Balkan Journal of Medical Genetics

ASSOCIATION OF GLUTATHIONE-S-TRANSFERASE (GSTM1 and GSTT1) AND FTO GENE POLYMORPHISMS WITH TYPE 2 DIABETES MELLITUS CASES IN NORTHERN INDIA
Raza ST, Abbas S, Ahmad A, Ahmed F, Zaidi ZH, Mahdi F
*Corresponding Author: Syed Tasleem Raza, Ph.D., Molecular Biology Laboratory, Department of Biochemistry, Era’s Lucknow Medical College and Hospital, Hardoi Road, Lucknow, Uttar Pradesh, India 226025. Tel.: +91-522- 240-8122; 240-8123. Fax: +91-522-240-7824. E-mail: tasleem24@gmail.com
page: 47

ANALYSIS OF POLYMORPHISMS

GSTM1 and GSTT1 Gene Polymorphisms. GSTM1 and GSTT1 genetic polymorphisms were evaluated using a multiplex polymerase chain reaction (PCR) technique (MJ Mini Thermal Cycler; Bio-Rad Laboratories, Hercules, CA, USA). The PCR primers were synthesized according to Arand et al. [21]. Primers for the GSTM1 gene were: 5’- GAA CTC CCT GAA AAG CTAA AGC-3’ and 5’- GTT GGG CTC AAA TAT ACG GTG G-3’ and for GSTT1: 5’-TTC CTT ACT GGT CCT CAC ATC TC-3’ and 5’-TCA CCG GACAT GGC CAG CA- 3’. The β-globin locus was used as an internal control to avoid false-negative readings. Primers for the β-globin locus were: 5’-CAA CTT CAT CCA CGT TCA CC-3’ and 5’-GAA GAG CCA AGG ACA GGT AC-3’. The PCR reaction was carried out in a total volume of 25 μL containing 10 pmol of each primer, 2.5 mmol/L of MgCl2, 0.2 mmol/L of each deoxynucleotide triphosphate, 1 unit of Taq polymerase (Bioline Ltd., London, UK) and 100 ng of genomic DNA. Amplification was performed by initial denaturation at 94 oC for 5 min., followed by 30 cycles at 94 °C for 1 min., 64 °C for 1 min. and 72 °C for 1 min., and a final extension of 72 °C for 7 min. The amplified products were identified by electrophoresis in a 1.5% agarose gel and stained with 0.5 mg/mL (concentration) ethidium bromide. The product lengths were 215, 480 and 268 bp for GSTM1, GSTT1 and β-globin locus, respectively. FTO Gene Polymorphism. The FTO SNP (rs993 9609) was genotyped by PCR (MJ Mini Thermal Cycler; Bio-Rad Laboratories) and restriction fragment length polymorphism (RFLP), analyses. Genomic DNA (20 ng) was incubated in a 10 μL solution containing 1 × NH4 buffer, 2.5 mmol/L magnesium, 200 mmol/L each dNTP, 20 pmol forward (5’-AAC TGG CTC TTG AAT GAA ATA GGA TTC AGA-3’) and reverse (5’-AGA GTA ACA GAG ACT ATC CAA GTG CAG TAC-3’) oligonucleotide primers [22], and 0.5 U Taq DNA polymerase (Bioline Ltd.). The PCR mix was denaturated at 94 °C for 5 min., followed by 20 cycles of 45 seconds at 94 °C, 45 seconds at 61 °C (dropping 0.5 °C per cycle), and 45 seconds at 72 °C. After this, the PCR mix was incubated for 15 cycles at 45 seconds at 94 °C, 45 seconds at 51 °C, and 45 seconds at 72 °C, followed by a final extension at 72 °C for 10 min. This was then incubated at 37 °C for 16 hours with 2 U ScaI (New England Biolabs, Hitchin, Hertfordshire, UK). Upon running the final products on a 3.0% agarose gel, the T allele produced a 182 bp band and the A allele produced 154 and 28 bp bands. Statistical Analyses. All the figures are presented as mean ± standard deviation (SD). The genotyping data were compared between patients and controls using the c2 test. Other variables were compared using the Student’s t-test for normally-distributed variables. All statistical tests were performed using the Statistical Package for the Social Sciences, version 12 software (SPSS Inc., Chicago, IL, USA).

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