The aim was to determine the genetic background of unknown muscular dystrophy in five French families.

The group of chondrodysplasia with multiple dislocations includes several entities, characterized by short stature, dislocation of large joints, hand and/or vertebral anomalies. Other features, such as epiphyseal or metaphyseal changes, cleft palate, intellectual disability are also often part of the phenotype. In addition, several conditions with overlapping features are related to this group and broaden the spectrum. The majority of these disorders have been linked to pathogenic variants in genes encoding proteins implicated in the synthesis or sulfation of proteoglycans (PG). In a series of 30 patients with multiple dislocations, we have performed exome sequencing and subsequent targeted analysis of 15 genes, implicated in chondrodysplasia with multiple dislocations, and related conditions. We have identified causative pathogenic variants in 60% of patients (18/30); when a clinical diagnosis was suspected, this was molecularly confirmed in 53% of cases. Forty percent of patients remain without molecular etiology. Pathogenic variants in genes implicated in PG synthesis are of major importance in chondrodysplasia with multiple dislocations and related conditions. The combination of hand features, growth failure severity, radiological aspects of long bones and of vertebrae allowed discrimination among the different conditions. We propose key diagnostic clues to the clinician.

Congenital muscular dystrophies (CMD) represent a set of primary neuromuscular disorders of very heterogeneous nature, both clinically and genetically. A growing number of defective genes have been identified in this group. Among them, INPP5K, a gene encoding a phosphoinositide 5-phosphatase, has recently been found mutated in twelve families of various ethnic origins. We report here two sisters living in the Reunion Island and born to a non-consanguineous couple of Creole descent. Both, aged 13 and 11 respectively, were investigated since early childhood for muscle weakness associated with learning difficulties. Of note was the presence of early bilateral cataracts in one sister that required surgery at age 4. The elevation of serum CK levels, up to roughly 5 times normal values, and a dystrophic pattern on muscle biopsy strongly suggested congenital muscular dystrophy. All immunofluorescence stains on muscle biopsies were expressed normally, even using semi-quantitative method [Western Blotting]. Various genes were subsequently screened, notably CAPN3, a gene frequently involved in the Reunion Island, DM1 due to the association with cognitive deficits and early cataracts, and FKRP. Interestingly, the mother developed early cataracts and underwent eye surgery at age 37. The family was included within the “Myocapture project”. The aim of this project is to identify new genes responsible for neuromuscular diseases by an exome sequencing approach in patients that were extensively investigated at clinical, muscle imaging and histopathological levels. Using this approach, we have identified a new variant in the INNP5K gene. The homozygous nucleotide change modifies an amino acid in the highly conserved catalytic 5-ptase domain of the protein. The description of this additional case confirms the involvement of INNP5K as a new candidate gene in CMDs with cataracts and learning deficits.

The Myocapture project using exome sequencing was set up to identify novel genes involved in neuromuscular disorders. The Institute of Myology's cohort of 98 families was selected as partner of this French project, after extensive characterisation of the clinical phenotype, muscle imaging and histopathology. Within the cohort, one consanguineous family presented two affected siblings. The index case, was first assessed at age 19 after a heart attack. He showed a first-degree atrioventricular (AV)-block associated with a CPK level as high as 4400 IU/L. Despite having a myopathic pattern at EMG and the dystrophic appearance of the muscle biopsy done two years later, no muscle weakness was reported. His elder sister had high CK levels (>1500 U/l) with a first-degree AV-block. However, she complained of muscle pain by the age of 30. Three other siblings were unaffected. Exome sequencing of the mother and the two affected children was performed. Variants in 8 candidate genes, including a homozygous one in POPDC1 (BVES), were identified after applying a filter of variant's frequency below 1% and a recessive mode of inheritance. POPDC1 (BVES) is a member of the Popeye-domain-containing (POPDC) gene family, which encodes transmembrane cAMP-binding proteins present in skeletal muscle and heart. Null mutations in animal models are associated with severe cardiac arrhythmia and muscular dystrophy. A single family with limb-girdle muscular dystrophy and AV-block has been described carrying a recessive missense mutation in POPDC1 (BVES). Here we describe a new homozygous variant. The nucleotide change is found in position +2 of intron 8 (c.816 + 2T>C). RT-PCR analyses of muscle RNA revealed exon 8 skipping and decreased expression of POPDC1 (BVES). Interestingly, plasma membrane localisation of POPDC1 and -2 protein was significantly reduced in the patient's muscle biopsies. These data are highly suggestive of a deleterious effect of this variant in this family.

According to the GeneTable, over 400 genes have been associated with neuromuscular diseases while 76 mapped loci are awaiting gene identification. At least 25% of patients remain without molecular diagnosis. The Myocapture project was designed to identify new genes responsible for myopathies by exome sequencing of 1000 individuals. In this context, we selected clinically well-characterised patients with limb girdle muscular dystrophies, congenital muscular dystrophies and hereditary sensory-motor neuropathies. The known genes had been previously excluded through routine diagnosis strategy. For each patient, we gathered all available information (biological analyses, electromyography, imaging and muscle biopsy histology). Out of 111 families, DNA samples from 161 patients and their relatives have been collected. Agilent SureSelectXT Human All Exon V5 was used for DNA capture before sequencing on Illumina HighSeq sequencer. Variants have been selected and filtered with the Polyweb software. We identified mutations in known genes (CAPN3, COL6A1, COL6A2 and TTN) that had either been missed or not screened during previous molecular investigations. Likewise, putative mutations in POMT2, RYR1 and BIN1 are awaiting further confirmation. A mutation in ASAH1 previously described in SMA with myoclonic epilepsy has been associated with a new phenotype. For the remaining cases, variants in non-previously reported genes for myopathies are under evaluation based on prioritisation pipeline focusing on genes encoding either skeletal muscle proteins or interacting with known disease proteins or implicated in biological processes related to striated muscle. New candidate genes were selected that are under confirmation based on functional studies. Overall, exome sequencing leads to 1) identification of new genes responsible for myopathies, 2) description of new phenotypic entities due to genes previously involved in other conditions and 3) identification of 10% of “missed” diagnoses.