PREVALENCE OF -56C/T REGULATORY POLYMORPHISM IN INTERFERON-γ RECEPTOR 1 PROMOTER REGION KNOWN TO BE POTENTIAL RISK FACTOR FOR HEPATITIS B VIRUS INFECTION IN NORTH INDIAN HEALTHY POPULATION

PREVALENCE OF -56C/T REGULATORY POLYMORPHISM IN INTERFERON-γ RECEPTOR 1 PROMOTER REGION KNOWN TO BE POTENTIAL RISK FACTOR FOR HEPATITIS B VIRUS INFECTION IN NORTH INDIAN HEALTHY POPULATION

Vikas Pahal ^*1, Rohit Randhawa ² and Krishan Swaroop Dadhich³

Department of Microbiology ¹, Department of Medical Biotechnology ²,  Dolphin (PG) College of Science and Agriculture, Chunnikalan, F.G. Sahib, Punjab, India

Department of Microbiology ³, Punjab University, Chandigarh (UT), India

ABSTRACT: Interferon-γ (IFN-γ), a defense molecule, mediates its response against viral infections like Hepatitis B virus by binding to interferon gamma receptor 1 (IFNGR1) on cytotoxic T lymphocytes (CTLs). Hence, interferon gamma receptor 1 (IFNGR1) gene expression is vital in the eradication of the HBV virus. It has been demonstrated by some studies that -56C/T SNPs in IFNGR1 gene promoter region can be correlated with HBV-infection resistance or susceptibility. It has also been shown that -56C SNP has protective effect against HBV infection in comparison to -56T SNP. This motivate us to find out the percentage prevalence of -56C/T SNPs in our healthy population. Genomic DNA was extracted using standard phenol-chloroform method from healthy blood donors. PCR was performed to amplify the 491 bp region of IFNGR1 gene promoter region and digested with AfeI restriction enzyme to observe the -56C/T SNPs. Allele ‘T’ has been observed as the overall dominant allele (65.71%) in our both male (71.79%) and female (58.06%) healthy population

Interferon gamma receptor 1,

IFN, HCC, Hepatitis B virus, SNPs

INTRODUCTION: There are more than 400 million chronic carriers of HBV infection worldwide. Depending upon the various genetic and viral factors this chronicity may progress to the end-stage infection-the hepatocellular carcinoma (HCC)^1-3. Eradication of the HBV infection from the host required both innate and adaptive immune system ⁴. MHC class-I restricted cytotoxic T lymphocytes (CTLs) are the main defense molecules against the HBV infection

In response to HBV-infection activated CTLs and natural killer (NK) cells produced interferon-γ (IFN-γ) molecules, which have ability to inhibit viral replication in the host ^4-9.

Interferon’s (IFN) are protein molecules which have several other functions including-induction of cell differentiation, inhibition of cell proliferation and angiogenesis, increase in the expression of class I and II MHC molecules, and increase the ability of healthy host cells to resist the new viral infection ¹⁰. It has been proved by many studies that CTLs which are activated by HBV-infection can eliminate the infection by inhibiting the viral gene expression and replication by releasing IFN-γ and TNF-α molecule ¹¹. Further, a microarray analysis demonstrated that IFN-γ and its receptor genes are the main fighter molecules in resolution of HBV-infection ^12-13.

Interferon-γ receptor 1 (IFNGR1) and interferon-γ receptor 2 (IFNGR2) are the part of a heterodimer receptor which mediate cellular response by IFN-γ molecule. IFNGR1 is responsible for binding of ligand and any change in the structure of this subunit may alter its sensitivity towards IFN-γ.It has been demonstrated by several studies that alterations of any kind in the gene of IFN-γ have an impact on HBV-mediated liver disease ¹⁴. Similarly, another study demonstrated the importance of genetic polymorphism of IFNGR1 receptor molecule with respect to the HBV clearance and persistence in their population. They observed a very significant association between the presence of -56C/T regulatory polymorphism in interferon-γ receptor 1 promoter and susceptibility to HBV infection ¹⁵. So, looking at the importance of this polymorphism we decided to study the prevalence of this polymorphism in our healthy population.

MATERIALS AND METHODS:

Samples collection:

As described earlier ^16-17 a total of 74 healthy volunteers were enrolled in the present study. All the isolates were negative to HCV, HBV and HIV infection with a median age 20.80+1.14 for male & 19.12+1.13 for female volunteers. All the isolates included in this study were from our college Dolphin (P.G) College of Science and Agriculture. The study was approved by the Institutional Ethics Committee (I.E.C) and a written consent was taken from each individual before inclusion in the present study.

