Biallelic Mutation of ARHGEF18, Involved in the Determination of Epithelial Apicobasal Polarity, Causes Adult-Onset Retinal Degeneration

Abstract

Mutations in more than 250 genes are implicated in inherited retinal dystrophy; the encoded proteins are involved in a broad spectrum of pathways. The presence of unsolved families after highly parallel sequencing strategies suggests that further genes remain to be identified. Whole-exome and -genome sequencing studies employed here in large cohorts of affected individuals revealed biallelic mutations in ARHGEF18 in three such individuals. ARHGEF18 encodes ARHGEF18, a guanine nucleotide exchange factor that activates RHOA, a small GTPase protein that is a key component of tight junctions and adherens junctions. This biological pathway is known to be important for retinal development and function, as mutation of CRB1, encoding another component, causes retinal dystrophy. The retinal structure in individuals with ARHGEF18 mutations resembled that seen in subjects with CRB1 mutations. Five mutations were found on six alleles in the three individuals: c.808A>G (p.Thr270Ala), c.1617+5G>A (p.Asp540Glyfs^∗63), c.1996C>T (p.Arg666^∗), c.2632G>T (p.Glu878^∗), and c.2738_2761del (p.Arg913_Glu920del). Functional tests suggest that each disease genotype might retain some ARHGEF18 activity, such that the phenotype described here is not the consequence of nullizygosity. In particular, the p.Thr270Ala missense variant affects a highly conserved residue in the DBL homology domain, which is required for the interaction and activation of RHOA. Previously, knock-out of Arhgef18 in the medaka fish has been shown to cause larval lethality which is preceded by retinal defects that resemble those seen in zebrafish Crumbs complex knock-outs. The findings described here emphasize the peculiar sensitivity of the retina to perturbations of this pathway, which is highlighted as a target for potential therapeutic strategies.

Keywords: ARHGEF18; apicobasal polarity; inherited retinal dystrophy; p114RhoGEF; retinal degeneration; retinitis pigmentosa.

Figure 1

Variant Analysis of ARHGEF18 in Individuals 1–3 (A) Pedigrees and cosegregation of mutations M1–M5 in families 1–3. (B) Schematic representation of mutation location in full-length ARHGEF18 including DBL homology (DH) and Plekstrin homology (PH) domains. (C) IGV visualization of 150 bp paired end reads spanning mutations ARHGEF18, c.2632G>T, and c.2738_2761del in individual 2, showing biallelic state. (D) Clustal Omega alignment of amino acid residues affected by M1 (missense) and M4 (in-frame deletion) mutations throughout vertebrate orthologues.

Figure 2

Retinal Abnormalities in ARHGEF18-Related Retinal Dystrophy Color fundus photographs, 55 degree fundus autofluorescence imaging, and optical coherence tomography (OCT). (A–C) Individual 1, right eye at age 38 years, showing (A) optic disc pallor, peripheral retinal pigment epithelium (RPE) atrophy, and nummular pigmentation; (B) peripheral patchy reduction of autofluorescence; and (C) reasonably preserved retinal layers on OCT with disruption of the inner segment ellipsoid band and intra-retinal cysts within the inner nuclear layer. (D–F) Individual 2, right eye at 48 years, showing (D) disc pallor and vessel attenuation with RPE atrophy within the macula and mid-periphery as well as peripheral nummular and dot lesions of hyperpigmentation; (E) extensive loss of autofluorescence in periphery and in a central ring in macula; and (F) loss of outer retina and RPE throughout the macula with small foci of preserved photoreceptors centrally. (G–I) Individual 3, right eye at 37 years, showing (G) vascular attenuation and occlusion, peripheral RPE atrophy, white dots, and nummular pigmentation; (H) loss of autofluorescence in periphery; and (I) reasonably preserved retinal layers on OCT with disruption of the inner segment ellipsoid band and intra-retinal cysts within the inner nuclear layer.

Figure 3

Signalling Activity of ARHGEF18 Variants HEK293T or HCE cells were transfected with cDNAs encoding wild-type or mutant VSV-tagged ARHGEF18. (A) Lysates of transfected HEK293T cells were assayed for RHOA-GTP levels by G-LISA assay, which measures binding of active RHOA to the GTPase binding domain of Rhotekin. (B) Serum response element (SRE) element activity was measured using a double luciferase assay with an SRE-containing promoter driving firefly luciferase expression and a CMV promoter expression of renilla luciferase. Firefly to renilla luciferase ratios were calculated and normalized to a plasmid control performed by co-transfecting an empty expression vector. The graphs show averages ± 1 standard deviations, n = 4, indicated are p values from ANOVA and t tests. (C) Transfected HCE cells were fixed and stained with anti-VSV and anti-phosphorylated myosin regulatory light chain (ppMLC) antibodies along Ato-647-labeled phalloidin to visualize F-actin and imaged by epifluorescence. (D) Cells transfected as in (C) were stained for the junctional markers alpha-catenin and Catenin delta-1 and imaged by confocal microscopy. Scale bars represent 20 μm in (C) and 10 μm in (D).