Uncoupling protein 2 gene polymorphisms in association with overweight and obesity susceptibility: A meta-analysis☆

A meta-analysis was performed to evaluate the associations of uncoupling protein 2 (UCP2) gene polymorphisms (Ala55Val, 45-bp insertion/deletion, and -866G/A) with overweight and obesity. A total of 42 studies were included in our analysis. Pooled effect estimates and 95% confidential intervals of each polymorphism were calculated under different inherited models. Fixed or random effect model was selected based on the between-study heterogeneity evaluated with I2. Source of heterogeneity was explored by subgroup analysis and meta-regression analysis. Potential publication bias was assessed using funnel plot and Peters test. After excluding studies that deviated from the Hardy–Weinberg equilibrium, T allele of Ala55Val polymorphism was associated with an increased risk of overweight and obesity under recessive model in the overall (OR = 1.24, 95%CI = 1.06–1.45) and Asian (OR = 1.28, 95%CI = 1.06–1.55) populations; and A allele of -866G/A polymorphism had a protective effect on overweight and obesity, especially for European populations (dominant model: OR = 0.88, 95%CI = 0.81–0.96, co-dominant 1 model: OR = 0.89, 95%CI = 0.81–0.98, co-dominant 2 model: OR = 0.85, 95%CI = 0.74–0.94, additive model: OR = 0.88, 95%CI = 0.80–0.95, and allelic model: OR = 0.91, 95%CI = 0.86–0.97). No evidence was observed in the association of 45-bp insertion/deletion polymorphism with overweight and obesity susceptibility. We failed to fully explore the between-study heterogeneity regarding the association of Ala55Val polymorphism with overweight and obesity. Further studies are required to provide more convincing evidence.


Introduction
Uncoupling proteins (UCP), a member of mitochondrial membrane transporters, were considered to be associated with energy homeostasis (Dalgaard and Pedersen, 2001). The UCP2, which is widely expressed in human tissues and serves as an uncoupler of oxidative phosphorylation, is involved in the regulation of lipid metabolism and ATP production (Diano and Horvath, 2012;Zhang et al., 2001). It has been reported that impaired expression of UCP2 mRNA in adipose tissue may be responsible for the pathophysiology of obesity (Oberkofler et al., 1998). Ala55Val (rs660339) in Exon 4, 45-bp insertion/deletion in Exon 8, and -866G/A (rs659366) in the promoter region, have been considered as the most interesting polymorphisms in UCP2 gene. The -866G/A and 45-bp insertion/deletion polymorphisms were found to be functional on mRNA expression (Esterbauer et al., 2001;Krempler et al., 2002). A variant allele of the Ala55Val polymorphism was reported to be associated with lower 24-h energy expenditure (Astrup et al., 1999). Associations of the three polymorphisms with the risk of overweight and obesity have been widely evaluated, but the conclusions remain controversial. In the present study, we performed a meta-analysis to assess the effect of UCP2 Ala55Val, 45-bp insertion/deletion, and -866G/A polymorphisms on the risk of overweight and obesity.

Eligibility criteria
A study that met the following criteria was included in the meta-analysis: (1) case-control, cohort study, or cross-sectional studies which evaluated the association of UCP2 gene polymorphisms with overweight and obesity; (2) provided sufficient data of genotypes in case (exposed) and control (unexposed) groups; (3) subjects were recruited from the same ethnicity during the same period; (4) if data were repetitively reported, the most complete one was included; (5) the genotype distribution of the controls obey Hardy-Weinberg equilibrium (HWE). The articles were independently assessed by two authors. Disagreements were resolved by consensus with a third reviewer.

Data extraction
Information of the studies was extracted by two independent authors, including first author, publication year, location, ethnicity of subjects, sample size, frequencies of genotype and allele, percentage of male subjects and mean age of subjects in case (exposed) and control (unexposed) groups.

