Association of IL-10 and IL-6 Gene Polymorphisms with Type 2 Diabetes Mellitus among Egyptian Patients

Recent findings indicate that certain cytokines are capable of interfering with insulin sensitive glucose uptake and induce insulin resistance. To test for the association of IL-10–1082 and IL-6–174 genetic polymorphisms with type 2 diabetes mellitus in Egyptian patients. Participants included 69 cases with type 2 DM (31 males and 38 females) with a median age of 57 years in addition to 98 healthy unrelated controls from the Nile Delta region, Egypt. Compared to controls, cases with type 2 diabetes mellitus showed a highly significant increase in the frequency of IL-10–1082 GG genotype (p<0.001, OR = 11.24); higher frequency of IL-10–1082 G allele (p<0.001, OR = 2.2) with a significantly lower frequency of IL-10–1082 GA genotype (p<0.001, OR = 0.22). On the other hand, the IL-6–174 CC genotype frequency was significantly higher in the diabetic group (p<0.001, OR = 5.41) in contrast to the IL-6–174 GC genotype that showed significantly lower frequency (p=0.005, OR = 0.31) with a non significant differences in either G or C allelic frequencies between diabetic and control groups (p>0.05). Polymorphisms related to IL-10–1082 GG and IL-6–174 CC genotypes may be considered as a risk factor for type 2 diabetes mellitus among Egyptian subjects with a potential impact on family counseling


INTRODUCTION
Diabetes mellitus is a growing disease that was estimated to affect roughly 6% of the world's population (1).Type 2 diabetes is by far the most common type of diabetes which accounts for about 90-95% of those with diabetes (2), and is characterized by variable degrees of insulin deficiency and resistance.It is a common disorder with a prevalence that rises markedly with increasing degree of obesity and sedentary lifestyle (3).Type 2 DM has also been recognized as an immune mediated disease leading to impaired insulin signaling and selective destruction of insulin producing beta cells in which cytokines play an important role (4).Cytokines are immunomodulatory proteins or glycoproteins which control or modulate the activities of cells within the immune system (5).The production of many cytokines is under genetic control and polymorphisms have been identified within a large number of these genes (6).Pro and anti-inflammatory cytokines play a key role in identifying patients at elevated risk for several diseases (7).Previous studies indicate that certain pro and/or anti-inflammatory cytokines are capable of interfering with insulin sensitive glucose uptake and induce insulin resistance (8).IL-6 is an interleukin that acts as both a pro-inflammotry and anti-inflammotry cytokine encoded by IL-6 gene.Its location at 7p21 and -174 polymorphism is located in its promoter region.It controls transcriptional activity of IL-6 which has an important role in type 1 DM (9).IL-10 is an anti-inflammatory and immunosuppressive substance produced within the body and plays a role in the regulation of immune responses (10).As its expression is relatively delayed, release of IL-10 provides an efficient autocrine mechanism for regulating proinflammatory cytokine production (11).The aim of the present work is to investigate the association of polymorphisms of IL-6-174 and IL-10-1082 genes to the susceptibility and severity of type 2 DM among cases from the Nile Delta region of Egypt.

MATERIALS AND METHODS
Participants included 69 cases with type 2 diabetes; 31 males and 38 females.Their age ranged between 40-78 years (mean±SD = 57.38 ± 7.67 years, median= 57 years).They were selected from the Department of Endocrinology, Specialized Medical Hospital, Mansoura University, Egypt.All patients had a diagnosis of estab-lished type 2 diabetes mellitus on the basis of medical history, clinical examination stressing on items as pulse, mean blood pressure (diastolic BP + 1/3 pulse pressure) (12), body mass index (BMI = weight (kg) / height2 (m) and laboratory tests as blood and urine glucose, insulin, glucagon, c-peptide and haemoglobin A1C (HBA1C).Exclusion criteria included any patient with gross or microscopic proteinuria, with renal affection related or not related to DM, with systemic or blood disease that may affect the kidneys as SLE, leukemia and lymphoma and cases with heavy urinary tract infection.Diagnosis of microalbuminuria was done by testing the first fasting mid-stream urine sample with Micral-2 test strips which is specific semi-quantitative test for detection of microalbuminuria.Cases were compared to a control group of 98 unrelated healthy subjects of matched age and sex from the same locality.They were proved healthy and euglycemic by clinical and laboratory tests.Written consent was taken from every participant in this study.3 ml venous blood sample on EDTA was taken from all patients and controls for analysis of cytokine gene polymorphisms by PCR.Another sample of 5 ml venous blood was taken fasting for assessment of glucose, insulin, glucagon, C-peptide and HBA1C.Quantitative determination of glucose was done by glucose oxidase method using spin react kit (Madrid, Spain) (13).Quantitative determination of insulin, C-peptide and glucagon was done by Amplified Sensitivity Immunoassay performed on microtitre plates (INS-EASIA Biosource, Belgium).The assay used monoclonal antibodies directed against distinct epitopes of the corresponding hormone.The amount of substrate turnover is determined colorimetrically by measuring the absorbance which is directly proportional to hormone concentration (14).HBA1C (glycosylated Haemoglobin) is measured by quantitative colorimetric method, determined as percent of glycohemoglobin in relation to total hemoglobin (Glycohemoglobin % = R of unknown/R of standard X concentration of glycohemoglobin of standard (Human GmbH, Germany).Homeostasis Model of Assessment of Insulin resistance (HOMA) was calculated as = Fasting glucose (mg/dl) X Fasting insulin /450 (15).

