Research Report
Association between interleukin 10 gene polymorphisms and risk of type 2 diabetes mellitus in a Chinese population
Journal of International Medical Research 2014, Vol. 42(3) 702–710 ! The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0300060513505813 imr.sagepub.com
Hua Bai1,*, Danqing Jing1, Aitao Guo2,* and Shinan Yin1
Abstract Objective: To investigate the relationship between the interleukin 10 (IL10) gene single nucleotide polymorphisms (SNP) 1082 G/A (rs1800896), 819 T/C (rs1800871) and 592 A/C (rs1800872) and risk of type 2 diabetes mellitus in a Chinese population. Methods: This case–control study recruited patients with type 2 diabetes mellitus and healthy control subjects. Genotyping of the 1082 G/A (rs1800896), 819 T/C (rs1800871) and 592 A/C (rs1800872) SNPs was conducted and genotype frequencies were compared between the two groups. Results: The study recruited 364 patients with type 2 diabetes mellitus and 677 healthy controls. Patients carrying the 1082 GG genotype had a significantly increased risk of type 2 diabetes mellitus (adjusted odds ratio [OR] 1.57, 95% confidence interval [CI] 1.03, 2.68), as did those patients carrying the 592 AA genotype (adjusted OR 1.63, 95% CI 1.06, 2.53). Subjects carrying both the 1082 GA þ GG and 592 AC þ AA genotypes had a significantly increased risk of type 2 diabetes mellitus (adjusted OR 2.03, 95% CI 1.24, 3.15). Conclusions: The SNPs 1082G/A and 592 A/C increased the risk for type 2 diabetes mellitus, and could be potential targets for screening for the early detection of the risk of type 2 diabetes mellitus.
Keywords Interleukin 10, type 2 diabetes mellitus, IL10 gene, polymorphism, predictive markers Date received: 9 August 2013; accepted: 29 August 2013 *These authors contributed equally to this work.
1
Department of Endocrinology, First Affiliated Hospital of the People’s Liberation Army, Beijing, China 2 Department of Pathology, General Hospital of the People’s Liberation Army, Beijing, China
Corresponding author: Dr Shinan Yin, Department of Endocrinology, First Affiliated Hospital of the General Hospital of the People’s Liberation Army, 51 Fucheng Road, Haidian District, Beijing 100048, China. Email: [email protected]
Creative Commons CC-BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 3.0 License (http://www.creativecommons.org/licenses/by-nc/3.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access page (http://www.uk.sagepub.com/aboutus/openaccess.htm).
Bai et al.
Introduction Type 2 diabetes mellitus is a common chronic disease, and its complications have become a major cause of morbidity, mortality and disability in both developed and developing countries.1 There were approximately 285 million people worldwide living with diabetes in 2010, 9.2 million of them in China.1 It is estimated that the number of patients with type 2 diabetes mellitus worldwide will reach 300 million by 2025.1 Although type 2 diabetes mellitus is induced by various environmental and genetic factors, its detailed aetiology is not yet fully understood.2 Several studies have reported that multiple cytokines may be involved in the regulation of the immune response, which has in turn been shown to play a role in the pathogenesis of type 2 diabetes mellitus.3,4 Interleukins are a diverse constellation of small cell signalling protein molecules, or cytokines, that regulate the function of the immune system in humans.5,6 Interleukins are produced predominantly by T cells, monocytes, macrophages and endothelial cells.7 Their functions include facilitating communication among immune cells, controlling genes, regulating transcription factors, and governing inflammation, differentiation, proliferation and the secretion of antibodies.8 Genetic polymorphisms of interleukins play a critical role in their activity, and could alter cytokine function and dysregulate their expression.9 Therefore, individual genetic differences may be closely related to the development of type 2 diabetes mellitus. Interleukin 10 (IL-10) is a multifunctional regulatory cytokine involved in the inflammatory response that functions as a general inhibitor of the proliferative and cytokine response of both type 1 and type 2 helper T cells.10 The gene encoding IL-10, IL10, is located on chromosome 1 (1q31– 1q32).11,12 Three functional promoter single
703 nucleotide polymorphisms (SNPs) in the IL10 locus at –1082G/A (rs1800896), 819 T/C (rs1800871) and 592 A/C (rs1800872) from the transcriptional start site have been confirmed, and there are indications that they influence IL10 gene transcription.13 One meta-analysis found there was an association between the IL10 gene 1082G/A polymorphism and type 2 diabetes mellitus, but no association was found for 819 T/C or 592 A/C.14 However, the results of various studies have been inconsistent.4,14 The aim of the present case–control study was to investigate the relationship between SNPs of the IL10 gene (1082 G/A [rs1800896], 819 T/C [rs1800871] and 592 A/C [rs1800872]) and the risk of type 2 diabetes mellitus in a Chinese population.
Patients and methods Study population This case–control study recruited consecutive patients diagnosed with type 2 diabetes mellitus according to World Health Organization criteria who were screened at the Department of Endocrinology, First Affiliated Hospital of the People’s Liberation Army, Beijing, China between January 2010 and November 2012.15 Patients with acute or chronic inflammatory disease, infections, cancer, or end-stage liver or kidney diseases were excluded from the study. Healthy control subjects were recruited from a health examination centre of the First Affiliated Hospital of the People’s Liberation Army during the same period, and were excluded if they had a history of acute or chronic inflammatory disease, infections, cancer, end-stage liver or kidney disease, coronary artery disease, or other metabolic disorders. A ‘super control group’ (age >75 years) was selected from the control group. Baseline clinical and demographic data for all study participants were collected from
704 the medical records. Body mass index (BMI) was calculated as weight (kg)/height2 (m2). Venous blood samples (5 ml) were obtained from all study participants after an 8-h fast. The blood samples were clotted at room temperature for 15 min and then serum was obtained by centrifugation at 2000 g for 15 min at 10 C (EppendorfÕ Centrifuge; Eppendorf [Shanghai] International Trade Company, Beijing, China). Serum samples were used for the determination of glucose, insulin, glycosylated haemoglobin (HbA1c), and lipid profile including serum total cholesterol (TC), triglycerides (TG), highdensity-lipoprotein cholesterol (HDL-C) and low-density-lipoprotein cholesterol (LDL-C), all of which were measured on the DimensionÕ RxL MaxÕ Integrated Chemistry System (Siemens, Erlangen, Germany). The Ethics Committee of the First Affiliated Hospital of the People’s Liberation Army reviewed and approved the study (no. 201001037). All participants provided written informed consent.
