Introduction
Regulation of serum urate concentration is central to the development of gout, with renal uric acid excretion a critical checkpoint [
1]. Genome-wide association scans examining the genetic control of serum urate concentrations have identified two renal urate transporters -
SLC2A9 and
ABCG2 - that have a strong effect on gout risk in multiple ethnic groups [
2]. Whilst other loci (
SLC22A11, GCKR, INHBC, SLC17A1, RREB1, PDZK1, SLC16A9, LRRC16A) have been associated with serum urate concentrations at a genome-wide level of significance in genome-wide association scans [
3,
4], only some of them (
SLC22A11, GCKR, INHBC, SLC17A1) were associated with gout at a nominal level of significance (
P < 0.05) in 1,100 cases nested within a large genome-wide association scan population-based cohort [
4]. To understand why some loci do not associate with gout, and to assess the weakly associated loci in clinical gout, it will be necessary to minimize heterogeneity owing to the type of gout (primary or secondary to other causes such as diuretic use) and to test for association in clinically proven cases.
The solute carrier family 17 member 1 (encoded by
SLC17A1), also known as sodium phosphate transport protein 1 (NPT1), is expressed on the apical membrane of renal tubular cells and mediates sodium and inorganic phosphate co-transport [
5]. Sodium-dependent transporter 1 has also been identified as a urate transport protein [
6,
7], probably secretory [
7] with the gout-protective allele of I269T [
8] leading to increased sodium-dependent transporter 1 activity [
6] and, presumably, increased secretion of uric acid. Genome-wide association scans have shown that genetic variants associate with serum urate concentration in a Caucasian sample [
3,
4].
SLC17A1 has been associated with gout in a Japanese sample set (I269T (
rs1165196), odds ratio (OR) = 0.55,
P = 0.005) [
8] but with conflicting results in Caucasian sample sets. Marker
rs1165205 in
SLC17A3 was first associated with gout (OR = 0.85,
P = 0.002) [
9]. A later study incorporating the same clinical material with additional cases and controls, however, reported reduced combined evidence for association with gout using a strongly correlated marker within
SLC17A1 (
rs1165196, r2 = 0.96; OR = 0.89,
P = 0.013) [
4] - in this study the markers most strongly associated with serum urate were within
SLC17A1 (
rs1165196 and other tightly correlated markers), suggesting that this gene was more likely than
SLC17A3 to harbor an etiological variant. A separate study reported no evidence in Caucasian for association with gout (
rs1183201, r2 with
rs1165196 = 0.87, OR = 0.97,
P = 0.68) [
10]. This equivocal evidence for association with gout in a Caucasian population is notable given the genome-wide evidence for association with serum urate concentration [
4]. Both studies had adequate power to detect association of a moderate effect size, but neither study used clinical criteria to define gout.
Here, we aimed to test the SLC17A1 locus for association with gout, in multiple ancestral groups, using cases defined as a diagnosis of gout by the 1977 American College of Rheumatology (ARA) clinical criteria. The variants tested were
rs1183201, demonstrated to influence serum urate concentration in Caucasian populations [
3], the maximally gout-associated SNP (
rs1165196 (I269T)) in Japanese [
8], and three other SNPs predicted to tag major variation in Polynesian populations.
Results
Association with gout was observed in the NZ Caucasian sample set for
rs1165196, rs1183201, rs3799344 and
rs12664474 (OR = 0.71 (95% confidence interval (CI) = 0.60 to 0.83),
P = 5.5 × 10
-5; OR = 0.67 (95% CI = 0.57 to 0.79),
P = 3.0 × 10
-6; OR = 0.69 (95% CI = 0.58 to 0.81),
P = 2.8 × 10
-5; and OR = 1.36 (95% CI = 1.12 to 1.66),
P = 1.3 × 10
-3, respectively), but not for
rs9358890 (OR = 1.31 (95% CI = 0.93 to 1.85),
P = 0.17) (Table
1). Given the low LD between
rs12664474 and
rs1183201 in CEU (
r2 = 0.16), suggesting the possibility of an independent effect at
rs12664474, we tested for association of
rs12664474 conditional on genotype at
rs1183201 in the NZ Caucasian samples; there was no evidence for a separate genetic effect on gout risk at
rs12664474 (
P = 0.37). We also tested for conditional associations at
rs1183201 and
rs1165196 (
r2 in controls = 0.90) - there was association at
rs1183201 conditional on genotype at
rs1165196 (
P = 0.007), but not at
rs1165196 when conditioned on genotype at
rs1183201 (
P = 0.14).
