Abstract
Purpose: The purpose of this study was to investigate the contribution and natural progression of ABCA4 deep intronic variants (DIVs) among a Chinese Stargardt disease (STGD) cohort. Methods: For unsolved STGD probands, DIVs in ABCA4 were detected by next-generation sequencing, and splicing effects were evaluated by in silico tools and validated through minigene experiments. Comprehensive ocular examinations, especially fundus changes, were carried out and analyzed. These and long-term follow-up data were compared with data of patients carrying biallelic coding variants of ABCA4. Results: Seven DIVs in ABCA4 were identified in 18 of 40 (45.0%) unsolved STGD probands, involving 2 novel and 5 known variants. Four DIVs were confirmed to effect splicing through minigene assay. The c.161-395G>A was the most prevalent DIV allele (30.6%, 11/36). In the early 5-year duration, localized maculopathy was predominant, accounting for 51.9% (14/27) of fundus recordings. Expanded macular lesion within the vascular arch, with or without flecks, was observed in 75.0% (12/16) of recordings beyond the 5-year duration, whereas generalized retinal dystrophy was rarely observed. Compared with those in the non-DIV group, the patients in the DIV group manifested milder fundus change at all disease stages (P < 0.05). Follow-up visits utilizing wide-field fundus autofluorescence (FAF) further validated the slower development of lesions. Optical coherence tomography angiography (OCTA) documented a gradual reduction in perfusion in each layer's capillaries and high-reflective deposits below the sub-RPE layer. Conclusions: DIVs contribute to nearly half of STGD cases with missing heritability, totally occupying 7.8% of all STGD families. Based on optimized grading criteria, patients with DIV alleles manifested localized macular lesions with slow progress. The amount of residual correctly spliced mRNA might play a role, suggesting that adding or enhancing normal ABCA4 expression might be a potential approach of intervention.