Landscapes of missense variant impact for human superoxide dismutase 1

Abstract

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease for which important subtypes are caused by variation in the Superoxide Dismutase 1 gene SOD1. Diagnosis based on SOD1 sequencing can not only be definitive but also indicate specific therapies available for SOD1-associated ALS (SOD1-ALS). Unfortunately, SOD1-ALS diagnosis is limited by the fact that a substantial fraction (currently 26%) of ClinVar SOD1 missense variants are classified as "variants of uncertain significance" (VUS). Although functional assays can provide strong evidence for clinical variant interpretation, SOD1 assay validation is challenging, given the current incomplete and controversial understanding of SOD1-ALS disease mechanism. Using saturation mutagenesis and multiplexed cell-based assays, we measured the functional impact of over two thousand SOD1 amino acid substitutions on both enzymatic function and protein abundance. The resulting 'missense variant effect maps' not only reflect prior biochemical knowledge of SOD1 but also provide sequence-structure-function insights. Importantly, our variant abundance assay can discriminate pathogenic missense variation and provides new evidence for 41% of missense variants that had been previously reported as VUS, offering the potential to identify additional patients who would benefit from therapy approved for SOD1-ALS.

Figure 1:. Generation of SOD1 missense variant effect maps.

(A) Overview of process to generate variant effect maps. (B) SOD1 variant effect maps measuring total enzymatic activity (top) and abundance (bottom). Box color either indicates the WT residue (yellow); a substitution with damaging (blue), tolerated (white), or above-WT (“hyper”; red) functional score; or missing data (gray). Consensus tracks summarize the map scores by position. β-strands are indicated by arrows, and alpha helices by loops.

Figure 2.. Modeling the effects of SOD1 missense variants on protein structure

(A) Distributions of median functional scores (where each median is of substitutions at each residue position) are shown for total enzymatic activity (left) and abundance (right) maps, summarizing sets of positions where the wild type residue is “core” (below 20% accessible surface area (ASA), “metal-binding”, “surface” (above 35% ASA), and “interface” (at the homodimerization interface). Boxes correspond to interquartile range, and bold bars indicate medians of the positional medians. Whiskers correspond to minima and maxima. P-values were calculated by Mann-Whitney U test. (B) Structural model of SOD1; colored according to the median functional score of substitutions at each position. (C) Venn diagram indicating whether amino acids found at SOD1 interfaces (either the homodimeric SOD1 interface or the SOD1-CCS interface with and without Zn2+-bound to SOD1) show for in each map whether the variant was tolerated (white) or intolerant (blue) to substitutions (see Methods for details on determination of threshold scores)

Figure 3.. Modeling effects of SOD1 missense variants on protein stability and structure

(A) For the subset of SOD1 missense variants with no predicted detrimental impact on stability (median predicted ΔΔG < −0.1), median total enzymatic activity score is plotted. Filled circles indicate residues: a) at the SOD1-CCS interface (orange); b) residues proximal to the active site (‘second-shell residues’; teal); c) at the SOD1 homodimeric interface (black). (B) To illustrate the structural impact of variants Asn87Lys and Gly128Pro on the metal-binding site, the average distance (Å) between the Cα atoms of residue pairs 125 (electrostatic loop) and 87 (βstrand residue proximal to the electrostatic loop) is shown in purple, along with the fraction of simulation time (blue) in which hydrogen bonds occurred between metal-binding residue pairs Asp125-His47 and Asp125-His72. (C) Mean-square fluctuation (MSF) of Cɑ atoms of WT SOD1, as well as Asn87Lys and Gly128Pro variants. MSF reflects the average deviation of atoms throughout the simulation (400 ns) relative to the initial structure.

Figure 4.. Genotype-phenotype analyses of SOD1 variants in both assays against a dataset of various phenotypes.

Scores for both activity and abundance maps (mean score for substitutions at each position) were compared with an assembled dataset of patient phenotypes including (A) Mouse protein half-life measurements, (B) Patient enzyme specific activity, (C) Patient protein abundance measurements, (D) Age of ALS onset, (E) Disease duration. NS = not significant. Grey indicates 95% confidence interval for significant correlations. See methods for details on regression and significance calculations.

Figure 5.. The SOD1 abundance map best distinguishes positive from negative reference variants, and provides evidence for 41% of VUS.

Evaluation of precision (fraction of variants in the positive reference set which scored below each threshold functional score) vs. recall (fraction of positive reference variants with functional scores below threshold) shown in (A). More specifically, we used “balanced precision” such that precision values reflect performance in a setting where positive and negative sets contain the same number of variants for reference sets with positive variants obtained from Labcorp Genetics. Balanced precision vs. recall curves are shown for the SOD1 total enzymatic activity (purple) and abundance maps (black), as well as for the best-performing computational predictor CPT (turquoise). (Performance for three additional predictors is shown in Figure S13.) Performance was summarized in terms of area under the balanced precision vs recall curve (AUBPRC) and recall at a balanced precision of 90% (R90BP). Plots indicate the number of variants in the positive (P/LP) and gnomAD negative (PB) reference sets used here (see Methods for details). Currently clinically annotated variants and their annotations are shown in (B). New evidence proposed based on calibrating our abundance map scores against the ClinVar or Labcorp Genetics reference sets is shown in the right column. (See main text and Methods for details on the derivation of evidence strength).