The functional impact of 1,570 individual amino acid substitutions in human OTC.

Deleterious mutations in the X-linked gene encoding ornithine transcarbamylase (OTC) cause the most common urea cycle disorder, OTC deficiency. This rare but highly actionable disease can present with severe neonatal onset in males or with later onset in either sex. Individuals with neonatal onset appear normal at birth but rapidly develop hyperammonemia, which can progress to cerebral edema, coma, and death, outcomes ameliorated by rapid diagnosis and treatment. Here, we develop a high-throughput functional assay for human OTC and individually measure the impact of 1,570 variants, 84% of all SNV-accessible missense mutations. Comparison to existing clinical significance calls, demonstrated that our assay distinguishes known benign from pathogenic variants and variants with neonatal onset from late-onset disease presentation. This functional stratification allowed us to identify score ranges corresponding to clinically relevant levels of impairment of OTC activity. Examining the results of our assay in the context of protein structure further allowed us to identify a 13 amino acid domain, the SMG loop, whose function appears to be required in human cells but not in yeast. Finally, inclusion of our data as PS3 evidence under the current ACMG guidelines, in a pilot reclassification of 34 variants with complete loss of activity, would change the classification of 22 from variants of unknown significance to clinically actionable likely pathogenic variants. These results illustrate how large-scale functional assays are especially powerful when applied to rare genetic diseases.