Saturation mapping of MUTYH variant effects using DNA repair reporters.

Variants of uncertain significance (VUSs) limit the actionability of genetic testing. A prominent example is MUTYH, a DNA repair factor underlying colorectal cancer with a pathogenic variant carrier rate of ∼1:50. To systematically interrogate MUTYH variant function, we coupled deep mutational scanning to DNA repair reporters containing its lesion substrate, 8OG:A. Our variant-to-function map covers 96.6% of possible MUTYH point variants (n = 10,941) and achieves 100% accuracy on known clinical variants (n = 247). Leveraging a large clinical registry, we observe significant associations with colorectal polyps and cancer, with more severely impaired missense variants conferring greater risk. We recapitulate functional differences between pathogenic founder alleles and highlight sites of complete missense intolerance, including residues that intercalate DNA and coordinate essential Zn²⁺ or Fe-S clusters. This map provides a resource to resolve the >1,100 existing missense VUSs in MUTYH and demonstrates a scalable strategy to interrogate other clinically relevant DNA repair factors.