First estimation of the scale of canonical 5’ splice site GT>GC mutations generating wild-type transcripts and their medical genetic implications

Abstract

ABSTRACTIt has long been known that canonical 5’ splice site (5’SS) GT>GC mutations may be compatible with normal splicing. However, to date, the true scale of canonical 5’SS GT>GC mutations generating wild-type transcripts, both in the context of the frequency of such mutations and the level of wild-type transcripts generated from the mutation alleles, remain unknown. Herein, combining data derived from a meta-analysis of 45 informative disease-causing 5’SS GT>GC mutations (from 42 genes) and a cell culture-based full-length gene splicing assay of 103 5’SS GT>GC mutations (from 30 genes), we estimate that ∼15-18% of the canonical GT 5’SSs are capable of generating between 1 and 84% normal transcripts as a consequence of the substitution of GT by GC. We further demonstrate that the canonical 5’SSs whose substitutions of GT by GC generated normal transcripts show stronger complementarity to the 5’ end of U1 snRNA than those sites whose substitutions of GT by GC did not lead to the generation of normal transcripts. We also observed a correlation between the generation of wild-type transcripts and a milder than expected clinical phenotype but found that none of the available splicing prediction tools were able to accurately predict the functional impact of 5’SS GT>GC mutations. Our findings imply that 5’SS GT>GC mutations may not invariably cause human disease but should also help to improve our understanding of the evolutionary processes that accompanied GT>GC subtype switching of U2-type introns in mammals.