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.

Figure 1

Autosomal-Recessive RP Families and Associated REEP6 Variants (A) Pedigrees of all families with RP in this study (families A to E). Proband II.1 from family A is compound heterozygous for REEP6 variants (M1/M2). All other affected individuals were homozygous for the respective REEP6 variants indicated (M3, M4, M5, M6). M3 c.−5835_115+936delinsAC027307.5:79083_79240inv is abbreviated to c.−5835_115+936delins. (B) REEP6 gene structure with nucleotide positions for M1 to M6 mapped on to exons (E1–E6). Exon 5 is present only in isoform REEP6.1 (absent from REEP6.2). Protein structure of REEP6.1 and REEP6.2 with position of the corresponding mutations indicated. (C) Sequence reads from individual A-II:1 aligned against + strand of hg19 showing heterozygous mutations in REEP6. Both mutations are in exon 4: c.T404C (p.Ala150Profs^∗2) and c.448delG (p.Leu135Pro) are in trans.

Figure 2

Clinical Findings for Individuals with Autosomal-Recessive RP Harboring REEP6 Variants (A) Retinal imaging of the left eye of individual II:1 at age 18 years from family A. (B) Retinal imaging of the left eye of individual II:8 at age 32 years from family B. (C) Right eye retinal imaging of individual II:5 at age 54 years from family D. (D) Retinal imaging of the right eye of individual II:4 from family E. Fundus imaging, fundus autofluorescence imaging, and optical coherence tomography (OCT) scan reveal typical retinitis pigmentosa features in all affected individuals. Color fundus photographs demonstrate severely constricted retinal arterioles, mid-peripheral retinal pigment epithelium (RPE) atrophy, retinal atrophy within and extending outward of vascular arcades, and intraretinal pigmented spicules. Fundus autofluorescence imaging demonstrates a hyperautofluorescent ring around fovea and areas of circumscribed hypoautofluorescence within and extending outward of vascular arcades. OCT demonstrates atrophy of the outer retina with small bands of retained photoreceptors (arrows) that correspond to the areas within the para-foveal rings.

Figure 3

Expression and Localization of REEP6 in Human iPSC Photoreceptors (A) Reverse-transcription PCR analysis of REEP6.1 and 6.2 isoforms in control adult fibroblasts (fibs) and control BJ optic cups during different weeks (wk) of photoreceptor differentiation. GAPDH used a loading control. (B) Maturing rhodopsin-positive rod cells (green) co-express REEP6 (red) in the IS and cell body (CB). At a later stage of development, mature rods develop rhodopsin-laden outer segments (green) in which REEP6 staining (red) is not detectable despite its continued expression in the IS and CB. (C) REEP6 (red) is not detectable in maturing cones, expressing cone arrestin (green). Mature cones (green) remain REEP6 (red) negative at this stage of development. Nuclei are counter-stained with DAPI (blue). (D) ER localization of REEP6.1. REEP6.1 WT (green) was expressed in SK-N-SH cells, stained with anti-REEP6, and counterstained with anti-KDEL (red). Scale bars represent 10 μm.

Figure 4

Reep6^L135P/L135P Mutant Mice Exhibit Photoreceptor Degeneration (A) Histological analysis of retinal sections from Reep6^+/L135P (left) and Reep6^L135P/L135P (right) mice was performed at 4 months and 6 months of age. Reep6^+/L135P retina shows normal layered organization compared to the Reep6^L135P/L135P retina that shows moderate thinning of the outer nuclear layer (ONL) at 4 months and marked thinning at 6 months of age. “INL” indicates inner nuclear layer. Scale bars represent 40 μm. (B) TEM images of P20 and 6-month-old retina from Reep6^+/L135P control retina and Reep6^L135P/L135P mutant retina shows thinning of ONL, outer segment (OS), and inner segment (IS) at 6 months of age indicative of progressive retinal degeneration. Scale bars represent 2 μm (top) and 10 μm (bottom). (C) Quantitative evaluation of scotopic a-wave and b-wave and photopic b-wave amplitude data for 4-month-old Reep6^+/L135P control retina and Reep6^L135P/L135P mutant mice. NS indicates not significant, ^∗∗significant at p < 0.01, ^∗∗∗significant at p < 0.001.