Supplementary Material for: Early-Onset Stargardt Disease Caused by Homozygosity of a Complex ABCA4 Allele from Eastern Africa: Two Case Reports

Introduction: Biallelic pathogenic variants in the ABCA4 gene are the leading cause of inherited retinal diseases (IRDs). Over 1,200 pathogenic or likely pathogenic ABCA4 variants have been reported, resulting in a broad clinical spectrum of ABCA4-retinal dystrophies (ABCA4-RD), with Stargardt disease being the most common. Most patients with ABCA4-RD are compound heterozygotes, carrying two pathogenic ABCA4 variants in trans. Case Presentation: We report two unrelated patients with early-onset (≤12 years) Stargardt disease, both found to be homozygous for a complex ABCA4 allele containing the hypomorphic c.5882G>A p.(Gly1961Glu) variant and the c.634C>T p.(Arg212Cys) variant. Both patients underwent detailed clinical assessment and genetic screening, including whole exome or genome sequencing. In vitro assays were performed to assess the individual and combined effect of these variants on the ABCA4 protein. The identified ABCA4 variants were expressed in HEK293FT and HeLa cells to assess their protein expression levels and intracellular localization compared to the wild type (WT) ABCA4 protein. Molecular analysis revealed that the Arg212Cys variant and the doubly mutated allele showed similarly reduced protein expression, while Gly1961Glu expressed close to WT level. Both variants, individually and combined, localized to intracellular vesicles similarly to WT ABCA4. Conclusion: This study highlights the genetic complexity of ABCA4-RD and the significance of pathogenic variants in cis. It also emphasizes the challenge of accurately predicting the functional consequences of specific ABCA4 alleles with in vitro assays.