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

Area of Science:

• Genetics
• Molecular Biology
• Ophthalmology

Background:

• Retinitis pigmentosa (RP) is a group of inherited retinal diseases.
• Multi-variant alleles are rare causes of RP.
• RPE65 gene mutations are associated with RP.

Purpose of the Study:

• To investigate the pathogenicity and function of a multi-variant allele of RPE65.
• To explore the genetic and molecular mechanisms underlying RP caused by rare multi-variant alleles.

Main Methods:

• Ophthalmic evaluations and genetic sequencing (NGS, Sanger) were performed.
• In silico analyses, plasmid construction, and cell-based assays (HEK293T) were utilized.
• Protein expression, stability, and ubiquitination levels were measured for wild-type and variant RPE65.

Main Results:

• A 9-year-old female with RP presented with five missense variants in RPE65.
• Four variants formed a multi-variant allele, with two being novel (c.490A>C, c.480G>C).
• In vitro, RPE65 variants and the multi-variant allele showed decreased expression and stability, with increased ubiquitination.

Conclusions:

• The RPE65 multi-variant allele and the novel variant c.16G>A may contribute to RP.
• Variants lead to RPE65 protein degradation via the proteasome-ubiquitin pathway.
• Understanding multi-variant alleles is crucial for diagnosing rare genetic eye diseases.