Increasing Role of Titin Mutations in Neuromuscular Disorders

Peer reviewe

Is Gene-Size an Issue for the Diagnosis of Skeletal Muscle Disorders?

Human genes have a variable length. Those having a coding sequence of extraordinary length and a high number of exons were almost impossible to sequence using the traditional Sanger-based gene-by-gene approach. High-throughput sequencing has partly overcome the size-related technical issues, enabling a straightforward, rapid and relatively inexpensive analysis of large genes. Several large genes (e.g. TTN, NEB, RYR1, DMD) are recognized as disease-causing in patients with skeletal muscle diseases. However, because of their sheer size, the clinical interpretation of variants in these genes is probably the most challenging aspect of the high-throughput genetic investigation in the field of skeletal muscle diseases. The main aim of this review is to discuss the technical and interpretative issues related to the diagnostic investigation of large genes and to reflect upon the current state of the art and the future advancements in the field. © 2020 - IOS Press and the authors. All rights reserved.Peer reviewe

SIRT6 polymorphism rs117385980 is associated with longevity and healthy aging in Finnish men

Background: Sirtuin-6 (SIRT6) is involved in various crucial cellular pathways, being a key regulator of telomere structure, DNA repair, metabolism, transcriptional control and the NF-kappa B pathway. Sirt6 knock-out mice have been reported to develop typical features of aging and senescence at the age of 2-3 weeks and die within 4 weeks. The aim of this study was to investigate whether sequence variations of SIRT6 are associated with aging and longevity in Finnish men. Methods: The sample of this study consisted of 43 longer-living and healthy males and 92 male control subjects who have died of natural causes at an average age of 66,6 (+/- 4,1) years and who belonged to the Helsinki Birth Cohort Study (HBCS). Single nucleotide polymorphisms (SNPs) in the exons and their surroundings of the SIRT6 were studied using direct PCR sequencing. Results: The SNP rs117385980 (C > T), situated 23 bases downstream of the exon 2 exon/intron border was found in heterozygous form in 1/43 longer-living healthy men (Minor allele frequency (MAF) 0,0116) and in 9/92 controls (MAF 0,0489). To replicate this finding, we studied a group of 63 healthy men at an average age of 83 years from the Helsinki Businessmen Study (HBS)-cohort. The heterozygosity of the same SNP was seen in 2/63 men from the HBS-cohort (MAF 0,0159). Fisher exact test was performed in our two combined study samples. The P-value for all samples combined was 0.07 and the odds ratio 3.53 (95% confidence interval 0.96-13.4). Conclusions: These results suggest an inverse association between the T allele of rs117385980 and longevity. The result needs to be confirmed in a larger study. It remains to be determined whether rs117385980 itself has an effect or if it is a mere genetic marker for some other yet undiscovered sequence variant causing a functional effect.Peer reviewe

The complexity of titin splicing pattern in human adult skeletal muscles

Background: Mutations in the titin gene (TTN) cause a large spectrum of diseases affecting skeletal and/or cardiac muscle. TTN includes 363 coding exons, a repeated region with a high degree of complexity, isoform-specific elements, and metatranscript-only exons thought to be expressed only during fetal development. Although three main classes of isoforms have been described so far, alternative splicing events (ASEs) in different tissues or in different developmental and physiological states have been reported. Methods: To achieve a comprehensive view of titin ASEs in adult human skeletal muscles, we performed a RNA-Sequencing experiment on 42 human biopsies collected from 12 anatomically different skeletal muscles of 11 individuals without any skeletal-muscle disorders. Results: We confirmed that the skeletal muscle N2A isoforms are highly prevalent, but we found an elevated number of alternative splicing events, some at a very high level. These include previously unknown exon skipping events and alternative 5' and 3' splice sites. Our data suggests the partial inclusion in the TTN transcript of some metatranscript-only exons and the partial exclusion of canonical N2A exons. Conclusions: This study provides an extensive picture of the complex TTN splicing pattern in human adult skeletal muscle, which is crucial for a proper clinical interpretation of TTN variants.Peer reviewe

Working out of the ‘toolbox’: an exploratory study with complementary therapists in acute cancer care

Aims: The aim of this research was to explore and capture therapists’ experiences of and preparation for working with patients in an acute cancer care setting. Method: Semi structured interviews with therapists (n=18) in an acute cancer hospital in the North West of England. The interviews were transcribed and analysed using thematic coding. Results: Key themes identified included; the need for a ‘tool box’ that goes beyond initial training, building confidence with adapting these new skills in practice, helping patients to become empowered, the need to support carers, research evidence and resources issues, and the role of supervision. Conclusion: This study was limited by being set in a single acute cancer site. Therapists valued having a ‘tool box’ but needed confidence and support to navigate the challenges of clinical practice.The authors would like to acknowledge the support of ‘Walk the Walk’ Charity, who help fund the complementary therapy services in the Radiotherapy and Chemotherapy Departments

Solve-RD : systematic pan-European data sharing and collaborative analysis to solve rare diseases

