Functional validation of GWAS gene candidates for abnormal liver function during zebrafish liver development

Genome-wide association studies (GWAS) have revealed numerous associations between many phenotypes and gene candidates. Frequently, however, further elucidation of gene function has not been achieved. A recent GWAS identified 69 candidate genes associated with liver enzyme concentrations, which are clinical liver disease markers. To investigate their role in liver homeostasis, we narrowed down this list to 12 genes based on zebrafish orthology, zebrafish liver expression, and disease correlation. To assess the function of gene candidates during liver development, we assayed hepatic progenitors at 48 hours post fertilization (hpf) and hepatocytes at 72 hpf using in situ hybridization following morpholino knockdown in zebrafish embryos. Knockdown of three genes (pnpla3, pklr, and mapk10) decreased expression of hepatic progenitor cells, while knockdown of eight genes (pnpla3, cpn1, trib1, fads2, slc2a2, pklr, mapk10, and samm50) decreased cell-specific hepatocyte expression. We then induced liver injury in zebrafish embryos using acetaminophen exposure and observed changes in liver toxicity incidence in morphants. Prioritization of GWAS candidates and morpholino knockdown expedites the study of novel genes impacting liver development and represents a feasible method for initial assessment of candidate genes to instruct further mechanistic analyses. Our analysis can be extended to GWAS for additional disease-associated phenotypes.