Use of animal models to understand titin physiology and pathology

In recent years, increasing attention has been paid to titin (TTN) and its mutations. Heterozygous TTN truncating variants (TTNtv) increase the risk of a cardiomyopathy. At the same time, TTNtv and few missense variants have been identified in patients with mainly recessive skeletal muscle diseases. The pathogenic mechanisms underlying titin-related diseases are still partly unknown. Similarly, the titin mechanical and functional role in the muscle contraction are far from being exhaustively clarified. In the last few years, several animal models carrying variants in the titin gene have been developed and characterized to study the structural and mechanical properties of specific titin domains or to mimic patients' mutations. This review describes the main animal models so far characterized, including eight mice models and three fish models (Medaka and Zebrafish) and discusses the useful insights provided by a thorough characterization of the cell-, tissue- and organism-phenotypes in these models.Peer reviewe

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

Identification of an intragenic deletion in the SGCB gene through a re-evaluation of negative next generation sequencing results

A large mutation screening of 504 patients with muscular dystrophy or myopathy has been performed by next generation sequencing (NGS). Among this cohort of patients, we report a case with a severe form of muscular dystrophy with a proximal weakness in the limb-girdle muscles. Her biopsy revealed typical dystrophic features and immunohistochemistry for α- and γ-sarcoglycans showed an absent reaction, addressing the clinical diagnosis toward a sarcoglycanopathy. Considering that no causative point mutation was detected in any of the four sarcoglycan genes, we re-evaluated the NGS data by careful quantitative analysis of the specific reads mapping on the four sarcoglycan genes. A complete absence of reads from the sixth exon of the β-sarcoglycan gene was found. Subsequent array comparative genomic hybridization (CGH) analysis confirmed the result with the identification of a novel 3.3 kb intragenic deletion in the SGCB gene. This case illustrates the importance of a multidisciplinary approach involving clinicians and molecular geneticists and the need for a careful re-evaluation of NGS data

Valosin-containing protein-related myopathy and Meige syndrome : Just a coincidence or not?

Non peer reviewe

Bi-Allelic DES Gene Variants Causing Autosomal Recessive Myofibrillar Myopathies Affecting Both Skeletal Muscles and Cardiac Function

Mutations in the human desmin gene (DES) may cause both autosomal dominant and recessive cardiomyopathies leading to heart failure, arrhythmias and atrio-ventricular blocks, or progressive myopathies. Cardiac conduction disorders, arrhythmias and cardiomyopathies usually associated with progressive myopathy are the main manifestations of autosomal dominant desminopathies, due to mono-allelic pathogenic variants. The recessive forms, due to bi-allelic variants, are very rare and exhibit variable phenotypes in which premature sudden cardiac death could also occur in the first or second decade of life. We describe a further case of autosomal recessive desminopathy in an Italian boy born of consanguineous parents, who developed progressive myopathy at age 12, and dilated cardiomyopathy four years later and died of intractable heart failure at age 17. Next Generation Sequencing (NGS) analysis identified the homozygous loss-of-function variant c.634C>T; p.Arg212*, which was likely inherited from both parents. Furthermore, we performed a comparison of clinical and genetic results observed in our patient with those of cases so far reported in the literature

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

Online Distribution Shift Detection via Recency Prediction

When deploying modern machine learning-enabled robotic systems in high-stakes applications, detecting distribution shift is critical. However, most existing methods for detecting distribution shift are not well-suited to robotics settings, where data often arrives in a streaming fashion and may be very high-dimensional. In this work, we present an online method for detecting distribution shift with guarantees on the false positive rate - i.e., when there is no distribution shift, our system is very unlikely (with probability $< \epsilon$) to falsely issue an alert; any alerts that are issued should therefore be heeded. Our method is specifically designed for efficient detection even with high dimensional data, and it empirically achieves up to 11x faster detection on realistic robotics settings compared to prior work while maintaining a low false negative rate in practice (whenever there is a distribution shift in our experiments, our method indeed emits an alert). We demonstrate our approach in both simulation and hardware for a visual servoing task, and show that our method indeed issues an alert before a failure occurs

Cryogenic Characterization of FBK HD Near-UV Sensitive SiPMs

We report on the characterization of near-ultraviolet high density silicon photomultiplier (\SiPM) developed at Fondazione Bruno Kessler (\FBK) at cryogenic temperature. A dedicated setup was built to measure the primary dark noise and correlated noise of the \SiPMs\ between 40 and 300~K. Moreover, an analysis program and data acquisition system were developed to allow the precise characterization of these parameters, some of which can vary up to 7 orders of magnitude between room temperature and 40~K. We demonstrate that it is possible to operate the \FBK\ near-ultraviolet high density \SiPMs\ at temperatures lower than 100~K with a dark rate below 0.01 cps/mm$^2$ and total correlated noise probability below 35\% at an over-voltage of 6~V. These results are relevant for the development of future cryogenic particle detectors using \SiPMs\ as photosensors