Variants identified by next-generation sequencing cause endoplasmic reticulum stress in Rhodopsin-associated...

Rhodopsin (RHO) is the most well-known genetic cause of autosomal dominant retinitis pigmentosa (adRP). This study aimed to investigate the genetic cause of a large Chinese adRP family and assess the pathogenicity of the detected RHO mutant.

Routine ocular examinations were conducted on all participants. Next-generation sequencing with targeted capture was performed to screen mutations in 179 genes associated with hereditary retinal diseases and 10 candidate genes. Variants detected by NGS were validated by Sanger sequencing and evaluated for pathogenicity. Fragments of mutant and wild-type RHO were cloned into the pEGFP-N1 vector and were transfected into different cell lines to observe the cellular localization of the Rhodopsin-GFP fusion protein and evaluate the expression of endoplasmic reticulum (ER) stress markers. RT-PCR analysis was used to detect transfected the splicing of X box-binding protein 1 (XBP1) mRNA, which is a critical factor affecting ER stress.

Genetic analysis identified a heterozygous missense variant, RHO, c.284 T > C (p.L95P) in this adRP family. Another RHO variant (p.P53R) that we reported previously was also included in further functional assessment. Both misfolded mutant proteins accumulated in the ER in a manner similar to that noted for the classic mutant P23H. Spliced XBP1 was observed in cells transfected with mutants, indicating an increase in ER stress.

Although the p.L95P variant is not a novel change, it was the first variant to be functionally evaluated and reported in Chinese RP patients. The results in our study provide significant evidence to classify the p.L95P mutation as a class II mutation.