Homozygous Variant in ARL3 Causes Autosomal Recessive Cone Rod Dystrophy

Abstract

Purpose: Cone rod dystrophy (CRD) is a group of inherited retinopathies characterized by the loss of cone and rod photoreceptor cells, which results in poor vision. This study aims to clinically and genetically characterize the segregating CRD phenotype in two large, consanguineous Pakistani families.

Methods: Funduscopy, optical coherence tomography (OCT), electroretinography (ERG), color vision, and visual acuity assessments were performed to evaluate the retinal structure and function of the affected individuals. Exome sequencing was performed to identify the genetic cause of CRD. Furthermore, the mutation's effect was evaluated using purified, bacterially expressed ADP-ribosylation factor-like protein 3 (ARL3) and mammalian cells.

Results: Fundus photography and OCT imaging demonstrated features that were consistent with CRD, including bull's eye macular lesions, macular atrophy, and central photoreceptor thinning. ERG analysis demonstrated moderate to severe reduction primarily of photopic responses in all affected individuals, and scotopic responses show reduction in two affected individuals. The exome sequencing revealed a novel homozygous variant (c.296G>T) in ARL3, which is predicted to substitute an evolutionarily conserved arginine with isoleucine within the encoded protein GTP-binding domain (R99I). The functional studies on the bacterial and heterologous mammalian cells revealed that the arginine at position 99 is essential for the stability of ARL3.

Conclusions: Our study uncovers an additional CRD gene and assigns the CRD phenotype to a variant of ARL3. The results imply that cargo transportation in photoreceptors as mediated by the ARL3 pathway is essential for cone and rod cell survival and vision in humans.

Figure 1

A missense variant of ARL3 is associated with cone rod dystrophy in two large consanguineous Pakistani families. (A) Segregation of ARL3 allele in families LUSG03 and LUSG04. Filled and empty symbols represent affected and unaffected individuals, respectively. Double lines indicate consanguineous marriages. The genotypes (wild type, heterozygous, and homozygous mutant) of the ARL3 mutant allele are also shown for each of the participating families members. Arrows indicate probands. (B) Human ARL3 has six exons. The c.296G>T variant is located in exon 5. The ARL3 protein has a GTP-binding domain, which harbors the p.(Arg99Ile) variant. ClustalW multiple amino acid sequence alignment of ARL3 orthologs shows that p.Arg99 (red) is highly conserved across species. Shown also is the p.Tyr90 variant (red), known to be mutated in two families with dominantly inherited retinitis pigmentosa. The positively charged guanidino side chain of Arg99 is required to form ionic bonds to stabilize the orientation of the negatively charged side chain of Asp26 (green), which is central to the canonical G-1 motif (aka P-loop) found in GTP-binding proteins, conserved in the ARF family in the sequence GLD²⁶X(A/S)GKT. (C) A homology model of the GTP-binding domain of ARL3. Replacement of arginine with isoleucine at residue 99 variant may alter/reduce the affinity of the protein for guanine nucleotides, resulting in an apoprotein that are much less stable in solution.

Figure 2

Color fundus photography of the right and left macula, respectively, of individuals with cone dystrophy from families LUSG03 (A) and LUSG04 (B). Myopic fundus changes were variably present in LUSG03 V:5, V:7 (A) and LUSG04 IV:1, IV:4, and IV:5 (B), including tilted optic disc, alpha zone atrophy, mild temporal disc pallor, and parapapillary RPE thinning consistent with early staphyloma formation. Macular atrophy was observed in LUSG03 V:5 and bull's eye lesions are visible in LUSG03 V:1, V:7 and LUSG04 IV:1, IV:2, and IV:4. Brain magnetic resonance imaging axial T2 image (C) and sagittal T2 image (D) of LUSG03 individual IV:5, demonstrating no increased interpeduncular fossa, normal cerebellar peduncles, and normal vermis.

Figure 3

Spectral domain OCT (Topcon) horizontal raster scans (B) with color fundus photography reference images (A), ETDRS retinal thickness maps (C), and en face infrared reference “shadowgram” imaging (D) of right eyes for individuals from families LUSG03 and LUSG04, demonstrating anatomic changes consistent with cone dystrophy. Reference image from LUSG03 V:5 shows frank geographic atrophy with overlying photoreceptor degeneration. The cone outer-segment tip (COST) line is variably diminished, from subtle (LUSG03 V:1, V:7 and LUSG04 IV:1, IV:5) to bull's eye parafoveal loss with outer nuclear layer thinning (LUSG04 IV:2, IV:4). Retinal thickness maps demonstrate diffuse retinal thinning in all patients, particularly in those with bull's eye lesions or macular atrophy (LUSG03 V:5). Increased posterior curvature from myopia is seen among LUSG03 V:5, V:7 and LUSG04 IV:1, IV:5. En face photoreceptor layer infrared shadowgram shows hypointense round lesions in LUSG04 IV:2, IV:4, isointense fovea in LUSG03 V:7 and LUSG04 IV:1, and central hyperintensity surrounded by a ring of hypointensity in LUSG03 V:1, V:5 and LUSG04 IV:5.

Figure 4

Full-field ERG responses for one normal and three affected individuals homozygous for ARL3 p.(Arg99Ile) variant. Two responses (green and orange) have been superimposed for each ERG condition to show reproducibility. ERG responses of affected individuals of both families revealed moderately to severe reduction in photopic amplitudes OU, but normal scotopic, except IV:05 of family LUSG04 who had poor scotopic rod responses but normal scotopic combined responses OU (possibly insufficient dark adaptation time) and IV:03 of family LUSG03.

Figure 5

The p.Arg99Ile variant affects the stability of the encoded protein. (A) SDS-PAGE stained with Coomassie blue revealed the vast majority of ARL3 to be soluble, as evident by its presence in the 100,000g supernatant (S100). In contrast, virtually all of the R99I mutant is in the pellet (P100), indicating an insoluble protein. (B) Western blot analysis revealed increased evidence of degradation of ARL3 harboring p.Arg99Ile variant when expressed in mammalian (COS7, and HeLa) cells. Immunoblotting with anti-ARL3 antibody revealed endogenous ARL3 (vector lane), whereas strong expression was observed when COS7 and HeLa were transiently transfected with ARL3 cDNA constructs encoding either wild-type or p.Arg99Ile mutant protein. The p.Arg99Ile mutant expresses to comparable levels as the wild-type protein in both cell types; however, it is all found to migrate faster than either of the endogenous or overexpressed wild-type bands. In addition, smaller immunoreactive bands, consistent with ARL3-p.Arg99Ile degradation, can be seen in the prolonged exposure (upper) image. These results are consistent with a less stable protein or a protein more accessible to proteases. (C) Expression of wild-type and mutated forms of human GFP-tagged ARL3 in COS7 and HeLa cells revealed no apparent difference in the distribution of encoded proteins. Scale bars apply to all panels and denote 10 μm.