Table 1
Association analysis in New Zealand case-control sample sets
rs1165196
| TT | CT | CC | C | TT | CT | CC | C | | | | | |
Caucasian | 167 (0.400) | 201 (0.482) | 49 (0.118) | 0.359 | 389 (0.318) | 590 (0.482) | 246 (0.200) | 0.442 | 1.0 × 10-4 | 5.5 × 10-5 | 0.71 (0.60 to 0.83) | 0.33 | 0.41 |
EP/N | 130 (0.570) | 86 (0.377) | 12 (0.053) | 0.241 | 98 (0.516) | 77 (0.405) | 15 (0.079) | 0.282 | 0.19 | 0.26 | 0.81 (0.60 to 1.11) | 0.65 | 0.98 |
EP/Z | 16 (0.364) | 22 (0.500) | 6 (0.136) | 0.386 | 53 (0.358) | 64 (0.432) | 31 (0.209) | 0.426 | 0.51 | 0.88 | 0.85 (0.52 to 1.38) | 0.72 | 0.16 |
WP | 138 (0.556) | 99 (0.399) | 11 (0.044) | 0.244 | 69 (0.496) | 54 (0.388) | 16 (0.115) | 0.309 | 0.05 | 0.06 | 0.72 (0.52 to 1.00) | 0.20 | 0.28 |
EP/WP | 10 (0.667) | 5 (0.333) | 0 (0.000) | 0.167 | 6 (0.316) | 10 (0.526) | 3 (0.158) | 0.421 | 0.02 | 0.07 | 0.28 (0.09 to 0.87) | 0.33 | 0.73 |
Combinedc | | | | | | | | | 5.7 × 10-7 | | 0.72 (0.64 to 0.82) | | |
rs1183201
d
| TT | TA | AA | A | TT | TA | AA | A | | | | | |
Caucasian | 158 (0.384) | 205 (0.499) | 48 (0.117) | 0.366 | 356 (0.291) | 608 (0.496) | 261 (0.213) | 0.461 | 2.0 × 10-6 | 3.0 × 10-6 | 0.67 (0.57 to 0.79) | 0.13 | 0.89 |
EP/N | 130 (0.570) | 86 (0.377) | 12 (0.053) | 0.241 | 96 (0.505) | 78 (0.411) | 16 (0.084) | 0.289 | 0.11 | 0.14 | 0.78 (0.57 to 1.06) | 0.65 | 0.98 |
EP/Z | 15 (0.349) | 21 (0.488) | 7 (0.163) | 0.407 | 52 (0.349) | 61 (0.409) | 36 (0.242) | 0.446 | 0.49 | 0.95 | 0.85 (0.52 to 1.39) | 0.94 | 0.04 |
WP | 132 (0.543) | 101 (0.416) | 10 (0.041) | 0.249 | 68 (0.479) | 57 (0.401) | 17 (0.120) | 0.320 | 0.03 | 0.03 | 0.70 (0.51 to 0.97) | 0.08 | 0.35 |
EP/WP | 9 (0.600) | 6 (0.400) | 0 (0.000) | 0.200 | 7 (0.350) | 10 (0.500) | 3 (0.150) | 0.400 | 0.07 | 0.11 | 0.38 (0.13 to 1.12) | 0.25 | 0.99 |
Combinedc | | | | | | | | | 3.0 × 10-8 | | 0.70 (0.62 to 0.79) | | |
rs9358890
| AA | AG | GG | G | AA | AG | GG | G | | | | | |
Caucasian | 366 (0.884) | 47 (0.114) | 1 (0.002) | 0.059 | 1114 (0.912) | 104 (0.085) | 4 (0.003) | 0.046 | 0.12 | 0.17 | 1.31 (0.93 to 1.85) | 0.69 | 0.35 |
EP/N | 97 (0.418) | 99 (0.427) | 36 (0.155) | 0.369 | 80 (0.419) | 78 (0.408) | 33 (0.173) | 0.377 | 0.80 | 0.71 | 0.96 (0.73 to 1.28) | 0.21 | 0.07 |
