Advances in Biological Research 10 (3): 167-174, 2016 ISSN 1992-0067 © IDOSI Publications, 2016 DOI: 10.5829/idosi.abr.2016.10.3.102174

Interleukin-6 Gene Polymorphism (-572 C>G) In Type 2 Diabetes of Bangladeshi Origin Subash Pandaya, Razwa Saleh, Md. Ashraful Alam, Md. Mamun Al-Amin, Preeti Jain and Hasan Mahmud Reza Department of Pharmaceutical Sciences, North South University, Dhaka-1229, Bangladesh Abstract: Interleukin-6, a major pro-inflammatory cytokine, is produced in various tissues, including activated leukocytes, adipocytes and endothelial cells. Several lines of evidence indicate a causal role of cytokine interleukin 6 (IL-6) in the development of type 2 diabetes in humans. Functional polymorphisms in the promoter sequence of IL-6 gene (-174G/C, -572C/G and -597 G/A) have been observed to alter expression levels of the cytokine. These polymorphisms have been investigated for association with type 2 diabetes mellitus; however the results are mostly unclear. This study was thus designed to explore the association of type 2 diabetes mellitus with the Single Nucleotide Polymorphism at the position -572 C>G. Our data showed no significant association between type 2 diabetes mellitus and prevalence of polymorphism in IL-6 in terms of allelic frequency for rs1800796 (odds ratio 1.5; 95% confidence interval 0.747-3.029; P =0.252). No association was observed with quantitative traits either. It can be concluded that IL-6 -572C>G was not associated with type 2 diabetes mellitus in the Bangladeshi population. Lack of association might be due to the presence of other haplotypic associations or it may require a larger sample size. Key words: IL-6 SNP reaction

Type 2 diabetes

Restriction fragment length polymorphism- polymerase chain

INTRODUCTION

development of the disease, but recent research has identified a number of promising candidates including IL-6 [2- 4]. IL-6 is a pleiotropic cytokine involved in the pathophysiology of various human diseases such as type 2 diabetes and obesity. It is released by the adipose tissue and this release is higher in obese people, especially in the ones with a greater waist-hip-ratio [5, 6]. IL-6 level also rises postprandially, along with the levels of glucose and insulin, in the interstitial fluid of adipose tissue suggesting that IL-6 may also modulate adipose glucose metabolism in the fed state [6-8]. Two other adipocyte derived factors, adiponectin [9] and leptin [10], have already been assessed for their correlation with type 2 diabetes in the Bangladeshi population. There is compelling evidence that augmented levels of IL-6 are associated not only with impaired glucose tolerance (IGT), but also in the prediction of the development of the disease. This indicates a potential role

Type 2 diabetes mellitus has been empirically shown to be a partially inheritable disease, in which a genetic component plays a significant role in disease aetiology. Although insulin resistance and progressive pancreatic -cell dysfunction have been established as the two fundamental features in the pathogenesis of type 2 diabetes, the specific molecular defects affecting insulin sensitivity and/or -cell function remain largely undefined. It has been estimated that 30-70% of type 2 diabetes risk can be attributed to genetics. Type 1 diabetes, with its autoimmune roots, has strong association with genetic factors responsible for the production of auto-antibodies [1], similarly in type 2 diabetes multiple genes are involved with different combination of genes playing roles in different subsets of individuals. It is not yet known how many genes are involved or how much control they exert over the Corresponding Author:

Hasan Mahmud Reza,Department of Pharmaceutical SciencesNorth South University, Dhaka 1229, Bangladesh. Tel: +880-255668200, ext. 1954, Fax: +880-255668211, Mob: +8801715885651.

