Pathogenicity and Functional Analysis of Multi-Variant Allele of RPE 65 Causing Retinitis Pigmentosa

Purpose: Multi-variant alleles causing retinitis pigmentosa (RP) are extremely rare. This study aimed to explore the pathogenicity and function of a multi-variant allele of RPE65 associated with retinitis pigmentosa. Methods: Ophthalmic evaluations were performed. Targeted next-generation sequencing was performed for the proband, and Sanger sequencing was conducted in the family members. In silico molecular genetics and bioinformatics analyses were performed. Wild-type RPE65 and variant plasmids were constructed and transferred into HEK293T cells. The expression levels, protein stability, and protein ubiquitination levels were measured. Results: A 9-year-old genotypic female showed signs of RP. Five missense variants were identified in the RPE65 exonic region of the proband. Four missense variants, c.1154C>T (p.T385D), c.571A>G (p.N191D), c.490A>C (p.K164Q), and c.480G>C (p.L160F), were arranged in the same allele, whereas the variant c.16G>A (p.E6K) was found on the other allele. c.490A>C, c.480G>C, and c.16G>A were novel variants. In vitro, variant proteins c.1154C>T, c.571A>G, c480G>C, and c.16G>A and the multi-variant allele exhibited decreased expression levels and decreased protein stability but increased ubiquitination levels. Conclusions: The multi-variant allele and RPE65 c.16G>A may cause RP. The novel variants (RPE65 c.16G>A and RPE65 c.480G>C) and the previously reported variants (RPE65 c.1154C>T and RPE65 c.571A>G) led to protein degradation and reduced the stability of RPE65, which appeared to be associated with the proteasome-ubiquitin pathway. Translational Relevance: Accurately assessing multi-variant alleles is important but challenging, and this study explored the pathogenicity and function of a multi-variant allele of RPE65.