EP/Z | 34 (0.753) | 11 (0.244) | 0 (0.000) | 0.122 | 129 (0.827) | 26 (0.167) | 1 (0.006) | 0.090 | 0.44 | 0.42 | 1.41 (0.67 to 2.96) | 0.35 | 0.80 |
WP | 110 (0.440) | 101 (0.404) | 39 (0.156) | 0.358 | 73 (0.507) | 61 (0.424) | 10 (0.069) | 0.281 | 0.03 | 0.06 | 1.43 (1.04 to 1.95) | 0.06 | 0.57 |
EP/WP | 8 (0.533) | 7 (0.467) | 0 (0.000) | 0.233 | 12 (0.571) | 7 (0.333) | 2 (0.095) | 0.262 | 0.78 | 0.87 | 0.86 (0.29 to 2.55) | 0.24 | 0.53 |
Combinedc | | | | | | | | | 0.05 | | 1.19 (1.00 to 1.41) | | |
rs3799344
| CC | CT | TT | T | CC | CT | TT | T | | | | | |
Caucasian | 165 (0.404) | 193 (0.473) | 50 (0.123) | 0.359 | 379 (0.309) | 592 (0.483) | 255 (0.207) | 0.450 | 5.9 × 10-6 | 2.8 × 10-5 | 0.69 (0.58 to 0.81) | 0.58 | 0.38 |
EP/N | 128 (0.561) | 88 (0.386) | 12 (0.053) | 0.246 | 97 (0.524) | 73 (0.395) | 15 (0.081) | 0.278 | 0.46 | 0.37 | 0.84 (0.62 to 1.15) | 0.53 | 0.80 |
EP/Z | 17 (0.378) | 22 (0.489) | 6 (0.133) | 0.378 | 53 (0.353) | 64 (0.427) | 33 (0.220) | 0.433 | 0.35 | 0.72 | 0.79 (0.49 to 1.29) | 0.79 | 0.49 |
WP | 136 (0.551) | 98 (0.397) | 13 (0.053) | 0.251 | 64 (0.457) | 59 (0.421) | 17 (0.121) | 0.332 | 0.02 | 0.02 | 0.67 (0.49 to 0.93) | 0.39 | 0.55 |
EP/WP | 10 (0.667) | 5 (0.333) | 0 (0.000) | 0.167 | 7 (0.350) | 10 (0.500) | 3 (0.150) | 0.400 | 0.04 | 0.08 | 0.30 (0.10 to 0.95) | 0.44 | 0.85 |
Combinedc | | | | | | | | | 7.4 × 10-8 | | 0.71 (0.62 to 0.80) | | |
rs12664474
| AA | AG | GG | G | AA | AG | GG | G | | | | | |
Caucasian | 264 (0.632) | 134 (0.321) | 20 (0.048) | 0.208 | 867 (0.707) | 321 (0.262) | 38 (0.031) | 0.162 | 0.01 | 1.2 × 10-3 | 1.36 (1.12 to 1.66) | 0.57 | 0.22 |
EP/N | 90 (0.383) | 104 (0.443) | 41 (0.174) | 0.396 | 74 (0.392) | 77 (0.407) | 38 (0.201) | 0.405 | 0.79 | 0.17 | 0.96 (0.73 to 1.27) | 0.25 | 0.03 |
EP/Z | 25 (0.543) | 18 (0.391) | 3 (0.065) | 0.261 | 107 (0.704) | 39 (0.257) | 6 (0.039) | 0.168 | 0.05 | 0.13 | 1.75 (1.00 to 3.05) | 0.92 | 0.32 |
WP | 102 (0.411) | 106 (0.427) | 40 (0.161) | 0.375 | 63 (0.444) | 67 (0.472) | 12 (0.085) | 0.320 | 0.13 | 0.10 | 1.27 (0.93 to 1.73) | 0.16 | 0.32 |
EP/WP | 7 (0.467) | 6 (0.400) | 2 (0.133) | 0.333 | 12 (0.571) | 7 (0.333) | 2 (0.095) | 0.262 | 0.51 | 0.38 | 1.41 (0.51 to 3.92) | 0.70 | 0.53 |
