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

RYR1-related disorders affect skeletal muscle and are caused by mutations or changes in the RYR1 gene. RYR1 variants are the most common cause of congenital myopathy – muscle weakness present from birth. For skeletal muscles to contract and produce force, calcium must be released from the sarcoplasmic reticulum (a calcium containing structure in the cell) in a highly controlled manner. This process is referred to as excitation-contraction coupling. RyR1 protein acts as a gatekeeper for muscle cell calcium stores. Improper functioning of RyR1 can therefore affect excitation-contraction coupling that result in muscle weakness. RyR1 protein is susceptible to oxidative stress which can make the existing dysfunction even worse. Certain RYR1 changes can also result in less RyR1 being produced. The goal of this project is to better understand how mutations in RYR1 affect the function of the protein and to identify promising treatments for this debilitating group of diseases. We introduced disease causing mutations into stem cells developed from skin cells. These cells were then transformed into muscle cells to allow us to compare the changes we see to those of cells obtained from patients with RYR1-related disorders. We also identified that a subset of individuals with RYR1-related disorders have a reduced concentration of a coenzyme called nicotinamide adenine dinucleotide (NAD). We are exposing these stem cell-generated cells and patient-derived cells to this potential therapeutic compound to see if it improves their function. We are using zebrafish as a disease model to test the impact of RYR1 changes on muscle function. We have successfully introduced multiple disease-causing mutations into zebrafish, which will allow us to better characterize their effects on muscle function. In addition, we are testing two antioxidants, alone and in combination, (Mitoquinol mesylate and n-3 polyunsaturated fatty acids) on mouse models of the disease. The initial results are promising, and experiments are in their final stages. Our lab successfully analyzed patient samples from the recently completed phase I study testing the safety and tolerability of Rycal – a compound that stabilizes the RyR1 protein channel.