IL-12 RB1 Genetic Variants Contribute to Human Susceptibility to Severe Acute Respiratory Syndrome Infection among Chinese

Tang, Fang; Liu, Wei; Zhang, Fang; Xin, Zhong-Tao; Wei, Mao-Ti; Zhang, Pan-He; Yang, Hong; Ly, Hinh; Cao, Wu-Chun

We typed the samples for 4 previously reported SNPs that cause missense mutations in the coding sequence of the IL-12RB1 gene [20] , namely +705A/G (Q214R, NCBI SNP ID:rs 11575934), +1158T/C(M365T, NCBI SNP ID:mrs 375947), +1196G/C (G378R, NCBI SNP ID: rs 401502), and +1664 C/T (P534S, HGBASE database ID: SNP001745641). For +705A/G genotyping, genomic DNA was amplified using the primer set 59ggttaagtgactggtgccaag-39 and 59-ctcaaaccactggcctcaag-39. The PCR fragment obtained was restricted with Bbv I (New England BioLabs, Beverly, Mass., US) at 37uC. For +1158T/C typing (M365T), 59-aacaaacgccatctgctacc-39 and 59-caacacctctctgggcctta-3 9, and Hsp 92II (Promega, Madison, Wisc., USA) were used at 37uC. For +1196G/C, 59 -aacaaacgccatctgctacc-3 9 and 59agagtgagaggccacctgag-3 9 , and Msp I (Promega, Madison, Wisc., USA) were used at 37uC. For +1664 C/T, 59-ggctgtggtagcccagcct-39 and 59-ggaagcgcagtgcagtgcatg-39 were used and restricted with Bsr I (New England BioLabs) at 65uC. PCR was carried out in PCR buffer (1.5 m M MgCl 2 , 10 m M Tris-HCl, pH 9.0, 50 m M KCl, 0.1% Triton H X-100), 100 mM of each dNTP, 25 mM primers and 1 unit of Taq polymerase in a final volume of 30 ml. Following an initial denaturation at 95uC for 5 min, samples were subjected to 30 cycles of PCR amplification with denaturation at 95uC for 30 s, annealing at 57-60uC for 30 s and elongation at 72uC for 30 s, followed by a final elongation step at 72uC for 10 min. The reaction products of the PCRs and enzyme restrictions were analyzed on 3% agarose gels (+705A/G, +1158T/C, +1664 C/T) or 8% polyacrylamide gels (+1196G/ C). The genotyping of all samples were performed in a blinded manner so that the case or control status was not known. Ten percent of the samples were chosen randomly from each polymorphism for resequencing in order to validate the genotyping method.