IL-18 GENE PROMOTER REGION 607C/A POLYMORPHISM IN HIV-1 INFECTED NORTH INDIAN POPULATION
Sobti RC1,*, Sharma VL2, Abitew AM2, Berhane N1, Mahdi SA1, Askari M1, Kuttiat VS3, Wanchu A3
*Corresponding Author: Ranbir C. Sobti, Department of Biotechnology, Panjab University, Chandigarh 160 014 India; Tel.: +172-2534087; +94-1-704-4523; Fax: +172-25341409; E-mail: rcsobti@ pu.ac.in
page: 41

MATERIALS AND METHODS

In this case control study 500 patients with HIV-1/ AIDS and an equal number of sex- and age-matched control groups were recruited from the Department of Internal Medicine, Post-Graduate Institute of Medical Education and Research at Chandigarh, India. Cases and controls were from the same geographical area and same ethnic backgrounds. Of the 500 cases, 174 were females and 326 males with a mean age of 35.4 7.9, while of the 500 controls, 183 were females and 317 males with a mean age of 36.2 9.8 years. The research protocols were reviewed and approved by the ethics committee of the institute. Written informed consent was obtained from all the participating individuals. Information about age, sex, occupation and other relevant clinical data was gathered from the immunological cards of the HIV-1/AIDS patients. Peripheral blood (2-3 mL) was drawn and collected in an EDTA-coated tube for genomic DNA extraction. The samples were kept at 80C until used for DNA isolation. Genomic DNA was extracted from peripheral blood leukocytes by sodium dodecyl sulphate lysis and proteinase K digestion followed by standard phenol-chloroform methods as previously described [15] and the extracts were subjected to a polymerase chain reaction (PCR). Genotyping of the IL-18 607C>A SNP was performed by (sequence specific primer)-SSP-PCR analysis [14]. Briefly, a common reverse primer 5-TAA CCT CAT TCA GGA CTT CC-3 and two sequence specific forward primers 5-GTT GCA GAA AGT GTA AAA ATT ATT AC-3 and 5-GTT GCA GAA AGT GTA AAA ATT ATT AA-3 were used. An amplification product of 196 bp was detected. A control forward primer 5-CTT TGC TAT CAT TCC AGG AA-3 was used to amplify a 301 bp fragment covering the polymorphic site as an internal positive amplification control. The reaction was performed in a final volume of 25 μL consisting of 2.5 μL of 10X PCR buffer, 0.25 μL of 10 mmol of dNTP, 1.5 μL of 25 mM MgCl2, 2 μL of genomic DNA, and 0.5 U of Taq polymerase. For every reaction mixture, 0.5 μL of the 607C allele-specific or the 607A allele-specific forward primer and 0.5 μL of the common reverse primer were included. The 0.15 μL of internal positive control primer was added to reaction mixture. The PCR products (Figure 1) were visualized by ethidium bromide stained electrophoresis on 2% agarose gel. Two separate PCRs were carried out for every individual DNA sample. Reactions were carried out in a Bio-Rad MyCycler (Bio-Rad Laboratories, Hercules, CA, USA). Denaturation for 2 min. at 94C was performed as the first step. This was followed by seven cycles of 20 seconds at 94C, 40 seconds at 64C and 40 seconds at 72C and 25 cycles of 20 seconds at 94C, 40 seconds at 57C and 40 seconds at 72C. All PCR products were visualized under UV light on ethidium bromide-stained 2% agarose gels. Statistical analysis. Results were analyzed with SPSS software, version 11 for windows 5 (SPSS Inc., Chicago, IL, USA). Hardy-Weinberg equilibrium between observed and expected genotype distributions was determined by means of the Pearson Chi-square (χ2) test. For the assessment of risk factors, ORs with 95% CIs were calculated according to Epi Info Version 3.5.1 2008; Centers for Disease Control and Prevention (CDC), Atlanta, GA, USA. The frequencies of genotypes and alleles were calculated by manual counting. The Pearson χ2 test was used to calculate the significance of the differences in frequency of genotypes and alleles between the patients and control subjects. p Values of <0.05 were considered to be statistically significant.



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