Abstract
Objective. To investigate the joint effects of alcohol consumption and ABCG2 gene variants on tophaceous gout occurrence.
Methods. The V12M (rs2231137), Q126X (rs72552713), and Q141K (rs2231142) of the ABCG2 gene were genotyped among controls, nontophaceous, and tophaceous gout cases in Taiwanese Han (n = 446, 77, 177) and Taiwan Aborigines (n = 1105, 203, 330).
Results. The missense variations V12M (C) and Q141K (T) significantly associated with tophaceous gout (p trend = 4.08 × 10−2, 9.00 × 10−12 in Han; 1.81 × 10−3, 9.34 × 10−10 in Aborigines). The nonsense variation Q126X (T) exerted a significant effect only in Han (p = 1.10 × 10−2), but not in Aborigines. In the prediction of tophaceous gout, the Q141K (T) OR were 1.51 in Han, 1.50 in Aborigines, and 1.55 (p = 7.84 × 10−5) in pooled analysis when compared to nontophaceous gout. We found the joint effects of alcohol consumption and Q141K (T/T) highly associated with tophaceous gout (adjusted OR ≥ 5.11; p ≤ 7.78 × 10−4); specifically the ever drinkers carrying the Q141K (T/T; adjusted OR 25.05, p = 9.21 × 10−4 in Han; adjusted OR 14.87, p = 1.08 × 10−8 in Aborigines).
Conclusion. Our findings showed alcohol consumption and ABCG2 Q141K, independently and jointly, associated with the risk of chronic tophaceous gout.
Gouty arthritis is characterized by the deposition of monosodium urate, which may subsequently form visible tophi surrounding the fibrous tissue in connective tissues, leading to destructive arthropathy when deposited in articular structures1,2. Large subcutaneous tophi can cause deformities and disabilities and often require lengthy treatments to “melt” the tophus or to remove the chalky mass of tophaceous deposits1,2,3.
Several epidemiologic studies have demonstrated that environmental factors (e.g., alcohol intake) and genetic predisposition [e.g., ATP-binding cassette, subfamily G, member 2 (ABCG2) gene] together contribute to the elevated urate levels in gout4,5,6. Alcohol intake is strongly associated with hyperuricemia and may partially explain the incidence and prevalence of gout4,7. The ABCG2 is a well-studied hyperuricemic gene that resides in the 4q22–q23; and proximal to the 4q21–25 region of gout-susceptibility gene GOUT1 (MIM ID %138900) that we previously associated in Taiwanese Han and Taiwan aborigines using genome-wide linkage study8,9.
Functionally, ABCG2 encodes for the urate transporter of the proximal tubule nephrons in the regulation of urate homeostasis5,10,11. A variant of ABCG2, for example, a loss-of-function variant at amino acid position 141 in the nucleotide-binding domain of the ABCG2 gene imparts a 50% reduction in urate transport velocity in vitro and is correlated with reduced renal function in the J-SHIPP Suita study5,10,11. Although ABCG2 gene variants are clearly associated with uric acid and gout, less is known about its influence on progression to tophaceous gout. We hypothesized that the joint effects of alcohol consumption and ABCG2 gene variants may contribute to an increased tophaceous gout occurrence.
MATERIALS AND METHODS
Study participants
Two case-control studies were conducted on the ethnic groups of Taiwan (Han and Aborigines). A total of 700 Han participated, comprising 446 controls and 254 patients with gout (77 tophaceous gout), and a total of 1638 Aborigines participated, comprising 1105 controls and 533 patients with gout (203 tophaceous gout). All patients satisfied the American College of Rheumatology gout survey criteria12. Tophaceous gout was diagnosed with confirmed crystals or with 1 or more tophi clearly visible and palpable from patients’ arms, legs, ears, or articular cartilage, accompanied by hyperuricemia at time of collection. The nontophaceous gout cases were recruited from the same hospitals as described13,14. We determined that all controls were gout-free using clinical history and routine physical examinations and further ascertained that none had received hypouricemics for medical conditions.
