A non-coding genetic variant maximally associated with serum urate levels is functionally linked to HNF4A-dependent PDZK1 expression.

Abstract

The precise molecular mechanisms by which urate-associated genetic variants affect urate levels are unknown. Here, we tested for functional linkage of the maximally associated genetic variant rs1967017 at the PDZK1 locus to elevated PDZK1 expression. We performed expression quantitative trait loci (eQTL) and likelihood analyses and gene expression assays. Zebrafish were used to evaluate tissue-specific gene expression. Luciferase assays in HEK293 and HepG2 cells measured the effect of rs1967017 on transcription amplitude. Probabilistic Annotation Integrator analysis revealed rs1967017 as most likely to be causal and rs1967017 was an eQTL for PDZK1 in the intestine. The region harboring rs1967017 was capable of directly driving green fluorescent protein expression in the kidney, liver and intestine of zebrafish embryos, consistent with a conserved ability to confer tissue-specific expression. Small interfering RNA depletion of HNF4A reduced endogenous PDZK1 expression in HepG2 cells. Luciferase assays showed that the T allele of rs1967017 gains enhancer activity relative to the urate-decreasing C allele, with T allele enhancer activity abrogated by HNF4A depletion. HNF4A physically binds the rs1967017 region, suggesting direct transcriptional regulation of PDZK1 by HNF4A. Computational prediction of increased motif strength, together with our functional assays, suggests that the urate-increasing T allele of rs1967017 strengthens a binding site for the transcription factor HNF4A. Our and other data predict that the urate-raising T allele of rs1967017 enhances HNF4A binding to the PDZK1 promoter, thereby increasing PDZK1 expression. As PDZK1 is a scaffold protein for many ion channel transporters, increased expression can be predicted to increase activity of urate transporters and alter excretion of urate.