Yuzo Takada

Common Defects of ABCG2, a High-Capacity Urate Exporter, Cause Gout: A Function-Based Genetic Analysis in a Japanese Population

Dysfunctional genotype combinations of polymorphic adenosine 5′-triphosphate–binding cassette transporter gene ABCG2/BCRP, which encodes a high-capacity urate secretion transporter in human gut and kidney, are major causes of gout. Gout, the “Disease of Kings” as it is often known, is a painful medical condition characterized by sharp acute pain in bone joints, due to the high deposition of uric acid crystals from the blood serum into the surrounding cartilage. It affects approximately 1% of the U.S. population and remains a significant public health concern. The prevalence of gout is much higher in certain Asian ethnic groups, and is also reportedly rising in African Americans. Current medical treatments are aimed at ameliorating pain severity, but as the underlying genetic etiology of the disease unfolds, new targets for future therapies are likely to be found. Although genome-wide association studies (GWAS) have enabled the calculation of risk predispositions for a wide variety of complex diseases, the relation of gene function to the causality of disease-related mutations has remained largely unclear. A recent U.S. population–based study supported an association between urate levels and gout in individuals carrying variants in a multifunctional transporter gene, ABCG2. This study identified Q141K as a high-risk variant in nearly 10% of gout cases in Caucasians. Now, a team led by Hirotaka Matsuo report that in a Japanese population, another risk variant in ABCG2, namely the Q126X nonfunctional mutation, confers an even higher risk associated with an increase in uric acid deposition in the blood and may cause gout in Asians. Because this gene is responsible for giving rise to a protein that transports harmful waste products and metabolites out of the kidney and gut, they extensively validate the biological activity of ABCG2 using functional assays in vitro that effectively recapitulate human data obtained from Japanese individuals afflicted with the disease. These findings lend weight to previously reported GWAS; moreover, these newly identified specific high-risk variants that block urate secretion may serve as potential intervention points for quelling the disease. Gout based on hyperuricemia is a common disease with a genetic predisposition, which causes acute arthritis. The ABCG2/BCRP gene, located in a gout-susceptibility locus on chromosome 4q, has been identified by recent genome-wide association studies of serum uric acid concentrations and gout. Urate transport assays demonstrated that ABCG2 is a high-capacity urate secretion transporter. Sequencing of the ABCG2 gene in 90 hyperuricemia patients revealed several nonfunctional ABCG2 mutations, including Q126X. Quantitative trait locus analysis of 739 individuals showed that a common dysfunctional variant of ABCG2, Q141K, increases serum uric acid. Q126X is assigned to the different disease haplotype from Q141K and increases gout risk, conferring an odds ratio of 5.97. Furthermore, 10% of gout patients (16 out of 159 cases) had genotype combinations resulting in more than 75% reduction of ABCG2 function (odds ratio, 25.8). Our findings indicate that nonfunctional variants of ABCG2 essentially block gut and renal urate excretion and cause gout.

ABCG2 dysfunction causes hyperuricemia due to both renal urate underexcretion and renal urate overload

Gout is a common disease which results from hyperuricemia. We have reported that the dysfunction of urate exporter ABCG2 is the major cause of renal overload (ROL) hyperuricemia, but its involvement in renal underexcretion (RUE) hyperuricemia, the most prevalent subtype, is not clearly explained so far. In this study, the association analysis with 644 hyperuricemia patients and 1,623 controls in male Japanese revealed that ABCG2 dysfunction significantly increased the risk of RUE hyperuricemia as well as overall and ROL hyperuricemia, according to the severity of impairment. ABCG2 dysfunction caused renal urate underexcretion and induced hyperuricemia even if the renal urate overload was not remarkable. These results show that ABCG2 plays physiologically important roles in both renal and extra-renal urate excretion mechanisms. Our findings indicate the importance of ABCG2 as a promising therapeutic and screening target of hyperuricemia and gout.

