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ATP‐binding cassette subfamily A, member 4 intronic variants c.4773+3A>G and c.5461‐10T>C cause Stargardt disease due to defective splicing
Author(s) -
Jonsson Frida,
Westin Ida Maria,
Österman Lennart,
Sandgren Ola,
Burstedt Marie,
Holmberg Monica,
Golovleva Irina
Publication year - 2018
Publication title -
acta ophthalmologica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.534
H-Index - 87
eISSN - 1755-3768
pISSN - 1755-375X
DOI - 10.1111/aos.13676
Subject(s) - stargardt disease , abca4 , minigene , biology , genetics , rna splicing , exon , hek 293 cells , compound heterozygosity , gene , alternative splicing , mutation , microbiology and biotechnology , rna , phenotype
Purpose Inherited retinal dystrophies ( IRD s) represent a group of progressive conditions affecting the retina. There is a great genetic heterogeneity causing IRD s, and to date, more than 260 genes are associated with IRD s. Stargardt disease, type 1 (STGD1) or macular degeneration with flecks, STGD1 represents a disease with early onset, central visual impairment, frequent appearance of yellowish flecks and mutations in the ATP‐binding cassette subfamily A, member 4 ( ABCA4 ) gene. A large number of intronic sequence variants in ABCA 4 have been considered pathogenic although their functional effect was seldom demonstrated. In this study, we aimed to reveal how intronic variants present in patients with Stargardt from the same Swedish family affect splicing. Methods The splicing of the ABCA 4 gene was studied in human embryonic kidney cells, HEK 293T, and in human retinal pigment epithelium cells, ARPE ‐19, using a minigene system containing variants c.4773+3A>G and c.5461‐10T>C. Results We showed that both ABCA 4 variants, c.4773+3A>G and c.5461‐10T>C, cause aberrant splicing of the ABCA 4 minigene resulting in exon skipping. We also demonstrated that splicing of ABCA 4 has different outcomes depending on transfected cell type. Conclusion Two intronic variants c.4773+3A>G and c.5461‐10T>C, both predicted to affect splicing, are indeed disease‐causing mutations due to skipping of exons 33, 34, 39 and 40 of ABCA 4 gene. The experimental proof that ABCA 4 mutations in STGD patients affect protein function is crucial for their inclusion to future clinical trials; therefore, functional testing of all ABCA 4 intronic variants associated with Stargardt disease by minigene technology is desirable.