A Novel ARL3 Gene Mutation Associated With Autosomal Dominant Retinal Degeneration

Abstract

Despite major progress in the discovery of causative genes, many individuals and families with inherited retinal degenerations (IRDs) remain without a molecular diagnosis. We applied whole exome sequencing to identify the genetic cause in a family with an autosomal dominant IRD. Eye examinations were performed and affected patients were studied with electroretinography and kinetic and chromatic static perimetry. Sequence variants were analyzed in genes (n = 271) associated with IRDs listed on the RetNet database. We applied a stepwise filtering process involving the allele frequency in the control population, in silico prediction tools for pathogenicity, and evolutionary conservation to prioritize the potential causal variant(s). Sanger sequencing and segregation analysis were performed on the proband and other family members. The IRD in this family is expressed as a widespread progressive retinal degeneration with maculopathy. A novel heterozygous variant (c.200A > T) was identified in the ARL3 gene, leading to the substitution of aspartic acid to valine at position 67. The Asp67 residue is evolutionary conserved, and the change p.Asp67Val is predicted to be pathogenic. This variant was segregated in affected members of the family and was absent from an unaffected individual. Two previous reports of a de novo missense mutation in the ARL3 gene, each describing a family with two affected generations, are the only examples to date of autosomal dominant IRD associated with this photoreceptor gene. Our results, identifying a novel pathogenic variant in ARL3 in a four-generation family with a dominant IRD, augment the evidence that the ARL3 gene is another cause of non-syndromic retinal degeneration.

Keywords: chromatic perimetry; ciliopathy; cones; electroretinography; retinal degeneration; rods; whole exome sequencing.

FIGURE 1

Analysis and filtering pipeline for RetNet genes.

FIGURE 2

(A) Pedigree of the family, showing members affected and unaffected by the IRD. The segregation of the ARL3 variant c.A200T is shown where mutation is indicated by “+/M,” and its absence is indicated by “+/+.” (B) Sanger sequencing electropherogram of the available family members confirming the exome sequencing results, as all affected are heterozygous (A/T) and the unaffected is homozygous for the wild-type allele (A/A) (shown in red rectangle).

FIGURE 3

Maculopathy at all stages of the IRD. (A–C) Fundus photographs of PIII-2, PIV-2, and PIII-1. Arrows point to maculopathy. (D) OCTs of normal subject (upper panels) compared with scans of PIV-3 (lower panels) along horizontal and vertical meridians (insets, upper right). Hatched bar shows the location of the optic nerve head in the horizontal scans. ONL, outer nuclear layer, highlighted in blue; INL, inner nuclear layer, purple; GCL, ganglion cell layer, orange; and RNFL, retinal nerve fiber layer, arrow. T, temporal; N, nasal; I, inferior, S, superior retina.

FIGURE 4

Electroretinography in the ARL3 family members. (A) Rod, mixed (maximum white flash) cone-rod (C-R), and cone ERGs from a subject with normal vision, PIV-3 (age 18) and PIII-2 (age 48). Note the different vertical scales for Normal and Patients. There are larger amplitudes, albeit abnormal, in the father (PIII-2) and the mixed C-R waveform is “negative.” (B) P2 and P3 component analyses of rod-isolated ERG photoresponses. (Upper) Rod-isolated a-waves to different stimulus intensities in a normal subject and PIII-2. The smooth curves represent a family of functions describing phototransduction activation best fit to the leading edges of the intensity series. (Lower) Normalized rod P2 components in response to the 3.9 log scot⋅td⋅s intensity stimulus in four normal subjects, and in PIII-2. (C) ERG results from four family members compared with normal limits (rectangle: ±2SD from the mean). (D) Relationship of rod b-wave amplitude to cone flicker amplitude. Amplitudes are normalized to mean normal values and expressed in log units. Line describes equal reduction of rod and cone amplitudes; gray zone represents greater cone than rod amplitude reduction (C < R). Patient data are larger symbols and small squares are data from normal subjects for comparison.

FIGURE 5

Kinetic and chromatic static perimetry in the family members. (A) Full kinetic fields to V-4e but reduced to I-4e test targets in PIII-2 at age 48. Static perimetry shows rod and cone sensitivity is mildly reduced in a large region of central field but there is greater peripheral reduction. (B) Serial maps in PIV-3 at age 13 are comparable in pattern to those of PIII-2, but over the subsequent 14 years, there is loss of function in regions of the central field. (C) PIII-1, at a similar age to the sibling, PIII-2, has only a small residual central island of vision. (D–G) Rod and cone perimetric profiles across the horizontal meridian provide more detail of the central function. (D,E) PIII-2 and PIV-2, despite different ages at the visit, show rod and cone sensitivities at the lower limit of normal for most of the central 50°, but there is a decline of function at greater eccentricities. (F) PIV-3, at ages 13 and 27, show a sequence of change from central field rod and cone sensitivities at the lower limit of normal to loss of rod and cone function extending from the fovea into the nasal field but some retained peripapillary sensitivities. (G) PIV-1 from ages 15–18 shows impaired rod function at the earlier age to barely measurable rod function at the later age. Cone sensitivity was measured at age 18 only and it was abnormally reduced across the horizontal meridian. Blue: 500 nm target, dark-adapted; orange: 600 nm, light-adapted (10 cd.m^–2 white background); N, nasal; T, temporal; S, superior; I, inferior visual field; R, C, M, rod-, cone- and mixed (rod and cone) -mediated. Shaded areas: normal range (±2SD).

FIGURE 6

Alignment of ARL3 amino acids surrounding the location corresponding to the D67V mutation in sequences from multiple species. The Asp (D) at residue 67 is indicated by the red rectangle and is conserved across all of the species shown, including human, rhesus, mouse, dog, elephant, chicken, X_tropicalis, and zebrafish. The sequences were aligned with use of the UCSC genome browser.