Serum Urate Concentrations and the Risk of Hyperuricemia

Serum Urate Concentrations and the Risk of HyperuricemiaCommon UCP2 varietys contribute to blood serum urate intentnesss and the peril of hyperuricemiaLuyu Yang, Zheng Dong, Jingru zhou, Yanyun Ma, Weilin Pu, Dongbao Zhao, Hongjun He, Hengdong Ji, Yajun Yang, Xiaofeng Wang, Xia Xu, Yafei Pang, Hejian Zou,Li Jin,Chengde Yang*, Jiucun Wang**Corresponding authorThese authors equally contributed to this study. riseElevated serum urate, which is regulated at multiple levels including genetic variates, is a guess factor for gout and opposite metabolic diseases. This study aimed to investigate the linkup between UCP2 variants and serum urate as well as hyperuricemia in a Chinese population. In broad(a), 4332 individuals were genotyped for ii coarse UCP2 variants, -866G/A and Ala55Val. These loci were not associated either serum urate level or with a risk of hyperuricemia in the total group of subjects. However, in feminines, -866G/A and Ala55Val were associated with a tear down serum urate (P = 0.006 and 0.014seperately) and played a preventative role against hyperuricemia (OR = 0.80, P = 0.018 OR = 0.79, P = 0.016). These intimacys were not discover in the males. After just stratification, the two loci were associated with serum urate in overweight, but not jaggy females. The haplotype A-T (-866G/A-Ala55Val) was a preventive factor for hyperuricemia in the female subgroup (OR = 0.80, P=0.017). This turn over study identify a novel gene, UCP2, that influences the serum urate concentration and the risk of hyperuricemia, and the stage of tie varies with sex and BMI levels.IntroductionUric pane of glass is the final product of purine oxidation in humans. Elevated serum urate, or hyperuricemia, has long been recognized as an independent risk factor for gout 1-2. There is a re modernisticed interest in hyperuricemia and its association with a number of other clinical disorders including hypertension, atherosclerosis, cardiovascular disease, chronic k idney diseases, and abdominal obesity, glucose intolerance, insulin resistance, and dyslipidemia, which are often subsumed under the term metabolic syndrome 3.Serum urate is balanced between uric acid ware in the colorful and its disposal via the kidney and gut 4. The occurrence of hyperuricemia could be caused by disruptions in any part of this metabolic process. Both genetic and environmental factors, much(prenominal) as gender and body mass index (BMI), have a strong put up on the risk of hyperuricemia 3. Among those factors, the attribution of genetic factors is estimated to be as high as 73% 5. Recent genome-wide association studies (GWAS) have identified 28 loci associated with serum urate concentration 6. However, only approximately 7% of the variation in serum urate concentration could be explained by those reported loci, suggesting the missing heritability remained to be explored 6.Human uncoupling proteins (UCPs) are mitochondrial transporters present in the inner memb rane of mitochondria 7. UCPs are capable of uncoupling ATP production from mitochondrial respiration by causing proton leak and preventing mitochondrial hyperpolarization and the formation of reactive oxygen species (ROS) 8. Among the five identified UCPs, UCP2 is widely expressed in almost all mammal tissues including white adipose tissue, liver, kidney, pancreatic islets, macrophages and retinal endothelial cells, indicating its employment in a variety of physiologic or pathologic events 9-12. Two of the most common polymorphisms of this gene, -866G/A (rs659366) in the actor and Ala55Val (rs660339) in codon 55, were identified as being associated with different phenotypes 7, 12, including obesity, insulin resistance, type 2 diabetes mellitus (T2D), low-density lipoprotein (LDL) particle size, coronary incidence and other metabolic disorders 9-10, 13-21.Given the involvement of UCP2 and hyperuricemia in a variety of metabolic disorders, we selected the two common loci -866G/A and Ala55Val to explore the association between genetic UCP2 variants and hyperuricemia in a Chinese population, offering a new diagnostic or therapeutic target for hyperuricemia.ResultsThere was noassociation between SNPs and serum urateThe two loci were proven in Hardy-Weinberg equilibrium (-866G/A P = 0.990 Ala55Val P = 0.690). For -866G/A, AA, AG, and GG genotypes accounted for 21.6%, 49.9%, and 28.6% of hyperuricemic patients, respectively in healthy harbours, the distribution was 21.2%, 49.6%, and 29.3%, respectively. As shown in evade 1, the -866G/A polymorphism was not found to be associated with serum urate (AA/GG important = -0.008, P = 0.644 AG/GGBeta = -0.012, P = 0.474) or with the risk of hyperuricemia (AA/GG OR = 1.05, P = 0.603 AG/GGOR = 1.03, P = 0.667). For Ala55Val, the TT, TC, and CC genotype distribution was 21.5%, 50.5% and 28.0% in hyperuricemic patients, respectively, and the distribution was 21.5%, 49.8% and 28.6% in healthy controls, respectively. No ass ociation was observed between Ala55Val polymorphism and serum urate (TT/CC