Interleukin-1 Receptor Antagonist and Interleukin-4 Genes Variable Number Tandem Repeats Are Associated with Adiposity in Malaysian Subjects

Interleukin-1 receptor antagonist (IL1RA) intron 2 86 bp repeat and interleukin-4 (IL4) intron 3 70 bp repeat are variable number tandem repeats (VNTRs) that have been associated with various diseases, but their role in obesity is elusive. The objective of this study was to investigate the association of IL1RA and IL4 VNTRs with obesity and adiposity in 315 Malaysian subjects (128 M/187 F; 23 Malays/251 ethnic Chinese/41 ethnic Indians). The allelic distributions of IL1RA and IL4 were significantly different among ethnicities, and the alleles were associated with total body fat (TBF) classes. Individuals with IL1RA I/II genotype or allele II had greater risk of having higher overall adiposity, relative to those having the I/I genotype or I allele, respectively, even after controlling for ethnicity [Odds Ratio (OR) of I/II genotype = 12.21 (CI = 2.54, 58.79; p = 0.002); II allele = 5.78 (CI = 1.73, 19.29; p = 0.004)]. However, IL4 VNTR B2 allele was only significantly associated with overall adiposity status before adjusting for ethnicity [OR = 1.53 (CI = 1.04, 2.23; p = 0.03)]. Individuals with IL1RA II allele had significantly higher TBF than those with I allele (31.79 ± 2.52 versus 23.51 ± 0.40; p = 0.005). Taken together, IL1RA intron 2 VNTR seems to be a genetic marker for overall adiposity status in Malaysian subjects.


Introduction
The prevalence of obesity worldwide is rising at alarming rate and has been described as a global pandemic. Malaysia has one of the highest rates of obesity in Asia-Pacific, where the combined prevalence of overweight and obesity was 43.8% and 48.6% among men and women above 20 years, respectively [1]. Obesity is closely associated with chronic and low-grade inflammation in the adipose tissue, signified by a lower level of anti-inflammatory cytokines and higher level of proinflammatory cytokines, which in turn differentially activate adipose-tissue macrophages (ATMs) [2]. Specifically, anti-inflammatory cytokines interleukin-(IL-) 13, IL-4, and IL-10 stimulate the alternatively activated ATMs (M2) in lean persons, while obesity induces a shift to the classically activated ATMs (M1) due to stimulation by proinflammatory cytokines TNF-, IL-1 , and IL-6 [3].
Interleukin-1 receptor antagonist (IL-1ra), also known as IL-1RN, is an endogenous competitive inhibitor of proinflammatory IL-1 and IL-1 [4] and is highly secreted by the white adipose tissue (WAT) [5]. IL-1ra is a proadipogenic factor as IL1RA knockout mice have reduced adipose storage, impaired adipogenesis, and decrease in adipocyte size [6], while its level is increased in the serum of obese patients, correlating with body mass index (BMI) and insulin resistance [7]. The human IL-1ra gene (IL1RA or IL1RN) has a Variable Number Tandem Repeat (VNTR) polymorphism within intron 2 due to variation in the number of copies of an 86 bp sequence. To date, six distinct alleles corresponding to 1, 2, 3, 4, 5, and 6 copies of the repeat sequence have been identified [8]. The 4-repeat (allele I) and 2-repeat (allele II) are most frequently found in the general population, while the other four alleles (alleles III, IV, V, and VI) are rarely observed [9]. This VNTR, particularly homozygosity for allele II, has been variably associated with various conditions such as obesity, inflammatory bowel disease, and coronary artery disease [reviewed in [10]] in different ethnic populations worldwide.
IL-4, secreted by activated Th2 lymphocytes, basophils, and mast cells, executes many biological roles such as induction of Th2 differentiation, immunoglobulin class switching, and B cell proliferation [11]. In animal studies, diet-induced obese mice had increased splenic lymphocytes production of IL-4 [12], rats receiving visceral fat removal surgery had decreased serum IL-4 [13], and mice treated with IL-4 had improved insulin sensitivity and glucose tolerance while lipid accumulation in adipose tissues was inhibited [14]. These suggest that IL-4 may participate in the processes of dietinduced obesity and metabolism. Similar to IL1RA, IL4 has a 70 bp VNTR polymorphism within intron 3, and two common alleles are B1 and B2 that have two and three tandem repeats, respectively [15]. There have been several reports on the association between the VNTR B1 allele and inflammatory diseases, such as multiple sclerosis [16], rheumatoid arthritis [17], and systemic lupus erythematosus [18]. With regard to obesity, there are limited studies on this VNTR, where two studies showed no association [19,20].
Since the association of IL1RA and IL4 VNTRs with obesity and its related parameters is still elusive especially in Asians, the objectives of this study are therefore to investigate the distribution of IL1RA and IL4 VNTRs genotypes and alleles and to determine whether they are associated with overall obesity (as measured by BMI), central adiposity (as measured by waist circumference, WC), and overall adiposity (as measured by total body fat, TBF) in Malaysian subjects. . The ethnicities of the subjects were self-identified. All subjects were pooled together for data analysis. This study has received ethical approvals from the UTAR Scientific and Ethical Review Committee (SERC). All subjects signed informed consent forms, and the study was conducted in accordance with the Declaration of Helsinki (amended in Brazil, 2013).