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of this cytokine in the aetiology of type 2 diabetes [11]. Low-grade inflammation has been reported to play a pivotal role in the pathogenesis of type 2 diabetes. Evidence also suggests that chronic activation of the innate immune system decreases insulin sensitivity and thus may herald the type 2 diabetes [12, 13]. In type 2 diabetes, molecular markers of inflammation include the acute-phase response proteins; sialic acid, -1 acid glycoprotein, serum amyloid A, C-reactive protein, cortisol and the cytokine IL-6 [4, 14]. Several SNPs throughout the IL-6 gene, located on chromosome 7q21, have been studied for their relevance in transcription regulation of IL-6 gene [15, 16]. Screening of both exon and intron sequences revealed multiple polymorphisms, following which the point mutations on the promoter region (namely -597 G>A, -572 C>G, previously known as -634 C>G and -174 G>C promoter polymorphisms) were deemed the most significant in disease pathogenesis [16, 17]. Several studies demonstrated that possessors of the homozygous G allele have higher IL-6 secretions by the peripheral blood mononuclear cells compared to homozygous C allele [15, 18]. In contrast, others observed the CC genotype to be associated with higher IL-6 levels [19, 20]. Caucasian GGG/GGG individuals were established as producing lower IL-6 protein in case of all three promoter polymorphisms [16]. These studies demonstrate the complex nature of IL-6 expression, which is influenced not by a single mutation, but multiple concurrently occurring polymorphisms (showing haplotypic association) as well as ligand binding at specific sites on promoter sequence [16, 17, 21]. A vast number of epidemiological, genetic and human in vivo and in vitro studies have investigated the putative role of IL-6 in the pathogenesis of underlying obesity, insulin resistance and -cell destruction associated with type 2 diabetes [22]. In a study conducted on Japanese population to determine the influence of -572C>G polymorphism on development of diabetic nephropathy;

no -174 G>C polymorphism was observed while it was widespread in diabetic populations of Caucasian origin [23]. Thus the current case-control study is aimed at determining the prevalence of -572 C>G in the Bangladeshi population. MATERIALS AND METHODS Study Participants and Measurements: Peripheral blood samples were collected from human subjects. Both patients and controls were of same ethnic origin. Patients also had similar disease severity because they were not suffering from any other diabetic complications. Anthropometric and certain biochemical parameters were recorded for each of the study subjects and the results are shown in Table 1. Prior to sample collection, all participants of this study filled out and signed the informed consent form. The study protocol complies with the Declaration of Helsinki and was approved by the Ethical Committee and the Institutional Review Board. DNA Extraction and Genotyping: Genomic DNA was extracted from whole blood by using Favorgen DNA extraction kit (Taiwan). Extracted DNA was aliquoted for each sample and stored at -20ºC for further analysis. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method as described by Bennermo et al. [24] was used to genotype the -572 C>G polymorphism in IL-6 gene. Genotyping was carried out using the following primers: Forward primer: 5'GGAGACGCCTTGAAGTAACTGC-3' and Reverse primer: 5'-GGGCTGACTCCATCGCAG-3'. The reaction mix for polymerase chain reaction contained 4 µl of DNA, 100 µmol of oligonucleotides and a 2x solution of master mix (Fermentas, USA). The PCR cycle began with an initial denaturation step at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 58°C for 45 sec and extension at 72°C for 1 min followed by a final extension at 72°C for 1 min. After PCR, 10 µL of

Table 1: Clinical and Biochemical Characteristics of the Study Subjects. N Variables Healthy Controls (N=29) Type 2 Diabetes Mellitus (N=38) 1 Age(years) 46.48±10.86 58.23±9.93 2 Gender (male/female) 19/10 21/17 3 Body mass index (Kg/m2) 24.56±3.36 27.04±3.90 4 Fasting Glucose (mmol/L) 5.56±0.96 7.85±1.75 5 Duration of disease(years) 13±9.15 6 Triglycerides (mg/dl) 197.6±35.76 142.1±38.93 7 HDL (mg/dl) 36.21±7.04 42.6±2.066 8 LDL (mg/dl) 164.8±29.51 120.4±43.61 9 HbA1c (%) 9.027±4.62 4.62±04185 Data are presented as mean±SD. HDL, High density lipoprotein, LDL, Low density lipoprotein, HbA1c, glycosylated hemoglobin.