Our questionnaire included sociodemographic characteristics such as age, sex, and ethnicity; medical history, including diabetes mellitus, hypertension (HTN), and gout status; the age of gout onset and duration (about 70% of gout cases provided information); and a complete history of alcohol consumption. Alcohol drinkers were categorized into those who currently consumed alcoholic beverages (irrespective of quantity) greater or equal to twice per week, and “ever drinker” was defined as those who consumed alcoholic beverages at least twice per week for more than 1 year habitually but had quit at least 1 year prior to our interview15. We do not have details on the type of alcohol used. Also, it is noteworthy that compared to Han gouty cases, the aboriginal medical history was limited regarding those receiving treatment (currently or in the past). The institutional review boards and ethics committees of the participating hospitals approved this study design. All participants gave their written informed consent.
Genotype determination
Total genomic DNA was obtained from peripheral blood leukocytes using a genomic DNA extraction kit (QIAGEN-Gentra Puregene Blood Kit, Gentra Systems). The ABCG2 gene polymorphisms V12M (rs2231137), Q126X (rs72552713), and Q141K (rs2231142)5,10,16 were genotyped using TaqMan single-nucleotide polymorphism (SNP) Genotyping Assays with ViiA 7 Real-Time PCR System (Applied Biosystems).
Statistical analysis
The differences of demographic and clinical information between gouty cases versus controls and tophaceous versus nontophaceous gout in the 2 ethnicities were analyzed by chi-square test for categorical variables (e.g., sex, comorbidity of diabetes mellitus or HTN, etc.) and t test for continuous variables [e.g., age, blood pressure, body mass index (BMI), etc.]. Fisher’s exact test was performed on small sample sizes and for sparse tables. The distribution of plasma triglyceride (TGC) levels was normalized by taking a log transformation of the original values because of the skewed distributions of the original values. Hardy-Weinberg equilibrium (HWE) was verified for all SNP by use of PLINK software17. Additive genetic effects were modeled by defining a continuous variable with levels 0, 1, 2 (e.g., G/G, G/T, and T/T for rs2231142) and also compared the T/G and T/T genotypes separately with the reference genotype G/G. Crude OR with 95% CI and p-values were determined for each case-control study separately, assuming an additive genetic model using a multinomial logistic regression. Multiple logistic regression adjusted for sex, BMI, log (TGC), creatinine, diabetes mellitus, and alcohol use, and in the pooled analysis, ethnicity, was used for inferring the risk of the rs2231142 on tophaceous gout. Pooled analysis was performed using the Cochran-Mantel-Haenszel test method in PLINK software, and a Breslow-Day test was used to assess the homogeneity of the OR from different populations. General linear regression adjusting for age, sex, BMI, total cholesterol, log (TGC), creatinine, alcohol use, and HTN, and in the pooled analysis, ethnicity, was used for inferring the influence of the rs2231142 on age of onset and duration of disease assuming an additive genetic model. A general linear regression model including an ethnicity × rs2231142 interaction term was applied. The potential independent effects of ABCG2 Q141K and alcohol use were evaluated using the multinomial logistic regression after adjustment for gout risk factors of age, sex, BMI, total cholesterol, log (TGC), creatinine, and HTN. For the joint analyses, we used 3 categories of ABCG2 Q141K genotypes (G/G, G/T, T/T) and 3 categories of alcohol use (nondrinker, ever drinker, current drinker). To calculate these measures in additive interaction between 2 risk factors, a new composite variable of 9 categories was computed and adjusted for gout risk factors by a multinomial logistic regression model. The multinomial logistic regression analysis then estimated the adjusted OR using this new indicator variable. This investigation at a single locus Q141K (rs2231142) for tophaceous gout when compared to nontophaceous gout demonstrated an 87.9% statistical power to detect an OR of 1.50 at α = 0.05. Power was calculated using Quanto v1.2.4. data handling, and associations were performed using the software packages SAS, version 9.3 (SAS Institute Inc.).