Genotyping All study participants provided 5 ml of venous blood, with 0.5 mg/ml ethylenediaminetetra-acetic acid used as the anticoagulant. The blood samples were kept at –20 C until use, Genomic DNA was extracted using the TIANamp Blood DNA Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. The genotyping of the IL10 gene SNPs 1082 G/A (rs1800896), 819 T/C (rs1800871) and 592 A/C (rs1800872) was conducted in a 384-well plate format on the MassARRAYÕ Analyzer 4 system (SequenomÕ , San Diego, CA, USA), which combines polymerase chain reaction (PCR) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technologies. The primers used for the IL10 gene SNPs 1082 G/ A (rs1800896), 819 T/C (rs1800871) and
Journal of International Medical Research 42(3) 592 A/C (rs1800872) for PCR amplification were designed using SequenomÕ Assay Design version 3.1 software (SequenomÕ ), according to the manufacturer’s instructions. For –1082 G/A (rs1800896), the forward and reverse primer sequences were 50 -AGAAGTCCTGATGTCACTGC30 and 50 -AAGTCAGGATTCCATGGAG30 , respectively. The forward and reverse primer sequences for 819 T/C (rs1800871) were 50 -ATGGTGTACAGTAGGGTGA G-30 and 50 -TTTCCACCTCTTCAGCTGT C-30 , respectively. The forward and reverse primer sequences for 592 A/C (rs1800872) were 50 -AAGAGGTGGAAACATGTGC C-30 and 50 -TACCCAAGACTTCTCCTT GC-30 , respectively. Each PCR reaction mix comprised 50 ng genomic DNA, 200 mM dNTP, 2.5 U Taq DNA polymerase (Promega, Madison, WI, USA) and 200 mM primers, in a total volume of 20 ml. The cycling programme involved preliminary denaturation at 94 C for 2 min, followed by 35 cycles of denaturation at 94 C for 30 s and annealing at 64 C for 30 s, with a final extension at 72 C for 3 min. The PCR products were verified by 1.0% agarose gel electrophoresis and visualized using ethidium bromide staining and ultraviolet light. The PCR products of rs1800896, rs1800871 and rs1800872 were 269 base pairs (bp), 238 bp and 300 bp, respectively. Reproducibility was verified by repeat analysis of a randomly chosen subgroup of 10% of the subjects.
Statistical analyses All statistical analyses were performed using the SPSSÕ statistical package, version 11.0 (SPSS Inc., Chicago, IL, USA) for WindowsÕ . Continuous and categorical variables were expressed as mean SD and n (%) of study participants, respectively. Categorical variables from patients and control subjects were compared using the 2-test and continuous variables were
Bai et al.
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compared using the Student’s t-test. The Hardy–Weinberg equilibriums between groups were compared using the 2-test. Unconditional logistic regression was conducted to assess the effects of the IL10 gene SNPs –1082 G/A (rs1800896), 819 T/C (rs1800871) and 592 A/C (rs1800872) on the risk of type 2 diabetes mellitus in codominant, dominant and recessive models, with results expressed as odds ratios (OR) and corresponding 95% confidence intervals (CI). All P-values were two sided and a P-value < 0.05 was considered statistically significant.
Results Of the 396 patients with type 2 diabetes who were screened, 364 (91.9%) were eligible and
were included in the study (148 female/216 male). For the control group, 725 healthy subjects were screened and 677 (93.4%) were recruited to the study (307 female/360 male). The super control group (age >75 years; n ¼ 37) selected from the control group comprised 22 women and 15 men. Baseline clinical characteristics of the study participants are shown in Table 1. Patients with type 2 diabetes had a significantly higher mean BMI than the healthy control subjects (P < 0.001). The mean fasting glucose, fasting insulin and HbA1c of the patients with type 2 diabetes were all significantly higher than those of the control and super control subjects (P < 0.001 for all comparisons). The serum lipid profile of the patients with type 2 diabetes was significantly different from that of the control and super control subjects,
Table 1. Clinical and demographic characteristics of patients with type 2 diabetes mellitus and healthy control subjects who were included in a study investigating the association between single nucleotide polymorphisms of the interleukin 10 gene and the risk of type 2 diabetes mellitus.
Characteristic Age, years Sex Female Male BMI, kg/m2 Time since diabetes diagnosis, years Fasting glucose, mmol/l Fasting insulin, pmol/l HbA1c, % TC, mg/dl TG, mg/dl HDL-C, mg/dl LDL-C, mg/dl
Patients with type 2 diabetes mellitus n ¼ 364
Control subjects n ¼ 677
Statistical significancea
Super control subjects (age >75 years) n ¼ 37
Statistical significancea
47.6 7.3
51.6 5.7
P < 0.001
75.4 7.9
P < 0.001
148 (40.7) 216 (59.3) 24.7 2.5 7.4 2.2
307 (45.3) 360 (53.2) 22.9 2.8 –
NS P < 0.001 –
15 22 23.2 2.6 –
NS P < 0.001 –
8.7 3.1 59.6 18.4 8.7 0.9 192.6 17.5 172.5 17.4 49.7 11.8 140.8 10.3
4.5 1.4 51.4 17.3 5.2 0.7 157.4 9.2 131.4 18.5 48.4 11.6 118.7 9.3
P < 0.001 P < 0.001 P < 0.001 P < 0.001 P < 0.001 P ¼ 0.04 P < 0.001
5.7 1.9 53.2 14.5 5.3 0.5 156.7 8.3 135.3 15.6 48.7 10.7 121.2 9.6
P < 0.001 P ¼ 0.02 P < 0.001 P < 0.001 P < 0.001 P ¼ 0.31 P < 0.001
Data presented as mean SD or n (%) of study participants. a Comparisons between patients and control subjects were made using the Student’s t-test for continuous variables and 2-test for categorical variables. BMI, body mass index; HbA1c, glycosylated haemoglobin; TC, total cholesterol; TG, triglycerides; HDL-C, high-densitylipoprotein cholesterol; LDL-C, low-density-lipoprotein cholesterol; NS, no statistically significant difference (P 0.05).
