Objectives Molecular biological approaches have recently identified urate transporters in renal proximal tubular cells. Human sodium-dependent phosphate cotransporter type 1 encoded by SLC17A1 is a urate transporter localised to the renal proximal tubular cells and candidate molecule to secret urate from renal tubular cells to urine. This study investigated the roles of SLC17A1 in the development of gout.
Patients and Methods Single nucleotide polymorphisms in the human SLC17A1 gene (rs1165176, rs1165151, rs1165153, rs1165196, rs1165209, rs1165215, rs1179086, rs3799344 and rs3757131) were selected, and an association study was conducted using male patients with gout (n=175) and male controls (n=595).
Results There were significant differences between gout and control groups in the distribution of genotypes at rs1165196 (T806C; Ile269Thr, odds ratio (OR) 0.55, p=0.0035), rs1179086 (OR 0.57, p=0.0018) and rs3757131 (OR 0.54, p=0.0026). In controls, T806C alone had no effect on serum uric acid (sUA) levels. However, T806C showed significant interaction with a reduction of sUA in obese individuals (body mass index ≥25) using multiple regression analysis.
Conclusions Our data suggest that SLC17A1 polymorphisms are associated with the development of gout.
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Hyperuricaemia is caused by either overproduction or renal underexcretion of urate. In the nephron, after filtration by the glomerulus, urates are reabsorbed and secreted in the proximal tubules and 10% of glomerular filtrate is excreted in the urine. Recent investigations suggest that renal urate underexcretion is the major mechanism of hyperuricaemia in the majority of patients with primary gout.1 In 2002, Enomoto et al2 reported the identification of the urate transporter, URAT1 encoded by SLC22A12, localised on the apical side of the proximal tubule in the human kidney. URAT1 is highly specific for urate and has been shown to exchange urate with these endogenous and exogenous anions. It has been reported that W258X in SLC22A12 lowers serum uric acid (sUA) levels and diminishes the risk of gout in Japanese men,3 suggesting the roles of urate transporters in the development of gout and hyperuricaemia.
Human sodium-dependent phosphate cotransporter type 1 (NPT1), a voltage-driven organic anion transporter encoded by SLC17A1, has been reported to localise to the apical membrane of the proximal tubule in the human kidney. It has been shown that NPT1 mediates the transport of p-aminohippuric acid (PAH) and also accepts uric acid as one of the substrates.4 Therefore, NPT1 is suggested to be responsible for tubular urate secretion in human kidney.
In the present study, we examined the roles of single nucleotide polymorphisms (SNP) in SLC17A1 in the development of primary gout in Japanese men.
Patients and methods
One hundred and eighty-one Japanese male patients with primary gout in the outpatient clinic at the Institute of Rheumatology, Tokyo Women's Medical University, were randomly selected for the study when they showed renal underexcretion of uric acid without significant renal functional impairment. The diagnosis of acute gouty arthritis was based on the preliminary classification criteria described by Wallace et al.5 The control group consisted of 595 healthy Japanese male subjects who registered for our previous study6 and gave consent that their samples could be used for other studies.
At the first visit, all patients underwent blood tests and renal function tests with 24-h urine collection without diet. All medications that might affect uric acid metabolism had been withdrawn at least 2 weeks before the 24-h urine collection, and creatinine clearance (CCR), uric acid clearance (CUA) and urinary output of uric acid were calculated. Fractional excretion of uric acid was determined as FEua=CUA/CCR×100, and expressed as a percentage. Underexcretion of uric acid was defined as the value of FEua<5.5%.
This research was reviewed and approved by the Hospital Ethics Committee for Human Genome Researches and all subjects participating in the study gave informed consent.
Genetic analysis of SCL17A1
Genomic DNA was prepared from peripheral blood using a genomic DNA isolation kit (Qiagen, Valencia, California, USA). The following nine SNP in the SCL17A were selected: rs1165176, rs1165151, rs1165153, rs1165196, rs1165209, rs1165215, rs1179086, rs3799344 and rs3757131. Of those, rs1165196 is a non-synonymous SNP at exon 7 and T to C changes at nucleotide 806 results in an Ile to Thr substitution. A detailed summary of nine SNP is displayed in supplemental table 1 (available online only).
Genotyping was done using TaqMan SNP genotyping assays (Applied Biosystems, Foster City, California, USA). Thermal cycling was performed on a GeneAmp 9700 PCR system (Applied Biosystems). The amplification mixture contained 7.5 µl TaqMan universal PCR master mix (Applied Biosystems), 0.375 µl primer–probe mixture, 6.375 µl RNase-DNase-free water (Sigma Chemical Co, St Louis, Missouri, USA) and 2 ng sample DNA, in a total volume of 15 µl per single tube reaction in 368-well microtitre plates. Assay conditions followed the manufacturers' instructions.
