An intronic REEP6 variant affecting the splicing of a retina-specific exon causes autosomal recessive retinitis pigmentosa in Palestinian patients

Abstract

Purpose : Inherited retinal diseases (IRDs) are a large group of heterogenous phenotypes caused by variants in over 350 genes. Recent studies reported that structural variants and intronic variants that do not affect the canonical splice-sites explain some of the missing heritability in IRDs. In the current study we aim to identify the cause of disease in multiple Palestinian families with autosomal recessive (AR) retinitis pigmentosa (RP) through in silico and in vitro analysis of a splice region variant in the receptor expression enhancing protein 6 (REEP6) gene.

Methods : A smMIPs targeted panel of 113 genes underlying RP, Leber congenital amaurosis (LCA), and other rod-diseases was used for sequencing the DNA samples. Variants were analyzed and annotated using the Franklin platform. We used SpliceAI tool that sets a cut-off value of >0.2 for delta score,for the prediction of the variants effect on splicing. In vitro splicing assays were performed on HeLa, HEK293 and 661W cell lines which were transfected with wild-type or mutant human constructs inserted into pcdhCMV plasmid. Next generation sequencing (NGS) was performed to visualize the splicing effect of the variant.

Results : We identified 6 patients who belong to 3 Palestinian families from Jerusalem. The patients are homozygous for c.518-9G>A in intron 4 of REEP6. SpliceAI prediction analysis for this variant revealed a 0.99 delta [DP1] score for exon 5 splice acceptor site loss. Exon 5 has been previously shown to only be included in the REEP transcript in the retina. An in vitro splicing assay revealed that this variant creates a new splice acceptor site, leading to the inclusion of 7 nucleotides of intron 4. Clinical analysis of 6 cases who are homozygous for c.518-9G>A revealed that the average age at first examination was 38 years and the mean visual acuity was 0.5. ERG testing was performed in one of the patients at the age of 55 and it was extinguished. All 6 patients were diagnosed with typical RP.

Conclusions : Only 13 REEP6 variants were previously reported worldwide. The variant we report here is the first REEP6 variant identified in the Israeli and Palestinian populations.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.