706 with significantly higher TC, TG and LDLC levels (P < 0.05 for all comparisons). The HDL-C of patients with type 2 diabetes was significantly higher than that in control subjects. The allele and genotype distributions of the IL10 gene SNPs 1082 G/A (rs1800896), 819 T/C (rs1800871) and 592 A/C (rs1800872) were found to be in Hardy–Weinberg equilibrium in the control group (Table 2). Unconditional logistic regression analyses showed that subjects carrying the IL10 gene 1082 GG genotype had a significantly increased risk of type 2 diabetes mellitus compared with AA genotype, with an adjusted OR of 1.57 (95% CI 1.03, 2.68) (P ¼ 0.036). Individuals carrying IL10 gene 1082 GG had a 1.82-fold increased risk of type 2 diabetes mellitus compared with GA þ AA genotype, with an adjusted OR of 1.82 (95% CI 1.09, 2.94) (P ¼ 0.021). Similarly, those patients carrying the IL10 gene 592 AA genotype had a significantly increased risk of type 2 diabetes mellitus in co-dominant and recessive models, with an adjusted OR of 1.63 (95% CI 1.06, 2.53) and 1.67 (95% CI 1.14, 2.75), respectively (P ¼ 0.026 and 0.001). Possibly due to the small size of the super control group, we did not find significant association between SNPs of the IL10 gene and risk of type 2 diabetes mellitus. Moreover, in determining whether the genotype distributions had sex-specific features, but we found IL10 1082 G/A and 592 A/C gene polymorphisms increased the risk for type 2 diabetes mellitus in both male and female subjects. Therefore, sex did not influence the association between IL10 1082 G/A and 592 A/C gene polymorphisms and the risk of type 2 diabetes mellitus. The results of the analyses of the interaction between the two IL10 gene 1082 G/ A and 592 A/C polymorphisms on the susceptibility to type 2 diabetes mellitus are shown in Table 3. Patients carrying both the 1082 GA þ GG and 592 AC þ AA
Journal of International Medical Research 42(3) genotypes had a significantly increased risk of type 2 diabetes mellitus (OR 2.03, 95% CI 1.24, 3.15; P < 0.001), indicating that the two genotypes had a cumulative effect on the risk of type 2 diabetes mellitus.
Discussion Interleukin 10 is an important immunoregulatory cytokine that is produced by activated T cells, monocytes, B cells and thymocytes.10 It plays an important role in stimulating and suppressing the immune response, and functions as an immune response modulator.16–18 Previous studies have reported several polymorphic sites in the IL10 gene promoter region upstream of the transcription start site, including three polymorphisms at positions 1082 A/G, 819 C/T and 592 A/C.13 Some previous studies reported that these IL10 gene promoter polymorphisms were associated with the risk of type 2 diabetes mellitus.19–21 This present case–control study investigated the association between genetic polymorphisms of the IL10 gene promoter region and the risk of type 2 diabetes mellitus. The main findings of the present study were that the 1082G/A and 592 A/C variants carried a significantly increased risk of type 2 diabetes mellitus, while the 1082 GA þ GG and 592 AC þ AA genotypes when carried together had a synergistic effect on the risk of type 2 diabetes mellitus. However, the 819 T/C gene polymorphism did not appear to be associated with the risk of type 2 diabetes mellitus. Previous studies have found an association between IL10 genetic variants and the risk of type 2 diabetes mellitus.10,19–22 These current findings are consistent with several studies.23,24 One study conducted in India indicated that the IL10 gene 1082 A/G genotype significantly increased the risk of type 2 diabetes mellitus.23 Another study from Iran found that the IL10 gene polymorphism 592 A/C was associated with
495 (73.1) 129 (19.1) 53 (7.8) 495 (73.1) 182 (26.9) 624 (92.2) 53 (7.8) 295 (43.6) 336 (49.6) 46 (6.8) 295 (43.6) 382 (56.4) 631 (93.2) 46 (6.8) 313 (46.2) 299 (44.2) 65 (9.6) 313 (46.2) 364 (53.8) 612 (90.4) 65 (9.6)
252 (69.2) 72 (19.8) 40 (11.0)
252 (69.2) 112 (30.8)
324 (89.0) 40 (11.0)
151 (41.5) 183 (50.3) 30 (8.2)
151 (41.5) 213 (58.5)
334 (91.8) 30 (8.2)
153 (42.0) 162 (44.5) 49 (13.5)
153 (42.0) 211 (58.0)
315 (86.6) 49 (13.5)
Control subjects n ¼ 677
1.00 (reference) 1.46 (0.96, 2.21)
1.00 (reference) 1.19 (0.91, 1.55)
1.00 (reference) 1.11 (0.83, 1.47) 1.54 (1.00, 2.39)
1.00 (reference) 1.23 (0.74, 2.04)
1.00 (reference) 1.09 (0.84, 1.42)
1.00 (reference) 1.06 (0.81, 1.40) 1.27 (0.74, 2.16)
1.00 (reference) 1.45 (0.92, 2.29)
1.00 (reference) 1.21 (0.90, 1.61)
1.00 (reference) 1.10 (0.78, 1.54) 1.45 (0.91, 2.30)
Crude OR (95% CI)
– NS
– NS
– NS P ¼ 0.04
– NS
– NS
– NS NS
– NS
– NS
– NS NS
Statistical significancea
1.00 (reference) 1.67 (1.14, 2.75)
1.00 (reference) 1.32 (0.96, 1.73)
1.00 (reference) 1.18 (0.89, 1.52) 1.63 (1.06, 2.53)
1.00 (reference) 1.42 (0.83, 2.25)
1.00 (reference) 1.22 (0.91, 1.58)
