H. Luque

Mutations affecting the cytoplasmic functions of the co-chaperone DNAJB6 cause limb-girdle muscular dystrophy

Limb-girdle muscular dystrophy type 1D (LGMD1D) was linked to chromosome 7q36 over a decade ago, but its genetic cause has remained elusive. Here we studied nine LGMD-affected families from Finland, the United States and Italy and identified four dominant missense mutations leading to p.Phe93Leu or p.Phe89Ile changes in the ubiquitously expressed co-chaperone DNAJB6. Functional testing in vivo showed that the mutations have a dominant toxic effect mediated specifically by the cytoplasmic isoform of DNAJB6. In vitro studies demonstrated that the mutations increase the half-life of DNAJB6, extending this effect to the wild-type protein, and reduce its protective anti-aggregation effect. Further, we show that DNAJB6 interacts with members of the CASA complex, including the myofibrillar myopathy–causing protein BAG3. Our data identify the genetic cause of LGMD1D, suggest that its pathogenesis is mediated by defective chaperone function and highlight how mutations in a ubiquitously expressed gene can exert effects in a tissue-, isoform- and cellular compartment–specific manner.

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

A study was undertaken to identify the molecular cause of Welander distal myopathy (WDM), a classic autosomal dominant distal myopathy.

The enigma of 7q36 linked autosomal dominant limb girdle muscular dystrophy

Introduction Two families with autosomal dominant limb girdle muscular dystrophy (LGMD) have previously been linked to a locus on chromosome 7q36 10 years ago. The locus has been termed both LGMD1D and 1E, but because of lack of additional families to narrow down the linked region of interest, this disease has remained elusive. Methods A large Finnish family was clinically and genetically investigated. Laboratory parameters were determined, including creatine kinase (CK) value, neurographic and electromyography studies, cardiac and respiratory function examinations, muscle biopsies and muscle imaging by CT or MRI. Results Patients had onset of muscle weakness in the pelvic girdle between the fourth and sixth decades with an autosomal dominant pattern of inheritance. CK values were slightly elevated and electromyography was myopathic only. Muscle biopsies showed myopathic and/or dystrophic features with very minor rimmed vacuolation and protein aggregation findings. Molecular genetic analysis indicates linkage of the disease to the locus on chromosome 7q36 completely overlapping with the previously reported locus LGMD1D/E. Discussion Advancement towards the causative gene defect in the 7q36 linked disease needs new additional families to narrow the region of interest. The phenotype in the previously linked families has not been reported in full detail, which may be one reason for the shortage of additional families. A comprehensive clinical and morphological phenotype of chromosome 7q36 linked autosomal dominant LGMD with a restricted and updated 6.4 Mb sized haplotype is reported here.

CAPN3-mediated processing of C-terminal titin replaced by pathological cleavage in titinopathy

Mutations in the extreme C-terminus of titin (TTN), situated in the sarcomeric M-band, cause tibial muscular dystrophy (TMD) and limb-girdle muscular dystrophy 2J (LGMD2J). The mutations ultimately cause a loss of C-terminal titin, including a binding site for the protease calpain 3 (CAPN3), and lead to a secondary CAPN3 deficiency in LGMD2J muscle. CAPN3 has been previously shown to bind C-terminal titin and to use it as a substrate in vitro. Interestingly, mutations in CAPN3 underlie limb-girdle muscular dystrophy 2A (LGMD2A). Here, we aimed to clarify the relationship of CAPN3 and M-band titin in normal and pathological muscle. In vitro analyses identified several CAPN3 cleavage sites in C-terminal titin that were defined by protein sequencing. Furthermore, cleavage products were detected in normal muscle extracts by western blotting and in situ by immunofluorescence microscopy. The TMD/LGMD2J mutation FINmaj proved to alter this processing in vitro, while binding of CAPN3 to mutant titin was preserved. Unexpectedly, the pathological loss of M-band titin due to TMD/LGMD2J mutations was found to be independent of CAPN3, whereas the involvement of ubiquitous calpains is likely. We conclude that proteolytic processing of C-terminal titin by CAPN3 may have an important role in normal muscle, and that this process is disrupted in LGMD2A and in TMD/LGMD2J due to CAPN3 deficiency and to the loss of C-terminal titin, respectively.

Spanish MYH7 founder mutation of Italian ancestry causing a large cluster of Laing myopathy patients.

