When splicing is not all or none: GT>GC 5' splice-site variants as a model for intermediate effects and challenges in...

Variants with intermediate functional effects-neither fully disruptive nor functionally neutral-represent an under-recognized source of genetic complexity and define a functional gray zone that complicates variant classification. Here, we address this issue using GT>GC (+2T>C) 5' splice-site variants as a tractable model, as approximately 15%-18% of such substitutions retain variable levels of residual wild-type (WT) transcript. Using residual WT transcript as a quantitative functional readout, we first revisited disease-associated GT>GC variants previously shown to retain substantial WT transcript, including SPINK1 c.194+2T>C, HBB c.315+2T>C, and BRCA2 c.8331+2T>C, illustrating how intermediate splicing effects complicate clinical interpretation across distinct genes and disease contexts. We then performed a locus-wide assessment of all 26 theoretically possible GT>GC substitutions in CFTR, integrating SpliceAI delta donor-loss scores with classifications from expert-curated databases. Minigene splicing analyses of four selected CFTR variants, together with full-length and minigene analyses of a BAP1 GT>GC variant with conflicting clinical interpretations, revealed heterogeneous and context-dependent splicing outcomes, underscoring both inter-assay variability and the inherent limitations of commonly used splicing assay systems. Collectively, our findings indicate that GT>GC variants capable of generating appreciable residual WT transcript exemplify a broader class of intermediate-effect alleles that expose the limitations of both computational prediction and experimental assessment. These observations highlight the need for classification frameworks that incorporate quantitative functional data and better capture the continuum of variant effects.