1.00 (reference) 1.09 (0.87, 1.53) 1.32 (0.81, 2.26)
1.00 (reference) 1.82 (1.09, 2.94)
1.00 (reference) 1.48 (0.96, 2.13)
1.00 (reference) 1.22 (0.84, 1.63) 1.57 (1.03, 2.68)
Adjusted OR (95% CI)b
– P ¼ 0.001
– NS
– NS P ¼ 0.026
– NS
– NS
– NS NS
– P ¼ 0.021
– NS
– NS P ¼ 0.036
Statistical significancea
Data presented as n (%) of study participants. a The Hardy–Weinberg equilibriums between groups were compared using the 2-test. Unconditional logistic regression was conducted to assess the effects of the IL10 gene SNPs 1082 G/A (rs1800896), 819 T/C (rs1800871) and 592 A/C (rs1800872) on risk of type 2 diabetes mellitus, with results expressed as odds ratios (OR) and corresponding 95% confidence intervals (CI). b Adjusted for age, BMI, fasting glucose, fasting insulin, HbA1c, TC, TG, HDL-C and LDL-C. BMI, body mass index; HbA1c, glycosylated haemoglobin; TC, total cholesterol; TG, triglycerides; HDL-C, high-density-lipoprotein cholesterol; LDL-C, low-density-lipoprotein cholesterol; NS, no statistically significant difference (P 0.05).
1082G/A AA GA GG Dominant model AA GGþGA Recessive model GAþAA GG 819 T/C CC TC TT Dominant model CC TCþTT Recessive model TCþCC TT 592 A/C CC AC AA Dominant model CC ACþAA Recessive model CCþAC AA
SNP of the IL10 gene
Patients with type 2 diabetes mellitus n ¼ 364
Table 2. Genotype frequencies of three single nucleotide polymorphisms (SNP) of the interleukin 10 (IL10) gene in patients with type 2 diabetes mellitus and healthy control subjects.
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(D871) and 592 A/C (rs1800872) on risk of type 2 diabetes mellitus, with results expressed as odds ratios (OR) and corresponding 95% confidence intervals (CI). b Adjusted for age, BMI, fasting.
– NS NS P < 0.001 1.00 (reference) 0.95 (0.62, 1.41) 1.04 (0.77, 1.42) 2.03 (1.24, 3.15) – NS NS P ¼ 0.003 (reference) (0.58, 1.33) (0.70, 1.34) (1.19, 3.09) 1.00 0.88 0.97 1.92 183 (27.0) 130 (19.2) 312 (46.1) 52 (7.7) (26.1) (15.9) (43.1) (14.8) CC CC ACþAA ACþAA AA GAþGG AA GAþGG
95 58 157 54
592 A/C 1082 G/A
SNP of the IL10 gene
Patients with type 2 diabetes mellitus n ¼ 364
Control subjects n ¼ 677
Crude OR (95% CI)
Statistical significancea
Adjusted OR (95% CI)b
Statistical significancea
Journal of International Medical Research 42(3) Table 3. Interaction between two single nucleotide polymorphisms (SNP) of the interleukin 10 (IL10) gene in patients with type 2 diabetes mellitus and healthy control subjects.
708
immune diseases, including type 2 diabetes mellitus with and without nephropathy.24 A recent large meta-analysis pooled ten case– control studies and concluded that the IL10 1082 A/G polymorphism was strongly associated with the risk of type 2 diabetes mellitus, but no association was found between the 819 C/T or 592 A/C polymorphisms and the risk of type 2 diabetes mellitus.4 However, results have been inconsistent. For example, several studies reported no significant association between the 1082 G/A and 592 A/C variants and the risk of type 2 diabetes mellitus.4,25 Since these studies were conducted in different populations, it is difficult to make direct comparisons between them. Discrepancies may be due to differences in variant frequencies among various ethnic groups, and IL10 gene polymorphisms may play different roles in the development of type 2 diabetes mellitus depending on the population. This present study had a number of limitations. First, it was conducted in a single hospital and the participants may not have been representative of other areas within China. Secondly, the aetiology of type 2 diabetes mellitus involves multiple genes and environmental factors, so other genetic and environmental factors should be considered in further studies. Secondly, the genotype distributions of 1082 G/A and 819 T/C in both the case and control subjects were not consistent with Hardy– Weinberg equilibriums, which suggests the sample of this study might not represent the Chinese population. The main reason might be the relative small sample size. Therefore, further studies with large sample sizes are needed to confirm our results. In conclusion, the findings of this present study suggest that the IL10 1082 G/A and 592 A/C gene polymorphisms increase the risk for type 2 diabetes mellitus, while carrying the combination of the 1082 GA þ GG and 592 AC þ AA genotypes
Bai et al. had a synergistic effect on the risk of type 2 diabetes mellitus in a Chinese population. However, the 819 T/C polymorphism was not associated with the risk of type 2 diabetes mellitus. These results suggest that these polymorphisms could be potential targets for screening for the early detection of the risk of type 2 diabetes mellitus.
Declaration of conflicting interest The authors declare that there are no conflicts of interest.
Funding This research received no specific grant from any funding agency in the public, commercial or notfor-profit sectors.
