Pathogenic impact of ABCA4 missense variants in the structurally uncharacterized ECD1 region: implications for Stargardt disease.

Pathogenic mutations in the ABCA4 gene cause several inherited retinal diseases, particularly Stargardt disease (STGD1). However, many missense variants remain classified as variants of uncertain significance (VUS) due to inconclusive evidence regarding their pathogenic impact. The missense VUS span across all the domains of ABCA4, with the majority found in the larger extracellular domains (ECDs). The largest uncharacterized region of ABCA4 is located in ECD1, where limited structural information and inconsistent computational predictions hinder clinical interpretation of missense VUS in this region. Here, we integrated in silico analysis with in vitro functional assays to evaluate the pathogenicity of VUS in this region and improve their diagnostic classification. Missense VUS in the ECD1 uncharacterized region were curated from ClinVar. Six multiallelic sites were identified in the uncharacterized region and 13 missense VUS on these multiallelic sites were characterized using the integrated analysis. In the in silico platform, the pathogenicity of the VUS were predicted using multiple algorithms, and the structural effects of the variants were analyzed compared to the wild type. Recombinant variants were expressed in virus-like particles (VLPs), and protein expression, membrane localization, and ATPase activity were quantified relative to wild type to identify potential disease-causing variants. From the integrated analysis, variants with pronounced structural destabilization, impaired membrane trafficking, and reduced or absent N-retinylidene-phosphatidylethanolamine (NRPE) substrate stimulated ATPase activities were identified as potentially deleterious. Notably, VUS at p.H193P and p.I214N showed loss of function, with p.I214N reflecting selectively impaired membrane targeting and p.H193P reflecting combined expression and trafficking defects. Additionally, NRPE-stimulated ATPase activities were impaired in VUS, p.V195L, p.V195I, p.D197H, p.I214F and p.N269S. Overall structural destabilization interfered with the NRPE-stimulated ATPase activities of p.N269S, while the lack of NRPE-stimulated ATPase activities of p.D197H, p.V195L, p.V195I and p.I214F are thought to be due to impaired NRPE interactions with ABCA4. All the VUS at p.R140, p.H193Y, p.D197N and p.N269H showed both the basal and NRPE-stimulated ATPase activities but less than that of the wild type, displaying a mild functional deficit. Together, these findings demonstrated that certain VUS within the unresolved ECD1 region disrupt ABCA4 stability and function, supporting their contribution to disease pathogenesis. This integrative approach highlights key residues likely to be pathogenic and advances the interpretation of VUS in inherited retinal disorders.