Beta = -0.013, P = 0.460 TC/CCBeta = -0.017, P = 0.324). There was no difference in the distribution of the genotypes or allelomorphs among hyperuricemic patients and healthy controls (TT/CC OR = 1.02, P = 0.824 TC/CCOR = 1.04, P = 0.652). Therefore, no statistically solid evidence supported the genetic effect of -866G/A and Ala55Val on serum urate or the risk of hyperuricemia in the total group of subjects.UCP2 variants were associated withserum urate andhyperuricemia in female subgroupsAs shown in Table 1, we stratified all subjects into male and female subgroups to further explore the gender-related genetic effects of the two polymorphisms. In the male subgroups, there were no pregnant associations between the two loci and serum urate or the risk of hyperuricemia (all P 0.025). However, some nominal significant associations were found between -866G/A and the hyperuricemia risk (genotype AA OR = 1.26, P = 0.038 allele A OR = 1.12, P = 0.035), indicating a possible risky effect of the -866G/A variant on hyperuricemia incidence in males.A significant association was found between SNPs and serum urate and hyperuricemia in the female subgroups. The -866G/A genotypes were associated with a lower serum urate (AA/GG Beta = -0.078, P = 0.015 AG/GG Beta = -0.104, P = 0.001) and a slump risk of hyperuricemia (AG/GG OR = 0.71, P = 0.025). The subjects carrying allele A had a lower serum urate and a decrease risk of hyperuricemia (A/G Beta = -0.054, P = 0.006 OR = 0.80, P = 0.018). For Ala55Val, genotype TT carriers showed a lower serum urate (TT/CC Beta = -0.075, P = 0.022) and a decreased risk of hyperuricemia (TT/CC OR = 0.64, P = 0.020). Genotype TC carriers only had a lower serum urate (TC/CC Beta = -0.082, P = 0.012) but no decreased risk of hyperuricemia (TC/CC OR = 0.77, P = 0.093). on the unscathedele T was associated with a lower serum urate (T/C Beta = -0.049, P = 0.016) and a decreased risk of hyperuricemia (T/C OR = 0.79, P = 0.016). except analysis of associationin femaleswith different BMI levelsFurther analysis was performed regarding the genetic effect of UCP2 variants on serum urate and the risk of hyperuricemia among females with different BMI levels (Table 2). The majority of the females enrolled were stratified into normal- or overweight group (Table 2). In the underweight subgroup, whose prototype size was limited after stratification, no significant association was observed between the two loci and serum urate or hyperuricemia risk (all P 0.025, Table 2). In the normal weight subgroup, -866G/A genotype AA+AG carriers were associated with a lower serum urate (AA+AG/GG Beta = -0.095, P = 0.022) but not with a decreased risk of hyperuricemia (AA+AG/GG OR = 0.65, P = 0.076). However, the Ala55Val genotypes or alleles showed no statistical association with serum urate (TT+TC/CC Beta = -0.070, P = 0.091 T/C Beta = -0.047, P = 0.106) or hyperurice mia (TT+TC/CC OR = 0.72, P = 0.173 T/C OR = 0.72, P = 0.051). In the overweight subgroup, the genotypes of both loci were associated a lower serum urate (AA+AG/GG Beta = -0.138, P = 0.001 TT+TC/CC Beta = -0.130, P = 0.003) and a significant, or at least marginal, decreased risk of hyperuricemia (AA+AG/GG OR = 0.62, P = 0.015 TT+TC/CC OR = 0.74, P = 0.027). However, the alleles of the loci were associated with a lower serum urate level (A/G Beta = -0.072, P = 0.019 T/C Beta = -0.072, P = 0.019) but not with a decreased risk of hyperuricemia (A/G OR = 0.75, P = 0.036 T/C OR = 0.74, P = 0.027). Our results suggested a stronger effect of UCP2 variants on overweight females than on normal weight females (Table 2). link between haplotypes and risk of hyperuricemiaAs listed in Table 3, the haplotypes of the two loci were estimated in the total group of subjects and after stratification by gender. The -866G/A and Ala55Val variants were in strong linkage disequilibrium (D = 0.974, r2 = 0.936 ). The wild type haplotype G-C (-866G/A-Ala55Val) was applied as the reference one. Haplotype A-T made up for the most frequent one, while single mutation at -866G/A or Ala55Val each accounted for less than 1 part (Table 3). In the total group of subjects, no haplotypes were correlated with susceptibility of hyperuricemia. In the female subgroups, haplotype A-T (-866G/A-Ala55Val) was associated with a decreased risk of hyperuricemia however, this association was null in males. No further significant associations between hyperuricemia and other two rare haplotypes were found in our study, partly due to the limited size of the rare haplotypes carriers (Table 3). These results correlated with the association between genotypes or alleles and hyperuricemia (Table 1).DiscussionUncoupling protein 2 (UCP2) is present in the inner mitochondrial membrane and mainly decreases the ATP level and ROS produced by electron transport therefore, UCP2 is convolute in a board range of pathological pr ocesses. In the present study, we first focused on the relationship between UCP2 variants and serum urate and hyperuricemia, potentially examining the reaching of the loci related to