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Association between interleukin 10 gene polymorphisms and risk of type 2 diabetes mellitus in a Chinese population
Journal of International Medical Research 2014, Vol. 42(3) 702–710 ! The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0300060513505813 imr.sagepub.com
Hua Bai1,*, Danqing Jing1, Aitao Guo2,* and Shinan Yin1
Abstract Objective: To investigate the relationship between the interleukin 10 (IL10) gene single nucleotide polymorphisms (SNP) 1082 G/A (rs1800896), 819 T/C (rs1800871) and 592 A/C (rs1800872) and risk of type 2 diabetes mellitus in a Chinese population. Methods: This case–control study recruited patients with type 2 diabetes mellitus and healthy control subjects. Genotyping of the 1082 G/A (rs1800896), 819 T/C (rs1800871) and 592 A/C (rs1800872) SNPs was conducted and genotype frequencies were compared between the two groups. Results: The study recruited 364 patients with type 2 diabetes mellitus and 677 healthy controls. Patients carrying the 1082 GG genotype had a significantly increased risk of type 2 diabetes mellitus (adjusted odds ratio [OR] 1.57, 95% confidence interval [CI] 1.03, 2.68), as did those patients carrying the 592 AA genotype (adjusted OR 1.63, 95% CI 1.06, 2.53). Subjects carrying both the 1082 GA þ GG and 592 AC þ AA genotypes had a significantly increased risk of type 2 diabetes mellitus (adjusted OR 2.03, 95% CI 1.24, 3.15). Conclusions: The SNPs 1082G/A and 592 A/C increased the risk for type 2 diabetes mellitus, and could be potential targets for screening for the early detection of the risk of type 2 diabetes mellitus.
Keywords Interleukin 10, type 2 diabetes mellitus, IL10 gene, polymorphism, predictive markers Date received: 9 August 2013; accepted: 29 August 2013 *These authors contributed equally to this work.
1
Department of Endocrinology, First Affiliated Hospital of the People’s Liberation Army, Beijing, China 2 Department of Pathology, General Hospital of the People’s Liberation Army, Beijing, China
Corresponding author: Dr Shinan Yin, Department of Endocrinology, First Affiliated Hospital of the General Hospital of the People’s Liberation Army, 51 Fucheng Road, Haidian District, Beijing 100048, China. Email: [email protected]
Creative Commons CC-BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 3.0 License (http://www.creativecommons.org/licenses/by-nc/3.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access page (http://www.uk.sagepub.com/aboutus/openaccess.htm).
Bai et al.
Introduction Type 2 diabetes mellitus is a common chronic disease, and its complications have become a major cause of morbidity, mortality and disability in both developed and developing countries.1 There were approximately 285 million people worldwide living with diabetes in 2010, 9.2 million of them in China.1 It is estimated that the number of patients with type 2 diabetes mellitus worldwide will reach 300 million by 2025.1 Although type 2 diabetes mellitus is induced by various environmental and genetic factors, its detailed aetiology is not yet fully understood.2 Several studies have reported that multiple cytokines may be involved in the regulation of the immune response, which has in turn been shown to play a role in the pathogenesis of type 2 diabetes mellitus.3,4 Interleukins are a diverse constellation of small cell signalling protein molecules, or cytokines, that regulate the function of the immune system in humans.5,6 Interleukins are produced predominantly by T cells, monocytes, macrophages and endothelial cells.7 Their functions include facilitating communication among immune cells, controlling genes, regulating transcription factors, and governing inflammation, differentiation, proliferation and the secretion of antibodies.8 Genetic polymorphisms of interleukins play a critical role in their activity, and could alter cytokine function and dysregulate their expression.9 Therefore, individual genetic differences may be closely related to the development of type 2 diabetes mellitus. Interleukin 10 (IL-10) is a multifunctional regulatory cytokine involved in the inflammatory response that functions as a general inhibitor of the proliferative and cytokine response of both type 1 and type 2 helper T cells.10 The gene encoding IL-10, IL10, is located on chromosome 1 (1q31– 1q32).11,12 Three functional promoter single
703 nucleotide polymorphisms (SNPs) in the IL10 locus at –1082G/A (rs1800896), 819 T/C (rs1800871) and 592 A/C (rs1800872) from the transcriptional start site have been confirmed, and there are indications that they influence IL10 gene transcription.13 One meta-analysis found there was an association between the IL10 gene 1082G/A polymorphism and type 2 diabetes mellitus, but no association was found for 819 T/C or 592 A/C.14 However, the results of various studies have been inconsistent.4,14 The aim of the present case–control study was to investigate the relationship between SNPs of the IL10 gene (1082 G/A [rs1800896], 819 T/C [rs1800871] and 592 A/C [rs1800872]) and the risk of type 2 diabetes mellitus in a Chinese population.
Patients and methods Study population This case–control study recruited consecutive patients diagnosed with type 2 diabetes mellitus according to World Health Organization criteria who were screened at the Department of Endocrinology, First Affiliated Hospital of the People’s Liberation Army, Beijing, China between January 2010 and November 2012.15 Patients with acute or chronic inflammatory disease, infections, cancer, or end-stage liver or kidney diseases were excluded from the study. Healthy control subjects were recruited from a health examination centre of the First Affiliated Hospital of the People’s Liberation Army during the same period, and were excluded if they had a history of acute or chronic inflammatory disease, infections, cancer, end-stage liver or kidney disease, coronary artery disease, or other metabolic disorders. A ‘super control group’ (age >75 years) was selected from the control group. Baseline clinical and demographic data for all study participants were collected from
704 the medical records. Body mass index (BMI) was calculated as weight (kg)/height2 (m2). Venous blood samples (5 ml) were obtained from all study participants after an 8-h fast. The blood samples were clotted at room temperature for 15 min and then serum was obtained by centrifugation at 2000 g for 15 min at 10 C (EppendorfÕ Centrifuge; Eppendorf [Shanghai] International Trade Company, Beijing, China). Serum samples were used for the determination of glucose, insulin, glycosylated haemoglobin (HbA1c), and lipid profile including serum total cholesterol (TC), triglycerides (TG), highdensity-lipoprotein cholesterol (HDL-C) and low-density-lipoprotein cholesterol (LDL-C), all of which were measured on the DimensionÕ RxL MaxÕ Integrated Chemistry System (Siemens, Erlangen, Germany). The Ethics Committee of the First Affiliated Hospital of the People’s Liberation Army reviewed and approved the study (no. 201001037). All participants provided written informed consent.