Statistical analysis
HWE for UCP2 Ala55Val, 45-bp insertion/deletion, and -866G/A genotype distributions in controls was tested using Pearson χ 2 test. Pooled odds ratios (ORs) with 95% confidence intervals (CIs) were calculated to assess the associations of the UCP2 Ala55Val, 45-bp insertion/deletion, and -866G/A polymorphisms with risk of overweight and obesity under dominant (CT+TT vs. CC for Ala55Val, ID+II vs. DD for 45-bp insertion/deletion, and GA+AA vs. GG for -866G/A), recessive (TT vs. CT+CC for Ala55Val, II vs. ID+DD for 45-bp insertion/deletion, and AA vs. GA+GG for -866G/A), co-dominant 1 (CT vs. CC for Ala55Val, ID vs. DD for 45-bp insertion/deletion, and GA vs. GG for -866G/A), co-dominant 2 (TT vs. CC for Ala55Val, II vs. DD for 45-bp insertion/deletion, and AA vs. GG for -866G/A), additive (2TT+CT vs. CC for Ala55Val, 2II+ID vs. DD for 45-bp insertion/deletion, and 2AA + vs. GG for -866G/A), over-dominant (CT vs. CC+TT for Ala55Val, ID vs. II+DD for 45-bp insertion/deletion, and GA vs. AA+GG for -866G/A) and allelic (T vs. C for Ala55Val, I vs. D for 45-bp insertion/deletion, and A vs. G for -866G/A) models, respectively. Pooled ORs in subgroups stratified by ethnicity were also calculated. In addition, cumulative analyses were performed to track the evidence over sample size. Z test was used to determine the significance of pooled OR. The I 2 was used to assess heterogeneity among studies (Higgins and Thompson, 2002). Heterogeneity was considered significant if I 2 N 50%, when random-effect model (REM) was adopted as the pooling method; otherwise, fix-effect model (FEM) was used. Meta-analysis regression was performed to identify potential covariates, including ethnicity, publication year, sex, age and sample size, which may contribute to between-study heterogeneity. Sensitive analysis was performed to identify 'outlying' studies and decrease statistical heterogeneity if I 2 N 50% using hetred module (Patsopoulos et al., 2008). Influence of each individual study on the pooled estimate was investigated with influence analysis. A study was suspected of excessive influence if the point estimate of its omitted meta-analysis was beyond the 95%CI of the full analysis. Publication bias was evaluated by funnel plot and Peters test (Peters et al., 2006). All statistical analyses were performed with STATA version 11.0 (Stata Corporation, College Station, TX, USA). P value b 0.05 was considered statistically significant (two-tailed).

Quantitative synthesis
The results of pooled analyses were summarized in Table 4.
Subgroup analysis stratified by ethnicity revealed a significant association in recessive (FEM: OR = 1.28, 95%CI = 1.06-1.55), co-dominant 2 (REM: OR = 1.50, 95%CI = 1.01-2.20), additive (REM: OR = 1.41, 95%CI = 1.04-1.93) and allelic (REM: OR = 1.32, 95%CI = 1.07-1.63) models for Asians. No significant association between Ala55Val polymorphism and risk of overweight and obesity was found in any of the models for Europeans. The pooled ORs for the association of Ala55Val polymorphism with overweight and obesity under recessive model in the main and cumulative meta-analysis were presented in Fig. 1.

UCP2 45-bp insertion/deletion polymorphism
The meta-analysis showed no significant association between the 45-bp insertion/deletion polymorphism and the risk of overweight and obesity in dominant (FEM: OR = 1.02, 95%CI = 0.92-1.

UCP2 -866G/A polymorphism
In the overall population, no significant association between -866G/A polymorphism and risk of overweight and obesity was found in any of the models.