DNA Extraction and Genotyping.
Participants were asked to rinse their vigorously with 5 mL of 3% sucrose solution for 1 min and the mouthwash samples were preserved in 3 mL TNE buffer [17 mM Tris/HCl (pH 8.0), 50 mM NaCl, and 7 mM EDTA]. DNA extraction protocol was then conducted following the protocol as outlined previously [26]. The IL1RA and IL4 VNTRs were genotyped by Polymerase Chain Reaction using the primers, reagents, and conditions adopted from [27] or [20], respectively, with minor modifications. PCR was performed in a 20 L total reaction mixture containing 1x PCR buffer (NH 4 ) 2 SO 4 without MgCl 2 (PhileKorea, Korea), 1.5 mM MgCl 2 , 0.2 mM dNTPs (PhileKorea, Korea), 0.5 M of forward primer, 0.5 M of reverse primer, 100 ng of DNA template, 0.5 U of Taq polymerase (PhileKorea, Korea), and an appropriate volume of sterile deionized water to top up. The PCR amplification protocol was carried out using the T1006 Thermal Cycler PCR machine (Bio-Rad, CA, USA) which began with initial denaturation at 95 ∘ C for 5 min, followed by 30 cycles of denaturation at 94 ∘ C for 30 sec, annealing at 60 ∘ C for 30 sec (for IL1RA) or 66 ∘ C for 45 sec (for IL4), extension at 72 ∘ C for 30 sec (for IL1RA) or 1 min (for IL4), and final extension at 72 ∘ C for 10 min. PCR products were electrophoresed on either 1.5% (for IL1RA) or 3% (for IL4) agarose gel, then stained with ethidium bromide, and visualized under UV light after electrophoresis. The sizes of the bands for IL1RA alleles were I: 410 bp; II: 240 bp; IL4 alleles were B2: 253 bp; B1: 183 bp ( Figure 1). Three genotypes from each VNTR were verified by DNA sequencing of PCR products (First BASE Laboratories Sdn. Bhd., Malaysia).

Statistical Analysis.
Statistical analysis was carried out using the IBM SPSS Statistics software version 16.0 (IBM, NY, USA). Allelic frequencies were estimated by gene counting and the distribution of genotypes was tested for Hardy-Weinberg equilibrium using the Chi-square ( 2 ) test. Data for continuous variables were presented as adjusted means ± standard error of the mean (SEM) and as frequency for categorical variables. The normality of distributions of continuous variables was tested with the Kolmogorov-Smirnov test and variables that were not distributed normally were logtransformed prior to statistical analysis. Genotype and allele frequencies of the polymorphism were assessed for association with demographic and anthropometric classes using Pearson's 2 test or Fischer's exact test. Logistic regression analysis (enter method) was performed for overall adiposity status with adjustment for covariate ethnicity. Analysis of covariance (ANCOVA) using the univariate General Linear Model with adjustment for covariate ethnicity was performed for anthropometric measurements and blood pressures. ANCOVA was also performed based on BMI status in  stratified analysis. A value of less than 0.05 was considered as statistically significant.

