Functional Characterization of ABCA4 Missense Variants Aids Variant Interpretation and Phenotype Prediction in Patients With ABCA4-Retinal Dystrophies

Abstract

Purpose: Biallelic pathogenic variants in the gene encoding the ATP-binding cassette transporter ABCA4 are the leading cause of irreversible vision loss in inherited retinal dystrophies (IRDs). Interpretation of ABCA4 variants is challenging, due to cis-modifying and hypomorphic variants. We have previously detected 10 missense variants of unknown significance (VUS) in patients with suspected ABCA4-retinal dystrophies (ABCA4-RDs) in Norway. In this study, we functionally characterized the VUS to aid interpretation of the variants and to determine if they are associated with the disease.

Methods: The ABCA4 VUS were expressed in HEK293T cells and the ABCA4 expression level and ATPase activity were determined and correlated with the patients' phenotype. The functional data further used for reclassification of the VUS following the American College of Medical Genetics and Genomics (ACMG) guidelines.

Results: Of the 10 VUSs, 2 variants, Cys205Phe and Asn415Thr, were categorized as functionally severe. The age at presentation in the 2 patients carrying these variants was divergent and seemed to be driven by the patients' second pathogenic variants Gly1961Glu and c.5461-10T>C, respectively. Three variants, Val643Gly, Pro799Leu, and Val1433Ile were categorized as functionally moderate, and were found in patients with intermediate/late age at presentation. The remaining five variants were categorized as functionally normal/mild. Based on our data, c.614G>T p.(Cys205Phe), c.1244A>C p.(Asn415Thr), and c.2396C>T p.(Pro799Leu) were reclassified to (likely) pathogenic, while 4 of the functionally normal/mild variants could be reclassified to likely benign.

Conclusions: Functional analyses of ABCA4 variants are a helpful tool in variant classification and enable us to better predict the disease severity in patients with ABCA4-RDs.

Figure 1.

(A) Topological models of ABCA4 showing the locations of the 10 missense variants examined in this study. There are two nonidentical tandem halves of ABCA4, each containing a N-glycosylated exocytoplasmic domain (ECD), a transmembrane domain (TMD) consisting of 6 transmembrane segments, a nucleotide-binding domain (NBD), and a regulatory domain (RD). (B) Structure of ABCA4 based on the sequence of ABCA4, the cryoEM structure of ABCA4 in its unbound state, and the AlphaFold model.

Figure 2.

Protein expression of ABCA4 missense variants relative to WT in transiently transfected HEK293 cells. (A) A representative Western blot labeled with Rho 1D4 antibody targeting WT ABCA4 and variants containing a 1D4 tag. Ezrin was used as a loading control for normalization. (B) Quantification of variants relative to WT (set to 100%), shown as mean of relative expression (n = 3). Each dot represents independent experiments.

Figure 3.

Intracellular localization of the ABCA4 variants in HeLa cells. Representative immunofluorescence images of transfected HeLa cells with plasmids encoding ABCA4 WT and variants or empty pCEP4 vector control (CTR). Rho1D4 antibody (green) was used to label ABCA4 containing the 1D4 tag, calnexin antibody (red) was used as an ER marker, and DAPI (blue) was used for nuclear counterstaining. The WT protein and variants expressed at WT levels localized to vesicles, whereas low-expressing variants (Cys205Phe and Pro799Leu) displayed reticular localization.

Figure 4.

ATPase activity of purified ABCA4 WT and missense variants in the presence and absence of N-ret-PE. By diluting ATR in an assay buffer containing PE, N-ret-PE is formed. The basal ATPase activity of the variants is normalized to WT basal ATPase activity (set to 100%), and the retinal-stimulated activity of each variant is relative to their basal activity. Bars indicate the mean of three independent experiments, each represented by a dot.

Figure 5.

OCT and UWF-FAF images of P4 and P5 with functionally severe variants characterized in this study. (A) P4: UWF-FAF image and OCT showing degeneration in the macula with limited change over a 12-year period. No peripheral degeneration is present. Para-macular retinal function was present 25 years after the disease onset of symptoms. (B) P5: OCT image of the macula demonstrates the extensive chorioretinal atrophy with pigment deposition and no foveal sparing. UWF-FAF and color fundus images of the right and left eye highlight the confluent atrophy of the posterior pole and the peripheral degeneration.

Figure 6.

OCT and UWF-FAF images of P6 and P7 with functionally moderate variants characterized in this study. (A) P6: UWF-FAF image shows mid-peripheral changes, foveal sparing, and some areas with complete atrophy (white arrow), 2 years after onset of symptom. New images could not be obtained due to cataract. (B) P7: UWF-FAF and OCT images showing mid-peripheral and peripheral changes of the retina at the time of diagnosis (2019). Foveal sparing was preserved four years after diagnosis, however, increasing atrophy para-macular (white arrow).

Figure 7.

Close-up images of the 3 residues Cys205, Asn415, and Pro799. (A) Cys205 (green) is located in the ECD1 domain and might form a disulfide bridge with the close cysteine residue at position 230 (blue). (B) Asn415 (green) is located in the ECD1 domain and is post-translationally modified with N-linked glycosylation (yellow). (C) Pro799 (green) is located in the TMD1 domain and causes a kink in the alpha-helix. N-Ret-PE (yellow) is bound to ABCA4 in the substrate binding pocket.