A high-resolution map of RHO missense variant trafficking using deep mutational scanning approaches, including a surface abundance immunoassay and a complementary membrane proximity assay, provides a valuable resource for pathogenicity assessment, genotype-phenotype correlations, and the development of targeted therapeutic strategies for RHO-adRP.
Abstract
Rhodopsin (RHO) missense variants are a leading cause of autosomal dominant retinitis pigmentosa (adRP), a progressive retinal degeneration. Interpreting RHO variant pathogenicity is challenging, and understanding their disease mechanisms is essential for developing therapeutics. We present a high-resolution map of RHO missense variant trafficking using deep mutational scanning approaches, including a surface abundance immunoassay and a complementary membrane proximity assay. This comprehensive, reproducible dataset encompassed all 6612 possible missense variants. Over 700 variants had pathogenic trafficking scores, substantially expanding the number of RHO variants with functional data. Trafficking scores correlated with the magnitude of ER stress markers and ClinVar pathogenicity classifications. Data also identified structurally clustered mutational intolerance around the intradiscal beta-plug region. Treatment with the chaperone YC-001 restored surface trafficking in most mistrafficking variants. This functional map of RHO variants provides a valuable resource for pathogenicity assessment, genotype-phenotype correlations, and the development of targeted therapeutic strategies for RHO-adRP.
The hypothesis that there is a biochemical link, most likely the UPR, between rhodopsin folding/misfolding status and metabolic homeostasis is supported and targeted metabolic modulation may offer a complementary therapeutic avenue for treating RP.
M. Murthy, Hannah Staggs-Sandy, Paniz Jasbi et al.· The FASEB Journal· 0 citations
Summary Inherited retinal diseases (IRDs) encompass a broad spectrum of genetic conditions leading to visual impairment. In this study, we identify KATNA1, encoding the catalytic p60 subunit of the microtubule-severing enzyme katanin, as a previously unrecognized cause of autosomal dominant macular dystrophy (adMD), a form of IRD. Specifically, we could ascertain the presence of 10 heterozygous missense changes affecting six conserved amino acids in 21 individuals from 16 unrelated families from various parts of the world, all presenting with non-syndromic MD of variable severity. Structure-guided analyses indicated that the identified variants potentially disrupt katanin’s assembly into hexamers or its ability to bind or hydrolyze ATP, thus compromising its microtubule-severing function. Characterization of patient-derived fibroblasts revealed accumulation of acetylated microtubules both in the cytoplasm and within the primary cilium, together with an altered subcellular distribution of KATNA1. Immunostaining of human retinal tissue showed that KATNA1 specifically localizes to photoreceptors, with distinct distribution patterns between rod and cone photoreceptors. Immunogold transmission electron microscopy confirmed this finding, revealing KATNA1 distribution along the rod axoneme and predominantly within the cone connecting cilium. Together, these results establish KATNA1 as a novel gene associated with adMD, possibly accounting for ~4% of all unresolved MD cases, and associate defective microtubule severing and cytoskeletal dysregulation with macular degeneration.
Carlo Rivolta, Karolina Kaminska, Abigail R. Moye et al.· Research Square· 0 citations
Pathogenic variants in SGCA, encoding α-sarcoglycan, cause an autosomal recessive limb-girdle muscular dystrophy, LGMDR3/2D, yet clinical interpretation of SGCA variants remains challenging due to the high prevalence of rare missense variants. α-sarcoglycan is an essential component of the sarcoglycan complex at the muscle cell membrane, and pathogenic variants frequently impair its membrane localization. Here, we systematically assess the effects of all possible single-nucleotide variants across the SGCA coding sequence using a saturation mutagenesis-based experimental assay that quantifies α-sarcoglycan surface expression. We generate a comprehensive functional atlas that distinguishes tolerated and damaging variants, aligning with independent genetic and clinical evidence, and reveals domain-specific properties of the cytoplasmic region, in which C-terminal truncating variants retain membrane localization, suggesting possible pathogenic mechanisms beyond impaired trafficking. This work provides a scalable functional framework to support genetic diagnosis and variant interpretation in sarcoglycanopathies. Graphical Abstract Schematic overview of saturation mutagenesis-based functional mapping of SGCA.
Shushu Huang, Kenneth K. Ng, Yanyu Lu et al.· bioRxiv· 0 citations
