Date of Award
Master of Science (MS) in Biology
Ashley N. Turner
Congenital stationary night blindness (CSNB) is a heterogeneous collection of genetic diseases affecting the eyes and vision in horses and humans. Current research has implicated several genetic mutations impacting different genes involved in phototransduction and signal transmission, including TRPM1. In horses, genetic mutations in TRPM1 also result in a leopard spotting pattern or leopard complex. The goal of this study is to examine the potential impact of CSNB associated TRPM1 missense variants of uncertain significance (VUS). Previous research in Caenorhabditis elegans have revealed an orthologous TRPM1 gene known as gon-2 that allows for comparative studies. The evolutionary relationship of TRPM1 and other orthologous genes were examined along with the evolutionary conservation of TRPM1 missense VUS. Three TRPM1 VUS were identified in conserved loci across human, horse, C. elegans, and other species. A gene mutational analysis, predictive missense variant analysis, and protein modeling predict TRPM1 the c.2572A>G (p.Ile875Val) to be likely pathogenic or damaging. These findings support further in vivo assessment of the VUS. To assess this, a CRISPR-Cas9-engineered C. elegans model containing the TRPM1 missense VUS in the nematode loci of gon-2 was proposed. Two sets of DNA primers were designed and tested to amplify the VUS region in gon-2 using polymerase chain reaction (PCR) and gel electrophoresis. CRISPR RNA guides were also designed to target gon-2 and will be used in future microinjection experiments. A PCR assay was optimized to be utilized for downstream screening and genotyping to identify the gon-2 VUS C. elegans strain. This mutant strain will allow for further in vivo investigation of the missense VUS in the C. elegans model.