RESULTS
Baseline demographics for Taiwan ethnic groups are presented in Appendix 1. There were 254 Han gout cases [77 (30%) tophaceous gout cases] and 446 controls [mean ages: 50.8 yrs (cases) and 54.6 yrs (controls), p = 0.001]. There were 533 aboriginal gout cases [203 (38%) tophaceous gout cases] and 1105 controls [ages, 51.1 (cases) and 52.1 (controls), p = 0.201]. Gout cases had higher mean TGC, creatinine, and uric acid levels as well as greater rates of comorbidity with hyperuricemia and HTN (p < 0.05). Han gout cases had higher BMI and total cholesterol levels (p < 0.001). We found that ABCG2 rs2231142 Q141K T/T significantly associated with tophaceous gout (Table 1). None of the SNP had deviated from HWE for the 2 control groups. There were significant associations of tophaceous gout susceptibility for the 2 variants in the 2 ethnicities (V12M C p trend = 4.08 × 10−2, Q141K T p trend = 9.00 × 10−12 in Han; V12M C p trend = 1.81 × 10−3, Q141K T p trend = 9.34 × 10−10 in Aborigines). In terms of the nonsense SNP Q126X, the T allele associated with tophaceous gout in Han, but its effect could not be tested in Aborigines (frequency 0%). The frequencies of controls and patients, without/with tophi, for the rs72552713 Q126X T allele were 0.11% (1/892) in controls, 0.28% (1/354) for non-tophi, and 1.95% (3/154) for tophi (p = 2.44 × 10−3; Fisher’s exact p = 1.08 × 10−2); allelic OR when compared to the C allele: the T allele was related to tophi (OR 17.70, 95% CI 1.83–171.19, p = 1.31 × 10−2) and non-tophi (OR 2.52, 95% CI 0.16–40.45, p = 0.513).
ABCG2 V12M, Q141K, and Q126X variants and tophaceous gout risk.
To better illustrate the association of the ABCG2 Q141K variant in tophaceous gout versus nontophaceous gout, we provided a summary in Table 2. The Q141K T OR for tophaceous gout were 1.51 (frequency 0.64 vs 0.52) in Han and 1.50 (frequency 0.61 vs 0.50) in Aborigines, when compared to nontophaceous gout after adjusting for covariates. The effect estimate from the pooled analysis was consistent with further evidence of homogeneity for the OR between the 2 populations (OR 1.55, 95% CI 1.25–1.92, p = 7.84 × 10−5; pBreslow-Day test = 0.85) after controlling for ethnicity and other gout risk factors. We can demonstrate that the number of risk alleles of Q141K showed additive effects on the risk of tophaceous gout, by comparing tophaceous gout cases carrying the reference Q141K G/G with Q141K G/T to show an OR 1.61 (95% CI 1.07–2.42, p = 2.18 × 10−2) and with Q141K T/T to show an OR 2.21 (95% CI 1.45–3.37, p = 2.20 × 10−4) after adjustment of covariates. Thus, the Q141K T/T was robust and informative on the risk of tophaceous gout.
ABCG2 Q141K (T) associate with tophaceous gout.
Aborigines who had tophaceous gout and carried Q141K T were better correlated with an earlier age of visit to the clinician (G/G = 45.22 yrs, G/T = 40.37 yrs, and T/T = 35.89 yrs; p trend = 0.0014) and a longer duration of the disease (G/G = 9.72 yrs, G/T = 10.64 yrs, and T/T = 14.58 yrs; p trend = 0.0019). However, this finding was not observed in the Han or in the nontophaceous gout cases in either ethnicity. Estimates per risk allele copy in the tophaceous gout cases, corrected for covariates, showed an earlier age of onset and longer duration of disease at rs2231142 T of −1.26 and −2.61 years and 1.24 and 2.61 years in both ethnicities, and −2.24 years (p trend = 0.0010) and 2.27 years (p trend = 0.0010) in the pooled group (Appendix 2). We also observed an ethnicity-specific interaction of Q141K with earlier age of visit to the clinician among the gout cases (p for interaction = 0.0286; Appendix 3).
In terms of alcohol consumption, Table 3 shows that Q141K T/T and ever drinker were 2 important predictors (OR 9.85, p = 2.18 × 10−8 and OR 7.03, p = 3.24 × 10−5 in Han; OR 3.60, p = 1.34 × 10−7 and OR 5.69, p = 9.22 × 10−12 in Aborigines). Being an ever drinker significantly influenced tophaceous gout risk, suggesting that alcohol use in patients with gout and tophi occurrence were linked. The Aborigines had a higher tendency toward gout as well as higher alcohol intake (75% vs 28% in Han). Further, for the current drinker, multiple logistic-regression analysis also verified alcohol use adjusted OR 1.62 (p = 2.10 × 10−5) to be more a significant predictor of tophaceous gout risk in Aborigines than in Han (OR 1.18, p = 0.6640).
ABCG2 Q141K (T/T) and alcohol use predict risk of tophaceous gout.
