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Updated: Feb 4, 2026
Determining the Likelihood of Variant Pathogenicity Using Amino Acid-level Signal-to-Noise Analysis of Genetic Variation
Published on: January 16, 2019
Pathogenicity and Functional Analysis of Multi-Variant Allele of RPE65 Causing Retinitis Pigmentosa
Guiyue Wu1,2,3,4, Qingge Guo1,2,4, Ya Li1,2,4
1Zhengzhou University People's Hospital, Henan Provincial People's Hospital, Zhengzhou, China.
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.
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