A homozygous mutation in the novel gene ARFGAP2 is associated with non-syndromic recessive inherited retinal degeneration (IRD).

Abstract

Purpose : To delineate the underlying genetic determinants of early-onset IRD in a large pedigree of Pakistani descent by whole exome sequencing (WES) and whole genome sequencing (WGS) analyses.

Methods : A pedigree with three consanguineous marriages, five affected and five unaffected members was enrolled and ophthalmic examination including fundus photography, ERG, and visual acuity measurements was performed. Genome-wide linkage and haplotype analyses were performed using STR markers. Subsequently, WES of one affected member was performed to identify the candidate variants. In addition, WGS of another affected member was performed to detect non-coding sequence alterations. Mapping and variant calling was performed using BWA and GATK. Structural variations were called using GenomeSTRiP and LUMPY. Candidate genes were prioritized by investigating their interactions with known retina-associated genes in the human protein interaction network. Effect of the potential mutation in the ADP Ribosylation Factor GTPase activating protein 2 (ARFGAP2) gene was evaluated by expressing the wild-type and mutant proteins in HeLa cells. Expression profile and localization of ARFGAP2 were studied by qRT-PCR, immunostaining, electron microscopy (EM), immunoblotting and GAP assay.

Results : Linkage (LOD score of 5.2 at θ = 0) and haplotype analysis mapped the disease locus to a 19Mb interval on chromosome 11. WES and WGS identified a rare homozygous mutation (p.R255C) in ARFGAP2. This is the only pathogenic and rare variant residing within the linkage interval that segregated with IRD. Protein interaction network analysis of ARFGAP2 showed a significant association with retinal genes. ARFGAP2 localizes to the photoreceptor inner segments, inner nuclear layer, and ganglion cell layer. HeLa cells expressing the mutant ARFGAP2 show trimerization of the protein, fragmentation of Golgi stacks, and high Arf1 activity, indicative of impaired GAP function. Such impairment could be an allosteric effect of mutant ARFGAP2 forming trimers on Golgi membrane that are stabilized by disulphide bonds, likely enabled by the mutant 255-Cys.

Conclusions : The mutant R255C-ARFGAP2 protein exhibits impaired GAP activity towards Arf1; hyperactivation of Arf1 perturbs Golgi structure. These findings provide mechanistic insights into how the mutant ARFGAP2 may contribute to degeneration of retina in patients with IRD.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.