الفهرس 
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Abstract
Hepatitis C virus (HCV) infection, a common chronic liver disease, continues to be a major public health problem. It is estimated that more than 185 million persons (2%–3% of the world population) are HCV infected, of them, around 71 million are chronically infected individuals and 34 million are chronically infected with HCV genotype 4. Approximately 399, 000 people die each year from hepatitis C, mostly from cirrhosis, hepatocellular carcinoma and liver failure.HCV has been classified into six major genotypes; HCV genotype 4 is common in the Middle East and in Africa including Egypt.
Egypt has the highest prevalence of HCV worldwide and ranks 5th amongst all countries for the burden of disease from viral hepatitis,; genotype 4 represents over 90% of HCV cases in Egypt. Recently, close to 15% of the adult population are seropositive for HCV and over four million individuals remain viraemic.
Type 2 diabetes is 1.8 to 2.5-fold higher in patients with chronic HCV infection and two to three folds more prevalent in HCV than in HBV infection. Moreover, hepatitis C viral eradication after antiviral treatment can lead to cure of type 2 diabetes mellitus. This offers a strong argument in favor of a causal relationship between HCV and T2DM.
Several hypotheses have been proposed to the mechanisms underlying HCV-mediated insulin resistance. HCV infection eventually leads to liver steatosis and fibrosis, increased oxidative stress and peroxidation, all of which trigger a cascade of inflammatory responses, thus contributing to the development of insulin resistance. Moreover, during HCV infection, tumor necrosis factor-α system is activated and interleukin-6 levels are increased, which disturb tyrosine phosphorylation of IRS-1.
Insulin-receptor (INSR) isan α2β2 heterotetramer disulfide-linked trans-membrane glycoprotein and a family member of tyrosine kinase receptors. It mediates the pleiotropic actions of insulin regulating glucose homeostasis.
Insulin receptor phosphorylates tyrosine residues of an intracellular cytoplasmic adaptor protein; the insulin receptor substrate (IRS). The IRS-1 is the first member of the family to be identified.The IRS-1 binding and phosphorylation leads to an increase in the high affinity glucose transporter-4 (GLUT4) molecules on the outer membrane of insulin-responsive tissues, including muscle cells and adipose tissue causing an increase in the uptake of glucose from blood into these tissues. Defects in muscle IRS-1 expression and function leads to impairment in the insulin signaling pathway resulting in insulin resistance and type 2 diabetes.
The insulin receptor is encoded by a single gene; INSR gene. The INSR gene is located on the short (p) arm of chromosome 19 and comprises 22 exons. Exons 17-22 encode the tyrosine kinase domain, and mutations in this region impair the function of the insulin receptor that may cause insulin resistance and hyperinsulinemia. Single nucleotide polymorphism (SNP) with C > T substitution at His 1058 position of INSR (rs 1799817) located in exon 17, was considered to be involved in insulin resistance.
The IRS-1 gene contains the entire 5’-untranslated region and protein coding region in a single exon and is localized on chromosome 2. A glycine to arginine substitution (GGG↔AGG substitutions) in codon 972 (Gly 972Arg) (rs 1801278) is the common polymorphism of the IRS-1 gene, which may have a pathogenic role in the development of type 2 diabetes due to insulin resistance and impaired insulin secretion.
Insulin receptor and the IRS proteins might be counter-regulated by degradation, differential expression, or modification by phosphorylation in cells expressing HCV core protein which inhibits the interactions of IRS-1 with both the insulin receptor and the downstream effectors of IRS-1. Taken together, these modulations may contribute, at least in part, to the induction of insulin resistance in hepatocytes expressing HCV core protein.
Single nucleotide polymorphisms in the insulin receptor and insulin receptor substrate-1 (IRS-1) gene were among the most frequently studied candidate genes in type 2 diabetes. To the best of our knowledge no much data are available on INSR H1085H
and IRS-1 G972R SNPs in HCV infected populations with type 2 diabetes. The present work aimed to study insulin receptorH1085H C > T (rs 1799817) and Insulin Receptor Substrate -1 G972R C > T (rs 1801278) single nucleotide polymorphisms in Egyptian patients suffering from chronic hepatitis C virus infection with Type 2 diabetes mellitus.
The present study was conducted on two hundred and two participants of 100 males and 102 females, divided as follow; the control group (group I) included 50 apparently healthyvolunteers of comparable age, sex and socioeconomic status as patients groups, group II included 50 type 2 diabetes mellitus patients without hepatitis C virus infection , group III included 52 chronic hepatitis C virus infected patients without type 2 diabetes mellitus and group IV included 50hepatitis C virus infected patients with type 2 diabetes mellitus.