Genotyping All study participants provided 5 ml of venous blood, with 0.5 mg/ml ethylenediaminetetra-acetic acid used as the anticoagulant. The blood samples were kept at –20 C until use, Genomic DNA was extracted using the TIANamp Blood DNA Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. The genotyping of the IL10 gene SNPs 1082 G/A (rs1800896), 819 T/C (rs1800871) and 592 A/C (rs1800872) was conducted in a 384-well plate format on the MassARRAYÕ Analyzer 4 system (SequenomÕ , San Diego, CA, USA), which combines polymerase chain reaction (PCR) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technologies. The primers used for the IL10 gene SNPs 1082 G/ A (rs1800896), 819 T/C (rs1800871) and
Journal of International Medical Research 42(3) 592 A/C (rs1800872) for PCR amplification were designed using SequenomÕ Assay Design version 3.1 software (SequenomÕ ), according to the manufacturer’s instructions. For –1082 G/A (rs1800896), the forward and reverse primer sequences were 50 -AGAAGTCCTGATGTCACTGC30 and 50 -AAGTCAGGATTCCATGGAG30 , respectively. The forward and reverse primer sequences for 819 T/C (rs1800871) were 50 -ATGGTGTACAGTAGGGTGA G-30 and 50 -TTTCCACCTCTTCAGCTGT C-30 , respectively. The forward and reverse primer sequences for 592 A/C (rs1800872) were 50 -AAGAGGTGGAAACATGTGC C-30 and 50 -TACCCAAGACTTCTCCTT GC-30 , respectively. Each PCR reaction mix comprised 50 ng genomic DNA, 200 mM dNTP, 2.5 U Taq DNA polymerase (Promega, Madison, WI, USA) and 200 mM primers, in a total volume of 20 ml. The cycling programme involved preliminary denaturation at 94 C for 2 min, followed by 35 cycles of denaturation at 94 C for 30 s and annealing at 64 C for 30 s, with a final extension at 72 C for 3 min. The PCR products were verified by 1.0% agarose gel electrophoresis and visualized using ethidium bromide staining and ultraviolet light. The PCR products of rs1800896, rs1800871 and rs1800872 were 269 base pairs (bp), 238 bp and 300 bp, respectively. Reproducibility was verified by repeat analysis of a randomly chosen subgroup of 10% of the subjects.
Statistical analyses All statistical analyses were performed using the SPSSÕ statistical package, version 11.0 (SPSS Inc., Chicago, IL, USA) for WindowsÕ . Continuous and categorical variables were expressed as mean SD and n (%) of study participants, respectively. Categorical variables from patients and control subjects were compared using the 2-test and continuous variables were
Bai et al.
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compared using the Student’s t-test. The Hardy–Weinberg equilibriums between groups were compared using the 2-test. Unconditional logistic regression was conducted to assess the effects of the IL10 gene SNPs –1082 G/A (rs1800896), 819 T/C (rs1800871) and 592 A/C (rs1800872) on the risk of type 2 diabetes mellitus in codominant, dominant and recessive models, with results expressed as odds ratios (OR) and corresponding 95% confidence intervals (CI). All P-values were two sided and a P-value < 0.05 was considered statistically significant.
Results Of the 396 patients with type 2 diabetes who were screened, 364 (91.9%) were eligible and
were included in the study (148 female/216 male). For the control group, 725 healthy subjects were screened and 677 (93.4%) were recruited to the study (307 female/360 male). The super control group (age >75 years; n ¼ 37) selected from the control group comprised 22 women and 15 men. Baseline clinical characteristics of the study participants are shown in Table 1. Patients with type 2 diabetes had a significantly higher mean BMI than the healthy control subjects (P < 0.001). The mean fasting glucose, fasting insulin and HbA1c of the patients with type 2 diabetes were all significantly higher than those of the control and super control subjects (P < 0.001 for all comparisons). The serum lipid profile of the patients with type 2 diabetes was significantly different from that of the control and super control subjects,
Table 1. Clinical and demographic characteristics of patients with type 2 diabetes mellitus and healthy control subjects who were included in a study investigating the association between single nucleotide polymorphisms of the interleukin 10 gene and the risk of type 2 diabetes mellitus.
Characteristic Age, years Sex Female Male BMI, kg/m2 Time since diabetes diagnosis, years Fasting glucose, mmol/l Fasting insulin, pmol/l HbA1c, % TC, mg/dl TG, mg/dl HDL-C, mg/dl LDL-C, mg/dl
Patients with type 2 diabetes mellitus n ¼ 364
Control subjects n ¼ 677
Statistical significancea
Super control subjects (age >75 years) n ¼ 37
Statistical significancea
47.6 7.3
51.6 5.7
P < 0.001
75.4 7.9
P < 0.001
148 (40.7) 216 (59.3) 24.7 2.5 7.4 2.2
307 (45.3) 360 (53.2) 22.9 2.8 –
NS P < 0.001 –
15 22 23.2 2.6 –
NS P < 0.001 –
8.7 3.1 59.6 18.4 8.7 0.9 192.6 17.5 172.5 17.4 49.7 11.8 140.8 10.3
4.5 1.4 51.4 17.3 5.2 0.7 157.4 9.2 131.4 18.5 48.4 11.6 118.7 9.3
P < 0.001 P < 0.001 P < 0.001 P < 0.001 P < 0.001 P ¼ 0.04 P < 0.001
5.7 1.9 53.2 14.5 5.3 0.5 156.7 8.3 135.3 15.6 48.7 10.7 121.2 9.6
P < 0.001 P ¼ 0.02 P < 0.001 P < 0.001 P < 0.001 P ¼ 0.31 P < 0.001
Data presented as mean SD or n (%) of study participants. a Comparisons between patients and control subjects were made using the Student’s t-test for continuous variables and 2-test for categorical variables. BMI, body mass index; HbA1c, glycosylated haemoglobin; TC, total cholesterol; TG, triglycerides; HDL-C, high-densitylipoprotein cholesterol; LDL-C, low-density-lipoprotein cholesterol; NS, no statistically significant difference (P 0.05).
