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

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. 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.

Keywords: OTC deficiency; Oxford Nanopore sequencing; SNV analysis/discovery; X-linked disease; metabolic disorder; model organisms; multiplexed assays of variant effect; rare disease; rare variants; urea cycle disorder; variant interpretation.

Graphical abstract

Figure 1

A yeast-based functional assay for human OTC amino acid substitutions (A) Comparison of the urea cycle in humans (left) and the arginine biosynthesis pathway in S. cerevisiae (right). (B) Yeast strain construction. The arg3Δ strain was transformed with PCR products containing either wild-type or variant alleles of the yeast codon-optimized human OTC coding sequence (yOTC), which is adjacent to a nourseothricin resistance drug marker (NAT). Homology-directed integration at the ARG3 locus places yOTC under the control of the yeast ARG3 promoter and TRP2 terminator. (C) Yeast growth in the absence of arginine as a quantitative assay for OTC function. In the absence of arginine, yeast cells harboring the native OTC ortholog (ARG3) grow robustly, but cells harboring a precise gene deletion (arg3Δ) do not. Yeast cells harboring wild-type yOTC or a benign variant (p.Lys46Arg) as the sole source of ornithine transcarbamylase activity grow at 77% and 69% of ARG3, respectively. Yeast cells harboring a pathogenic variant (p.Arg141Gln) are unable to grow. Growth on minimal medium is calculated as the product of the area and the intensity from plates imaged after 3 days. Growth estimates for each genotype ± standard deviations are displayed relative to ARG3 (set to 1.0) and arg3Δ (set to 0). Six independent isolates of each strain (ARG3, arg3Δ0, wild-type yOTC, yOTC-Arg141Gln, and yOTC-Lys46Arg) were assayed in triplicate.

Figure 2

Quantitative growth measurements of 1,570 OTC variants (A) Frequency of yOTC variants exhibiting varying levels of normalized growth. Variant growth is reported relative to that of the arg3 deletion (normalized growth value = 0, marked in purple) and the wild-type yOTC (normalized growth value = 1, marked in green). The 0.05 and 0.9 lines used to demarcate the amorphic, hypomorphic, and unimpaired growth ranges are indicated with dashed gray lines. (B) Relative growth of each ordered yOTC variant plotted with ± SE of the growth estimates.

Figure 3

Missense variant effect map across the length of human OTC compared to structural features and conservation (A) The top plot depicts the binned growth of the 2–7 substitutions introduced at each amino acid position in our library, ordered from highest (top) to lowest (bottom) growth. Below are the positions of OTC functional residues (substrate binding and catalysis), ConSurf evolutionary conservation scores (PDB: 1OTH), and OTC secondary structure. More negative ConSurf scores indicate more conserved positions. Helices and beta-sheets are depicted as gray cylinders and arrows, respectively, and turns are shown as upward half-coils. (B) Protein structure of OTC (PDB: 1OTH) colored by binned ConSurf conservation score. (C) Protein structure of OTC (PDB: 1OTH) colored by binned median yeast functional assay score.

Figure 4

Effect of amino acid substitutions at functionally important residues (A) The distribution of variant growth scores for residues in OTC motifs involved in substrate binding and catalysis. Each circle is colored according to the amino acid that is substituted as shown (variant). The median growth score for each position is shown as a horizontal gray bar. (B) Protein structures of OTC highlighting the SMG loop at residues 264–276 (green and blue, left and right, respectively). The left structure shows OTC binding CP in orange (PDB: 1FVO). The structure on the right shows OTC binding PALO, an analog of CP-ornithine, with PALO in orange (PDB: 1OTH).

Figure 5

Yeast assay results for amino acid substitutions corresponding to variants with clinical significance calls (A) Strip charts of normalized growth for all amino acid substitutions and those corresponding to ClinVar classification groups. Colored dots correspond to amino acid substitutions in the SMG loop (green) or that correspond to pathogenic/likely pathogenic missense SNVs predicted to impair splicing (pink). (B) Comparison of the proportion of all amino acid substitutions and those with either ClinVar pathogenic/likely pathogenic or benign/likely benign annotations falling under a given level of normalized growth. Dotted lines indicate boundaries of amorphic, hypomorphic, and functionally unimpaired assay ranges.

Figure 6

Clinical stratification of yeast assay results (A) Comparison of the proportion of all amino acid substitutions falling under a given level of normalized growth versus those associated with neonatal, female, or late presentation. Table insert gives the proportion of each class falling in each growth range. Blue shading indicates enrichment and depletion relative to the whole population. (B) Strip charts of normalized growth for all amino acid substitutions and those corresponding to disease presentation groups. Colored dots corresponding to amino acid substitutions in the SMG loop (green) or to missense SNVs predicted to impair splicing (pink) are shown.

Figure 7

Age of onset in late-onset males (A) Cumulative frequency versus age of onset/diagnosis for symptomatic, late-onset males. (B) Age of diagnosis/onset among late-onset males with missense SNVs and yeast growth scores for the matching amino acid substitutions. Substitutions corresponding to missense SNVs predicted to impair splicing or that are present in the SMG loop were omitted from this plot.