Full history was taken from all subjects then the following laboratory investigations were done including: determination of fasting and postprandial serum glucose, glycated hemoglobin (HbA1C), triglycerides, cholesterol ( total, high and low density lipoprotein fractions), urea, creatinine ,uric acid levels, alanine aminotransferase (ALT), aspartate amino- transferase (AST) and gamma glutamyl transferase (GGT) activities. Insulin level was determined then HOMA IR and QUICKI index were calculated. Polymerase chain reaction (PCR) for hepatitis C virus (HCV) for groups III, IV, HCV antibodies for groups I and II and hepatitis B surface antigen for all groups were done.
Genomic DNA was extracted from peripheral blood leukocytes by using ion-exchange column chromatography. Genotyping of INSR H1085H (rs1799817) and IRS-1 G972R (rs 1801278) using polymerase chain reaction (PCR-RFLP) techniques with restriction enzymes PmlI and BstNI respectively were done.
The present study revealed that BMI and WHR were significantly higher in type 2 diabetes without HCV infection (group II) and HCV infected patients with T2DM (group IV) than control group. Serum levels of urea, creatinine and uric acid were significantly increased in diabetic groups (II and IV) than control group.
In the present work, Alanine aminotransferase (ALT) activity was significantly higher in patients groups (II,III and IV) than control group, while aspartate amino- transferase (AST) and gamma glutamyle transferase (GGT) activities were significantly higher in HCV infected patients ( groups III and IV) than control group.
In the current study, type 2 diabetic patients without HCV infection (group II)
and HCV infected patients with T2DM (group IV) showed significant increase in LDL-cholesterol levels than control group. Whereas, HDL- cholesterol level was significantly lower in diabetic groups (Groups II and IV) than control group. Total cholesterol and triglycerides levels were significantly higher in type 2 diabetes without HCV infection (group II) only than control group.
Fasting serum glucose, fasting insulin and HOMA-IR index were significantly higher in diabetic groups, (groups II and IV) than controls. Whereas, QUICKI index was significantly lower in these groups than controls. Moreover, there was a significant positive correlation between BMI and HOMA-IR in the studied patient groups (II, III and IV) and between HOMA-IR and ALT activity in type 2 diabetic patients without HCV infection ( group II). Whereas, there was a significant negative correlation between BMI and QUICKI index in the patient groups (II, III and IV).
In the present study, the molecular studies INSR H1085H genotype distribution did not deviate from the Hardy-Weinberg equilibrium among the participants. As regard INSR H1085H C > T genotype distribution, type 2 diabetes patient without HCV infection (group II) and HCV infected patients with T2DM (group IV) showed significant increase in TT genotypes and significant decrease in CC genotypes than the control group (group I). Also, the minor allele (T) frequency of both (group II) and (group IV) was significantly higher than control group. Moreover, testing the dominant model of inheritance revealed a statistically higher frequency of exposed genotypes ‘’CT&TT’’ among type 2 diabetes without HCV infection (group II) than control group with substantial increased in insulin resistance and T2DM risk among the exposed group (CT and TT) with adjusted Odds ratio for BMI, age and sex.
In the current work, testing the dominant model of inheritance revealed a statistically higher frequency of exposed genotypes ‘’CT&TT’’ among the HCV infected patients with type 2 diabetes (group IV) than control group but it became insignificant after adjustment of odds ratio for significant confounders as BMI , age and sex. Testing the recessive model, where the exposed group was composed of only subjects with the homo-mutant TT genotype was not applicable as the odds ratio cannot be estimated because of the absence of (TT) genotype among the control subjects.
In the present study, there was significant association between INSR H1085H heterozygous and homozygous mutant genotypes with high fasting serum glucose > 126 mg/dL even with diabetic drug intake , fasting insulin and HOMA-IR> 4.5, so it might be an indicator of poor glycemic control.
In the current study, IRS-1 G972R genotype distribution did not deviate from the Hardy-Weinberg equilibrium among the participants. Type 2 diabetes patient without HCV infection (group II), HCV infected patients without diabetes (group III) and HCV infected patients with T2DM (group IV) showed significant increase in AA and GA genotypes and significant decrease in GG genotypes than the control group. The minor allele (A) frequency of patient groups (groups II, III and IV) was significantly higher than controls.
In the present study, testing the dominant model of inheritance revealed a statistically higher frequency of exposed genotypes ‘’GA&AA’’ among T2DM
without HCV infection (group II) than control group with substantial increase in risk of insulin resistance and T2DM among the exposed group (GA and AA) with significant adjusted Odds ratio for BMI, age and sex.
In the current work, testing the dominant model of inheritance revealed a statistically higher frequency of exposed genotypes ‘’GA&AA’’ among the HCV infected patients without T2DM (group III) and HCV infected patients with T2DM (group IV) than control group but odds ratio became insignificant after adjustment for significant confounders
as BMI, age and sex. Testing the recessive model, where the exposed group was composed of only subjects with the homo-mutant AA genotype, was not applicable as the odds cannot be estimated because of the absence of (AA) genotype among the control subjects. Moreover, there was no significant association between INSR H 1085H C > T (rs 1799817) and IRS-1 G972R (rs 1801278)polymorphisms.