The Predicted Splicing Variant c.11+5G>A in RPE65 Leads to a Reduction in mRNA Expression in a Cell-Specific Manner

Abstract

Pathogenic variants in RPE65 lead to retinal diseases, causing a vision impairment. In this work, we investigated the pathomechanism behind the frequent RPE65 variant, c.11+5G>A. Previous in silico predictions classified this change as a splice variant. Our prediction using novel software’s suggested a 124-nt exon elongation containing a premature stop codon. This elongation was validated using midigenes-based approaches. Similar results were observed in patient-derived induced pluripotent stem cells (iPSC) and photoreceptor precursor cells. However, the splicing defect in all cases was detected at low levels and thereby does not fully explain the recessive condition of the resulting disease. Long-read sequencing discarded other rearrangements or variants that could explain the diseases. Subsequently, a more relevant model was employed: iPSC-derived retinal pigment epithelium (RPE) cells. In patient-derived iPSC-RPE cells, the expression of RPE65 was strongly reduced even after inhibiting a nonsense-mediated decay, contradicting the predicted splicing defect. Additional experiments demonstrated a cell-specific gene expression reduction due to the presence of the c.11+5G>A variant. This decrease also leads to the lack of the RPE65 protein, and differences in size and pigmentation between the patient and control iPSC-RPE. Altogether, our data suggest that the c.11+5G>A variant causes a cell-specific defect in the expression of RPE65 rather than the anticipated splicing defect which was predicted in silico.

A Molecular Genetic Analysis of RPE65-Associated Forms of Inherited Retinal Degenerations in the Russian Federation

“…The c.11+5G>A variant has been described multiple times as pathogenic in various worldwide populations; however, there were no more data on its prevalence among other alterations in the RPE65 gene [22,37,40]. A study by Vázquez-Domínguez I. et al shows that the c.11+5G>A variant does not affect splicing as expected according to prediction programs, but leads to a significant decrease in the expression of RPE65, specific for the pigment epithelium, with an undetermined mechanism [41].…”

The qMini assay identifies an overlooked class of splice variants

“…The RPE65 :c.11+5G>A variant has been observed in at least five probands in the homozygous state and in at least 27 probands in the heterozygous state together with another RPE65 pathogenic variant in the literature (Supplementary Table 3). A recent study applied the midigene approach and found that the variant produces a minor mRNA species with a 124-nt intron retention and a majority of mRNA from normal splicing 10 . Interestingly, RPE65 mRNA and protein levels are markedly diminished in patient-derived retinal pigment epithelial (RPE) cells, which was not rescued by inhibition of nonsense-mediated decay, leading to the conclusion that the variant causes diminished RPE65 expression independent of splicing 10 .…”

“…The copyright holder for this preprint this version posted November 3, 2023. ; https://doi.org/10.1101/2023.11.02.23297963 doi: medRxiv preprint intron retention and a majority of mRNA from normal splicing 10 . Interestingly, RPE65 mRNA and protein levels are markedly diminished in patient-derived retinal pigment epithelial (RPE) cells, which was not rescued by inhibition of nonsense-mediated decay, leading to the conclusion that the variant causes diminished RPE65 expression independent of splicing 10 .…”

“…The copyright holder for this preprint this version posted November 3, 2023. ; https://doi.org/10.1101/2023.11.02.23297963 doi: medRxiv preprint intron retention and a majority of mRNA from normal splicing 10 . Interestingly, RPE65 mRNA and protein levels are markedly diminished in patient-derived retinal pigment epithelial (RPE) cells, which was not rescued by inhibition of nonsense-mediated decay, leading to the conclusion that the variant causes diminished RPE65 expression independent of splicing 10 . Our in vitro qMini assay data supports the conclusion that the variant leads to a lower splicing efficiency and subsequently diminished mRNA and protein levels in the patient derived RPE cells.…”

Stargardt disease-associated missense and synonymous ABCA4 variants result in aberrant splicing

“…In fact, some studies have highlighted inconsistencies between splicing aberrations detected using midigenes or simpler cellular models, as opposed to more sophisticated models derived from reprogrammed induced pluripotent stem cells, such as photoreceptor precursor cells or retinal organoids. As the genomic context of the complex models closely resembles that of the native retinal environment, these are more representative of the actual splicing process in the retina ( 29 , 32 , 39 , 40 ). These findings point to the limits imposed by the midigene system.…”