The intronic ABCA 4 c.5461‐10T>C variant, frequently seen in patients with Stargardt disease, causes splice defects and reduced ABCA 4 protein level

Purpose Despite being the third most common ABCA 4 variant observed in patients with Stargardt disease, the functional effect of the intronic ABCA 4 variant c.5461‐10T>C is unknown. The purpose of this study was to investigate the molecular effect of this variant. Methods Fibroblast samples from patients carrying the ABCA 4 variant c.5461‐10T>C were analysed by isolating total RNA , followed by real‐time polymerase chain reaction (RT‐PCR) using specific primers spanning the variant. For detection of ABCA 4 protein, fibroblast samples were lysed and analysed by SDS ‐ PAGE followed by immunoblotting using a monoclonal ABCA 4 antibody. Results The ABCA 4 variant c.5461‐10T>C causes a splicing defect resulting in the reduction of full‐length mRNA in fibroblasts from patients and the presence of alternatively spliced mRNA s where exon 39–40 is skipped. A reduced level of full‐length ABCA 4 protein is observed compared to controls not carrying the variant. Conclusions This study describes the functional effect and the molecular mechanism of the pathogenic ABCA 4 variant c.5461‐10T>C. The variant is functionally important as it leads to splicing defects and a reduced level of ABCA 4 protein.