 Chemicals:

All the chemicals used in this study were of analytical grade. PCR reagents, DNA ladders and MilliQ water were purchased from Chromous Biotech (INDIA). Trisbuffers, EDTA, agarose, glacial acid, ethidium bromide were purchased from Himedia (INDIA) and plastic-wares were purchased from AXYGEN (AXYGEN, Scientific Pvt. Ltd., New Delhi). Restrictive enzyme (AfeI) and Taq Polymerase enzyme were purchased from New England Bio Labs.

DNA extraction & amplification of IFNGR1 gene:

DNA was extracted from the blood of our isolates using standard chloroform-phenol method as described earlier ^16-17. Primers were designed on the basis of available literature ^18-19 and accuracy confirmation was done by using Primer 3 software. The primers were synthesized by Chromous Biotech on payment basis. To validate the amplification results a negative control (using MilliQ water in place of DNA) and a positive control (comprises the amplified 228-bp fragment of LDLR gene) were used.

For amplification of IFNGR1 gene(419 bp)from human DNA, we used single round PCR. For this single round of PCR, final master mixture had 25 μl volume and contained 0.1 µl (5U/µl) DNA Taq polymerase enzyme, 200 µM each of dNTP’s, 1 µM of each primer (Forward primer: 5’-GCCCATCTCAGCCCTGCTCA-3’ & reverse primer: 5’- ATTGTGGCTCGGCTGTGGCC-3’), 3.0 mM MgCl_2, 2.5µl of  10x Buffer (NH₄⁺) and 2.5 µl target DNA. The PCR reactions were performed in a mastercyclerusing a cycle profile of 94⁰C for 50 seconds  (denaturation), 63⁰Cfor 50 seconds (annealing) and 72⁰C for 55 seconds (extension) for 36 cycles followed by a final extension at 72⁰C for 8 minutes. Initial denaturation step was performed at 95⁰C for 7 minutes.

 Genotyping of IFNGR1 gene product for respective allele:

IFNGR1 gene has two alleles- allele ‘C’ and allele ‘T’. They can be differentiated by the analysis of restriction fragment length polymorphisms (RFLPs) pattern generated by specific restriction endonuclease enzyme (AfeI) on 419 bp product of IFNGR1 gene. Banding pattern for genotype 56T remains as an intact 419 bp band and for 56C it was 115 & 304 bp bands. The protocol was followed as per the manufacturer’s instructions. Briefly, 11 µl of PCR product of IFNGR1 gene was dispensed in 0.5 ml sterile eppendorf tubes containing 8.5µlMilliQ water followed by 3µl 10X buffer and 2.5µlrestriction endonuclease enzyme (AfeI) and contents were mixed gently by pipetting up and down. After centrifugation for few seconds tubes were incubated at 37⁰C. The reaction was terminated by adding 4µl loading dye and restricted products were run on ethidium bromide stained 3% agarose gel.

 Statistical analysis:

For comparison of two independent proportions, the chi square (χ²) or Fischer exact test was used and statistical differences were calculated using Sigma Plot software (version 11.00). Differences were considered significant if P values obtained were found to be less than 0.05 (P<0.05).

 RESULTS:

Seventy isolates were genotyped successfully (Fig. 1) for IFNGR1 gene promoter polymorphism.

FIG.1: [A] AMPLIFIED PRODUCTS OF IFNGR1 PROMOTERREGION (419 bp) ON 2.0% ETHIDIUM BROMIDE STAINED AGAROSE GEL. LANE 1 SHOWS MOLECULAR WEIGHT MARKEr (100 bp), LANE 2-6 SHOWS 419 bp AMPLIFIED PRODUCTS FROM DIFFERENT ISOLATES. 228 BP INTERNAL CONTROL BAND IS ALSO SHOWN IN EACH WELL.

[B]

RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP) ANALYSIS OF THE IFNGR1 PROMOTER REGION (419 bp) AMPLIFIED PRODUCTS ON 3.0 % ETHIDIUM BROMIDE STAINED AGAROSE GEL. LANE1 SHOWS100 bp MOLECULAR WEIGHT MARKERS. LANE 2, 5 & 7 SHOWS 228, 304 & 115 bp BAND AND LANES 3, 4 & 6 SHOWS UNDIGESTED PATTERN 419 bp & 228 bp PRODUCT FROM DIFFERENT ISOLATES RESTRICTED BY AFEI RESTRICTION ENZYME

Allele ‘T’ was found to be most prevalent (46/70=65.71%) in our healthy population followed by allele ‘C’ (24/70=34.28%) as shown in Fig.2.

