Frequent hypomorphic alleles account for a significant fraction of ABCA4 disease and distinguish it from age-related macular degeneration

Abstract

Background: Variation in the ABCA4 gene is causal for, or associated with, a wide range of phenotypes from early onset Mendelian retinal dystrophies to late-onset complex disorders such as age-related macular degeneration (AMD). Despite substantial progress in determining the causal genetic variation, even complete sequencing of the entire open reading frame and splice sites of ABCA4 identifies biallelic mutations in only 60%-70% of cases; 20%-25% remain with one mutation and no mutations are found in 10%-15% of cases with clinically confirmed ABCA4 disease. This study was designed to identify missing causal variants specifically in monoallelic cases of ABCA4 disease.

Methods: Direct sequencing and analysis were performed in a large familial ABCA4 disease cohort of predominately European descent (n=643). Patient phenotypes were assessed from clinical and retinal imaging data.

Results: We determined that a hypomorphic ABCA4 variant c.5603A>T (p.Asn1868Ile), previously considered benign due to high minor allele frequency (MAF) (~7%) in the general population, accounts for 10% of the disease, >50% of the missing causal alleles in monoallelic cases, ~80% of late-onset cases and distinguishes ABCA4 disease from AMD. It results in a distinct clinical phenotype characterised by late-onset of symptoms (4th decade) and foveal sparing (85%). Intragenic modifying effects involving this variant and another, c.2588G>C (p.Gly863Ala) allele, were also identified.

Conclusions: These findings substantiate the causality of frequent missense variants and their phenotypic outcomes as a significant contribution to ABCA4 disease, particularly the late-onset phenotype, and its clinical variation. They also suggest a significant revision of diagnostic screening and assessment of ABCA4 variation in aetiology of retinal diseases.

Keywords: ABCA4; Age-related macular degeneration; Stargardt disease; foveal sparing; hypomorphic variant.

Figure 1

Summary of disease trajectories associated with autofluorescent fleck accumulation in ABCA4 disease. (A) Autofluorescence images illustrating milestones of fleck accumulation beginning with confined or bull’s eye lesions with no visible flecks, to early accumulations within and around the macula, state of absolute confluence with fleck atrophy and end-stage of multiple coalescing lesions across the posterior pole. (B) Timeline of milestones (mean age) disease trajectories with respect to genotype groups. Symptomatic onset (black dashed line) varies across genotypic groups. Notably, patients harbouring c.5882G>A (p.Gly1961Glu) and c.5603A>T (p.Asn1868Ile) present at a significantly later age as compared with patients harbouring other ABCA4 variant combinations. Both c.5882G>A (p.Gly1961Glu) and c.5603A>T (p.Asn1868Ile) patient groups exhibit a delayed onset of visible flecks in the retina (blue line) and, notably, do not progress to the absolute confluence (red line) or end-stage stage (black line) phenotypes as seen in all other cases.

Figure 2

Summary of phenotypic distinction in patients harbouring the c.5603A>T (p.Asn1868Ile) allele of ABCA4. (A) Patients harbouring biallelic ABCA4 variants, including null alleles, and c.5882G>A (p.Gly1961Glu) report visual symptoms at a mean age of 19.7 and 22.7 years, respectively, while the disease is significantly delayed (to 36.3 years, p<0.0001) in patients with the c.5603A>T (p.Asn1868Ile) allele. (B) The prevalence of foveal sparing is highest among patients with p.Asn1868Ile at 84.7% while observed only in ~33% of cases with other ABCA4 variants including p.Gly1961Glu. (C) Autofluorescence imaging across the macula in a p.Asn1868Ile patient exhibiting foveal sparing within an area of retinal pigment epithelium (RPE) and photoreceptor cell atrophy. (D) A spectral domain-optical coherence tomographic scan across the fovea of the same patient reveals the presence of outer retinal layers: RPE, ellipsoid zone (EZ) and external limiting membrane (ELM) in the fovea. SD-OCT, spectral domain-optical coherence tomography.

Figure 3

Hypothetical structural model of ABCA4 transmembrane and nucleotide-binding domains. Two symmetrical structural motifs are shown by different colours. Ribbon structure of motif 1, which include transmembrane and nucleotide-binding domains localised in the first half of the protein, is shown in orange. Motif 2 is localised in the second half of the ABCA4 protein and shown in cyan. Transmembrane and nucleotide-binding domains are labelled according to Dawson and Locher as the TMDs and NBDs, respectively. Residues G863 and N1868 are exposed to disk lumen. Energetic (ΔΔG, kcal/mol) and protein folding effects of structural perturbations p.Asn1868Ile and p.Gly863Ala were predicted from the model using the unfolding mutation screen.