Pathogenic mechanisms of RPGR mutations in X-linked retinitis pigmentosa: integrating clinical pedigree and...

This study aims to identify pathogenic retinitis pigmentosa GTPase regulator (RPGR) mutations in a Chinese pedigree with X-linked retinitis pigmentosa (XLRP) and elucidate the cellular and molecular mechanisms underlying RPGR-associated photoreceptor degeneration through the integrated analysis of clinical data and single-cell transcriptomics.

A three-generation Chinese XLRP pedigree was enrolled for comprehensive ophthalmic examinations, including BCVA, OCT, FAF, and ERG. Whole-exome sequencing was performed on the proband to identify the pathogenic variants, followed by Sanger sequencing for validation in family members. To analyze the downstream molecular mechanisms, we analyzed a public single-cell RNA sequencing dataset (SRP535874) of RPGR mutant retinal organoids across four developmental time-points (D40-D200). Bioinformatics analyses included cell clustering, differential expression analysis, GO/KEGG enrichment, protein-protein interaction (PPI) network construction, and pseudotime trajectory analysis.

A hemizygous frameshift mutation (c.2476_2477del; p.R826Gfs*8) in the ORF15 region of RPGR was identified in the proband and confirmed in his two sons by Sanger sequencing. Clinical examinations revealed severe retinal degeneration in the affected male, intermediate phenotype in female carriers, and early-stage changes in the young affected male. Single-cell transcriptomic analysis of RPGR mutant retinal organoids revealed a paradoxical increase in photoreceptor transcriptional activity at late developmental stages (D150 and D200) despite the loss of the outer retinal structure in the patients, which may reflect aberrant differentiation and impaired functional maturation of photoreceptor precursors. Differential expression analysis showed upregulation of the stress-response genes and downregulation of phototransduction and ciliary transport genes. GO and KEGG enrichment analyses implicated disrupted ribosome biogenesis, RNA metabolism, ubiquitin-mediated proteolysis, and neurodegenerative disease pathways. PPI network analysis indicated decoupling of the core "ciliary transport-phototransduction axis" and activation of a coordinated stress-response module. Pseudotime trajectory analysis showed arrested photoreceptor differentiation at an intermediate stage, preventing the progression to functional maturity.

We identify a previously reported but extremely rare RPGR ORF15 frameshift mutation (c.2476_2477del; p.R826Gfs*8) in a Chinese XLRP pedigree. Single-cell transcriptomic analysis indicates that RPGR loss-of-function mutation may disrupt the ciliary transport-phototransduction axis, activate stress responses, and block photoreceptor differentiation. These findings expand the RPGR mutation spectrum, provide mechanistic insights into XLRP pathogenesis, and have implications for genetic counseling and targeted therapy.