These results are in contrast to predictions, where SIFT categorized 4 of the PRYSPRY variants as deleterious, PolyPhen-2 predicted three to be possibly damaging and the other algorithms only F446I. Open in a separate window Figure 3. PRYSPRY missense variants maintain antibody binding.(A) Representative Isothermal titration calorimetry (ITC) trace of IgG Fc against WT TRIM21 PRYSPRY fitted to the one set of sites model. (Kneut) and signaling activity (Ksig). Kneut is usually a measure of the efficiency of TRIM21-mediated adenovirus neutralization as defined by the exponential decay constant calculated from Physique 6A. Ksig is usually a measure of signaling ability defined by the fold-change in NF-B reporter activity upon adenovirus contamination in the presence of antibody (Physique 8). elife-48339-supp1.xlsx (12K) DOI:?10.7554/eLife.48339.027 Transparent reporting form. elife-48339-transrepform.docx (247K) DOI:?10.7554/eLife.48339.028 Data Availability StatementAll data generated or analysed during this study are included in the manuscript and supporting files. Previously published data from your 100 Genomes Project (2015; and the Genome Aggregation Datatbase (2016; was used as part of this work. Abstract The genetic basis of most human disease cannot be explained by common variants. One answer to this missing heritability problem may be rare COL24A1 missense variants, which are individually scarce but collectively abundant. However, the phenotypic impact of rare variants is usually under-appreciated as gene function is normally analyzed WZ811 in the context of a single wild-type sequence. Here, we explore the impact of naturally occurring missense variants in the human population around the cytosolic antibody receptor TRIM21, using volunteer cells with variant haplotypes, CRISPR gene editing and functional reconstitution. In combination with data from a panel of computational predictors, the results suggest that protein robustness and purifying selection ensure that function is usually amazingly well-maintained despite coding variance. mutations (Keinan and Clark, 2012) on which selection has not yet acted. Multiple different rare mutations are thought to underlie the genetics of many complex human disorders including schizophrenia, epilepsy, lipid metabolism disorder, and inflammatory disease?(Andrews et al., 2013; McClellan and King, 2010). Estimates from your 1000 Genomes Project suggest that 40% of rare missense mutations are damaging compared to 5% of common variants?(Abecasis et al., 2010). While the introduction of next-generation sequencing (NGS) has made obtaining human WZ811 sequence data straightforward and WZ811 inexpensive, linking genotype to phenotype is usually far less trivial. Sophisticated computational tools have been produced in order to predict the functional impact of missense variants. Early prediction methods typically utilized a combination of sequence conservation and amino-acid properties while newer tools typically employ?ensemble methods that integrate a large number of diverse features using machine learning. Regrettably, these predictions are not usually prognostic of disease severity or end result. A study of the cystic fibrosis gene CFTR found a poor correlation between predicted functional impact and disease?(Dorfman et al., 2010), while in silico classification of rare BRCA1/2 mutations was not predictive of pathogenicity (Ernst et al., 2018). A direct assessment of multiple computational methods, carried out as part of the Crucial Assessment of Genome Interpretation, compared phenotypic predictions with an empirical dataset quantifying the ability of SUMO-conjugating enzyme UBE2I variants to rescue the growth of missense mutations by random mutagenesis into immune genes and measured the impact on lymphocyte subsets in homozygous mice?(Miosge et al., 2015). Strikingly, only 20% of variants predicted by computational methods to be deleterious gave an observable phenotype. The same study found that WZ811 while approximately 50% of missense mutations within the same species were predicted to be functionally impaired, this compared with only 5% of the variants found between-species. This would?suggest that many variants possess near neutral phenotypes not discernible yet sufficiently impactful to undergo purifying selection. Moreover,?it highlights a crucial general question: are predicted deleterious mutations actually often neutral or does phenotypic characterization fail to capture their impact? We decided to address this question by investigating how naturally occurring variants impact the cytosolic antibody receptor TRIM21, using multiple molecular and cellular assays that independently quantify protein stability, function and phenotype. TRIM21 intercepts incoming antibody-coated pathogens during cellular contamination and causes them to be degraded by the proteasome. TRIM21 also activates immune signaling pathways, including NF-B, although this is tightly regulated to prevent inopportune inflammation. These disparate complex functions are achieved using multiple component domains and by recruiting a range of cofactors. We decided the protein stability, ligand binding, viral neutralization, innate immune signaling, protein expression and dominant-negative potential of rare missense variants in TRIM21 identified from your gnomAD consortium and 1000 Genomes Project?(Lek.