Source of heterogeneity and sensitive analysis
As shown in Table 4, significant heterogeneity (I 2 N 50%) was observed among studies of the Ala55Val, 45-bp insertion/deletion, and -866G/A polymorphisms in some inherited models. Univariate meta-regression with the covariates of ethnicity, publication year, sex (ratio of male in case group to that in control group) and age (ratio of mean age in case group to that in control group) revealed that no covariates had excessive influence on between-study heterogeneity regarding the association of each polymorphism with overweight and obesity.
Significant heterogeneity was observed among studies of the Ala55Val polymorphism under all the inherited models except for the recessive one in the overall population. When stratified by ethnicity, the heterogeneity still existed in participants of Asian descent. Studies that were key contributors to between-study heterogeneity were indicated in Table 4. After sensitive analysis, the associations between T allele and risk of overweight and obesity were no longer significant in the co-dominant 2, additive, and allelic inherited models among the overall population.
Significant heterogeneity was observed among studies regarding the associations of the 45-bp insertion/ deletion polymorphism with overweight and obesity in additive and allelic models among the overall population. When stratified by ethnicity, the heterogeneity was significantly decreased in the population of Asian descent, but still existed in that of European descent. Results of sensitive analysis indicated that one study (Evans et al., 2000) was mainly responsible for the between-study heterogeneity in additive and allelic models for the overall population, and that in dominant, co-dominant 2, additive and allelic models for the population of European descent, considering the association of the 45-bp insertion/deletion polymorphism with overweight and obesity. After exclusion of the study, the associations still remained non-significant in all the inherited models.
Significant heterogeneity was observed among studies regarding the associations of the -866G/A polymorphism with overweight and obesity in dominant, co-dominant 2, additive, and allelic models among the overall population. When stratified by ethnicity, the heterogeneity was significantly decreased in the population of European descent, but still existed in that of Asian descent. After sensitive analysis, the associations between A allele and risk of overweight and obesity were significant in the co-dominant 2 (FEM: OR = 0.99, 95%CI = 0.80-0.98) and allelic (FEM: OR = 0.94, 95%CI = 0.89-0.99) models in the overall population. However, the associations remained non-significant in all the aforementioned inherited models among the population of Asian descent.

Influence analysis
With articles that were key contributors to heterogeneity being excluded, no individual study was found to have excessive influence on the pooled effect under all the mentioned inherited models for any of the polymorphisms (data not shown).

Publication bias evaluation
After exclusion of studies that were key contributors to between-study heterogeneity, no publication bias was detected in all the aforementioned inherited models using funnel plot and Peters test regarding the associations between the three polymorphisms with overweight and obesity (data not shown).

