCLUSIONS: We hypothesize that LGMD could be better described as oligogenic disorders in which dominant clinical presentation can result from the combined effect of mutations in a set of genes. In this view, the inter- and intrafamilial variability could reflect a specific genetic background and the presence of sets of phenotype-influencing or co-causative mutations in genes that either interact with the known LGMD-associated genes or are a part of the same pathways or structures.

Abnormal spontaneous activity in primary myopathic disorders.

INTRODUCTION: Reproducible non-insertional spontaneous activity (SA), with the exception of endplate activity, is an unequivocal sign of abnormality and is one of the most useful findings obtained on electromyography. METHODS: In this retrospective study we analyzed occurrence and distribution of abnormal SA in 151 patients with genetically confirmed myopathies. RESULTS: Complex repetitive discharges (CRDs) occurred more frequently than fibrillation potentials (fibs) and positive sharp waves (PSWs) in centronuclear myopathy (CNM) and limb-girdle muscular dystrophy type 2A (LGMD-2A), whereas fibs/PSWs were observed more often in desminopathy and facioscapulohumeral dystrophy (FSHD). Abnormal SA was commonly found in CNM (66.7%) and desminopathy (61.5%), occasionally in Duchenne (DMD) and Becker muscular dystrophy (BMD) (45.2% and 27.6%, respectively), but rarely in FSHD (14.9%) and LGMD-2A (12.0%). CONCLUSIONS: Abnormal SA probably occurs more frequently in disorders associated with structural changes in muscle fibers. Screening for SA may be a valuable tool for diagnosis of non-myotonic myopathies. Muscle Nerve 56: 427-432, 2017.

CAV3 mutation in a patient with transient hyperCKemia and myalgia.

Mutations in caveolin-3 (CAV3) can lead to different clinical phenotypes affecting skeletal or cardiac muscles. Here, we describe a patient with Klinefelter syndrome, ulcerative colitis and Sjögren syndrome, who developed transient hyperCKemia, myalgia and mild muscular weakness. Using whole exome sequencing (WES), a missense mutation G169A was found in the CAV3 gene. In addition, we identified a homozygous frameshift deletion in MS4A12 that may contribute to inflammatory bowel disease, further demonstrating usefulness of WES in dual molecular diagnoses.