Combinedc | | | | | | | | | 2.0 × 10-3 | | 1.25 (1.09 to 1.43) | | |
Table 2
Association of four-marker rs9358890-rs3799344-rs1183201-rs12664474 haplotypes with gout
Caucasian | | | | |
A-T-A-A | 270 (0.339) | 1044 (0.428) | 0.66 (0.56 to 0.78) | 1.5 × 10-6 |
A-C-T-A | 327 (0.410) | 876 (0.360) | 1.22 (1.03 to 1.44) | 0.014 |
A-C-T-G | 121 (0.152) | 284 (0.117) | 1.34 (1.06 to 1.69) | 0.015 |
G-C-T-G | 47 (0.059) | 104 (0.043) | 1.39 (0.98 to 1.98) | 0.067 |
EP/N | | | | |
A-T-A-A | 107 (0.239) | 101 (0.274) | 0.84 (0.61 to 1.15) | 0.27 |
A-C-T-A | 160 (0.356) | 109 (0.297) | 1.33 (0.99 to 1.78) | 0.06 |
G-C-T-G | 161 (0.360) | 140 (0.380) | 0.93 (0.70 to 1.23) | 0.60 |
EP/Z | | | | |
A-T-A-A | 32 (0.372) | 120 (0.414) | 0.84 (0.51 to 1.38) | 0.49 |
A-C-T-A | 32 (0.372) | 111 (0.384) | 0.95 (0.58 to 1.56) | 0.84 |
G-C-T-G | 9 (0.105) | 26 (0.090) | 1.19 (0.53 to 2.64) | 0.68 |
A-C-T-G | 10 (0.116) | 22 (0.075) | 1.63 (0.74 to 3.59) | 0.23 |
WP | | | | |
A-C-T-A | 171 (0.357) | 96 (0.343) | 1.07 (0.78 to 1.45) | 0.70 |
A-T-A-A | 116 (0.241) | 89 (0.318) | 0.68 (0.49 to 0.94) | 0.021 |
G-C-T-G | 171 (0.356) | 77 (0.275) | 1.46 (1.06 to 2.02) | 0.021 |
A-C-T-G | 12 (0.025) | 12 (0.043) | 0.57 (0.25 to 1.29) | 0.17 |
EP/WP | | | | |
A-T-A-A | 5 (0.167) | 16 (0.400) | 0.30 (0.10 to 0.95) | 0.035 |
A-C-T-A | 15 (0.500) | 13 (0.324) | 2.08 (0.78 to 5.50) | 0.14 |
G-C-T-G | 6 (0.200) | 11 (0.275) | 0.66 (0.21 to 2.05) | 0.47 |
The five variants were then tested for association in the Polynesian sample sets (Table
1), with the only evidence for association in individual sample sets coming from WP at
rs1183201 (OR = 0.70,
P = 0.03) and
rs3799344 (OR = 0.67,
P = 0.02). However, meta-analysis of the Polynesian sample sets - carried out to increase power - replicated the association observed in Caucasian at
rs1165196 (OR = 0.75 (95% CI = 0.60 to 0.94),
P = 0.013,
PHet = 0.33),
rs1183201 (OR = 0.74 (95% CI = 0.61 to 0.91),
P = 0.003,
PHet = 0.57) and
rs3799344 (OR = 0.74 (95% CI = 0.61 to 0.90),
P = 0.003,
PHet = 0.33), but not at
rs9358890 (OR = 1.15 (95% CI = 0.95 to 1.40),
P = 0.16,
PHet = 0.28) or
rs12664474 (OR = 1.16 (95% CI = 0.96 to 1.40),
P = 0.13,
PHet = 0.23).
