Functional profiling of 2,193 ASS1 missense variants: Insights into variant pathogenicity and epistatic interactions in citrullinemia type I

Deep mutational scanning of 2,193 ASS1 missense variants, revealing intragenic complementation

A high-throughput yeast functional assay measured the impact of 2,193 ASS1 missense variants (90% of all SNV-accessible substitutions). The assay reaches PS3 ACMG level and enables definitive reclassification of all 25 ClinVar VUS in the functionally impaired range. An unexpected finding was intragenic complementation of the ASS1 homotetramer, where deleterious variants from different subunits concentrate in a subset of active sites, partially restoring enzymatic function. This positive epistasis has direct implications for interpreting compound heterozygous genotypes in citrullinemia.

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This paper illustrates the power of deep mutational scanning applied to the urea cycle: virtually all ASS1 VUS can now be classified. Intragenic complementation is a major mechanistic discovery, potentially applicable to other homotetrameric proteins, challenging standard interpretation of compound heterozygous states in molecular genetics.