Table 4 shows the joint effects of rs2231142 Q141K T/T and alcohol consumption. To explore the extent to which rs2231142 Q141K T/T was an important risk factor for tophaceous gout occurrence, we performed a multiple logistic-regression analysis on tophaceous gout cases according to alcohol intake categories, compared to nondrinkers carrying wild-type genotype Q141K G/G as reference. We found that Q141K T/T increased from OR 6.21 without alcohol use to OR 12.69 with alcohol use in Han and from OR 2.63 without alcohol use to OR 5.11 with alcohol use in Aborigines. We found especially that the ever drinkers with carriers of rs2231142 Q141K T/T were the most deterministic of tophaceous gout out of both ethnicities (OR 25.05, p = 9.21 × 10−4 in Han; OR 14.87, p = 1.08 × 10−8 in Aborigines).
Different alcohol use status and rs2231142 Q141K in association with tophaceous gout.
DISCUSSION
We studied 2338 persons from 2 different Taiwan ethnic groups and found that an important variant, Q141K T, in the ABCG2 gene, significantly associated with the odds of nontophaceous gout risk (p = 3.34 × 10−10 and 3.57 × 10−3 in Han and Aborigines), the odds of tophaceous gout occurrence (p = 9.00 × 10−12 and 9.34 × 10−10 in Han and Aborigines), and in comparing tophaceous gout versus nontophaceous gout (OR 1.55; p = 7.84 × 10−5). Further, we found that Q141K associated with tophaceous gout across the alcohol consumption categories, with a stronger association in ever drinkers (OR 25.05 and OR 14.87 in Han and Aborigines) than in current drinkers (OR 12.69 and OR 5.11 in Han and Aborigines).
The variants V12M and Q141K, associated with tophaceous gout in Han and Aborigines owing to missense polymorphisms, were correlated (D′ = 0.94 in Han; D′ = 0.99 in Aborigines). The other variant, Q126X T, is also associated with tophaceous gout (OR 18.03, p = 1.28 × 10–2) but only in Han (T 2% vs 0.1%). The ABCG2 mediates high-capacity urate transport even under high-urate conditions from its ATP dependence and kinetic analysis. Of patients with gout, 10.1% (vs 0.9% of controls) possessed the genotype combinations Q126X and Q141K (OR 25.8) and reduced > 75% of the ABCG2 function, indicating that nonfunctional variants of ABCG2 can essentially block gut and renal urate excretion, to elevate gout risk5. Our study verified this effect in the Han but not in Aborigines.
The genetics and mechanism of how gout progresses into tophaceous gout are poorly understood. We posited that the worst clinical outcome category, tophaceous gout versus nontophaceous gout cases, could validate our hypothesis that the Q141K sufficiently predisposes patients with existing gout to develop tophi. Our findings showed that Q141K T contributed to about a 50% increase (OR ≥ 1.50) in tophaceous gout compared to nontophaceous gout, across ethnic groups. This effect was persistently higher for tophaceous gout versus controls than for nontophaceous gout versus controls. The tophaceous and nontophaceous gout carriers of Q141K T from hospital-based and population-based settings shared similar allele frequencies; thus our analysis of Q141K T should have no misclassification bias as a true susceptibility locus for gouty arthritis.
The frequency of Q141K T is highly variable in the human population. The T allele ranges 1–3% in Africans, 11% in whites, 9% in Eastern Polynesians, 29% in Western Polynesians, 30% in Asians, and 44% in Taiwan Aborigines16,18,19. Gout prevalence in US individuals of Asian ancestry (T allele 30%) is about 3 times higher than in US individuals of European ancestry (T allele 11%)20. The prevalence of gout in individuals of European ancestry is 1.4%21; in Taiwanese Han it is 2.1–4.4%22. For Taiwanese Aborigines, hyperuricemia can be as prevalent as 41% to 82% and gout as much as 12%22,23. In our study, the risk allele Q141K T has a frequency of 44% in Aborigines and 31% in Han. Other supporting evidence is that the aboriginal patients with tophaceous gout who were carriers of Q141K T/T presented at an earlier age to the clinician. This remained consistent with a Q141K T allele estimated OR of 1.94 and a 3.4-fold increased risk of tophaceous gout compared to noncarriers.
