The functional impact of 1,570 SNP-accessible missense variants in humanOTC

Abstract

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. Neonatal onset patients appear normal at birth but rapidly develop hyperammonemia, which can progress to cerebral edema, coma and death, outcomes ameliorated by rapid diagnosis and treatment. Existing biochemical assays have limitations, including the sensitivity of the citrulline assays used in newborn screening panels. With prior knowledge of variant pathogenicity, DNA sequence-based diagnostics would provide an alternative screening method. Here, we develop a high throughput functional assay for human OTC and measure the impact of 1,570 variants, 84% of all SNP-accessible missense mutations. Our assay scores agree well with existing clinical significance calls, distinguishing known benign from pathogenic variants and variants with neonatal onset from late-onset disease presentation. Further, use of an intronless expression construct allows us to measure the impact of amino acid changes at splice sites independent of their effect on splicing, thereby separating the contribution of splicing and protein coding changes to aid the analysis of molecular mechanisms underlying pathogenicity. Finally, we assess the utility of our functional data on OTC variant curation by using the current ACMG/AMP guidelines to reclassify variants. Inclusion of our data as PS3/BS3 substantially improves variant interpretation. Thus, our dataset is of high clinical utility and illustrates the power of functional assays to inform interpretation of existing and novel genetic variation.

NAGS, CPS1, and SLC25A13 (Citrin) at the Crossroads of Arginine and Pyrimidines Metabolism in Tumor Cells

“…Gain-of-function missense variants that result in a higher enzymatic activity of mutant proteins have been observed in urea cycle enzymes. A high-throughput functional assay of all single nucleotide variant (SNV)-accessible amino acid replacements in human OTC revealed that around 5% of all OTC variants have up to 30% higher activity than wild-type human OTC [ 65 ]. Furthermore, human OTC with two common p.R46K and p.Q270R sequence variants had 40% higher enzymatic activity than the wild-type or either of the single mutant proteins [ 66 ].…”

Predicting the functional effect of compound heterozygous genotypes from large scale variant effect maps

“…For each yPSAT1 transformant, we know which codon is mutated (target codon), but not which specific variant is present. To determine this, we used a custom MinION (Oxford Nanopore Technologies) sequencing pipeline, as described in Lo et al [32]. Briefly, individual transformants were pooled in groups of 12, so that no target codon was represented more than once in a single pool.…”

“…For haploid strains, raw phenotypic values were normalized, quality control filters were applied to each isolate, and a final relative growth estimate for each variant was determined, as described in Lo et al [47]. Briefly, normalization steps were carried out to account for the effects of plate-to-plate variation, relative growth of neighboring patches, and plate edge effects.…”