706 with significantly higher TC, TG and LDLC levels (P < 0.05 for all comparisons). The HDL-C of patients with type 2 diabetes was significantly higher than that in control subjects. The allele and genotype distributions of the IL10 gene SNPs 1082 G/A (rs1800896), 819 T/C (rs1800871) and 592 A/C (rs1800872) were found to be in Hardy–Weinberg equilibrium in the control group (Table 2). Unconditional logistic regression analyses showed that subjects carrying the IL10 gene 1082 GG genotype had a significantly increased risk of type 2 diabetes mellitus compared with AA genotype, with an adjusted OR of 1.57 (95% CI 1.03, 2.68) (P ¼ 0.036). Individuals carrying IL10 gene 1082 GG had a 1.82-fold increased risk of type 2 diabetes mellitus compared with GA þ AA genotype, with an adjusted OR of 1.82 (95% CI 1.09, 2.94) (P ¼ 0.021). Similarly, those patients carrying the IL10 gene 592 AA genotype had a significantly increased risk of type 2 diabetes mellitus in co-dominant and recessive models, with an adjusted OR of 1.63 (95% CI 1.06, 2.53) and 1.67 (95% CI 1.14, 2.75), respectively (P ¼ 0.026 and 0.001). Possibly due to the small size of the super control group, we did not find significant association between SNPs of the IL10 gene and risk of type 2 diabetes mellitus. Moreover, in determining whether the genotype distributions had sex-specific features, but we found IL10 1082 G/A and 592 A/C gene polymorphisms increased the risk for type 2 diabetes mellitus in both male and female subjects. Therefore, sex did not influence the association between IL10 1082 G/A and 592 A/C gene polymorphisms and the risk of type 2 diabetes mellitus. The results of the analyses of the interaction between the two IL10 gene 1082 G/ A and 592 A/C polymorphisms on the susceptibility to type 2 diabetes mellitus are shown in Table 3. Patients carrying both the 1082 GA þ GG and 592 AC þ AA
Journal of International Medical Research 42(3) genotypes had a significantly increased risk of type 2 diabetes mellitus (OR 2.03, 95% CI 1.24, 3.15; P < 0.001), indicating that the two genotypes had a cumulative effect on the risk of type 2 diabetes mellitus.
Discussion Interleukin 10 is an important immunoregulatory cytokine that is produced by activated T cells, monocytes, B cells and thymocytes.10 It plays an important role in stimulating and suppressing the immune response, and functions as an immune response modulator.16–18 Previous studies have reported several polymorphic sites in the IL10 gene promoter region upstream of the transcription start site, including three polymorphisms at positions 1082 A/G, 819 C/T and 592 A/C.13 Some previous studies reported that these IL10 gene promoter polymorphisms were associated with the risk of type 2 diabetes mellitus.19–21 This present case–control study investigated the association between genetic polymorphisms of the IL10 gene promoter region and the risk of type 2 diabetes mellitus. The main findings of the present study were that the 1082G/A and 592 A/C variants carried a significantly increased risk of type 2 diabetes mellitus, while the 1082 GA þ GG and 592 AC þ AA genotypes when carried together had a synergistic effect on the risk of type 2 diabetes mellitus. However, the 819 T/C gene polymorphism did not appear to be associated with the risk of type 2 diabetes mellitus. Previous studies have found an association between IL10 genetic variants and the risk of type 2 diabetes mellitus.10,19–22 These current findings are consistent with several studies.23,24 One study conducted in India indicated that the IL10 gene 1082 A/G genotype significantly increased the risk of type 2 diabetes mellitus.23 Another study from Iran found that the IL10 gene polymorphism 592 A/C was associated with
495 (73.1) 129 (19.1) 53 (7.8) 495 (73.1) 182 (26.9) 624 (92.2) 53 (7.8) 295 (43.6) 336 (49.6) 46 (6.8) 295 (43.6) 382 (56.4) 631 (93.2) 46 (6.8) 313 (46.2) 299 (44.2) 65 (9.6) 313 (46.2) 364 (53.8) 612 (90.4) 65 (9.6)
252 (69.2) 72 (19.8) 40 (11.0)
252 (69.2) 112 (30.8)
324 (89.0) 40 (11.0)
151 (41.5) 183 (50.3) 30 (8.2)
151 (41.5) 213 (58.5)
334 (91.8) 30 (8.2)
153 (42.0) 162 (44.5) 49 (13.5)
153 (42.0) 211 (58.0)
315 (86.6) 49 (13.5)
Control subjects n ¼ 677
1.00 (reference) 1.46 (0.96, 2.21)
1.00 (reference) 1.19 (0.91, 1.55)
1.00 (reference) 1.11 (0.83, 1.47) 1.54 (1.00, 2.39)
1.00 (reference) 1.23 (0.74, 2.04)
1.00 (reference) 1.09 (0.84, 1.42)
1.00 (reference) 1.06 (0.81, 1.40) 1.27 (0.74, 2.16)
1.00 (reference) 1.45 (0.92, 2.29)
1.00 (reference) 1.21 (0.90, 1.61)
1.00 (reference) 1.10 (0.78, 1.54) 1.45 (0.91, 2.30)
Crude OR (95% CI)
– NS
– NS
– NS P ¼ 0.04
– NS
– NS
– NS NS
– NS
– NS
– NS NS
Statistical significancea
1.00 (reference) 1.67 (1.14, 2.75)
1.00 (reference) 1.32 (0.96, 1.73)
1.00 (reference) 1.18 (0.89, 1.52) 1.63 (1.06, 2.53)
1.00 (reference) 1.42 (0.83, 2.25)
1.00 (reference) 1.22 (0.91, 1.58)
1.00 (reference) 1.09 (0.87, 1.53) 1.32 (0.81, 2.26)
1.00 (reference) 1.82 (1.09, 2.94)