Muelas N, Hackman P, Luque H, Suominen T, Espinós C, Garcés‐Sánchez M, Sevilla T, Azorín I, Millán JM, Udd B, Vílchez JJ. Spanish MYH7 founder mutation of Italian ancestry causing a large cluster of Laing myopathy patients.

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.

Diagnostic anoctamin-5 protein defect in patients with ANO5-mutated muscular dystrophy

Previously, detection of ANO5 protein has been complicated by unspecific antibodies, most of which have not identified the correct protein. The aims of the study were to specify ANO5 protein expression in human skeletal muscle, and to investigate if the ANO5 protein levels are affected by different ANO5 mutations in anoctaminopathy patients.

P.3.12 Characterization of CAPN3-dependent proteolysis of C-terminal titin

The titinopathies tibial muscular dystrophy (TMD) and limb-girdle muscular dystrophy 2J (LGMD2J) are caused by mutations in the C-terminus of titin, residing in the sarcomeric M-band. Mutations identified so far affect the last Ig domain M10 or the preceding is7 region. The FINmaj mutation, underlying TMD/LGMD2J in patients of Finnish origin, causes the exchange of four amino acids in M10 and presumably leads to domain unfolding. The other known mutations cause missense changes of single amino acids or truncation of the ultimate C-terminus. Loss of C-terminal titin epitopes in IF microscopy and reduced amount of C-terminal titin fragments in western blotting suggest that increased or abnormal proteolytic turnover of mutant titin may contribute to the pathomechanism. The muscle-specific protease calpain 3 (CAPN3) binds M-band titin at the is7 region; the interaction is thought to regulate the autolytic activation of CAPN3. LGMD2J patients and FINmaj knock-in mice show secondary CAPN3 deficiency, reflecting loss of the binding site and consequent dysregulation of CAPN3 activity. To elucidate the proteolytic events involved in the pathogenesis of M-band titinopathies, we investigated the cleavage of C-terminal titin by CAPN3. Cleavage fragments generated by CAPN3 were identified by coexpressing various wild-type and mutant titin constructs with active or inactive CAPN3 in cell culture, and comparing the resulting fragment patterns. Active CAPN3 produced several titin fragments, which were characterized by western blotting, protein sequencing and mass spectrometry for identification of cleavage sites. Furthermore, targeted mutagenesis of predicted CAPN3 recognition sites was utilized for understanding the sequence determinants of CAPN3 cleavage and studying the order of cleavage events.

P.5.14 The co-chaperone DNAJB6 – mutated in LGMD1D – and its role in handling of aggregation-prone muscle proteins

Mutations in the co-chaperone DNAJB6 causing LGMD1Dhave so far been identified in Finnish, Italian, North American, Japanese, and Korean populations. All identified mutations are localized in a small area of the G/F-rich linker domain of DNAJB6. We have earlier studied the effect of wild-type and mutant DNAJB6 on the aggregation of polyQ-huntingtin and all the tested mutants show a significant loss of anti-aggregation effect. The molecular details of DNAJB6 functions in muscle are largely unknown. DNAJB6 is apparently very important for correct handling of proteins in muscle as the mutations all cause dystrophy with protein aggregations and autophagic rimmed vacuolar pathology. The importance of DNAJB6 in muscle protein turnover and anti-aggregation was studied in transiently transfected mammalian cell cultures using co-expression of wild-type and mutant DNAJB6 and aggregation-prone proteins, such as mutant desmin and myotilin. Aggregated proteins were filter trapped and visualised using antibodies. Soluble proteins were measured by western blotting. The anti-aggregation activity was calculated as the ratio of aggregated vs. soluble protein amounts. Both lysosomal or proteasomal activity was blocked using inhibitors (E64d/pepstatin A and MG132 respectively) for the assessment of their involvement in the aggregation processes. The studies are ongoing and detailed results will be presented.

G.O.1 Welander distal myopathy is caused by a mutated RNA binding protein

Abstract Welander distal myopathy (WDM) is a late onset, autosomal dominant distal myopathy prevalent in Finland and Sweden. First symptoms of finger extensor weakness occur after age 40, with progression to all hand muscles and ankle dorsiflexion. Rare homozygotes show also proximal muscular weakness, earlier onset and faster progression. WDM is linked to chromosome 2p13 with a maximal linked region of interest of