Discussion
UCP2 gene was first identified as a candidate gene for obesity by Fleury et al. (1997). Since then, the associations of UCP2 gene polymorphisms (Aal55Val, 45-bp insertion/deletion, and -866G/A) with overweight and obesity have been widely studied; however, the results were conflicting. Considering the small effect of a genetic variant, a large sample size is certainly required in the primary genetic association study with adequate power. In order to obtain a robust conclusion regarding the role of UCP2 gene polymorphisms in the risk of overweight and obesity based on a larger scale of population, we performed the meta-analyses consisting 42 studies to evaluate the associations. The pooled ORs from our meta-analyses revealed a risk effect of T allele in Ala55Val polymorphism in populations of Asian descent, a protective effect of A allele in -866G/A polymorphism in populations of European descent and no effect of 45-bp insertion/deletion polymorphism on overweight and obesity. Cumulative meta-analyses showed an increased accuracy in the estimate of effects with larger samples aggregated for the three polymorphisms in UCP2 gene.    Between-study heterogeneity is a usual concern in meta-analyses (Munafo and Flint, 2004), which was also observed in our analysis under most of the inherited models regarding the association of UCP2 polymorphisms with overweight and obesity. After stratification analysis by ethnicity, the heterogeneity in some comparison groups remained significant. To further explore the source of heterogeneity, we performed a meta-regression analysis involving covariates such as publication year, sex, age, and sample size. However, none of the covariates was found to contribute to the between-study heterogeneity regarding the associations of aforementioned polymorphisms with overweight and obesity. It suggested that some other confounding factors, such as design quality, representativeness and general characteristics of the participants, environmental exposures, and non-uniform genotyping methods may be responsible for the heterogeneity. However, we failed to evaluate the role of these factors in the source of heterogeneity in our analysis due to inadequate information in the original articles.
No significant between-study heterogeneity (I 2 b 50) was found in the recessive model regarding the association of Ala55Val polymorphism with overweight and obesity. Available data suggested a stable association of T allele of Ala55Val polymorphism with an increased risk of overweight and obesity in the recessive model for the Asian population. Studies indicated that the Ala55Val polymorphism had effects on metabolic rate and nocturnal physical activity (Astrup et al., 1999;Klannemark et al., 1998;Walder et al., 1998). T allele of the polymorphism was reported to be associated with lower energy expenditure and lower fat oxidation (Astrup et al., 1999), which may be implicated in the pathogenesis of overweight and obesity. In this meta-analysis, we failed to fully explore the source of between-study heterogeneity observed in most of the inherited models. Although significant associations were revealed in the co-dominant 2, additive and allelic models in the meta-analysis, the conclusions were limited because of the unsolved heterogeneity.
Significant between-study heterogeneity was found in the additive and allelic models regarding the association of 45-bp insertion/deletion polymorphism with overweight and obesity in the overall population. One study (Evans et al., 2000) that was a key contributor to the heterogeneity was identified using sensitive analysis. The between-study heterogeneity largely decreased after the key contributor was excluded, and the conclusions were consistent with those before sensitive analysis. In our meta-analysis, no evidence was observed on the association of UCP2 45-bp insertion/deletion polymorphism with overweight and obesity both in European and Asian populations.
Associations of -866G/A polymorphism with the risk of obesity were evaluated using meta-analysis by Andersen et al. (2013) and Liu et al. (2013) before. In this study, we performed a meta-analysis based on 15,976 individuals from 18 studies with strict eligible criteria to provide robust evidence. Available data in our meta-analysis suggested a stable association of -866G/A polymorphism with overweight and obesity in the population of European descent. The pooled ORs for GA+AA vs. GG, GA vs. GG, AA vs. GG and A vs. G for Europeans were 0.88, 0.89, 0.85 and 0.91, respectively, which were consistent with previous researches (Andersen et al., 2013;Liu et al., 2013). Besides, we identified a significant association of -866G/A polymorphism with overweight and obesity under additive model (2AA+GA vs GG) for the population of European descent. However, no evidence was observed in all the aforementioned models for the population of Asian descent. Oberkofler et al. indicated a reduced UCP2 mRNA level in intraperitoneal adipose tissue in obese individuals (Oberkofler et al., 1998). A allele of -866G/A polymorphism was reported to be associated with enhanced UCP2 mRNA expression in adipose tissue in European population (Esterbauer et al., 2001), which may explain its protective effect against overweight and obesity.
Obesity is a heterogeneous disorder caused by complex gene × gene and gene × environment interactions. The effects of Ala55Val and -866G/A polymorphisms on overweight and obesity differed in Europeans and Asians, which may be attributed to different genetic backgrounds. Significant linkage disequilibrium has been observed among Ala55Val, 45-bp insertion/deletion, and -866G/A polymorphisms in overweight and obese populations (Esterbauer et al., 2001;Ochoa et al., 2007;Oktavianthi et al., 2012;Wang et al., 2007). Lack of consideration of the effects of adjacent loci may partially explain the discrepancies among individual studies. Since limited information was provided in the included studies, effects of haplotypes on overweight and obesity could not be evaluated in our meta-analysis, which require further investigation.
The conclusions of our meta-analysis should be considered under the following limitations. First, the outcome of interest was not uniform in all the included studies. Cases were defined as morbidly obese population, obese population, or combined population of overweight and obesity in different studies. The cutoff used for overweight or obesity differed in studies with the same target population. Second, subgroup analysis was performed to decrease potential between-study heterogeneity; however, limited studies and small sample size in some subgroups may lead to an underpowered result. Third, since insufficient information was provided in the original studies, we failed to fully explore the source of between-study heterogeneity under some circumstances. Last, language was restricted to English and Chinese in the search strategy, which may limit the result.

Conclusions
In conclusion, meta-analyses of currently available studies supported that T allele of Ala55Val polymorphism was associated with an increased risk of overweight and obesity under recessive model, especially for populations of Asian descent; and A allele of -866G/A polymorphism had a protective effect on overweight and obesity, especially for European populations. However, 45-bp insertion/deletion polymorphism is not a susceptibility locus to overweight and obesity. Additional association studies with large sample size and sufficient information allowing powerful stratification analyses are needed to provide more robust evidence to the conclusions. The roles of haplotypes of UCP2 gene in the pathogenesis of overweight and obesity require to be clarified in future researches.

Funding
The study was sponsored by a grant from the Ministry of Science and Technology of People's Republic of China (2006BA105A01).