D′ and r2, linkage disequilibrium measures, were calculated using the LDSUPPORT.7 In this study, the haplotype blocks were defined as the region whose SNP had an r2 value of more than 0.5. The comparisons between the gout and control groups were performed by χ2 test for categorical variables and by the Mann–Whitney U test for continuous variables. Odds ratios and 95% CI were calculated when possible. We used a Bonferroni correction of p<0.00625, equivalent to a 0.05 significance, to correct for multiple testing, assuming independent tests of four SNP in tables 1 and 2.
Multiple regression was conducted to evaluate the interaction between sUA and various factors such as body mass index (BMI), serum triglycerides and T806C in controls. The significance level was set at 0.05.
Demographic data of the study population are shown in supplemental table 2 (available online only). DNA samples from 175 of 181 gout patients were successfully amplified and were subjected to genotyping. Allele frequencies for each SNP were in Hardy–Weinberg equilibrium in both the patients and the controls (data not shown). Linkage disequilibrium patterns in SCL17A1 were illustrated by their D′ and r2 values in control groups (supplemental figure 1, available online only). The r2 values indicate that the six SNP (rs1165176, rs1165151, rs1165153, rs1165196, rs1165209 and rs1165215) are located in the same haplotype block. Based on that observation, we selected rs1165196 (T806C) as a tag SNP in this block and the following studies were conducted using rs1165196, rs1179086, rs799344 and rs3757131. Table 1 shows the distribution of genotypic and allelic frequencies of the four SNP in each group. Significant associations between gout and three SNP (rs1165196, rs1179086 and rs3757131) were indicated. Then, we investigated the effect of the SNP on sUA levels in controls. However, no association was found between each SNP and sUA levels (table 2). Some medical factors such as obesity and dyslipidaemia have been indicated to be associated with sUA levels. Therefore, we conducted a multiple regression analysis to examine the interaction between sUA and SLC17A1 polymorphisms together with age, BMI and serum triglyceride levels. BMI (≥25 vs <25) and serum triglycerides were significantly associated with sUA, whereas the genotype status of four SNP (rs1165196, rs1179086, rs799344 and rs3757131) showed no significant association. However, in subjects with a BMI of 25 or greater, the CC genotype at rs1165196 (T806C) was significantly associated with a reduction in sUA (table 3). SNP other than T806C showed no interaction with age, BMI or serum triglycerides.
Recent genome-wide association studies identified substantial associations between SLC2A9,8,–,11 ABCG212 and SLC17A312 and hyperuricemia or gout. In the present study, it was suggested that SLC17A1 is one of the genetic factors for the development of gout. Each polymorphism in SLC17A1 did not show any association with sUA levels in controls. However, the CC genotype at T806C was significantly associated with a reduction in sUA in individuals with a BMI greater than 25. In Japan, obesity is defined as a BMI greater than 25.13 Therefore, the T allele at T806C may play a role in the increase in sUA in obese individuals. This result is interesting because the TT genotype and the T allele at T806C were associated with the development of gout (table 1), and obesity is a known risk factor for the development of gout.14 T806C may thus be associated with the development of gout through the effect on sUA in obese individuals. Interestingly, Dehghan et al12 have reported the association between rs1165196 (T806C) and sUA using a genome-wide association study. This study confirmed the result in Japanese individuals.
There is an increasing interest in the association between hyperuricaemia and the metabolic syndrome. Obesity is one of the most important conditions associated with the metabolic syndrome. Diminished uric acid excretion is reported in the metabolic syndrome and is mediated by proximal tubular sodium reabsorption enhanced by hyperinsulinaemia.15 Taken together, it is suggested that hyperinsulinaemia in obese individuals influences the function of NPT1, leading to reduced uric acid excretion. The precise mechanisms at the molecular level remain to be clarified. The T806C polymorphism leads to an Ile to Thr substitution at 269 amino acid. The analysis of urate transport activity in oocytes injected with mutant complementary RNA of T806C will reveal the effect of this substitution.
We previously reported that W258X in SLC22A12 (URAT1) was a suppressing factor for the development of gout.3 In this study, there was no interaction between the W258X polymorphism and the T806C polymorphism in SLC17A1 (data not shown).
The present study indicated that SLC17A1 polymorphisms including T806C were associated with the development of gout in Japanese men. The effect of T806C on sUA may be enhanced in obese individuals. Further studies at the molecular level will be needed to confirm the association.
Funding This work was supported by Health and Labour Sciences Research grants for Research on Human Genome Tailor Made and a grant for the Research for the Future programme from the Japan Society for the Promotion of Science.
Competing interests None.
Patient consent Obtained.
Ethics approval This study was conducted with the approval of the Hospital Ethics Committee for Human Genome Researches of Tokyo Women's Medical University.
Provenance and peer review Not commissioned; externally peer reviewed.
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