Common dysfunctional variants in ABCG2 are a major cause of early-onset gout

Gout is a common disease which mostly occurs after middle age, but more people nowadays develop it before the age of thirty. We investigated whether common dysfunction of ABCG2, a high-capacity urate transporter which regulates serum uric acid levels, causes early-onset gout. 705 Japanese male gout cases with onset age data and 1,887 male controls were genotyped, and the ABCG2 functions which are estimated by its genotype combination were determined. The onset age was 6.5 years earlier with severe ABCG2 dysfunction than with normal ABCG2 function (P = 6.14 × 10−3). Patients with mild to severe ABCG2 dysfunction accounted for 88.2% of early-onset cases (twenties or younger). Severe ABCG2 dysfunction particularly increased the risk of early-onset gout (odds ratio 22.2, P = 4.66 × 10−6). Our finding that common dysfunction of ABCG2 is a major cause of early-onset gout will serve to improve earlier prevention and therapy for high-risk individuals.

Genome-wide association study of clinically defined gout identifies multiple risk loci and its association with clinical subtypes

Objective Gout, caused by hyperuricaemia, is a multifactorial disease. Although genome-wide association studies (GWASs) of gout have been reported, they included self-reported gout cases in which clinical information was insufficient. Therefore, the relationship between genetic variation and clinical subtypes of gout remains unclear. Here, we first performed a GWAS of clinically defined gout cases only. Methods A GWAS was conducted with 945 patients with clinically defined gout and 1213 controls in a Japanese male population, followed by replication study of 1048 clinically defined cases and 1334 controls. Results Five gout susceptibility loci were identified at the genome-wide significance level (p<5.0×10−8), which contained well-known urate transporter genes (ABCG2 and SLC2A9) and additional genes: rs1260326 (p=1.9×10−12; OR=1.36) of GCKR (a gene for glucose and lipid metabolism), rs2188380 (p=1.6×10−23; OR=1.75) of MYL2-CUX2 (genes associated with cholesterol and diabetes mellitus) and rs4073582 (p=6.4×10−9; OR=1.66) of CNIH-2 (a gene for regulation of glutamate signalling). The latter two are identified as novel gout loci. Furthermore, among the identified single-nucleotide polymorphisms (SNPs), we demonstrated that the SNPs of ABCG2 and SLC2A9 were differentially associated with types of gout and clinical parameters underlying specific subtypes (renal underexcretion type and renal overload type). The effect of the risk allele of each SNP on clinical parameters showed significant linear relationships with the ratio of the case–control ORs for two distinct types of gout (r=0.96 [p=4.8×10−4] for urate clearance and r=0.96 [p=5.0×10−4] for urinary urate excretion). Conclusions Our findings provide clues to better understand the pathogenesis of gout and will be useful for development of companion diagnostics.

Identification of ABCG2 Dysfunction as a Major Factor Contributing to Gout

The ATP-binding cassette, subfamily G, member 2 gene ABCG2/BCRP locates in a gout-susceptibility locus (MIM 138900) on chromosome 4q. Recent genome-wide association studies also showed that the ABCG2 gene relates to serum uric acid levels and gout. Since ABCG2 is also known as a transporter of nucleotide analogs that are structurally similar to urate, and is an exporter that has common polymorphic reduced functionality variants, ABCG2 could be a urate secretion transporter and a gene causing gout. To find candidate mutations in ABCG2, we performed a mutation analysis of the ABCG2 gene in 90 Japanese patients with hyperuricemia and found six non-synonymous mutations. Among the variants, ATP-dependent urate transport was reduced or eliminated in five variants, and two out of the five variants (Q126X and Q141K) were frequently detected in patients. Haplotype frequency analysis revealed that there is no simultaneous presence of Q126X and Q141K in one haplotype. As Q126X and Q141K are a nonfunctional and half-functional haplotype, respectively, their genotype combinations are divided into four estimated functional groups. The association study with 161 male gout patients and 865 male controls showed that all of those who had dysfunctional ABCG2 had an increased risk of gout, and that a remarkable risk was observed in those with ≤1/4 function (OR, 25.8; 95% CI, 10.3–64.6; p = 3.39 × 10−21). In 2,150 Japanese individuals, the frequency of those with dysfunctional ABCG2 was more than 50%. Our function-based clinicogenetic analysis identified the combinations of dysfunctional variants of ABCG2 as a major contributing factor in Japanese patients with gout.