hyperuricemia.The present study revealed no association between the two polymorphisms of UCP2 and serum urate or hyperuricemia in the total group of subjects. However, because serum urate is extensively influenced by gender differences, we stratified the total group of subjects and determined that -866G/A and Ala55Val were associated with serum urate and hyperuricemia in females 25-26. Females with the -866G/A genotype AA+AG or allele A had lower serum urate and a decreased risk of hyperuricemia, indicating a protective role of -866G/A for hyperuricemia in females.The -866G/A variant is a functional polymorphism laid in the promoter region and putatively changes the transcription factor binding sites 7. The wild type G allele in -866G/A was associated with lower UCP2 mRNA brass 19, 27. Increased UCP2 mRNA expression from the A allele was translated into an increased amount of UCP2 protein, with corresponding induced proton leak, decreased ATP/ADP ratio and enhanced body waste of ROS 10, 19. Hypermethylation in the promoter region could affect the binding of transcripation factors, causing aberrant gene expression. Consistent with our expectations, we found a typical CpG island in the UCP2 promoter region, which include the locus of the -866G/A variant, using information from the University of California-Santa Cruz (UCSC Santa Cruz, CA, USA) database (http//genome.ucsc.edu/cgi-bin/hgGateway). We believe the UCP2 promoter variant -866G/A could shape this CpG island and protect the UCP2 promoter region from DNA methylation, uncovering a novel underlying mechanism that determines -866G/A increases UCP2 transcription.Uric acid accumulation is caused by the acceleration of ATP degradation to AMP, a precursor of uric acid, and UCP2 could decrease the ATP level and lower redundant AMP for uric acid formation 7, 28. Moreover, an elevation of serum urate concentration occurs as a physiologic response to increased oxidative stress 31. Because the ROS level could be down-regulated by UCP2, a counter-regulatory increase of serum urate as an antioxidant defense is less urgent. Therefore, the -866G/A variant in the promoter region might serve as a protective factor through a higher UCP2 mRNA level and increased definition of the UCP2 protein, which might regulate ROS and modify the ATP/ADP ratio.The other locus, Ala55Val, is a missense variant in exon 4 and is associated with an altered degree of uncoupling 7. In our study, a protective effect for hyperuricemia and lower serum urate were observed in genotype TT and allele T in the female subgroups. However, the genetic effect of the Ala55Val variant was less clear. Several researchers identified an association of Ala55Val with the BMI level and type 2 diabetes mellitus (T2D), with controversial conclusions within coh orts, and few functional studies were performed 14, 32-33. Similar to -866G/A, the protective role of the Ala55Val variant for hyperuricemia might also be attributed to altered UCP2 transcription.In the male subgroups, a less statistically significant but possible effect of -866G/A and Ala55Val was observed for hyperuricemia risk and higher serum urate. Similar gender-associated genetic effects of UCP2 variants were more or less observed for diseases other than hyperuricemia 7. For example, Heidema et al. suggested a genetic effect of UCP2 on weight gain was regulated through different mechanism in males and females 34. Lee, et al. demonstrated that the association between UCP2 variants and BMI was more apparent among female subjects 35. Cheurfa et al. confirmed the association of UCP2 variants with coronary arterial blood vessel diseases in males but not females 36. In the present study, we found UCP2 variants -866G/A and Ala55Val had a stronger effect on females with hyperuricemia . One possible accounting for the gender-associated genetic effects of UCP2 might be a regulation role of sex hormones such as estrogen. Estrogen was reported to repress UCP2 in a breast cancer cell line and papillary thyroid cancer cells 37-38. Taken together, these results suggest the UCP2 protein level was down-regulated by estrogen in females but transposed by the variants of -866G/A and Ala55Val, providing a plausible explanation for the specific protective effects of UCP2 variants on females 37.Genetic effects on hyperuricemia and obesity have been widely recognized 3. In the present study, we found that -866G/A and Ala55Val were associated with lower serum urate and a decreased risk of hyperuricemia in overweight, but not underweight, females (Table 2). The relative pure sample size might limit the correlation analysis in the underweight group. However, we did observe females with higher BMI level were more likely to benefit from the protective genetic effect of -866G/A an d Ala55Val, where the association was significant between the two SNPs and serum urate level of risk of hyperuricemia. In the contrast, among the normal weight females, -866G/A, but not Ala55Val, showed a significant association with a low risk of hyperuricemia, indicating a less contribution