TNF-α-308 and INF-γ + 874 Gene Polymorphisms in Relation to Susceptibility and Severity of Type 2 Diabetes Mellitus among Egyptian Cases

To explore the association of TNF-α-308 and IFNγ +874 genetic polymorphisms with type 2 diabetes mellitus (DM) in Egyptian cases. Participants included 207 cases with type 2 DM, 93 males and 114 females. Their mean age was 57 years. They were compared to a control group of 212 healthy unrelated subjects from the same locality. DNA was amplified using PCR with sequence specific primers for detection of polymorphism related to TNF-α-308 (G/A) and IFN-γ+874 (A/T). The combined homozygote pattern of TNF-α-308 (AA) and IFN-γ+874 (A/A) was significantly higher in diabetic cases versus controls (p=0.023) that was manifest (although non-significant) among complicated, uncontrolled cases and cases with marked insulin resistance. Polymorphism related to TNF-α-308 AA and IFN-γ+874 AA may be considered as a genetic biomarker for type 2 DM in Egyptian subjects with potential impact on family counseling and management.


INTRODUCTION
The main cause of type 2 diabetes mellitus (DM) is unclear.Recently, it has been recognized as an immune mediated disease in which cytokines play an important role (1,2).Cytokines are key mediators which regulate immune response; and their expression by immune cells depends on several factors such as infection, inflammation, hormonal conditions and also relevant gene polymorphisms (3).
TNF-α-308 is a multifunctional cytokine primarily produced by macrophages and fat cells.It can directly inhibit phosphorylation of insulin receptors' substrate and reduce glucose uptake by peripheral tissues (4).In human, the TNF-α gene is located within the highly polymorphic major histocompatibility complex region on chromosome 6p21.3(5).Many studies have shown that single nucleotide polymorphism (SNP) at position -308 G/A was associated with various inflammatory conditions including DM (6).IFN-γ is a Th1 cytokine which supports the immune system to perform cytolysis of target cells and also was reported to be increased in DM (7).IFN-γ gene intron-1 polymorphisms was speculated to influence immune complex disease susceptibility which is characterized by an imbalance of various immunoregulatory systems (8).
The aim of the present work is to investigate the association of polymorphisms of TNF-α-308 and IFN-γ +874 genes to the susceptibility and severity of type 2 DM among Egyptian cases from the Nile Delta region of Egypt.

MATERIAL AND METHODS
This study was conducted on 207 diabetic patients (93 males and 114 females) with an age range between 40-78 years (57.38±7.67 years).They were selected from the Department of Endocrinology and Diabetes, Specialized Medical Hospital, Mansoura University, Egypt.All patients had a diagnosis of established type 2 diabetes mellitus on the basis of medical history, clinical examination and laboratory tests.Fasting and post-prandial blood glucose were estimated using the glucose oxidase method (spin react kit, Madrid, Spain) (9).Quantitative determination of insulin, C-peptide and glucagon were done by amplified sensitivity immunoassay performed on microtitre plates (INS-EASIA Biosource, Belgium) using monoclonal antibodies directed against distinct epitopes of the corresponding hormone (10).Glycosylated hemoglobin (HbA1c) was measured by quantitative colorimetric method, determined as percent of glycohemoglobin in relation to total hemoglobin (Human GmbH, Germany).Homeostasis model of assessment of insulin resistance (HOMA) was calculated as =Fasting glucose (mg/dl) x Fasting insulin (IU/ml)/405 (11).Exclusion criteria included cases with systemic or blood diseases that may affect the kidney other than DM such as SLE, leukemia and lymphoma and cases with heavy urinary tract infection.
Cases were compared to a control group of 212 unrelated subjects of matched age and sex from the same locality.They were proven healthy and euglycemic by clinical and laboratory tests.A written consent was taken from every participant in this study.

