Functional Characterization of Clinically-Relevant Rare Variants in ABCG2 Identified in a Gout and Hyperuricemia Cohort

Abstract

ATP-binding cassette subfamily G member 2 (ABCG2) is a physiologically important urate transporter. Accumulating evidence demonstrates that congenital dysfunction of ABCG2 is an important genetic risk factor in gout and hyperuricemia; recent studies suggest the clinical significance of both common and rare variants of ABCG2. However, the effects of rare variants of ABCG2 on the risk of such diseases are not fully understood. Here, using a cohort of 250 Czech individuals of European descent (68 primary hyperuricemia patients and 182 primary gout patients), we examined exonic non-synonymous variants of ABCG2. Based on the results of direct sequencing and database information, we experimentally characterized nine rare variants of ABCG2: R147W (rs372192400), T153M (rs753759474), F373C (rs752626614), T421A (rs199854112), T434M (rs769734146), S476P (not annotated), S572R (rs200894058), D620N (rs34783571), and a three-base deletion K360del (rs750972998). Functional analyses of these rare variants revealed a deficiency in the plasma membrane localization of R147W and S572R, lower levels of cellular proteins of T153M and F373C, and null urate uptake function of T434M and S476P. Accordingly, we newly identified six rare variants of ABCG2 that showed lower or null function. Our findings contribute to deepening the understanding of ABCG2-related gout/hyperuricemia risk and the biochemical characteristics of the ABCG2 protein.

Keywords: ABCG2/BCRP; European cohort; WGA.; common disease; exon sequence; functional study; gout susceptibility; heritability of serum uric acid; multiple rare variant; urate transporter.

Figure 1

Family history of gout and the numbers of allelic variants in ABCG2. Depending on the presence or absence of family gout history, proportion of gout patients with or without any of the 11 non-synonymous alleles identified in ABCG2 is summarized. The presence of ABCG2 allelic variants was associated with the gout family history (odds ratio = 1.91, 95% CI: 1.10, 3.34; P = 0.0176, Fisher’s exact test).

Figure 2

Schematic illustration of a putative topological model of human ABCG2 protein. Red box, rare variants analyzed in the present study; Black box, common variants. Helices in the transmembrane domain are numbered (1a to 6b) according to a previous study [34]. Asn596 is an N-linked glycosylation site. Unique motifs common to ABC proteins: Walker A (amino acids 80–86), Walker B (amino acids 205–210), and signature C (amino acids 186–200) in ABCG2 protein are indicated by colors.

Figure 3

Effects of each mutation on the maturation status and protein levels of ABCG2 in transiently transfected 293A cells. (A) Immunoblot detection of ABCG2 wild-type (WT) and its variants in the whole cell lysate samples that were prepared 48 h after the transfection. Arrowhead, matured ABCG2 as a glycoprotein; α-Tubulin, a loading control. (B) Relative protein levels of ABCG2 WT and its variants. The signal intensity ratio (ABCG2/α-tubulin) of the immunoreactive bands was determined and normalized to that in ABCG2 WT-expressing cells. Data are expressed as the mean ± SD. n = 3.

Figure 4

Effects of each mutation on the cellular localization of ABCG2 protein in transiently transfected 293A cells. (A) Intracellular localization of ABCG2 variants. Q126X (a stop gain variant that is deficient in the plasma membrane localization [22]) and Q141K are controls. (B) High magnification images of cells transfected with R147W and S572R variants indicate that these mutations impaired localization to the plasma membrane of the cells. Framed areas in the panels of top lane were observed under a higher magnification. Confocal microscopic images were obtained 48 h after the transfection. Nuclei were stained with TO-PRO-3 iodide (gray). Plasma membrane (PM) was labeled with Alexa Fluor^® 594-conjugated wheat germ agglutinin (WGA) (red). Bars indicate 5 μm.

Figure 5

Functional validation of each ABCG2 variant as ATP-dependent urate transporters. (A) Immunoblot detection of ABCG2 WT and its variants expressed in plasma membrane vesicles prepared from 293A cells. Na⁺/K⁺ ATPase, a loading control. (B) ATP-dependent transport of urate by ABCG2 WT and its variants. The data are shown as % of WT; data are expressed as the mean ± SD. n = 3. Statistical analyses for significant differences were performed using Bartlett’s test, followed by a Dunnett’s test (*, P < 0.05; **, P < 0.01 vs WT).

Figure 6

ABCG2 amino acids evolutionary conserved among seven mammalian species. The positions of non-synonymous substitutions conserved among seven species examined in the present study are grey labelled. Regarding Abcg2 protein in each species, NCBI Reference Sequence ID and amino acid sequence identity (vs human ABCG2, NM_004827.3) are summarized as below: Pan troglodytes (Chimpanzee, GABE01009237.1), 99%; Macaca mulatta (Rhesus macaque, NM_001032919.1, 96%; Sus scrofa (Pig, NM_214010.1), 84%; Bos taurus (Bovine, NM_001037478.3), 84%; Rattus novergicus (Rat, NM_181381.2), 81%; Mus musculus (Mouse, NM_011920.3), 81%. Multiple sequence alignments and homology calculations were carried out using the GENETYX software (GENETYX Co., Tokyo, Japan) with the ClustalW2.1 Windows program according to our previous study [27].