Results
The demographic and anthropometric characteristics of the 315 subjects are as shown in Table 1. There was no significant association between gender and the demographic and anthropometric classes. Table 2 shows the genotype and allele distribution of IL1RA and IL4 VNTRs, which did not deviate from the Hardy-Weinberg equilibrium and are categorized under different demographic and anthropometric classes. The overall minor allele frequencies (MAFs) of IL1RA and IL4 were 0.02 and 0.25, respectively. The allelic distribution of IL1RA was significantly different between Chinese and Indians and for IL4, it was significantly different between all ethnicities ( Table 2). IL1RA and IL4 allele distributions were also significantly associated with TBF class, but not other demographic and anthropometric classes. Genotype distribution of IL4 was also not significantly associated with gender, ethnicity, BMI, WC, and TBF classes.
Since TBF class was significantly associated with both IL1RA and IL4 VNTRs, logistic regression analysis was carried out to study the association of IL1RA and IL4 VNTRs with overall adiposity (TBF) status ( Table 3). As TBF was significantly different between ethnicities (data not shown), ethnicity was considered as a covariate. The IL1RA VNTR genotype and allele were significantly associated with overall adiposity before and after adjusting for ethnicity. Particularly, subjects with heterozygous I/II genotype had 14.45 times higher risk to have high adiposity compared with subjects with homozygous I/I genotype, while those with allele II had 6.81 times higher risk to have high adiposity compared with subjects with allele I. After controlling for ethnicity, the association of IL1RA genotype and allele with adiposity status remained significant with I/II subjects having 12.21 times higher risk compared with I/I subjects and II subjects having 5.78 times higher risk compared with I subjects (Table 3). However, IL4 VNTR B2 allele was only significantly associated with overall adiposity status before adjusting for ethnicity [OR = 1.53 (CI = 1.04, 2.23; = 0.03)], and this association was abolished after controlling for ethnicity [OR = 1.13 (CI = 0.74, 1.74; = 0.57)] (Table 3).
Indeed, covariate analysis of variance after controlling for ethnicity also showed similar result, where subjects carrying IL1RA II allele had 8.28% significantly higher TBF than the those with I allele (Table 4). However, stratified analysis based on BMI status showed that this was only true among nonobese subjects, but not obese subjects (data not shown). All other anthropometric measurements and blood pressures were not significantly different between IL1RA and IL4 VNTR alleles.