Statistical analysis
Data were processed and analyzed using the Statistical Package of Social Science (SPSS, version 15).The frequency of studied allelic polymorphisms among cases was compared to that of controls describing number and percent of each and tested for positive association using Fisher's exact test and odds ratio (OR) with 95% confidence intervals (CI).A minimum level of p < 0.05 is considered significant.
On the other hand, comparing the combined genotype frequencies among cases and controls, it was noted that cases showed a significantly higher frequencies of the combined genotypes having homozygosity for both rare alleles (AA with AA) or either one of them (AA with AG or AG with AA) (Table 2, p= 0.023).Also, the homozygotic pattern TNF-α-308 (AA) with IFN-γ (AA) was higher in complicated versus non complicated cases, in patients with marked insulin resistance (HOMA >20 (15) ) and also higher in uncontrolled patients (HbA1c >7 ( 16) ) but didn't reach the statistical significance (Table 2).

DISCUSSION
Type 2 DM is the most common type of diabetes that is characterized by variable degree of insulin deficiency and resistance.Its prevalence rises markedly with increasing degree of obesity and sedentary life (17).The association of genetic polymorphism of inflammatory cytokines with type 2 DM is largely debatable, but re-cent findings indicate that certain proinflammatory cytokines are capable of interfering with insulin sensitive glucose uptake and can induce insulin resistance (18).
This study showed that the presence of the rare alleles of TNF-α-308 and IFN-γ +874 genes in combination either in a homozygosus of heterozygous forms was significantly higher in Egyptian cases with type 2 DM compared to controls.Also their frequency was higher in complicated cases, and in those with marked insulin resistance and with poor controlled glycaemia..These results probably point to the potential impact of the combined association of rare alleles of TNF-α (A) and INF-γ (A) on the susceptibility and severity of type 2 DM.As an agreement to this study, Kubaszek et al., reported -in their study among Finnish population-that the -308 A allele of the TNF-α gene was associated with two fold higher risk for type 2 DM, and was also a predictor for the conversion from impaired glucose tolerance (IGT) to type 2 DM (19).
Given that approximately 84% of the Egyptian type 2 diabetic cases were carriers of the TNF-α -308 A allele, we can speculate that these cases are high producers of TNF-α as the -308A allele had been shown to increase the transcription and expression of TNF-α and was also found to inhibit insulin signaling and impair insulin secretion ( 3,20).Furthermore, the gene frequency of A allele of TNF-α in Egyptian diabetic cases was relatively higher than that reported among other populations like Swedish (21), Northern Irish (22) , and Chinese diabetics (23).It is worth mentioning that testing the association of TNF-α -308 with type 1 diabetes among Egyptian cases showed similar results of significantly higher frequency of the AA genotype and A allele in cases compared to controls (24).

Table 1. Frequencies of TNF-α-308 (G/A) and IFN-γ +874 (A/T) genotype and allelic polymorphisms among type 2 diabetic Egyptian patients compared to control subjects
Nonetheless, these results are not in agreement with Furuta et al. who did not find any differences in allelic frequencies of the TNF-α -308 (G-A) polymorphism between type 2 diabetes and unrelated controls in Japanese patients (25).This probably points to the potential positive impact of the analysis of combined polymorphic variants of inflammatory genes -as in our casesrather than of one important single gene polymorphism.
As regard IFN-γ+874 (A/T) gene polymorphism, this study showed that the frequency of AA genotype was higher in Egyptian diabetic cases versus control subjects particularly among uncontrolled cases.These results predict that the Egyptian cases were low producers of IFN-γ as previously reported by Pravica et al. (14).This may draw the attention to the effect of the low In conclusion, considering the fact that genetic polymorphisms are population specific, it might be speculated that the rare A allele of both TNF-α-308 and INFγ +874 is associated with type 2 DM predisposing to complications, increased insulin resistance and poor control.These can be considered potential biomarkers with a probable impact on diagnosis and management.

Table 2 .
Distribution of cases in relation to combined genotypes with the rare alleles of TNF-α (A) and IFN-g (A) genes # Complications included diabetic neuropathy, nephropathy and retinopathy *significant p<0.05