DNA Extraction and amplification
DNA was extracted from anticoagulated whole blood by Axygen kit (Axygen Scientific, USA).The IL-6-174 (G/C) SNP genotypes were determined in an allele-specific PCR including the G and C alleles.Primers sequences were (IL-6 F): 5-GAGCTTCTCTTTCGTTCC-3 and either

Statistical analysis
Data were processed and analyzed using the Statistical Package of Social Science (SPSS, version 15).Quantitative variables were described by their mean and standard deviation and compared using student t-test.On the other hand, the frequency of studied allelic polymorphisms among cases was compared to that of controls using Fisher's Exact test and odds ratio (OR) with 95% confidence intervals (CI).A minimum level of significance is considered at a p level of < 0.05.

RESULTS
Investigations related to the diagnosis of type 2 DM showed a highly significant increase in the fasting glucagon level compared to controls (222.48±48.30ng ⁄ L vs. 73.77±28.92ng ⁄ L, p<0.001) with a significantly lower C-peptide level (3.06±1.18ng⁄ml vs. 6.49±1.60 ng⁄ml, p<0.001).Regarding studied genetic polymorphisms, cases with type 2 DM showed a significantly higher frequency of IL-10-1082 GG genotype in comparison to controls (37.7% vs. 5.1%, P < 0.001) with a significantly lower frequency of the GA genotype (59.4% vs. 86.7%,p<0.001).Cases showed also a significant increase in ** p highly significant <0.001

DISCUSSION
Type 2 DM has been recognized as an immune mediated disease leading to impaired insulin signaling and selective destruction of insulin producing beta cells in which cytokines play an important role (4).The most prominent feature from the current study is the significant higher frequency of IL-10-1082 GG genotype with higher frequency of G allele among Egyptian type 2 DM cases versus control non-diabetic group.This was apparent in patients with poor control and cases with marked insulin resistance.These findings are in agreement with Kolla et al. who found significant increase in IL-10-1082 GG genotype in Indian patients with type 2 diabetes mellitus ( 16).The higher level of IL-10 GG genotype that is known to be a high producer may emphasize the immune modulatory action of IL-   (25).Taking into consideration the fact that genetic polymorphisms are population specific, we can come to the conclusion that IL-10-1082 GG and IL-6-174 CC gene polymorphisms are associated with type 2 DM among Egyptian population.In this respect, we recommend further wide scale studies in multiplex families using segregation and linkage analysis to document these observations.

Table 1 .
Frequencies of IL-10-1082 (G /A) genotype and allelic polymorphisms among type 2 diabetic patients compared to control subjects (IL-6 R1): 5-CCTATTGTGTCTTGCC-3 or (IL-6 R2):5-CCCTAGTTGTGTCTTGCG-3 Reactions contained 1 umol/L of each primer, 0.5 U Taq polymerase, 200 umol/L dNTPs mixture, and 1.5 mmol/L MgCl2 in 1X NH4 Cl buffer in addition to test DNA, to a final volume of 25 ul.Aamplification was performed on thermal cycler with 30 cycles each of 94 o C for 30 seconds, 54 o C for 60 seconds, and 72 o C for 60 seconds, followed by a final extension of 72 o C for 7 minutes.PCR products were resolved by agarose gel electrophoresis (3%) and visualized ethidium bromide with a positive band at 234 bp.IL-10-1082 polymorphism were determined in an IL-10 allele specific PCR including the IL-10 G or A alleles.Primers sequences were: (IL-10 F) 5-AGCAACACTCCTCGTCGCAAC-3 with (IL-10 R1) 5-CCTATCCCTACTTCCCCC-3 or (IL-10 R2) 5-CCTATCCCTACTTCCCCT-3 Reactions contained 20 umol/L of each primer, 0.5 U Taq Polymerase and 200 umol/L dNTPs mixture with 1.5 mmol/L MgCl2 in 1X NH4 Cl buffer in addition to test DNA, to a final volume of 25 ul.Amplification was performed on thermal cycler with 30 cycles each of 94 o C seconds, 60 o C for 60 seconds, 72 o C for 60 seconds, followed by a final extension of 72 o C for 7 minutes.PCR products were resolved by agarose electrophoresis (3%) and visualized ethidium bromide with a positive band at 179 bp.

Table 2 .
Frequencies (18)t was reported byWogensen et al.that IL-10 should not simply be regarded as an immune inhibitory cytokine, since, it possesses, powerful immunostimulatory properties as well(17).Nonetheless, Settin et al found no association with IL-10-1082 polymorphic variants in type 1 DM(18).

Table 3 .
(24)0-1082 and IL-6-174 genotype frequencies among cases with controlled versus uncontrolled diabetes and cases with variable degrees of insulin resistance significant increase in risk of type 2 DM in British subjects compared to other genotypes.Also they stated that IL-6-174 CC and GC genotypes is protective against type 2 DM, that is partially in agreement with our finding that IL-6-174 GC polymorphism was associated with decreased risk of type 2 DM(23).Similarly, Qi et al. stated that IL-6-174 G/C polymorphisms were not associated with risk of type 2 DM in American population(24).Vozarova et al. also -in contrast to our results-reported that IL-6-174 GC polymorphism and G allele was associated with increased risk of type 2 DM in Native Americans and Caucasians