Mutations in REEP6 Cause Autosomal-Recessive Retinitis Pigmentosa

Abstract

Retinitis pigmentosa (RP) is the most frequent form of inherited retinal dystrophy. RP is genetically heterogeneous and the genes identified to date encode proteins involved in a wide range of functional pathways, including photoreceptor development, phototransduction, the retinoid cycle, cilia, and outer segment development. Here we report the identification of biallelic mutations in Receptor Expression Enhancer Protein 6 (REEP6) in seven individuals with autosomal-recessive RP from five unrelated families. REEP6 is a member of the REEP/Yop1 family of proteins that influence the structure of the endoplasmic reticulum but is relatively unstudied. The six variants identified include three frameshift variants, two missense variants, and a genomic rearrangement that disrupts exon 1. Human 3D organoid optic cups were used to investigate REEP6 expression and confirmed the expression of a retina-specific isoform REEP6.1, which is specifically affected by one of the frameshift mutations. Expression of the two missense variants (c.383C>T [p.Pro128Leu] and c.404T>C [p.Leu135Pro]) and the REEP6.1 frameshift mutant in cultured cells suggest that these changes destabilize the protein. Furthermore, CRISPR-Cas9-mediated gene editing was used to produce Reep6 knock-in mice with the p.Leu135Pro RP-associated variant identified in one RP-affected individual. The homozygous knock-in mice mimic the clinical phenotypes of RP, including progressive photoreceptor degeneration and dysfunction of the rod photoreceptors. Therefore, our study implicates REEP6 in retinal homeostasis and highlights a pathway previously uncharacterized in retinal dystrophy.

A novel nonsense variant in REEP6 is involved in a sporadic rod‐cone dystrophy case

“…Variants identified in TTC1 and APC2 , coding for tetratricopeptide repeat domain‐containing protein 1 and adenomatous polyposis coli protein 2, respectively, were predicted to be pathogenic only by 1 program, and THOC3 , coding for THO complex subunit 3, was not found in a homozygous region. Although these variants could contribute to the retinal or to an unrecognized extra‐ocular phenotype in our patient, the homozygous mutation in REEP6 was prioritized after recent reports on mutations in this gene underlying RCD in human and mouse models . The nonsense mutation in REEP6 was confirmed by Sanger sequencing (Figure F).…”

“…Although these variants could contribute to the retinal or to an unrecognized extra-ocular phenotype in our patient, the homozygous mutation in REEP6 was prioritized after recent reports on mutations in this gene underlying RCD in human and mouse models. [15][16][17] The nonsense mutation in REEP6 was confirmed by Sanger sequencing ( Figure 1F). While no additional blood sample was available for familial segregation, copy number variation and SNP analysis from WES data confirmed that the variant is indeed homozygous in the patient and did not reveal a deletion or duplication in REEP6.…”

“…Similarly, the non‐sensitive NMD variant, p.Val187Glyfs*13, would lead to a truncated protein with an impaired function. In addition, missense mutations may lead to hypomorphic alleles as suggested by the study of knock‐in animal . The absence of the protein or its decreased function impact on the synthesis, stability and/or trafficking of phototransduction proteins such as PDE6 and GCs to the outer segment of rods as recently reported in REEP6 knock‐out mice .…”

“…To date, the mutation spectrum in RCD includes 3 missense variants, 3 frameshifts secondary to deletions or duplications, one large homozygous deletion of 6886 bps encompassing PCSK4 and exon 1 of REEP6 and the novel nonsense variant reported herein (Figure ). The large homozygous deletion was predicted to abolish the expression of both genes . Two of the three frameshifts and the novel nonsense variant may lead to an absence of protein product secondary to nonsense‐mediated mRNA decay (NMD), leading to a loss of protein function .…”

Missense variants in the X-linked genePRPS1cause retinal degeneration in females

“…Targeted NGS sequencing of known genes can identify disease‐causing variants in an estimated 60% of nonsyndromic cases and 80% of syndromic cases (Bravo‐Gil et al., ; Ellingford et al., ; Glockle et al., ). In patients that lack a molecular diagnosis, whole exome sequencing (WES) and whole genome sequencing (WGS) are effective tools to identify new potential causative genes and variants (Audo et al., , ; Chaki et al., ).…”