Exploring Genetic Mechanisms in Nebulin-Based Nemaline Myopathy and Rhabdomyolysis

Abstract

Skeletal muscle is a highly organized tissue housing components of contraction, metabolic, and regulatory machinery. These elements function coordinately to facilitate efficient energy production and maintain cellular homeostasis, thereby generating stability and power for all body movements. Any perturbations (e.g., genetic or environmental) to this coordination, result in loss of muscle health and function. Skeletal muscle diseases are genetically and clinically heterogenous which makes both accurate diagnosis and treatment challenging. Understanding molecular mechanism of these diseases is important, both for disease management and for the development of therapeutic strategies. This study has focused on studying molecular mechanisms in two types of skeletal muscle disease, nebulin-based nemaline myopathy and rhabdomyolysis. Nebulin, encoded by NEB gene, is a giant ~800 KDa filamentous protein and a crucial component of the thin filament in skeletal muscle, which play a critical role in various important processes in skeletal muscle such as maintaining Z-disk structure, myofibril alignment and crossbridge cycling and thin filament length measurement. The mutations in NEB gene lead to NEB-based nemaline myopathy (NEM2), a rare, clinically and genetically heterogeneous disorder, characterized by hypotonia and muscle weakness which does not have any curative treatment. The mutations causing NEM2 are of different types including splice site, truncation, frameshift, deletion or duplication. Splice site mutations are the most prevalent type of mutation within the NEB gene. Although characterization of some type of NEB mutations has partly revealed the disease mechanism in NEM2 such as shorter thin filament, structurally altered thin filament or altered cross-bridge cycling which explains force deficit, however, they cannot explain disease mechanism by all NEB mutations. In this study we characterized NEB mutations in 10 NEM2 patients with different type of mutations at RNA, protein and muscle function level. Results showed that truncation mutations affect NEB mRNA stability and lead to nonsense-mediated decay of the mutated transcript. Moreover, a high incidence of cryptic splice site activation was found in patients with splicing mutations that are expected to disrupt the actin-binding sites of nebulin, a novel pathogenic mechanism for NEM2. Determination of protein levels revealed patients with either relatively normal or markedly reduced nebulin. We observed a positive relation between the reduction in nebulin and a reduction in thin filament length (TFL), or reduction in tension (both maximal and submaximal tension). Interestingly, our study revealed a duplication mutation in nebulin that resulted in a larger nebulin protein and longer TFL, indicating that pathogenic mechanism of NEM2 involves not only shortened but also elongated thin filaments. Additionally, we investigated the effect of Omecamtiv mecarbil (OM), a small-molecule activator of cardiac myosin, on force production of type I muscle fibers of NEM2 patients. Results showed that OM treatment substantially increased submaximal tension across all NEM2 patients ranging from 87-318%, with the largest effects in patients with the lowest level of nebulin, highlighting the potential of OM treatment to improve skeletal muscle function in NEM2 patients, especially those with large reductions in nebulin levels. To deepen our understanding of genotype-phenotype correlations in NEM2, we characterized five homozygous NEB mutations in zebrafish strains previously generated. The results showed that these mutants recapitulated most aspects of NEM2, showing drastically reduced survival, defective muscle structure with disorganized muscle fibers, misaligned and defective sarcomeres, reduced contraction force, shorter TFL, presence of electron-dense structures in myofibers, and thickening of the Z-disks. Additionally, we generated a zebrafish strain with one full super-repeat deletion and characterized it to investigate if deletion of full super-repeat which carries NEB mutation could be therapeutic strategy. Interestingly, we found that homozygous zebrafish strain for full super-repeat deletion did not show significant differences in force production compared to wild type indicating the potential of this approach to treat NEM2 patients.The other part of study was to understand disease etiology in rhabdomyolysis. Rhabdomyolysis (RM) is a complex condition characterized by the release of intracellular substances into the bloodstream due to skeletal muscle injury, leading to a spectrum of complications, ranging from mild to severe and life-threatening. Despite proposed acquired and inherited causes, the etiology of RM remains unknown. In this study we investigated the external and genetic triggers of RM by examining 833 patients with RM diagnoses and comparing identified clinical and genetic conditions with appropriate control samples. Our findings revealed a variety of conditions as external triggers of RM, with long-term drug therapy ranking as the most prevalent trigger (27%), followed by infection (23%), diabetes (19.5%), psychoactive substance use (19%), blood-related disorders (15%), muscle-related disorders (11%), convulsions (7%), malignancy (4.7%), surgery (2.4%), and trauma (1.8%). In addition, our genetic analysis identified 21 pathogenic variants in 9 genes among 26 patients. Furthermore, we identified 61 patients with homozygous/hemizygous (HOM/HEM) states for damaging mutations in 21 unique genes, along with 238 patients with heterozygous (HET) states in autosomal genes with dominant inheritance. However, only 4 genes with damaging mutations were significantly more frequent and had odds ratio greater than 1 when compared with controls exhibiting a similar incidence of RM-associated comorbidities. Additionally, 27% of patients with available genetic data had damaging mutations in more than one gene, suggesting the potential oligogenic nature of rhabdomyolysis.