FIG. 2: PERCENTAGE PREVALENCE OF DIFFERENT ALLELES OF IFNGR1 GENE PROMOTER REGION

Out of 70 accurately genotyped isolates, 39 were male and 31 were female. In male, out of 39, 28 had presence of allele ‘T’(71.79%), whereas allele ‘C’ was observed in 11 (28.20%) isolates (P>0.05). In female population, allele ‘T’ was observed in 18 isolates (58.06%), whereas 13 isolates(P>0.05) showed the presence of allele ‘C’ (41.93%) as shown in Fig.3.

FIG.3: PERCENTAGE PREVALENCE OF DIFFERENT ALLELES OF IFNGR1 GENE PROMOTER REGION IN MALE AND FEMALE POPULATION

Allele ‘T’ was found to be more prevalent in male population than female (28/46=60.86% vs. 39.13%), but not up to the mark of significance (P>0.05). Similarly, allele ‘C’ was found to be more prevalent in female population than male population (13/24=54.16% vs. 11/24=45.83%) in our isolates, but again not up to the mark of significance (P>0.05) as shown in Fig.4.

FIG. 4: PERCENTAGE PREVALENCE OF BOTH ALLELES OF IFNGR1 GENE PROMOTER REGION IN MALE AND FEMALE POPULATION

 DISCUSSION: Literature showed that IFN-γ/IFNGR1 signaling pathway has a very important role to eradicate the HBV infection ^18-19. Polymorphism in IFNGR1 has a direct effect on number of diseases including HBV pathogenesis ¹⁹. Some reports demonstrated that -56C/T SNP has a direct effect on IFNGR1 promoter activity ^{14, 15, 19}. It has been shown by a study using reporter gene analysis that allele ‘C’ (-56C promoter variant) has been found to have high transcriptional activity as compared to allele ‘T’ , and hence allele ‘C’ variant have high IFNGR1 molecules which is essential for the accurate and specific working of IFN-γ/IFNGR1 signaling pathway ¹⁹. Effect of IFNGR1 polymorphism is context specific. Its effect differed in different cell lines, for example, in human B cell line allele ‘C’ is responsible for low transcriptional activity, have nearly no effect in myeloid cell lines, and increased transcriptional activity in hepatocytes. Hence, with respect to HBV infection pathogenesis allele ‘C’ can provide a better protection than allele ‘T’.

So, allele ‘T’ can be considered as HBV susceptible and allele ‘C’ as HBV resistant allele with respect to counter-attack the HBV infection. In this study we have demonstrated the prevalence of different genotypes of IFNGR1 gene in our healthy isolates. Allele ‘T’ has been observed as the dominant allele (65.71%) in our both male (71.79%) and female (58.06%) healthy population. Allele ‘T’ was found to be more prevalent in male population than female (60.86% vs. 39.13%), whereas allele ‘C’ was found to be more prevalent in female population than male population (54.16% vs. 45.83%).

CONCLUSION: HBV pathogenesis severity not only depends on the viral strain but also on the host genetic factors. This complex combination between the virus-host is responsible for different spectrum of disease. In some cases infection resolved and in some others host met with the final deadly stage of infection i.e.  HCC. Although the different spectrum of HBV-related liver disease is multifactorial, SNPs in different immunogenes have their own role for the observed differences in disease manifesto in different ethnic groups and also at the level of individual ^20-21.

So, if we consider polymorphism in IFNGR1 gene as one of the factor for this multifactorial dependent disease, then we are at the risk because more than 65% of our healthy population has HBV-susceptible allele.

This is a small sample size study which is based on the previous observation of this polymorphism in different ethnic population. Hence a large study, which includes patients having persistent HBV infection and resolved disease, must be needed to confirm the same results in our ethnic population.

 CONFLICT OF INTEREST: We declare that we have no conflict of interest.

 ACKNOWLEDGMENTS: The authors are very thankful to the Dr. Vinod Mittal (M.D), Dolphin (PG) college of Science and Agriculture, Chunni Kalan, F.G. Sahib, for all the financial support to this study.