The Caucasian and Polynesian sample sets were combined in meta-analysis for
rs1165196 (OR = 0.72 (95% CI = 0.64 to 0.82),
P = 5.7 × 10
-7),
rs1183201 (OR = 0.70 (95% CI = 0.62 to 0.79),
P = 3.0 × 10
-8,
PHet = 0.64),
rs9358890 (OR = 1.19 (95% CI = 1.00 to 1.41),
P = 0.05,
PHet = 0.37),
rs3799344 (OR = 0.71 (95% CI = 0.62 to 0.80),
P = 7.4 × 10
-8,
PHet = 0.43), and
rs12664474 (OR = 1.25 (95% CI = 1.09 to 1.43),
P = 2.0 × 10
-3,
PHet = 0.23). Of the five SNPs,
rs1183201 was the only one significant at a genome-wide level of significance (
P < 5 × 10
-8). None of the SNPs were significantly associated with serum urate in either the Caucasian controls (for whom there were serum urate data available; see Supplemental Table
1 in Additional file
1) or the less admixed combined WP and EP/N controls (all
P > 0.28).
Because haplotypes are multi-allelic we analyzed association of haplotypes with gout, with the purpose of investigating the mechanism of effect - that is, whether risk and/or protective variants are present and comparing association pattern between populations. Analysis of four-marker haplotypes (
rs9358890-
rs3799344-
rs1183201-
rs12664474; Table
2) revealed the most consistent evidence for association to come from the A-T-A-A haplotype (OR = 0.30 to 0.84), with significant association in the Caucasian, WP and EP/WP sample sets (
P = 1.5 × 10
-6 to 0.035).
Discussion
Genetic regulators of serum urate concentration that have been previously associated with gout at a genome-wide level of significance (
P < 5 × 10
-8) in Caucasian samples are
SLC2A9 [
4,
9,
20] and
ABCG2 [
4,
9,
16]. Here, we provide strong evidence for a role of the
SLC17A1 locus in gout in a Caucasian population (
rs1183201, OR = 0.67,
P = 3.0 × 10
-6; Table
1) that was replicated in Polynesian samples, with the minor allele of
rs1183201 also conferring a similar degree of risk (OR = 0.74,
Pmeta-analysis = 3.0 × 10
-3). The haplotype data (Table
2) are consistent with the presence of at least one genetic variant influencing the risk of gout at the
SLC17A1 locus. We hypothesize that the variant is protective of gout and is contained on a common haplotype (27 to 43%; A-T-A-A), conferring significant protection in three out of the five sample sets (also with OR < 1 in both EP sample sets). There were no haplotypes consistently conferring risk. Combining the populations provided a genome-wide level of significance for association of
rs1183201 with gout (OR = 0.70,
P = 3.0 × 10
-8). This confirms the
SLC17A1 locus as the third associated with gout.
The role of
SLC17A1 has been previously evaluated in gout in a Japanese sample set [
8], with the nonsynonymous variant I269T (
rs1165196) having the strongest evidence for association (OR = 0.55,
P = 0.004, minor allele (269T) protective).
rs1165196 is in strong LD with
rs1183201 -the maximally associated variant in our study - in Japanese (HapMap JPT) and Caucasian (HapMap CEU) samples (
r2 = 0.92 and
r2 = 0.87, respectively). Given that I269T has been shown to affect the function of SLC17A1, with the protective variant (269T, minor allele of
rs1165196) leading to increased activity in
Xenopus oocytes and, presumably, increased renal elimination of urate [
6], it is therefore possible that
rs1165196 is an etiological variant. However, we found no evidence in the Caucasian sample set supporting association at
rs1165196 when conditioned on genotype at
rs1183201, and association was weaker at
rs1165196 than
rs1183201 in combined Caucasian and Polynesian meta-analysis (OR = 0.72,
P = 5.7 × 10
-7 and OR = 0.70,
P = 3 × 10
-8, respectively) and in Polynesian alone (OR = 0.75,
P = 0.013 and OR = 0.74,
P = 0.003, respectively) (we did not conditionally analyze the small Polynesian sample sets). Ostensibly this observation argues that
rs1183201 (or a variant in strong LD) is more likely than
rs1165196 to be an etiological variant within
SLC17A1. Given that
rs1165196 has a stronger effect in serum urate levels in Caucasian ([
4] β = 6.205 vs. 6.050 for
rs1183201) populations, however, this interpretation should await further testing in larger gout and serum urate sample sets.
