Characterizing common loss-of-function genes and their potential utility in assessing population variability and chemical susceptibility in vitro

Identification of the genes and variants underlying inter-individual and population variability in susceptibility to chemical exposures has been challenging due to complex real-world exposures, large sample size requirements for association studies, and limitations in the translation of model system genetic variability to people. These constraints have precluded comprehensive genome wide consideration of the human genetic variants that could play a significant role in population level differences in genetic susceptibility. And thus, most of identified variants which play a demonstrated role in human chemical toxicity were identified in candidate gene studies. As an alternative to current genetic association approaches, we suggest a functional approach to identify the genes in which common human variants could underlie genetic variability in toxicant response. As a first step, we focus on the genes with the most common aggregate predicted loss-of-function (pLoF) mutation alleles in human genome as of gnomAD v3.0 which we designated as the PopVarLoF ( Pop ulation Var iant L oss o f F unction) set. Unexpectedly, enrichment analysis of these genes found significant overrepresentation of gene products playing important known functional roles in toxicology. Interrogation of GWAS and PheWAS databases found that these genes are associated with diverse metabolic phenotypes consistent with the relevance of the PopVarLoF set in studying variability of toxicant response in human populations. We further characterized the PopVarLoF set by developing custom lentiviral CRISPR knockout libraries targeting the PopVarLoF genes to assess their functional essentiality in the HepG2/C3A cell line. Functional disruption of only 14 of the PopVarLoF genes (∼1%) without toxicant exposure resulted in significant growth defects in this cell line, consistent with the majority of PopVarLoF gene products having non-essential roles, which also supports its potential application for toxicant susceptibility screening. Incorporation of empirical assessment of common population level significant genetic sources of population variability in susceptibility to chemical exposure into new approach methodologies (NAMs) such as human cell-based in vitro models of cellular apical endpoint of toxicological relevance could contribute to more robust risk assessment which protects vulnerable populations while reducing uncertainty.