ABCG2 Dysfunction Increases the Risk of Renal Overload Hyperuricemia

ATP-binding cassette transporter, sub-family G, member 2 (ABCG2/BCRP) is identified as a high-capacity urate exporter, and its dysfunction has an association with serum uric acid levels and gout/hyperuricemia risk. Generally, hyperuricemia has been classified into urate “overproduction type,” “underexcretion type,” and “combined type” based on only renal urate excretion, without considering an extra-renal pathway such as gut excretion. In this study, we investigated the effects of ABCG2 dysfunction on human urate handling and the mechanism of hyperuricemia. Clinical parameters for urate handling including urinary urate excretion (UUE) were examined in 644 Japanese male outpatients with hyperuricemia. The severity of their ABCG2 dysfunction was estimated by genotype combination of two common ABCG2 variants, nonfunctional Q126X (rs72552713) and half-functional Q141K (rs2231142). Contrary to the general understanding that ABCG2 dysfunction leads to decreased renal urate excretion, UUE was significantly increased by ABCG2 dysfunction (P = 3.60 × 10−10). Mild, moderate, and severe ABCG2 dysfunctions significantly raised the risk of “overproduction” hyperuricemia including overproduction type and combined type, conferring risk ratios of 1.36, 1.66, and 2.35, respectively. The present results suggest that common dysfunctional variants of ABCG2 decrease extra-renal urate excretion including gut excretion and cause hyperuricemia. Thus, “overproduction type” in the current concept of hyperuricemia should be renamed “renal overload type,” which is caused by two different mechanisms, “extra-renal urate underexcretion” and genuine “urate overproduction.” Our new concept will lead to a more accurate diagnosis and more effective therapeutic strategy for hyperuricemia and gout.

Common dysfunctional variants of ABCG2 have stronger impact on hyperuricemia progression than typical environmental risk factors

Gout/hyperuricemia is a common multifactorial disease having typical environmental risks. Recently, common dysfunctional variants of ABCG2, a urate exporter gene also known as BCRP, are revealed to be a major cause of gout/hyperuricemia. Here, we compared the influence of ABCG2 dysfunction on serum uric acid (SUA) levels with other typical risk factors in a cohort of 5,005 Japanese participants. ABCG2 dysfunction was observed in 53.3% of the population investigated, and its population-attributable risk percent (PAR%) for hyperuricemia was 29.2%, much higher than those of the other typical environmental risks, i.e. overweight/obesity (BMI ≥ 25.0; PAR% = 18.7%), heavy drinking (>196 g/week (male) or >98 g/week (female) of pure alcohol; PAR% = 15.4%), and aging (≥60 years old; PAR% = 5.74%). SUA significantly increased as the ABCG2 function decreased (P = 5.99 × 10−19). A regression analysis revealed that ABCG2 dysfunction had a stronger effect than other factors; a 25% decrease in ABCG2 function was equivalent to “an increase of BMI by 1.97-point” or “552.1 g/week alcohol intake as pure ethanol” in terms of ability to increase SUA. Therefore, ABCG2 dysfunction originating from common genetic variants has a much stronger impact on the progression of hyperuricemia than other familiar risks. Our study provides a better understanding of common genetic factors for common diseases.

Significant association of serum uric acid levels with SLC2A9 rs11722228 among a Japanese population

Genome-wide association studies identified that SLC2A9 (GLUT9) gene polymorphisms were associated with serum uric acid (SUA) levels. Among the Japanese, a C/T polymorphism in intron 8 (rs11722228) was reported to be highly significant, though the function and strength of association were unknown. This study aimed to confirm the association, estimating the means of SUA according to the genotype, as well as OR of the genotype. Subjects were 5024 health checkup examinees (3413 males and 1611 females) aged 35 to 69 years with creatinine <2.0 mg/dL. Since SLC22A12 258X allele and ABCG2 126X allele are known to influence SUA levels strongly, the subjects with SLC22A12 258WW and ABCG2 126QQ (3082 males and 1453 females, in total 4535 subjects) were selected. The genotype frequency of SLC2A9 rs11722228 was 2184 for CC, 1947 for CT, and 404 for TT, being in Hardy-Weinberg equilibrium (p=0.312). Mean SUA was 6.10 mg/dL for CC, 6.25 mg/dL for CT, and 6.45 mg/dL for TT among males (p=1.5E-6), and 4.34 mg/dL, 4.59 mg/dL, and 4.87 mg/dL among females (p=4.6E-11), respectively. Males with SUA less than 5.0 mg/dL were 14.7% for CC, 10.6% for CT, and 7.8% for TT (p=2.3E-4), and females with SUA less than 4.0 mg/dL were 34.1%, 25.5%, and 15.4% (p=3.7E-6), respectively. This study was the first report to estimate the impact of SLC2A9 rs11722228 on SUA levels. Since the allele frequency of rs11722228 is similar among different ethnic groups, the impact remains to be examined in other ethnic groups.