from the protective effect of UCP2 variants than seen in overweight females. It was also implied from our results that the tw1functional 866GA promoter variant displayed a stronger effect. The interactions between obesity, uric acid and UCP2 were complicated. BMI has long been viewed as an essential factor influencing uric acid 3. UCP2 transcription was activated by juicy acids 16. A recent meta-analysis revealed that UCP2 -866G/A and Ala55Val are associated with a risk of obesity 32. Subtle intermediary obesity related phenotypes such as elevated triglycerides, total cholesterol concentrations, increased the risk of dyslipidemia and circulating leptin levels were also observed to be correlate d with UCP2 variants 40. Based on these results, we assumed lipid metabolism material such as fatty acids participated in and enhanced the genetic effect of UCP2 variants on serum urate regulation, explaining the stronger genetic effect of UCP2 variants on females with higher BMI levels observed in the present study.The -866G/A and Ala55Val variants were in strong linkage disequilibrium (D = 0.974, r2 = 0.936). The haplotype frequency analysis revealed that variants of the two loci were more in co-variant haplotype A-T (-866G/A-Ala55Val) compared with the single variant forms of G-T or A-C (Table 3). Haplotype A-T was associated with a decreased risk of hyperuricemia only in females, which was consistent with the genotype or alleles results. However, the small size of the two rare haplotypes might limit the power of association analysis with hyperuricemia risk to a certain extent. The susceptibility of hyperuricemia in the two rare haplotype carriers required validation in a larger cohort.ConclusionThe present study identified a novel gene, UCP2, with two loci, -866G/A and Ala55Val this gene influenced the serum urate concentrations and the risk of hyperuricemia in females. The associations of those loci were affected by gender and BMI. This study supported the potential involvement of this gene in the prevention, prediction and treatment of hyperuricemia.Materials and methodsExperimental designA total of 4332 subjects were enrolled from the Taizhou longitudinal Study 22 and included 1387 hyperuricemic patients and 2945 healthy controls. The associations of common UCP2 variants with serum urate and hyperuricemia were well-tried by linear regression and logistic regression with or without gender stratification, respectively. A body mass index (BMI) subgroup was also used for further analysis.ParticipantsAll subjects were enrolled from Taizhou Longitudinal Study 22, of which 1387 individuals had serum urate level over 7 mg/dl and were treat as hyperuricemic pa tients, and 2945 individuals had normal serum urate ( 7 mg/dl) and were treated as healthy controls 23. The subjects were divided into subgroups (underweight BMI 18.5 normal weight 18.50 BMI Genetic analysisGenetic analysis was carried out in accordance with the written informed assent and guideline offered by the Ethical Committees of the School of feeling Science of Fudan University. For genetic analysis, peripheral blood was collected from all the individuals included in this study. Genomic DNA was extracted from whole blood using the QIAamp DNA Blood Mini kit (QIAGEN, Germany) and was stored at -20. The DNA concentration and quality (including optical density (OD) 260/280 and 260/230 measurements) were determined using a Nanodrop Lite spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Genotyping of -866G/A and Ala55Val in UCP2 were performed by SNPscan according to the manufacturers instructions.Statistical analysisThe clinical characteristics were presented as the mean SD. Students t- mental test was used to test for a significant difference in the mean age, BMI and serum urate between hyperuricemic patients and healthy controls. The chi-square test was used to describe the gender distribution difference between hyperuricemic patients and healthy controls.The chi-square test was used to test Hardy-Weinberg equilibrium (HWE) of the two loci. We conducted a logistic regression analysis to calculate adjusted odd ratio (OR) with 95% confidence time interval (95% CI) and P-values to describe the distribution of -866G/A and Ala55Val adjusted for age and gender between hyperuricemic patients and healthy controls. A linear regression was performed to calculate Beta and P-values to estimate the effect on serum urate in different genotypes and alleles. Genotype GG, allele G of -866G/A and genotype CC, allele C of Ala55Val were used as references, respectively. Stratification into subgroups was performed on the basis of gender and different BMI va lues for further analysis. Haplotype frequencies between the hyperuricemic patients and controls were estimated by OR (95% CI) and chi-square test. The haplotype of the most frequent (-866G/A-Ala55Val, G-C) was used as the reference. A 2-sided P-value less than 0.025 was considered statistically significant after multiple correlation by Bonferroni method. 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