Mechanism of the unstable proteins generated by P346H-BEST1 and P233L-BEST1 mutations and their role in causing RP50 and BVMD

BEST1 mutations are associated with a spectrum of ocular disorders collectively termed Bestrophinopathies, including autosomal dominant Best vitelliform macular dystrophy (BVMD), adult-onset vitelliform macular dystrophy (AVMD), autosomal dominant vitreoretinochoroidopathy (ADVIRC), retinitis pigmentosa type 50 (RP50), and autosomal recessive bestrophinopathy (ARB). The molecular mechanisms underlying their distinct retinal phenotypes remain largely unknown. In this study, we characterized disease subtypes, causative genes, and mutation sites through comprehensive ophthalmic evaluations, whole-genome sequencing, and Sanger sequencing validation. Cellular immunofluorescence assays were performed to assess the impact of BEST1 mutations on the subcellular localization of Bestrophin-1. Protein stability of wild-type and mutant Bestrophin-1 was investigated in transiently transfected MDCK II cells treated with proteasome or lysosome inhibitors. We identified BEST1 c.1037C>A (p.P346H) and BEST1 c.698C>T (p.P233L) mutations in two families diagnosed with RP50 and BVMD, respectively. These mutations induced protein destabilization and subsequent degradation via the ubiquitin-proteasome-dependent pathway. Additionally, both p.P346H and p.P233L caused cytoplasmic mislocalization of the mutant Bestrophin-1 protein. Our findings suggest that the pathogenic mechanisms associated with p.P233L and p.P346H likely involve mislocalization of the mutant protein, protein instability and its targeted degradation via ubiquitin-proteasome-dependent pathway.