Molecular insights into ABCA4-related retinal disease: an integrated approach using in silico and in vitro tools
Date
2024
Authors
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Publisher
University of Delaware
Abstract
The retina-specific ATP-binding cassette transporter, ABCA4, is crucial for maintaining retinal health by clearing toxic byproducts generated during phototransduction. Pathogenic variants in ABCA4 disrupt this function, leading to blinding inherited retinal diseases such as Stargardt disease, retinitis pigmentosa, and cone-rod dystrophy, collectively affecting millions worldwide. Despite the identification of over 4,000 ABCA4 variants, a significant portion remains unclassified in terms of pathogenicity, posing substantial challenges for accurate diagnosis, clinical management, and therapeutic intervention. The increasing number of variants with uncertain clinical significance, combined with the challenges of experimentally studying the large transmembrane protein ABCA4, points to the urgent need for high-throughput, practical, yet accurate methods. This dissertation employs an integrated approach, combining in silico protein structural analysis with a virus-like particle-based in vitro characterization platform to systematically investigate the pathogenic impact of missense ABCA4 variants of uncertain significance (VUS) at the protein level. The efficacy of both methods was validated using variants with known pathogenicity, and this combined approach was then applied to a set of ABCA4 VUS, which effectively led to their reclassification under American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines. Overall, the in silico methods serve as a first step in prioritizing variants and elucidating the molecular mechanisms underlying protein dysfunction, while the VLP platform provides a streamlined, higher-throughput method for in vitro functional characterization of variants. This integrated methodology advances our understanding of ABCA4-related retinal diseases and offers a scalable framework that can be adapted to study other membrane proteins involved in genetic disorders.
Description
Keywords
ATP-binding cassette, Human molecular genetics, Pathogenicity prediction, Retinal degeneration, Variants of uncertain significance