ABCA4 midigenes reveal the full splice spectrum of all reported noncanonical splice site variants in Stargardt disease

Abstract

Stargardt disease is caused by variants in the gene, a significant part of which are noncanonical splice site (NCSS) variants. In case a gene of interest is not expressed in available somatic cells, small genomic fragments carrying potential disease-associated variants are tested for splice abnormalities using in vitro splice assays. We recently discovered that when using small minigenes lacking the proper genomic context, in vitro results do not correlate with splice defects observed in patient cells. We therefore devised a novel strategy in which a bacterial artificial chromosome was employed to generate midigenes, splice vectors of varying lengths (up to 11.7 kb) covering almost the entire gene. These midigenes were used to analyze the effect of all 44 reported and three novel NCSS variants on pre-mRNA splicing. Intriguingly, multi-exon skipping events were observed, as well as exon elongation and intron retention. The analysis of all reported NCSS variants in allowed us to reveal the nature of aberrant splicing events and to classify the severity of these mutations based on the residual fraction of wild-type mRNA. Our strategy to generate large overlapping splice vectors carrying multiple exons, creating a toolbox for robust and high-throughput analysis of splice variants, can be applied to all human genes.

Deep Scleral Exposure: A Degenerative Outcome of End-Stage Stargardt Disease

“…Other variants included a stop-gain, c.6088C>T (p.Arg2030*) variant in Patient 3 and 8 noncoding variants: 5 variants in canonical splice site sequences (c.4773+3A>G, c.2160+1G>C, c.768G>T, c.3050+5G>A, and c.5714+5A>G) and 3 deepintronicvariants(c.302+68C>T, c.4539+2028C>T and c.4539+2001G>A) that likely have a negative effect on exon splicing. 36 The sibling pair Patient 11 and Patient 12 harbored a deep intronic variant, c.4539+2028C>T, and a deletion/insertion, c.6148–698_c.6670del/insTGTGCACCTCCCTAG, described in a previous report. 37 Patient 5 harbors another deep intronic variant, c.4539+2001G>A.…”

Expanding the Mutation Spectrum in ABCA4: Sixty Novel Disease Causing Variants and Their Associated Phenotype in a Large French Stargardt Cohort

“…Indeed, we cannot exclude the possibility that this variant may affect splicing in vivo since it has already been proven that ABCA4 can have non-canonical splice sites variant with important effects on protein [19,20,21,22,23,59]. …”

Cost‐effective molecular inversion probe‐based ABCA4 sequencing reveals deep‐intronic variants in Stargardt disease

“…The effect of seven NCSS and three deep‐intronic variants was assessed by midigene‐based splicing assays employing wild‐type (WT) BA constructs described elsewhere (Sangermano et al, ) and a newly designed BA31 construct (Table S3). Details of mutagenesis primers and sequencing primers are given in Table S4.…”