Balkan Journal of Medical Genetics

ETHNIC VARIATION IN INTERLEUKIN-6 –174 (G/C) POLYMORPHISM IN THE MALAYSIAN POPULATION
Gan G-G*, Subramaniam R, Lian L-H, Nadarajan VS
*Corresponding Author: Professor Dr. Gin-Gin Gan, Department of Medicine, Faculty of Medicine, University Malaya, Lembah Pantai, Kuala Lumpur, Malaysia; Tel.: +603-79-492-429; Fax: +603-79-556-936; E-mail: gangg@ummc.edu.my
page: 53

MATERIALS AND METHODS

Subjects. Blood was obtained from healthy blood donors residing in Kuala Lumpur, capital city of Malaysia. The study was approved by the local institution ethics committee and informed consent was obtained from all individuals. DNA was extracted from the blood samples by a standard phenolchloroform method and QiAmp DNA Blood Mini Kit (Qiagen GmbH, Hilden Germany). DNA concentration was measured using an ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA) to assess the quantity of the product. Cytokine Gene Polymorphism Gene Analyses. The –174 (G/C) (rs1800795) polymorphism in the 5’ regulatory region of the IL-6 gene was performed by a custom TaqMan® single nucleotide polymorphism (SNP) genotyping assay (Applied Biosystems, Foster City, CA, USA) system on the LightCycler® 480 Real-Time polymerase chain reaction (PCR) 384 well plate system (Roche Applied Science, Mannheim, Germany). It discriminates the (SNP) by detecting differences in the melting temperatures of the products (Tm). The forward primer was 5’-CGA CCT AAG CTG CAC TTT TCC-3’ and reverse primer was 5’-GGG CTG ATT GGA AAC CTT ATT AAG ATT G-3’; the probes for the −174C allele was 5’- CCT TTA GCA TGG CAA GAC-3’ and the −174G allele was 5’-CCT TTA GCA TCG CAA GAC-3’. The 5’ nuclease assay was performed using 10 to 30 ng genomic DNA, 2X TaqMan® GTXpress™ Master Mix (Applied Biosystems), and 20X TaqMan® genotyping assay. The PCR cycle consisted of hold for 10 min. at 95 °C, 40 cycles of denaturing for 15 seconds at 92 °C and annealing for 1 min. at 60 °C. Negative, non template controls and known positive controls were included in each experimental run. Statistical Analyses. Allele and genotype frequencies of the three ethnic groups were compared using the c2 contingency table. The data were tested for Hardy-Weinberg equilibrium. A p value of <0.05 was considered as statistically significant. All statistical tests were performed using the Statistical Package for the Social Sciences (SPSS) version 17 (SPSS Inc., Chicago, IL, USA).

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