Splicing Outcomes of 5′ Splice Site GT>GC Variants That Generate Wild-Type Transcripts Differ Significantly Between Full-Length and Minigene Splicing Assays

Abstract

Combining data derived from a meta-analysis of human disease-associated 5′ splice site GT>GC (i.e., +2T>C) variants and a cell culture-based full-length gene splicing assay (FLGSA) of forward engineered +2T>C substitutions, we recently estimated that ∼15–18% of +2T>C variants can generate up to 84% wild-type transcripts relative to their wild-type counterparts. Herein, we analyzed the splicing outcomes of 20 +2T>C variants that generate some wild-type transcripts in two minigene assays. We found a high discordance rate in terms of the generation of wild-type transcripts, not only between FLGSA and the minigene assays but also between the different minigene assays. In the pET01 context, all 20 wild-type minigene constructs generated the expected wild-type transcripts; of the 20 corresponding variant minigene constructs, 14 (70%) generated wild-type transcripts. In the pSPL3 context, only 18 of the 20 wild-type minigene constructs generated the expected wild-type transcripts whereas 8 of the 18 (44%) corresponding variant minigene constructs generated wild-type transcripts. Thus, in the context of a particular type of variant, we raise awareness of the limitations of minigene splicing assays and emphasize the importance of sequence context in regulating splicing. Whether or not our findings apply to other types of splice-altering variant remains to be investigated.

Counteracting the Common Shwachman–Diamond Syndrome-Causing SBDS c.258+2T>C Mutation by RNA Therapeutics and Base/Prime Editing

“…In accordance, a residual extent of correct splicing was further demonstrated by expression of the SBDS mutant minigenes, which also revealed, besides the exon 2 skipping event, the usage of a cryptic splice site which was not appreciable ex vivo likely because of the nonsense-mediated decay effect. Altogether, these data on the residual usage of the correct 5′ss, which has key pathophysiological implications for SDS, are in agreement with recent research showing that GT>GC variants at 5’ss can be compatible with correctly processed mRNA [ 30 , 31 , 32 , 33 ].…”

Minigene splicing assays reveal new insights into exonic variants of the SLC12A3 gene in Gitelman Syndrome.

“…Variants of c.2755A>T and c.2863A>T, predicted by BDGP and HSF to have a significant impact on ESE / ESS motifs, did not cause exon 24 skipping, and the WT minigene of exon 12 only produced the exon 12-excluded transcript. Coincidentally, the same results that candidate variants did not alter pre-mRNA splicing were revealed in other studies (Suarez-Artiles et al, 2018;Wang et al, 2020;Zhang et al, 2021) and there was a previous study that suggested only 18 of the 20 wild-type minigene constructs produced the desired wild-type transcripts in the pSPL3 context, improving the awareness of the limitations of minigene assays and emphasizing the importance of sequence context in regulating splicing (Lin et al, 2021). The reasons for these results may be related to the defects of software, the limited transferability of minigenes, the differential expression of splicing factors in cells, the interference of the mRNA secondary structure, etc.…”

Characterization of a possible founder synonymous variant in TECTA in multiple individuals with autosomal recessive hearing loss

“…For this reason, we used an in vitro minigene assay to characterize the effect of this synonymous variant on splicing. Although there is an inherent limitation to the minigene‐based analysis of splicing (Lin et al, 2021), our experiment successfully demonstrates the activation of a cryptic splice site with no leaky expression of the wild‐type transcript. This was used as supportive evidence in clarifying the clinical significance of this TECTA synonymous variant.…”

“…For this reason, we used an in vitro minigene assay to characterize the effect of this synonymous variant on splicing. Although there is an inherent limitation to the minigene-based analysis of splicing (Lin et al, 2021) Our analysis thus strongly suggests this variant arose as a founder variant in Latinos of African ancestry, which explains why this variant is rare or absent in other subpopulations of gnomAD and our internal databases. Disease-relevant variants occurring at elevated frequencies due to bottleneck events in a population's history are expected to occur in the LAI data, and several studies have identified founder variants in genes associated with other diseases in the Latino ancestry (Gonzaga-Jauregui et al, 2015).…”