Identification of a urate transporter, ABCG2, with a common functional polymorphism causing gout

Abstract

Genome-wide association studies (GWAS) have successfully identified common single nucleotide polymorphisms (SNPs) associated with a wide variety of complex diseases, but do not address gene function or establish causality of disease-associated SNPs. We recently used GWAS to identify SNPs in a genomic region on chromosome 4 that associate with serum urate levels and gout, a consequence of elevated urate levels. Here we show using functional assays that human ATP-binding cassette, subfamily G, 2 (ABCG2), encoded by the ABCG2 gene contained in this region, is a hitherto unknown urate efflux transporter. We further show that native ABCG2 is located in the brush border membrane of kidney proximal tubule cells, where it mediates renal urate secretion. Introduction of the mutation Q141K encoded by the common SNP rs2231142 by site-directed mutagenesis resulted in 53% reduced urate transport rates compared to wild-type ABCG2 (P < 0.001). Data from a population-based study of 14,783 individuals support rs2231142 as the causal variant in the region and show highly significant associations with urate levels [whites: P = 10(-30), minor allele frequency (MAF) 0.11; blacks P = 10(-4), MAF 0.03] and gout (adjusted odds ratio 1.68 per risk allele, both races). Our data indicate that at least 10% of all gout cases in whites are attributable to this causal variant. With approximately 3 million US individuals suffering from often insufficiently treated gout, ABCG2 represents an attractive drug target. Our study completes the chain of evidence from association to causation and supports the common disease-common variant hypothesis in the etiology of gout.

Fig. 1.

ABCG2 is a urate transporter. (A) C-14 urate accumulation data from Xenopus oocytes injected with either H₂O or mRNA coding for MRP4 or ABCG2. (B) Urate accumulation in oocytes injected with H₂O, ABCG2 WT mRNA (incubated with or without 5 μM FTC), or the nonfunctional ABCG2 mutant S187T (N = 10 samples each and n = 20 oocytes each for all accumulation experiments). (C) Urate accumulation is dependent on the extracellular urate concentration (H₂O [blue circles]; S187T [black squares]; WT [red triangles]; N = 10 and n = 20 each). (D) Urate efflux in oocytes incubated overnight in 500 μM C-14 urate as relative efflux over time (H₂O [blue circles]; ABCG2 [red triangles]; N = 5 and n = 50 each). (E) Urate efflux depends on the intracellular urate concentration (H₂O [blue circles]: slope = 0.01, N = 55; ABCG2 [red triangles]: slope = 0.03, N = 55; P < 0.001). (F) FTC (5 μM) inhibits urate export in native LLC-PK₁ renal proximal tubule cells as shown by increased urate accumulation in FTC-treated cells compared to non-treated control cells (N = 6). (G) LLC-PK₁ cells express endogenous ABCG2 at the apical brush border membrane. Merged Z sections of LLC-PK₁ cells stained with BCRP1 antibody (green) and nuclear DAPI stain (blue; scale bar, 5 μm). Mean ± SEM; **, P < 0.001; *, P < 0.01.

Fig. 2.

The ABCG2 Q141K mutation results in reduced urate transport. (A) Across-species comparison of the ABCG2 protein sequence. Sequence is also compared with the cystic fibrosis transmembrane conductance regulator (CFTR). Note the close proximity of Q141 in ABCG2 to the mutational hot spot F508 in CFTR. (B) Western blot of oocytes expressing ABCG2 WT or Q141K (monomers and dimers of ABCG2 are detected). Actin was used as a loading control. (C) Accumulation rates in oocytes expressing ABCG2 WT or Q141K normalized to ABCG2 expression level. (D) Efflux rates for ABCG2 WT and Q141K. (WT [red triangles]: slope = 0.01, N = 55; Q141K [black squares]: slope = 0.02, N = 55). Mean ± SEM; **, P < 0.001.

Fig. 3.

Analysis of the ABCG2 locus and rs2231142 in population-based samples. (A) Association of urate levels and 602 SNPs in the ABCG2 region. Color legend: rs2231142 (blue), other SNPs coded by LD with rs2231142 based on HapMap CEU: red (r² with rs2231142 0.8–1.0), orange (r² = 0.5–0.8), yellow (r² = 0.2–0.5), and white (r²<0.2). Gene annotations are based on Build 36.1, and arrows correspond to direction of transcription and gene size. (B) Analyses as in A, conditional on genotype at rs2231142. (C) Mean serum urate levels by genotype at rs2231142 in white and black participants. *, P trend = 2 × 10⁻¹⁷, **, P trend = 0.015. (D) Prevalence of gout by genotype at rs2231142. *, P trend = 4 × 10⁻⁶, **, P trend = 0.04. Gout cases/overall sample in whites: GG (327/6792), GT (118/1601), TT (10/96); blacks: GG (187/2198), GT/TT (20/152).

Fig. 4.

Model of urate handling by human renal proximal tubule cells. The physiological relevance of urate transporters (named by their gene symbols) in humans is established by genetic variation causing hyper-/hypouricemia and gout for URAT1 (23) and SLC2A9 (, –28). Transporters expressed in human kidney and shown to transport urate in model systems: ¹OAT4; ²OAT1, OAT3; ³MRP4; ⁴OAT1, OAT3 (1, 29, 30). Arrows indicate the direction of urate transport (gray, reabsorption; black, secretion). Abbreviations: U⁻, urate.