Publisher Copyright: © 2021, The Author(s).For the first time in Europe hundreds of rare disease (RD) experts team up to actively share and jointly analyse existing patient’s data. Solve-RD is a Horizon 2020-supported EU flagship project bringing together >300 clinicians, scientists, and patient representatives of 51 sites from 15 countries. Solve-RD is built upon a core group of four European Reference Networks (ERNs; ERN-ITHACA, ERN-RND, ERN-Euro NMD, ERN-GENTURIS) which annually see more than 270,000 RD patients with respective pathologies. The main ambition is to solve unsolved rare diseases for which a molecular cause is not yet known. This is achieved through an innovative clinical research environment that introduces novel ways to organise expertise and data. Two major approaches are being pursued (i) massive data re-analysis of >19,000 unsolved rare disease patients and (ii) novel combined -omics approaches. The minimum requirement to be eligible for the analysis activities is an inconclusive exome that can be shared with controlled access. The first preliminary data re-analysis has already diagnosed 255 cases form 8393 exomes/genome datasets. This unprecedented degree of collaboration focused on sharing of data and expertise shall identify many new disease genes and enable diagnosis of many so far undiagnosed patients from all over Europe.Peer reviewe

Comprehensive transcriptomic analysis shows disturbed calcium homeostasis and deregulation of T lymphocyte apoptosis in inclusion body myositis

Objective Inclusion body myositis (IBM) has an unclear molecular etiology exhibiting both characteristic inflammatory T-cell activity and rimmed-vacuolar degeneration of muscle fibers. Using in-depth gene expression and splicing studies, we aimed at understanding the different components of the molecular pathomechanisms in IBM. Methods We performed RNA-seq on RNA extracted from skeletal muscle biopsies of clinically and histopathologically defined IBM (n = 24), tibial muscular dystrophy (n = 6), and histopathologically normal group (n = 9). In a comprehensive transcriptomics analysis, we analyzed the differential gene expression, differential splicing and exon usage, downstream pathway analysis, and the interplay between coding and non-coding RNAs (micro RNAs and long non-coding RNAs). Results We observe dysregulation of genes involved in calcium homeostasis, particularly affecting the T-cell activity and regulation, causing disturbed Ca2+-induced apoptotic pathways of T cells in IBM muscles. Additionally, LCK/p56, which is an essential gene in regulating the fate of T-cell apoptosis, shows increased expression and altered splicing usage in IBM muscles. Interpretation Our analysis provides a novel understanding of the molecular mechanisms in IBM by showing a detailed dysregulation of genes involved in calcium homeostasis and its effect on T-cell functioning in IBM muscles. Loss of T-cell regulation is hypothesized to be involved in the consistent observation of no response to immune therapies in IBM patients. Our results show that loss of apoptotic control of cytotoxic T cells could indeed be one component of their abnormal cytolytic activity in IBM muscles.Peer reviewe

Abnormal Splicing of NEDD4 in Myotonic Dystrophy Type 2 Possible Link to Statin Adverse Reactions

Myotonic dystrophy type 2 (DM2) is a multisystemic disorder caused by a (CCTG)n repeat expansion in intron 1 of CNBP. Transcription of the repeats causes a toxic RNA gain of function involving their accumulation in ribonuclear foci. This leads to sequestration of splicing factors and alters pre-mRNA splicing in a range of downstream effector genes, which is thought to contribute to the diverse DM2 clinical features. Hyperlipidemia is frequent in DM2 patients, but the treatment is problematic because of an increased risk of statin-induced adverse reactions. Hypothesizing that shared pathways lead to the increased risk, we compared the skeletal muscle expression profiles of DM2 patients and controls with patients with hyperlipidemia on statin therapy. Neural precursor cell expressed, developmentally downregulated-4 (NEDD4), an ubiquitin ligase, was one of the dysregulated genes identified in DM2 patients and patients with statin-treated hyperlipidemia. In DM2 muscle, NEDD4 mRNA was abnormally spliced, leading to aberrant NEDD4 proteins. NEDD4 was down-regulated in persons taking statins, and simvastatin treatment of C2C12 cells suppressed NEDD4 transcription. Phosphatase and tensin homologue (PTEN), an established NEDD4 target, was increased and accumulated in highly atrophic DM2 muscle fibers. PTEN ubiquitination was reduced in DM2 myofibers, suggesting that the NEDD4-PTEN pathway is dysregulated in DM2 skeletal muscle. Thus, this pathway may contribute to the increased risk of statin-adverse reactions in patients with DM2

Adult-onset dominant muscular dystrophy in Greek families caused by Annexin A11

Objective: Mutations in the prion-like domain of RNA binding proteins cause dysfunctional stress responses and associated aggregate pathology in patients with neurogenic and myopathic phenotypes. Recently, mutations in ANXA11 have been reported in patients with amyotrophic lateral sclerosis and multisystem proteinopathy. Here we studied families with an autosomal dominant muscle disease caused by ANXA11:c.118G > T;p.D40Y. Methods: We performed deep phenotyping and exome sequencing of patients from four large Greek families, including seven affected individuals with progressive muscle disease but no family history of multi-organ involvement or ALS. Results: In our study, all patients presented with an autosomal dominant muscular dystrophy without any Paget disease of bone nor signs of frontotemporal dementia or Parkinson's disease. Histopathological analysis showed rimmed vacuoles with annexin All accumulations. Electron microscopy analysis showed myofibrillar abnormalities with disorganization of the sarcomeric structure and Z-disc dissolution, and subsarcolemmal autophagic material with myeloid formations. Molecular genetic analysis revealed ANXA11:c.118G > T;p.D4OY segregating with the phenotype. Interpretation: Although the pathogenic mechanisms associated with p.D4OY mutation in the prion-like domain of Annexin All need to be further clarified, our study provides robust and clear genetic evidence to support the expansion of the phenotypic spectrum of ANXA11.Peer reviewe