Discussion
In this study, as expected, only allele I and allele II of IL1RA VNTR can be found. The other alleles are too rare to be   found in most of the populations [27]. The IL1RA VNTR is considered a rare genetic polymorphism, as the MAF was 0.02 (less than 0.05). There is significant proof to show that IL1RA VNTR is highly influenced by ethnicity [reviewed in [27]]. A study in 19 Chinese populations found out that allele I had higher frequency than allele II, which were 0.913 and 0.064, respectively, indicating that the prevalence of allele I in China was significantly higher, and the prevalence of allele II was significantly lower than American and European Caucasian populations [28]. Similarly in African and African-American people, the frequency of allele II homozygotes is considerably lower than that in the Caucasian population [29]. Alike with IL1RA, the allele and genotype frequencies for IL4 VNTR in our population are strikingly different from those of the Caucasian population [17,[30][31][32], with our MAF of 0.25 being more similar to the Japanese population (MAF = 0.33) [33] and a previous Malaysian study (MAF = 0.37) [34]. IL-1ra serum level is increased in human obesity and is under strong genetic control [5], partly by the IL1RA VNTR polymorphism. Indeed, IL1RA allele II has a clear influence on IL-1ra circulating levels since, in normal human subjects, its carrier individuals had higher levels than the noncarrier individuals (745 ng/mL versus 627 pg/mL) [35]. With regard to obesity, two previous Asian studies with relatively small sample sizes found no significant association of IL1RA VNTR with BMI in Koreans ( = 261) [36] and North Indians ( = 103) [37]. Similarly, our study found no association with BMI value or overall obesity status, but IL1RA VNTR was associated with both TBF value and overall adiposity status. In stratified analysis, TBF value was found to be higher in subjects carrying IL1RA II allele than those with I allele, only among nonobese subjects. This could be attributed to the low BMI and high TBF paradox among Malays/Chinese/Indians, as shown previously [38]. The IL1RA VNTR may have functional significance as the repeated sequence contains possible binding sites for transcription factors [9]. A review by Witkin et al. 2002 [10] summarized that individuals homozygous for allele II have more prolonged and severe proinflammatory immune response than persons with other IL1RA genotypes, which might be beneficial when combating 6 Journal of Obesity infectious agents [39] or neoplasms [40], but is detrimental for those with chronic inflammatory conditions [41] or those who are pregnant [42].
The IL4 VNTR which is located in IL4 intron 3 could be a functional polymorphism as it could affect mRNA splicing, leading to different splice variants [43]. Indeed, the B2 allele has been associated with reduced amount of peripheral Th cells which produce IL-4 [33]. Our study showed that this VNTR was associated with overall adiposity status (TBF class), but not with TBF value after adjustment for ethnicity. However, consistent with two previous studies which showed no association of this VNTR with obesity status in North Indian [19] and Turkish [20] populations, we found no association with both BMI value and overall obesity status. The role of IL-4 in modulating adipogenesis has been established by previous studies. Tsao et al. (2014) [44] showed IL-4 inhibited adipogenesis via STAT6 pathway or by influencing the cell proliferation at the mitotic clonal expansion phase. IL-4 also enhances lipolysis via the PKA pathway in mature adipocytes [44]. Other study carried out by Rao et al. (2014) [45] revealed that IL-4 signaling activates macrophages in WAT, leading to the production of nervous system molecules required for converting WAT to beige fat. Taken together, the direct functional significance of IL1RA and IL4 VNTRs particularly on affected tissues in obesity (like adipose tissue) and other metabolic disease (like type 2 diabetes mellitus) warrants further investigation.
Limitations of the present study include the small sample size of Malay and Indian subjects and the lack of other indigenous ethnic groups especially from East Malaysia (Sabah and Sarawak); hence the results from this study may not be fully representative of the general Malaysian population. Other common genetic variants like the IL-1 promoter region and exon-5 and IL4 -590 T/C single nucleotide polymorphisms, which were not screened in this study, could have association with obesity and its related parameters. The direct phenotypegenotype correlation could also be determined by measuring the serum levels of IL-1ra and IL-4 using ELISA in the future.
Obesity results in a proinflammatory state which involves the release of cytokines and adipokines by adipose tissue. Indeed, the main finding of this study shows that VNTR genetic polymorphisms in two genes, IL1RA and IL4, encoding for anti-inflammatory cytokines IL-1ra and IL-4, respectively, are associated with overall adiposity status (TBF) in Malaysian subjects, before controlling for ethnicity. IL1RA VNTR shows a more prominent effect, as TBF was significantly higher in those with IL1RA II allele compared with I allele, even after controlling for ethnicity. Individuals with IL4 VNTR B2 allele had higher risk of having higher adiposity, but the association was abolished after controlling for ethnicity. Taken together, IL1RA intron 2 VNTR seems to be a genetic marker for overall adiposity status in Malaysian subjects.

Disclosure
Preliminary results of this study were presented as a poster presentation at the 6th Malaysian Symposium of Biomedical Science, May 16-17, 2015.