Pathogenicity and Functional Analysis of Multi-Variant Allele of RPE65 Causing Retinitis Pigmentosa

  • Transl Vis Sci Technol. 2026 Feb 2;15(2):1. doi: 10.1167/tvst.15.2.1.
Guiyue Wu  1  2  3  4 ,  Qingge Guo  1  2  4 ,  Ya Li  1  2  4 ,  Shun Yao  1  2  3  4 ,  Ya You  1  2  4 ,  Leilei Hao  5 ,  Bo Lei  1  2  3  4
Affiliations
  • 1. Zhengzhou University People's Hospital, Henan Provincial People's Hospital, Zhengzhou, China.
  • 2. Henan Eye Institute, Henan Eye Hospital, Zhengzhou University People's Hospital, Henan Provincial People's Hospital, Zhengzhou, China.
  • 3. Eye Institute, Henan Academy of Innovations in Medical Science, Zhengzhou, China.
  • 4. Henan Clinical Research Center for Ocular Diseases, People's Hospital of Zhengzhou University, Zhengzhou, China.
  • 5. Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
PMID: 41626883 DOI: 10.1167/tvst.15.2.1
Abstract

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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