1.00 (reference) 1.48 (0.96, 2.13)
1.00 (reference) 1.22 (0.84, 1.63) 1.57 (1.03, 2.68)
Adjusted OR (95% CI)b
– P ¼ 0.001
– NS
– NS P ¼ 0.026
– NS
– NS
– NS NS
– P ¼ 0.021
– NS
– NS P ¼ 0.036
Statistical significancea
Data presented as n (%) of study participants. a The Hardy–Weinberg equilibriums between groups were compared using the 2-test. Unconditional logistic regression was conducted to assess the effects of the IL10 gene SNPs 1082 G/A (rs1800896), 819 T/C (rs1800871) and 592 A/C (rs1800872) on risk of type 2 diabetes mellitus, with results expressed as odds ratios (OR) and corresponding 95% confidence intervals (CI). b Adjusted for age, BMI, fasting glucose, fasting insulin, HbA1c, TC, TG, HDL-C and LDL-C. BMI, body mass index; HbA1c, glycosylated haemoglobin; TC, total cholesterol; TG, triglycerides; HDL-C, high-density-lipoprotein cholesterol; LDL-C, low-density-lipoprotein cholesterol; NS, no statistically significant difference (P 0.05).
1082G/A AA GA GG Dominant model AA GGþGA Recessive model GAþAA GG 819 T/C CC TC TT Dominant model CC TCþTT Recessive model TCþCC TT 592 A/C CC AC AA Dominant model CC ACþAA Recessive model CCþAC AA
SNP of the IL10 gene
Patients with type 2 diabetes mellitus n ¼ 364
Table 2. Genotype frequencies of three single nucleotide polymorphisms (SNP) of the interleukin 10 (IL10) gene in patients with type 2 diabetes mellitus and healthy control subjects.
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(D871) and 592 A/C (rs1800872) on risk of type 2 diabetes mellitus, with results expressed as odds ratios (OR) and corresponding 95% confidence intervals (CI). b Adjusted for age, BMI, fasting.
– NS NS P < 0.001 1.00 (reference) 0.95 (0.62, 1.41) 1.04 (0.77, 1.42) 2.03 (1.24, 3.15) – NS NS P ¼ 0.003 (reference) (0.58, 1.33) (0.70, 1.34) (1.19, 3.09) 1.00 0.88 0.97 1.92 183 (27.0) 130 (19.2) 312 (46.1) 52 (7.7) (26.1) (15.9) (43.1) (14.8) CC CC ACþAA ACþAA AA GAþGG AA GAþGG
95 58 157 54
592 A/C 1082 G/A
SNP of the IL10 gene
Patients with type 2 diabetes mellitus n ¼ 364
Control subjects n ¼ 677
Crude OR (95% CI)
Statistical significancea
Adjusted OR (95% CI)b
Statistical significancea
Journal of International Medical Research 42(3) Table 3. Interaction between two single nucleotide polymorphisms (SNP) of the interleukin 10 (IL10) gene in patients with type 2 diabetes mellitus and healthy control subjects.
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immune diseases, including type 2 diabetes mellitus with and without nephropathy.24 A recent large meta-analysis pooled ten case– control studies and concluded that the IL10 1082 A/G polymorphism was strongly associated with the risk of type 2 diabetes mellitus, but no association was found between the 819 C/T or 592 A/C polymorphisms and the risk of type 2 diabetes mellitus.4 However, results have been inconsistent. For example, several studies reported no significant association between the 1082 G/A and 592 A/C variants and the risk of type 2 diabetes mellitus.4,25 Since these studies were conducted in different populations, it is difficult to make direct comparisons between them. Discrepancies may be due to differences in variant frequencies among various ethnic groups, and IL10 gene polymorphisms may play different roles in the development of type 2 diabetes mellitus depending on the population. This present study had a number of limitations. First, it was conducted in a single hospital and the participants may not have been representative of other areas within China. Secondly, the aetiology of type 2 diabetes mellitus involves multiple genes and environmental factors, so other genetic and environmental factors should be considered in further studies. Secondly, the genotype distributions of 1082 G/A and 819 T/C in both the case and control subjects were not consistent with Hardy– Weinberg equilibriums, which suggests the sample of this study might not represent the Chinese population. The main reason might be the relative small sample size. Therefore, further studies with large sample sizes are needed to confirm our results. In conclusion, the findings of this present study suggest that the IL10 1082 G/A and 592 A/C gene polymorphisms increase the risk for type 2 diabetes mellitus, while carrying the combination of the 1082 GA þ GG and 592 AC þ AA genotypes
Bai et al. had a synergistic effect on the risk of type 2 diabetes mellitus in a Chinese population. However, the 819 T/C polymorphism was not associated with the risk of type 2 diabetes mellitus. These results suggest that these polymorphisms could be potential targets for screening for the early detection of the risk of type 2 diabetes mellitus.
Declaration of conflicting interest The authors declare that there are no conflicts of interest.
Funding This research received no specific grant from any funding agency in the public, commercial or notfor-profit sectors.
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