REFERENCES:

1. Kramvis A, Arakawa K, Yu MC, Nogueira R, Stram DO, Kew MC. Relationship of serological subtype, basic core promoter and precore mutations to genotypes /subgenotypes of hepatitis B virus. J Med Virol 2008; 80: 27–46.
2. Pahal V, Singh J. Prevalence of different genotypes/subgenotypes and precore mutations in chronically infected Hepatitis B Virus isolates of Haryana in North India. International Journal of Applied Microbiology Science 2014; 3(2):1-14.
3. Pahal V, Singh J. Hepatitis B Virus core promoter mutations and their relatedness to genotypes and liver disease severity in HBV-infected chronic isolates from the state of Haryana in India. International Journal of Applied Microbiology Science 2014; 3(3):1-11.
4. Rehermann B. Immune responses in hepatitis B virus infection. Semin Liver Dis2003; 23:21e38.
5. Guidotti LG, Ishikawa T, Hobbs MV, et al. Intracellular inactivation of the hepatitis Bvirus by cytotoxic T lymphocytes. Immunity1996; 4:25e36.
6. McClary H, Koch R, Chisari FV, et al. Relative sensitivity of hepatitis B virus and other hepatotropic viruses to the antiviral effects of cytokines. J Virol2000; 74:2255e64.
7. Van Nunen AB, Pontesilli O, Uytdehaag F, et al. Suppression of hepatitis B virus replication mediated by hepatitis A-induced cytokine production. Liver2001; 21:45e9.
8. Guidotti LG, Chisari FV. Noncytolytic control of viral infections by the innate andadaptive immune response. Annu Rev Immunol 2001; 19:65e91.
9. Kakimi K, Guidotti LG, Koezuka Y, et al. Natural killer T cell activation inhibits hepatitis B virus replication in vivo. J Exp Med 2000; 192:921e30.
10. Chen GY, Chen Z, Li MW, Liu HM, Hu ZR. Modulation and implications in pathogenesis of interferon gamma receptor 1 in patients with chronic hepatitis B. Zhonghua Gan Zang Bing ZaZhi2003; 11:14–
11. Guidotti LG, Ishikawa T, Hobbs MV, Matzke B, Schreiber R, Chisari FV. Intracellular inactivation of the hepatitis B virus by cytotoxic T lymphocytes. Immunity1996; 4:25–
12. Wieland S, Thimme R, Purcell RH, Chisari FV. Genomic analysis of the host response to hepatitis B virus infection. Proc Natl AcadSci2004; 101:6669–
13. Basturk B, Karasu Z, Kilic M, Ulukaya S, Boyacioglu S, Oral B. Association of TNF-alpha -308 polymorphism with the outcome of hepatitis B virus infection in Turkey. Infect Genet Evol2008; 8:20–
14. Liu M, Cao B, Zhang H, Dai Y, Liu X, Xu C. Association of interferon-gamma gene haplotype in the Chinese population with hepatitis B virus infection. Immunogenetics 2006; 58:859–
15. Zhou J, Lu L, Yuen MF, Lam TW, Chung CP, Lam CL, Zhang B, Wang S, Chen Y, Wu SH, Poon VK, Ng F, Chan CC, Jiang S, Yuen KY, Zheng BJ. Polymorphisms of type I interferon receptor 1 promoter and their effects on chronic hepatitis B virus infection. J Hepatol2007; 46:198–
16. Pahal V, Randhawa R, Singh G, Chandel A, Dadhich KS (2015). Prevalence of different alleles of apolipoprotein E (ApoE) gene with respect to the genetic risk for Hepatitis B virus infection in healthy north Indian population. International journal of recent research aspects 2015; 2: 69-73.
17. Pahal V, Randhawa R, Singh G, Chandel A, Dadhich KS (2015). Genetic polymorphisms of LMP7&TAP1 human-immunogenes as genetic risk factor for Hepatitis B Virus infection in north Indian healthy population. International journal of recent research aspects 2015; 2: 74-78.
18. Abbott W, Gane E, Winship I, Munn S, Tukuitonga C. Polymorphism in intron 1 of the interferon-gamma gene influences both serum immunoglobulin E levels and the risk for chronic hepatitis B virus infection in Polynesians. Immunogenetics2007; 59:187–
19. Zhou J, Chen DQ, Poon VKM, Zeng Y, Ng F et. al. A regulatory polymorphism in interferon-γ receptor 1 promoter is associated with the susceptibility to chronic hepatitis b virus infection. Immunogenetics2009; 61:423-430.
20. Wang FS. Current status and prospects of studies on human genetic alleles associated with hepatitis B virus infection. World J Gastroenterology 2003; 9:641–4.
21. Thio CL, Thomas DL, Carrington M. Chronic viral hepatitis and the human genome. Hepatology2000; 31: 819-827.
    

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    How to cite this article:

    Pahal V, Randhawa R and Dadhich KS: Prevalence of -56C/T regulatory polymorphism in interferon-γ receptor 1 promoter region known to be potential risk factor for Hepatitis B virus infection in north Indian healthy population. Int J Pharm Sci Res 2016; 7(7): 3112-16.doi: 10.13040/IJPSR.0975-8232.7(7).3112-16.

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