In the Caucasian analysis,
rs1183201 was strongly associated with gout (OR = 0.67 (95% CI = 0.57 to 0.79)). This SNP, or SNPs in strong LD, has been studied for association with gout in two previous studies: Yang and colleagues [
4], with OR = 0.89 (95% CI = 0.82 to 0.98); and Stark and colleagues [
10], with OR = 0.97 (95% CI = 0.86 to 1.11). The strength of effect in our study is considerably greater than the previous studies, with a 95% CI that does not overlap with either study. Given that the control allele frequencies were similar between our study and those of Yang and colleagues [
4] and Stark and colleagues [
10] (0.461 (
rs1183201), 0.46 (
rs1165196), and 0.487 (
rs1183201), respectively), the differences in effect size are therefore caused by differences in allele frequency in case sample sets. Differences in ascertainment of cases are a possible reason for this effect. Here, cases were clinically ascertained by ARA criteria with exclusion of patients suspected of having diuretic-induced gout. In Yang and colleagues' study, cases were drawn from five population-based cohorts and were ascertained by: self-report or allopurinol treatment (AGES Reykjavik Study); self-report (Atherosclerosis Risk in Communities Study); receiving gout medication (allopurinol, colchicine, probenecid; Cardiovascular Health Study); self-report (Framingham Heart Study); and receiving gout medication (allopurinol, colchicine, probenecid, benzbromarone; Rotterdam Study) [
4]. In Stark and colleagues' study, cases were ascertained by self-report and review of medical history [
10].
In the study by Yang and colleagues no details were included about the inclusion, or otherwise, of diuretic-induced cases [
4]; and in the study by Stark and colleagues 36.1% of cases were taking diuretic medication [
10]. The use of self-reported gout probably results in participants without clinical evidence for gout being included in case sample sets; for example, only 69% of men who self-reported as new cases of gout met the ARA classification criteria for gout [
21], and reliability and sensitivity for self-reported gout have been estimated at 63 to 73% and 84%, respectively [
22]. Although the reliability of use of medications such as allopurinol, colchicine, probenecid and benzbromarone has not been extensively investigated for gout classification, the use of allopurinol prescription gives a positive predictive value of 39% for probable/definite gout [
23]. Certainly, gout case sample sets ascertained using such indirect criteria had lower effect sizes reported at
SLC2A9, compared with sets using ARA criteria [
20]. The method of ascertainment in the previous studies [
4,
10] would thus reduce power to detect association at
SLC17A1 owing to inclusion of nongout participants in the case sample sets. The use of diuretic medications is well established as a gout risk factor [
24], perhaps by inhibition of urate excretion mediated by human organic anion transporter 4 [
25]. In Stark and colleagues' study [
10], this could reduce power to detect association by studying cases with secondary gout, since the inhibitory effect of diuretic medication on organic anion transporter 4-mediated renal urate excretion would predominate over the genetic effect on gout risk mediated by the
SLC17A1 locus. It is also conceivable that diuretics directly influence the function of urate transporters encoded in the locus. The loop diuretic bumetanide has recently been shown to be a transport substrate for sodium-dependent transporter 4 (encoded by
SLC17A3), and functional polymorphic variants are likely to influence transport ability [
26]. Given the likelihood that gene-diuretic interactions exist, one would be prudent to exclude gout cases taking diuretic medication as a potential confounding factor in order to evaluate the direct effect of genetic variation in the
SLC17A1 locus on primary gout.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
JEH-M, AJP-G and TRM helped to design the study, oversee its execution, and prepare the manuscript. GTJ, AvR, PJG, AAH, JH, PBJ, LKS and ND helped to provide clinical recruitment and prepare the manuscript. BC and GWM helped to collect data and prepare the manuscript. All authors read and approved the final manuscript.