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P Value 0.00001 0.008 0.000000001 0.475 0.007 0.001 0.00002

Advan. Biol. Res., 10 (3): 167-174, 2016

RESULTS

the reaction mixture was digested with 1U of BsrBI restriction endonuclease (Thermoscientific, USA) in a 30µL reaction volume at 37°C for 2 hrs. The digested products were electrophoresed (Takara Mupid Electrophoresis Systems) on a 2.0% agarose gel, stained with ethidium bromide and visualized on a UV-Trans illuminator (Uvitech, Cambridge).

A total number of 67 unrelated subjects were screened for glycemic status (Table 1). Of them 38 were type 2 diabetes and 29 were healthy controls. The number of males and females in the patient group was 21 and 17 respectively, while in control group their respective number was 19 and 10. The amplified PCR products of IL-6 gene covers the -572 C>G region and had a molecular size of 154 bp (Figure 1) After digestion with the enzyme, the 154 bp product yielded two bands at 94 and 60 bp (Figure: 2). Evaluation of the -572 (G>C) polymorphism in IL-6 by BsrBI digestion revealed the prevalence of GG,CG and CC genotypes in both diabetics and controls (Table 2). The observed genotypic frequencies were in Hardy Weinberg Equilibrium. The frequency of G and C alleles did not differ significantly between the diabetic and control groups (0.46/0.36 vs. 0.54/0.64, P=0.252). The dominant and recessive models were adjusted for age and sex (Table 3). The adjusted recessive model could not provide any association for the SNP (OR 0.55, 95% confidence interval [CI] 0.12-2.53; P= 0.43). However significant association was observed for dominant model (OR 0.28, 95% confidence interval [CI] 0.08-1.01; P= 0.044) which was found to be not significant after analysis of the relative risk data. The SNP rs1800796 was also analyzed against type 2 diabetes quantitative traits using multivariate two-way ANOVA. The polymorphism was not found to be associated with any of the phenotypic traits namely, fasting plasma glucose, body mass index, high density

Statistical Analysis: Hardy-Weinberg equilibrium was assessed using genotype data. Allele and genotype frequencies were calculated in patients and healthy controls by direct gene counting. Statistical analysis of the categorical variables (genotype and allelic frequency difference between diabetic and controls) was performed by ÷2 test. Comparison of the mean values of quantitative traits (continuous independent variables) between diabetic and control subjects was performed using t-test. The association between type 2 diabetes risk and -572 C>G SNP was estimated by the odds ratio and corresponding 95% confidence intervals. The ORs were obtained from combination of single studies by the comparisons of allelic model (G allele vs. C allele) using chi-square on SPSS and also for co-dominant model, dominant model (G/G+G/C vs. CC) and recessive model (GG vs. G/C+C/C). Association analysis measured the dominant, recessive and co-dominant effect for each polymorphism using SNPassoc version 1.9-2. Differences between groups and genotypes versus the individual characteristics (continuous data) were measured using two-way ANOVA. SPSS software version 17 for windows was used for the above statistical analysis. P-values of less than 0.05 were considered significant.

Fig. 1: A photograph of gel electrophoresis showing PCR product for SNP -572 G>C at 154 bp. Lane 1-7 are diabetic DNA sample and Lane 8 is DNA ladder = 100 base pair. 169

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Fig. 2: A photograph of RFLP-PCR products of IL-6 SNP-572 G>C run on 2% agarose gel. A 100bp marker DNA is used in lane 8. Lane 1 and 2 were cut by restriction enzyme showing bands below 100bp while lane 3-7 remains uncut showing band at 154bp. Table 2: Frequency of Polymorphism within the – 572 G>C region of IL-6 Gene in Type 2 Diabetic Patients and Controls Genotype

NGT

Type 2 diabetes

GG n (%) CG n (%) CC n (%)

5 (17%) 11 (37%) 13 (44%)

8 (21%) 19 (50%) 11 (29%)

