Functional characterization of genetic variants of uncertain significance in genes associated with excitation-contraction coupling disorders

National Institute Of Nursing Research

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Abstract

Ryanodine receptor-1 related myopathies (RYR1-RM) are the most frequently diagnosed congenital myopathies. An estimated 1 in 90,000 children in the United States is diagnosed with this rare neuromuscular disorder. Clinical presentations of RYR1-RM are notoriously diverse and range from mild to severe, including muscle weakness, hypotonia, scoliosis, ophthalmoplegia, and respiratory distress. Both dominant and recessive modes of inheritance have been documented, and the RYR1 disease spectrum has now expanded to include adult-onset phenotypes. The ryanodine receptor type 1 (RyR1) protein is a skeletal muscle calcium release channel responsible for effective skeletal muscle contractile activation. RyR1 releases intracellular calcium from the sarcoplasmic reticulum (SR) in response to an action potential of the surface membrane, resulting in muscle contraction. Variations in the ryanodine receptor-1 (RYR1) gene can result in dysregulation of calcium release from the SR, mitochondrial oxidative stress, hyper- or hypo-sensitivity to channel agonists, and decreased RyR1 protein expression. Based on the residual variant intolerance score (RVIS) of -8.29 (0.01%), the RYR1 gene is highly intolerant to change. Despite the strong association of genetic variations with disease, RYR1-RM may not always be confidently and precisely diagnosed through the use of genetic sequencing alone. Our work uses precise single-nucleotide genome editing experiments and generation of in vitro and in vivo RYR1-RM disease models to characterize the impact of VUS on RyR1 function, thereby providing additional information for their reclassification as either likely pathogenic or likely benign. This research will thereby accelerate screening and testing of several specific therapeutic candidates that may alleviate some RYR1-RM associated symptoms, such as muscle weakness and fatigue, and improve muscle function. This leverages leverages advances in CRISPR-Cas9 gene-editing technology to functionally characterize RYR1 genetic variations with the goal of bringing potential therapies to affected individuals in a timely manner. For disease modeling, the application of molecular biology and gene editing to generate missense point mutants in zebrafish can be significantly more cost-effective and scalable than in other vertebrate model animals. Data and patient samples collected from completed RYR1-RM clinical trials add to the feasibility of our work. Additionally, results will be evaluated in the context of the affected individuals clinical phenotype collected from clinical trial participants (only for participants that provided consent for future use of data).