Identification and functional characterization of ABCA4 gene variants in three patients with Stargardt disease or retinitis pigmentosa

Qi Luo¹Juan Huang²Lu Shi^3,4Guanghong Zhang¹Linping Xue²Kehu Wu¹Xiaoyu Li¹Lei Yang¹Dujun Li¹Liangwei Mao⁴Jihong Luo^1*

• ¹Department of Ophthalmology, Hubei Provincial Hospital of Traditional Chinese Medicine, Wuhan, China
• ²The First Clinical Medical School, Hubei University of Chinese Medicine, Hubei, China
• ³Hubei Provincial Key Laboratory of Agricultural Bioinformatics, College of Informatics, Huazhong Agricultural University, Wuhan, Hebei Province, China
• ⁴Wuhan Primbio Medical Laboratory, Wuhan, China

The diversity of phenotypes-ranging from inherited retinal dystrophies (such as Stargardt disease 1, cone-rod dystrophy 3, retinitis pigmentosa 19) to late-onset age-related macular degeneration 2-has been attributed to loss-of-function variants in ABCA4 gene. In this study, we aimed to identify and analyze potential pathogenic ABCA4 variants in patients with Stargardt disease or retinitis pigmentosa and to explore the impact of an intronic variant (NM_000350.3:c.6386+4A>G) on mRNA splicing. We enrolled three patients from unrelated families with Stargardt disease or retinitis pigmentosa after comprehensive ophthalmological evaluations were performed. Whole-exome sequencing followed by Sanger sequencing were applied for mutation screening, focusing on inherited retinal dystrophies related genes. Additionally, the splicing alteration caused by c.6386+4A>G was functionally characterized by a minigene splicing assay. Five ABCA4 germline variants were detected in three patients: one frameshift, one nonsense, one splicing, and two missense variants. Furthermore, two pathogenic, two likely pathogenic variants and one variant of uncertain significance were determined according to ACMG/AMP and ClinGen SVI guidelines. The result of minigene splicing assay proved that c.6386+4A>G affected wild type donor splice site recognition of intron 46 and yielded a truncated transcript with 47 bp deletion in exon 46. In our study, the identification of two novel ABCA4 variants expanded the mutational spectrum of the ABCA4 gene in Stargardt disease and retinitis pigmentosa, while providing new insights into the molecular pathology of ABCA4 splicing defects.