Alleles G n (%) C n (%)

21(0.36) 37(0.64)

35(0.46) 41(0.54)

P Value

0.252

Table 3: Different models for SNP association analysis SNP association with response status (n=67, adjusted by age + sex) Model

Genotype Status

Case Status

Control

OR (CI 95%)

P-Value

Co-dominant

C/C C/G G/G C/C

11 (28.9%) 19 (50%) 8 (21.1%) 11 (28.9%)

13 (44.8%) 11 (37.9%) 5 (17.2%) 13 (44.8%)

0.29 (0.07-1.11)

0.13

Dominant

C/C C/G-G/G

27 (71%) 30 (79%)

16 (55.2%) 24 (82.8%)

0.28 (0.08-1.01)

0.044

Recessive

C/C-C/G G/G

8 (21.1%) 19 (50%)

5 (17.2%) 18 (62.1%)

0.55 (0.12-2.53)

0.43

lipoprotein (HDL), low density lipoprotein (LDL), total cholesterol, triglycerides or glycated haemoglobin (HbA1c) levels.

0.26 (0.04-1.54)

alleles and their frequencies at many loci vary widely among population groups. Gene-gene or geneenvironment interactions could lead to the varying genetic effects of IL-6 observed in different population. Several lines of evidence indicate a causal role of the cytokine IL-6 in the development of type 2 diabetes in humans [26]. The main aetiological features of type 2 diabetes and its inflammatory complications, such as nephropathy, are yet to be identified. But it seems that the immune system plays important roles in aetiology and pathogenesis of type 2 diabetes and its associated complications. Polymorphism in the promoter of IL-6 gene, -572 C>G (rs1800796) has been investigated for

DISCUSSION Type 2 diabetes is a multi-factorial disease in which environmental triggers interact with genetic variants in the predisposition to the disease. Insulin resistance and/or progressive -cell dysfunction have been implicated in the pathogenesis of type 2 diabetes [25]. Although the human chromosomes and the loci that they contain are identical throughout the species, the nature of different 170

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association with type 2 diabetes, in numerous studies and substantial differences were shown between ethnic groups that are at risk, the alleles and the haplotypes [15, 26]. Thus it is important to determine the role of IL-6 in various populations. In recent years, a large amount of evidence has accumulated indicating that insulin resistance and type 2 diabetes is closely related to a chronic low-grade inflammatory state of the body. Since subclinical inflammatory reactions have been implicated in the pathogenesis of type 2 diabetes the various inflammatory cytokines such as IL-6, IL-1 and TNF- have been studied to determine their possible roles in type 2 diabetes [27]. The circulating level of IL-6 remains elevated in people with type 2 diabetes and furthermore, this value serves as indirect measure for condition of insulin resistance. Links between IL-6 gene promoter polymorphisms (597/-572/-174) and Type 2 diabetes or diabetic nephropathy [4, 15, 28, 29]; direct impact of promoter polymorphisms on IL-6 secretion; effect of other mediators on said polymorphisms as well as IL-6 secretion; and haplotypic association between these promoter allelic variants have all been explored in various studies [17, 30, 31]. The -174 G>C polymorphism has been rare or absent from Asian populations albeit it’s high prevalence in the Caucasian population [15, 24, 26, 29]. The propensity for G allele in the promoter region -572C>G was found to be much higher in Asians, which acts as a basis for this study [29]. The homozygous GG genotype has been associated with increased IL-6 secretion [15]. The findings of this study suggested that G allele stimulates higher IL-6 and other inflammatory mediators in vivo compared with C allele carriers while the GC heterozygote individuals were observed to release reduced IL 6 whilst the CC carriers showed least IL 6 secretion in the Japanese. The study conducted by Koh et al. [29]also concluded that higher IL-6 levels were observed in healthy G allele carriers compared to C allele carriers. Two other studies performed on the Chinese Han population also corroborated the above data on IL-6 secretion. On the contrary, Müller-Steinhardt et al. [16]showed that healthy GG carriers produce lower level of IL-6 when stimulated by lipopolysaccharide. Results of this study might not be entirely consistent with in vivo conditions. These differences among Asian populations and their counterparts in the Caucasians are likely due to diverse ethnic and geographical influence on promoter polymorphism prevalence. The IL-6 promoter

