It is well known that the mean serum uric acid (SUA) is lower in females than in males. In addition, it is fully documented that age, menopause, food consumption, alcohol intake, obesity, a sedentary lifestyle, dyslipidemia, insulin resistance, blood pressure, renal function, and drug use for hypertension were associated with SUA levels [1–6]. Meanwhile, recent studies have elucidated that genotypes are also influential factors of SUA.

SUA is reabsorbed in renal tubules through uric acid transporter 1 (URAT1) encoded by SLC22A12 in chromosome 11q13 [7, 8]. SLC22A12 was documented to have functional polymorphisms, among which W258X was found among those with renal hypouricemia [9–11]. However, the distribution of SUA among those with 258X allele was not reported in a general population. This study aimed to examine SUA distribution according to SLC22A12 W258X genotye, elucidating the overall effect of SLC22A12 W258X on SUA among Japanese.

This study was approved by the Ethics Committee of Nagoya University School of Medicine (approval number 288). Subjects who gave written informed consent to participate in the study were enrolled.

Genotyping

DNA was extracted from buffy coat conserved at -80°C using a BioRobot^® M48 (QIAGEN Group, Tokyo). SLC22A12 W258X polymorphism was genotyped by a polymerase chain reaction with confronting two-pair primers (PCR-CTPP) [14]. Each 25 μl reaction tube contained 30-80 ng DNA, 0.12 mM dNTP, 12.5 pmol of each primer, 0.5 U AmpliTaq Gold (Perkin-Elmer, Foster City, CA) and 2.5 μl of 10x PCR buffer including 15 mM MgCl₂. The PCR-CTPP was conducted with initial denaturation at 95°C for 10 minutes, 35 cycles of denaturation at 95°C for 1 minute, annealing at 62°C for 1 minute, and extension at 72°C for 1 minute, and a final extension at 72°C for 5 minutes. The primers were F1: 5'- TCC ATG CAG GCT CCA GG -3', R1: 5'- ACC ACC AGC TGC AGC AGT GTT -3', F2: 5'- TAC GGT GTG CGG GAC TGG -3', and R2: 5'- GGC AGG ATC TCC TCT GAG G -3'. The amplified DNA fragments were 117-base pairs (bp) for the W allele (G allele), 176-bp for the X allele (A allele), and 255-bp for a common band, as demonstrated in Figure 1.

This study demonstrated the SUA distribution according to SLC22A12 W258X genotype in a general Japanese population. The SUA of all five subjects with XX genotype was less than 1 mg/dL, while SUA of those with WX genotype distributed widely; from 0.8 mg/dL to 7.8 mg/dL in males and from 2.0 mg/dL to 6.5 mg/dL in females. The difference in the mean SUA between WX and WW genotypes was 2.26 mg/dL in males and 1.19 mg/dL in females, indicating that the reduction of the mean SUA due to possessing X allele was significantly larger in males than in females. Since the distribution among the males with WX genotype was closer to that among the females with WW genotype (Table 3), the effect of X allele on SUA was similar to the effect of sex difference. BMI did not significantly modify the effect of the X allele on SUA both for males and females. These findings were actually new for the associations between SLC22A12 W258X and SUA.

Among Japanese, the frequency of 258X allele was 0.024 among 1,875 participants of a cohort study [11], 0.023 among 980 controls in a case-control study [15], and 0.025 among 5,165 participants from another cohort study [16], which were quite similar to the estimate in the present study (0.023). Among Koreans, 258X allele was found in 3 of 5 hypouricemia patients [17], and was 1.1% in a general population [18]. Since this allele has not been reported among other ethnic groups to date, the origin was thought to be in East Asia [19].

Several genotypes affecting SUA have been reported to date. ABCG2 in chromosome 4q22 coding ATP-binding cassette subfamily G member 2 has functional polymorphisms, Q126X (rs72552713) and Q141K (rs2231142), with a minor allele frequency of 0.018 and 0.281 in Japanese, respectively [20]. Although the genotypes with reduced function increase the risk of hyperuricemia [20–22], 126X was rare and 141K was less influential on SUA. SLC2A9 in chromosome 4p16-p15.3 coding glucose transporter 9 (GLUT9) was reported to have mutations (R380W and R198C in Japanese [23], L75R in an Israeli-Arab family, and exon 7 deletion in Ashkenazi-Jewish [24]) causing hypouricemia. Their allele frequency was very rare. Common polymorphisms including MTHFR C677T have been reported to have an association with SUA [25, 26], but the impact was limited in comparison with the above genotypes. Accordingly, SLC22A12 258X seemed to be one of the important genetic traits influencing SUA among Japanese.

The present study discovered that the X allele had a significantly larger impact in males than in females. The OR of possessing the X allele was larger in males than in females; 102.5 vs 25.6 for SUA < 3 mg/dL and 58.2 vs 11.9 for SUA < 4 mg/dL. The differences in mean SUA between males and females were 0.20 mg/dL among those with XX genotype, 0.63 mg/dL among those with WX genotype, and 1.71 mg/dL among those with WW genotype. There was no biological explanation for these phenomena.

In the present study, since the subjects with XX genotype were few, the distriubtion of those with XX genotype might not reflect the distribution of the population with XX genotype. Another limitation was that the medication influencing SUA was not taken into account for the genotype frequency according to the SUA level. Since the medication was common for hyperuricemia, but not for hypouricemia, the effect due to the medication might be limited for low SUA.

In conclusion, this study demonstrated the SUA distribution according to SLC22A12 W258X genotype in a large study. The effect of X allele was larger in males than in females. Since the X allele was influential and relatively common among Japanese, the information on the genotype would be useful for the interpretation of individual SUA. Since SUA distributes widely among Japanese with WX genotype, further studies are warranted to elucidate the determinants of the SUA distribution among those with WX genotype, as well as among those with WW genotype.