Combinatorial genetics reveals a scaling law for the effects of mutations on splicing

Despite a wealth of molecular knowledge, quantitative laws for accurate prediction of biological phenomena remain rare. Alternative pre-mRNA splicing is an important regulated step in gene expression frequently perturbed in human disease. To understand the combined effects of mutations during evolution, we quantified the effects of all possible combinations of exonic mutations accumulated during the emergence of an alternatively spliced human exon. This revealed that mutation effects scale non-monotonically with the inclusion level of an exon, with each mutation having maximum effect at a predictable intermediate inclusion level. This scaling is observed genome-wide for cis and trans perturbations of splicing, including for natural and disease-associated variants. Mathematical modelling suggests that competition between alternative splice sites is sufficient to cause this non-linearity in the genotype-phenotype map. Combining the global scaling law with specific pairwise interactions between neighbouring mutations allows accurate prediction of the effects of complex genotype changes involving >10 mutations. Overall design: Combinatorially complete genotype-phenotype landscape of a model human alternatively-spliced exon (FAS exon 6) by deep mutation scanning