Martin Krahn

Constitutive Activation of the Calcium Sensor STIM1 Causes Tubular-Aggregate Myopathy

Tubular aggregates are regular arrays of membrane tubules accumulating in muscle with age. They are found as secondary features in several muscle disorders, including alcohol- and drug-induced myopathies, exercise-induced cramps, and inherited myasthenia, but also exist as a pure genetic form characterized by slowly progressive muscle weakness. We identified dominant STIM1 mutations as a genetic cause of tubular-aggregate myopathy (TAM). Stromal interaction molecule 1 (STIM1) is the main Ca(2+) sensor in the endoplasmic reticulum, and all mutations were found in the highly conserved intraluminal Ca(2+)-binding EF hands. Ca(2+) stores are refilled through a process called store-operated Ca(2+) entry (SOCE). Upon Ca(2+)-store depletion, wild-type STIM1 oligomerizes and thereby triggers extracellular Ca(2+) entry. In contrast, the missense mutations found in our four TAM-affected families induced constitutive STIM1 clustering, indicating that Ca(2+) sensing was impaired. By monitoring the calcium response of TAM myoblasts to SOCE, we found a significantly higher basal Ca(2+) level in TAM cells and a dysregulation of intracellular Ca(2+) homeostasis. Because recessive STIM1 loss-of-function mutations were associated with immunodeficiency, we conclude that the tissue-specific impact of STIM1 loss or constitutive activation is different and that a tight regulation of STIM1-dependent SOCE is fundamental for normal skeletal-muscle structure and function.

COMPLETE FATTY DEGENERATION OF LUMBAR ERECTOR SPINAE MUSCLES CAUSED BY A PRIMARY DYSFERLINOPATHY

Magnetic resonance imaging of the thoracolumbar spine showed complete fatty degeneration of the lumbar erector spinae muscles in a woman who had complained of chronic lower back pain for 5 years and of progressive weakness of the lower limbs for 1 year. Neuromuscular examination of the lower limbs showed no obvious anomaly, and there was no camptocormia. Serum creatine kinase levels were increased (six‐ to ninefold); electrodiagnostic examination revealed no activity at rest or during effort in the erector spinae muscles and was normal in proximal and distal muscles of the limbs. Muscle computed tomography revealed mild fatty degeneration of thigh and gastrocnemius muscles, and histopathology of the deltoid muscle showed dystrophic features and complete lack of dysferlin. Molecular analysis identified a homozygous disease‐causing mutation in the gene encoding dysferlin. Because there were no similar cases in the family, the final diagnosis was sporadic limb‐girdle muscular dystrophy type 2B. Overall, this case report shows that the lumbar and lower thoracic of erector spinae muscles may display complete fatty degeneration without the occurrence of camptocormia, with primary dysferlin deficiency as a possible cause. Muscle Nerve, 2007

Identification of Variants in the 4q35 Gene FAT1 in Patients with a Facioscapulohumeral Dystrophy-Like Phenotype

Facioscapulohumeralmuscular dystrophy (FSHD) is linked to copy‐number reduction (N < 10) of the 4q D4Z4 subtelomeric array, in association with DUX4‐permissive haplotypes. This main form is indicated as FSHD1. FSHD‐like phenotypes may also appear in the absence of D4Z4 copy‐number reduction. Variants of the SMCHD1 gene have been reported to associate with D4Z4 hypomethylation in DUX4‐compatible haplotypes, thus defining FSHD2. Recently, mice carrying a muscle‐specific knock‐out of the protocadherin gene Fat1 or its constitutive hypomorphic allele were shown to develop muscular and nonmuscular defects mimicking human FSHD. Here, we report FAT1 variants in a group of patients presenting with neuromuscular symptoms reminiscent of FSHD. The patients do not carry D4Z4 copy‐number reduction, 4q hypomethylation, or SMCHD1 variants. However, abnormal splicing of the FAT1 transcript is predicted for all identified variants. To determine their pathogenicity, we elaborated a minigene approach coupled to an antisense oligonucleotide (AON) assay. In vitro, four out of five selected variants induced partial or complete alteration of splicing by creating new splice sites or modifying splicing regulators. AONs confirmed these effects. Altered transcripts may affect FAT1 protein interactions or stability. Altogether, our data suggest that defective FAT1 is associated with an FSHD‐like phenotype.

Transcriptional explorations of CAPN3 identify novel splicing mutations, a large-sized genomic deletion and evidence for messenger RNA decay

Mutations in the gene encoding calpain‐3 (CAPN3) cause autosomal recessive limb‐girdle muscular dystrophy type 2A (LGMD2A) and idiopathic eosinophilic myositis. Accurate diagnosis and genetic counselling are based on the identification of disease‐causing mutations on both alleles of CAPN3 in the patients. In the present study, we used transcriptional analysis as a complementary approach for patients suspected of being affected with LGMD2A, in whom initial denaturing high‐performance liquid chromatography genomic mutation screening evidenced no or only one CAPN3 mutation obviously considered as disease causing. This allowed to identify and characterize cDNA deletions. Further genomic analysis allowed to determine the origin of these deletions, either as splicing defects caused by intronic mutations or as an internal multi‐exonic deletion. In particular, we report two novel CAPN3 mutations (c.1745 + 4_1745 + 7delAGTG in IVS13 and c.2185‐16A>G in IVS20) and a recurrent large‐sized genomic deletion including exons 2–8 for which genomic breakpoints have been characterized. In addition, our results indicate nonsense‐mediated messenger RNA decay as a mechanism for under‐expression of CAPN3 associated to some specific variations.