Ethnic Differences in ATP-binding Cassette Transporter, Sub-Family G, Member 2 (ABCG2/BCRP): Genotype Combinations and Estimated Functions

ATP-binding cassette transporter, sub-family G, member 2 (ABCG2/BCRP) is a xenobiotic transporter and also regulates serum uric acid levels as a urate transporter. We have shown that the severity of ABCG2 dysfunction can be estimated by simple genotyping of two dysfunctional variants, Q126X (rs72552713) and Q141K (rs2231142). This genotyping method is widely accepted for the risk analysis of hyperuricemia/gout, but there is no report on ethnic differences in ABCG2 dysfunctions. Here, we estimated ABCG2 dysfunctions by its genotype combination (Q126X and Q141K) and compared them in three different ethnic groups (500 Japanese, 200 Caucasians and 100 African-Americans). The minor allele frequencies of Q126X and Q141K in Japanese (0.025 and 0.275, respectively) were significantly higher than those in Caucasians (0.005 and 0.085, respectively) and African-Americans (0 and 0.090, respectively). Additionally, the rates of mild, moderate and severe ABCG2 dysfunctions in Japanese (35.4%, 12.4% and 1.6%, respectively) were higher than those in Caucasians (14.0%, 2.5% and 0%, respectively) and African-Americans (14.0%, 2.0% and 0%, respectively). Because ABCG2 dysfunctional diplotypes were commonly observed in both Caucasians (16.5%) and African-Americans (16.0%), the genotyping of the two ABCG2 dysfunctional variants is useful for evaluating individual differences in the ABCG2 dysfunction which affect the pharmacokinetics of substrate drugs and hyperuricemia risk in all three ethnic groups.

Common Variant of PDZK1, Adaptor Protein Gene of Urate Transporters, is Not Associated with Gout

To the Editor: Gout, a multifactorial disease characterized by acute inflammatory arthritis, is caused as a consequence of hyperuricemia. Previous genetic studies have revealed that gout and serum uric acid (SUA) levels have associations with various genes such as ATP-binding cassette transporter, subfamily G, member 2 ( ABCG2/BCRP )1,2,3, glucose transporter 9 ( GLUT9/SLC2A9 )1, organic anion transporter 4 ( OAT4/SLC22A11 )1,4, monocarboxylate transporter 9 ( MCT9/SLC16A9 )1,5, and leukine-rich repeat-containing 16 A ( LRRC16A/CARMIL )1,6. PDZ domain containing 1 ( PDZK1 , also known as NHERF3 ) plays a pivotal role as a scaffolding protein that forms urate transportsome6,7,8,9 with URAT1, ABCG2, and OAT4 (Figure 1). A single-nucleotide polymorphism (SNP), rs12129861, was first reported to have an association between PDZK1 gene and SUA1, which was confirmed by a replication study10. Although the minor allele of rs12129861 is shown to decrease SUA1,10, to the best of our knowledge, no study to date has investigated its association with clinically defined patients with gout. Figure 1. Urate transportsome in the renal tubular cells. PDZK1 (also known as NHERF3) is a scaffolding protein that binds to several urate transporters such as URAT1, OAT4, and NPT1. As for ABCG2, the interaction with PDZK1 is shown to be weak (dotted line)9. … Address correspondence to Dr. H. Matsuo, Department of Integrative Physiology and Bio-Nano Medicine, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan. E-mail: hmatsuo{at}ndmc.ac.jp