Structure-based analysis of missense mutations impacting the catalytic and substrate binding sites of hRPE65

hRPE65 is a critical enzyme in the retinoid visual cycle and is implicated in retinal diseases caused by missense mutations that affect its function. However, many hRPE65 variants of uncertain significance (VUS) remain unclassified, hindering their clinical interpretation. This study aims to develop a molecular dynamics (MD)-based protocol to evaluate the pathogenicity of missense mutations located within the catalytic and substrate pockets of hRPE65. Using a full-length hRPE65 model complexed with all-trans-retinylpalmitate, we assessed 15 VUS for their structural and functional impacts. Our findings provide insights into the deleterious effects of these mutations, offering a framework for reclassifying VUS and identifying patients eligible for gene therapy. This approach may support clinicians in improving diagnostic precision and therapeutic decision-making for retinal diseases.