It is estimated that 5–10% of adult Americans have hyperuricemia, of whom 20% develop gout7. Thus hyperuricemia alone is often not sufficient for the expression of gout. Environmental factors (high dietary purine intake and alcohol) and genetic predisposition contribute to higher serum uric acid and gout risk4,6,24. Our study showed that alcohol intake and Q141K T/T, independently and jointly, associated with the risk of tophaceous gout. The joint risk among tophaceous gout carriers of Q141K T/T who are ever drinkers (OR ≥ 14.87) was significantly higher than for current drinkers (OR ≥ 5.11). The alcohol self-reporting is roughly accurate and is in accordance with previous reports that alcohol-associated disorders affect 42.2–54.7% of Taiwanese aboriginals25 and 22.1% of Taiwanese Han15. Thus alcohol consumption by ethnicity has been similarly represented in our study.
There are limitations to our study that warrant attention. First, the record of current and past treatment of patients with gout or those with lower serum urate among aboriginals was limited because of the relatively poor primary healthcare for the Aborigines. It is reported that aboriginal patients with tophaceous gout were not being treated for their disease on a regular basis26. In our study, the proportion of tophi in aboriginals with gout was 38% (203/533, Appendix 1) and higher compared to other reports (9.2%–17.2%)26. This may affect the gout onset and duration because the rate of formation of tophaceous deposits in primary tophaceous gout is correlated with the degree and duration of hyperuricemia. However, it should also be noted that progression to chronic tophaceous gout has numerous causes such as poor compliance with, ineffectiveness of, or inability to tolerate prescribed regimens, and serum urate levels have not been shown to directly correlate with tophi occurrence.
Second, we do not have details about the alcohol types consumed (e.g., beer or wine); they may contain varying amounts of purines being metabolized into uric acid. However, acute alcohol intake causes lactate production, and because lactate is an antiuricosuric agent, it will reduce renal urate excretion and exacerbate hyperuricemia7. Taking uricosuric medications (probenecid, sulfinpyrazone, and benzbromarone) in conjunction with alcohol consumption may alter the effectiveness of urate-lowering therapies. Last, a strong sex specificity of Q141K in men has been described10,16; however, we found no evidence for an interaction considering sex in our studied groups (p > 0.05), which might be due to the small number of women with gout, resulting in low power to detect the effect size of the variants. We found that ABCG2 Q141K T have similar allele frequencies between men and women (test of homogeneity, pBreslow-Day test > 0.3) by ethnicity and associated with gout among women in the 2 ethnic groups (p < 0.05; Appendix 4).
Our results corroborate with previous finding that ABCG2 increases gout risk. Additionally, we showed the Q141K ABCG2 variant increases severe or tophaceous gout. Further, alcohol consumption has an additive increase in the OR of alcohol drinkers who harbor the variant Q141K T/T. Our findings were consistently observed in 2 distinct ethnic groups from Taiwan, and highlight potentially exciting new clinical intervention and gout treatment opportunities.
Acknowledgment
The authors thank the medical staff members at the Jianshih and Wufong Township Health Station (Dr. Xian-Min Qiu and nurses), Kaohsiung Medical University Hospital (Dr. Gau-Tyan Lin), and the Kaohsiung Chang-Gung Hospital (clinical rheumatologist, Dr. Chung-Jen Chen) for the ascertainment of clinical phenotypes and for data collection.
APPENDIX 1.
Characteristics of the study participants.
APPENDIX 2.
ABCG2 rs2231142 [T/T] association with the age of visit to clinician and duration among tophaceous gout cases
APPENDIX 3.
Interaction of ethnicity and rs2231142 Q141K with age of onset among gout cases. A general linear model including ethnicity × rs2231142 Q141K interaction term was applied. P for interaction was adjusted for age, sex, body mass index, total cholesterol, log (triglycerides), creatinine, alcohol use, and hypertension. Error bars indicate 95% CI.
APPENDIX 4.
ABCG2 rs2231142 Q141K[T] association with risk of gout
Footnotes
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Supported by the Taiwan National Health Research Institutes (NHRI-98A1-PDCO-0307101), the Taiwanese National Science Council (NSC97-2314-B-039-007-MY3 and NSC99-2628-B-037-039-MY3), and the Kaohsiung Medical University (KMU-Q102006, KMU-M103018).
- Accepted for publication December 13, 2013.
REFERENCES
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