First estimate of the scale of canonical 5′ splice site GT>GC variants capable of generating wild‐type transcripts

It has long been known that canonical 5′ splice site (5′SS) GT>GC variants may be compatible with normal splicing. However, to date, the actual scale of canonical 5′SSs capable of generating wild‐type transcripts in the case of GT>GC substitutions remains unknown. Herein, combining data derived from a meta‐analysis of 45 human disease‐causing 5′SS GT>GC variants and a cell culture‐based full‐length gene splicing assay of 103 5′SS GT>GC substitutions, we estimate that ~15–18% of canonical GT 5′SSs retain their capacity to generate between 1% and 84% normal transcripts when GT is substituted by GC. We further demonstrate that the canonical 5′SSs in which substitution of GT by GC‐generated normal transcripts exhibit 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 capable of reliably distinguishing 5′SS GT>GC variants that generated wild‐type transcripts from those that did not. Our findings imply that 5′SS GT>GC variants in human disease genes may not invariably be pathogenic. Based upon complementary data from the meta‐analysis of 45 disease‐causing 5′SS GT>GC variants and the cell culture‐based full‐length gene splicing analysis of 103 5′SS GT>GC substitutions, we have provided a first estimate of ~15–18% for the proportion of canonical GT 5′SSs that are capable of generating between 1% and 84% normal transcripts in case of the substitution of GT by GC. Given that even the retention of 5% normal transcripts can significantly ameliorate a patient's clinical phenotype, our findings imply the potential existence of hundreds or even thousands of disease‐causing 5′SS GT>GC variants that may underlie relatively mild clinical phenotypes. As 5′SS GT>GC variants can also give rise to relatively high levels of wild‐type transcripts, our findings imply that 5′SS GT>GC variants may not invariably be pathogenic in disease‐causative or disease‐associated genes.