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

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.

The Surviving, Not Thriving, Photoreceptors in Patients with ABCA4 Stargardt Disease.

Stargardt disease (STGD1), associated with biallelic variants in the ABCA4 gene, is the most common heritable macular dystrophy and is currently untreatable. To identify potential treatment targets, we characterized surviving STGD1 photoreceptors. We used clinical data to identify macular regions with surviving STGD1 photoreceptors. We compared the hyperreflective bands in the optical coherence tomographic (OCT) images that correspond to structures in the STGD1 photoreceptor inner segments to those in controls. We used adaptive optics scanning light ophthalmoscopy (AO-SLO) to study the distribution of cones and AO-OCT to evaluate the interface of photoreceptors and retinal pigment epithelium (RPE). We found that the profile of the hyperreflective bands differed dramatically between patients with STGD1 and controls. AO-SLOs showed patches in which cone densities were similar to those in healthy retinas and others in which the cone population was sparse. In regions replete with cones, there was no debris at the photoreceptor-RPE interface. In regions with sparse cones, there was abundant debris. Our results raise the possibility that pharmaceutical means may protect surviving photoreceptors and so mitigate vision loss in patients with STGD1.