Improving molecular diagnosis of distal myopathies by targeted next-generation sequencing

Distal myopathies are a heterogeneous group of muscle diseases sharing the clinical pattern of predominant weakness in the feet and/or hands. The classical approach for molecular diagnosis is based on targeted gene-by-gene analysis guided by currently existing combinatorial algorithms.1 Many patients remain undiagnosed. Within the last 5 years, next-generation sequencing (NGS) has emerged as a successful and rapid approach to simultaneously analyse multiple genes in neuromuscular disorders.2 Our objective was to evaluate the efficiency of a targeted NGS approach using a panel of neuromuscular genes on patients with distal myopathies. We first tested its validity in a control group of six index cases (IC) with known molecular diagnosis. Then, we prospectively evaluated this approach by testing a group of 17 IC without molecular diagnosis. ### Patients We prospectively included 54 patients (37 IC and 17 relatives) with a diagnosis of distal myopathy, followed at the Neuromuscular Diseases and ALS Reference Centre of La Timone Hospital, Marseille, France, between 1989 and 2014. Among these 37 IC previously explored by Sanger sequencing, 20 IC had an identified molecular diagnosis: six IC constituted the control group. The remaining 17 undiagnosed IC constituted the test group. A targeted-NGS approach was used to search mutations in 298 neuromuscular genes in both groups. Samples analysed in this study have been prepared and stored by the Center of Biological Resources, Department of Medical Genetics, La Timone Hospital, Marseille, and used following the ethical recommendations of our institution and according to the Declaration of Helsinki. All included patients gave their written consent prior to …

Identification of Splicing Defects Caused by Mutations in the Dysferlin Gene

Missense, iso‐semantic, and intronic mutations are challenging for interpretation, in particular for their impact in mRNA. Various tools such as the Human Splicing Finder (HSF) system could be used to predict the impact on splicing; however, no diagnosis result could rely on predictions alone, but requires functional testing. Here, we report an in vitro approach to study the impact of DYSF mutations on splicing. It was evaluated on a series of 45 DYSF mutations, both intronic and exonic. We confirmed splicing alterations for all intronic mutations localized in 5′ or 3′ splice sites. Then, we showed that DYSF missense mutations could also result in splicing defects: mutations c.463G>A and c.2641A>C abolished ESEs and led to exon skipping; mutations c.565C>G and c.1555G>A disrupted Exonic Splicing Enhancer (ESE), while concomitantly creating new 5′ or 3′ splice site leading to exonic out of frame deletions. We demonstrated that 20% of DYSF missense mutations have a strong impact on splicing. This minigene strategy is an efficient tool for the detection of splicing defects in dysferlinopathies, which could allow for a better comprehension of splicing defects due to mutations and could improve prediction tools evaluating splicing defects.

Toward an objective measure of functional disability in dysferlinopathy.

Understanding the natural history of dysferlinopathy is essential to design and quantify novel therapeutic protocols. Our aim in this study was to assess, clinically and functionally, a cohort of patients with dysferlinopathy, using validated scales.

Clinical heterogeneity and a high proportion of novel mutations in a Chinese cohort of patients with dysferlinopathy.

BACKGROUND AND AIMS Dysferlinopathies are a group of autosomal recessive muscular dystrophies caused by mutations in the dysferlin gene. This study presents clinical features and the mutational spectrum in the largest cohort of Chinese patients analyzed to date. PATIENTS AND METHODS A total of 36 unrelated Chinese patients with diagnostic suspicion of dysferlinopathy were clinically and genetically characterized. RESULTS Patients were divided into five phenotypes: 19 patients with limb girdle muscular dystrophy (LGMD) type 2B, 10 with Miyoshi myopathy (MM), 1 with distal anterior compartment myopathy (DACM), 3 with exercise intolerance, and 3 with asymptomatic hypercreatine phosphokinasemia (hyperCPKemia). Thirty-one patients showed an absence or drastic reduction of dysferlin expression by Westernblot. Forty-three mutations were identified in DYSF, including 31 novel. CONCLUSION Our study underlines clinical heterogeneity and a high proportion of novel mutations in Chinese patients affected with dysferlinopathy.

Respiratory and cardiac function in Japanese patients with dysferlinopathy

We retrospectively reviewed respiratory and cardiac function in patients with dysferlinopathy, including 2 autopsy cases with respiratory dysfunction.

Genetic Characterization of a French Cohort of GNE‐mutation negative inclusion body myopathy patients with exome sequencing

Hereditary inclusion body myopathy (hIBM) refers to a group of clinically and genetically heterogeneous diseases. The overlapping histochemical features of hIBM with other genetic disorders lead to low diagnostic rates with targeted single‐gene sequencing. This is true for the most prevalent form of hIBM, GNEpathy. Therefore, we used whole‐exome sequencing (WES) to determine whether a cohort of clinically suspected GNEpathy patients undiagnosed by targeted GNE analysis could be genetically characterized.