In the human genome, most 5 ' splice sites (similar to 99%) employ the canonical GT dinucleotide whereas a small minority (similar to 1%) use the noncanonical GC dinucleotide. The functionality and pathogenicity of 5 ' splice site GT>GC (+2T>C) variants have been extensively studied but we know very little about 5 ' splice site GC>GT (+2C>T) variants. Herein, we have addressed this deficiency by performing a meta-analysis of reported +2C>T "pathogenic" variants together with a functional analysis of engineered +2C>T substitutions using a cell culture-based full-length gene splicing assay. Our results establish proof of concept that +2C>T variants are qualitatively different from +2T>C variants in terms of their functionality and suggest that, in sharp contrast to +2T>C variants, most if not all +2C>T variants have no pathological relevance. Our findings have important implications for interpreting the clinical relevance of +2C>T variants and understanding the evolutionary switching between GT and GC 5 ' splice sites in mammalian genomes.