polymorphisms have also been associated with progression to diabetic complications such as diabetic nephropathy, endothelial dysfunction leading to coronary artery disease [29] and even coronary heart disease and MI [32]. According to the results of our current study, we did not find any association between type 2 diabetes and allele frequency of SNPs -572 C>G (P value was 0.252). This result is different from the study conducted by Yin et al [31] that study showed evidence for significant association between the IL-6 gene -572 C>G polymorphism and type 2 diabetes risk (OR=1.29, CI=95%). Koh et al [29] and Hamid et al [30] also demonstrated positive association consistent with the outcome of Yin et al. [31]. Kitamura et al [15] further showed the association of -572 C>G polymorphism with development of diabetic nephropathy. This finding, however, is in agreement with the study of Huth et al. [26] which is a meta-analysis on unpublished data mostly obtained from Caucasian populations. A study conducted on the Finnish population demonstrated that interaction between promoter sequence polymorphisms of IL-6 and TNF- was successful in predicting the conversion of IGT patients to type 2 diabetics [28]. On the other hand, in the meta-analysis by Qi et al. [33], there was insufficient evidence from case-control studies to support the relationship between the common IL-6 gene polymorphism and type 2 diabetes. This may happen due to ethnic variation. In our study three different genetic models were also used to determine the association of 572 C>G IL-6 polymorphism and type 2 diabetes. Both codominant and recessive models demonstrated no significant association. Although a possible association is indicated by the dominant model (P=0.044) with a relatively small increase in risk of developing diabetes with inheritance of the G allele, however, the relative risk data (OR=0.28, 95% CI – 0.08-1.01) showed that this association is not statistically significant. The -572 SNP, despite not being in linkage disequilibrium with other promoter polymorphisms, showed haplotypic association with -174 and -597. This haplotypic association might demonstrate a stronger predisposition to the disease rather than a single polymorphism. The GGG haplotype has been associated with higher gene expression, but transcription may be impaired or suppressed by impact of varied transcription factor levels working at different promoter regions, as the expression pattern of this gene is complicated. The alteration in base pair at a singular functional promoter site is also likely to generate changes in transcription 171

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factor binding in the other associated site [17]. Thus predicting the influence of a single promoter polymorphism on genotypic expression and disease susceptibility is difficult. Further studies are required to explore these haplotypic differences and transcription factor influences on Interleukin -6 gene expression. The other interleukins such as IL-1 which has already been studied for association with rheumatoid arthritis [34] and congenital cytomegalovirus [35] may also serve as potential diabetes susceptibility genes in the Bangladeshi population and require further study.

2.

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CONCLUSIONS In summary, we stated that conflicting data still exist demonstrating the association between IL-6 polymorphism and type 2 diabetes. In this study, genotyping based analysis was performed to understand the relationship between -572 C>G polymorphism and type 2 diabetes in Bangladeshi population. However, further studies are required with larger population size to establish the firm association of -572 C>G polymorphism and type 2 diabetes. List of Abbreviations: IL-6 : Interleukin 6 T2D : Type 2 diabetes IGT : Impaired glucose tolerance SNP : Single nucleotide polymorphism PCR-RFLP: Polymerase chain reaction- restriction fragment length polymorphism LDL : Low density lipoprotein HDL : High density lipoprotein HbA1c : Glycated haemoglobin TNF: Tumor necrosis factor-alpha : Interleukin-1beta IL-1 ACKNOWLEDGEMENT We acknowledge Dr. Md. Mahbubur Rahman for his suggestion with the statistical analysis and Dr. Omar Faroque, BIHS, Dhaka for his kind assistance with patient sample collection. There is no financial conflict of interest. REFERENCES 1.

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