Table 1. Primers, Probes, and Restriction Enzymes

Restriction Gene Variant Primer*,y Enzymez IL-10 (cid:1)1082 A/G F 59-acactactaaggcttccttggga-39 EcoNI R 59-gatggggtggaagaagttgaaat-39 (cid:1)819 T/C FO 59-acactactaaggcttccttggga-39 RO 59-tgcacttgctgaaagcttcttat-39 FI 59-tgtacccttgtacaggtgatgtcaT-39 RI 59-gagcaaactgaggcacagaggtG-39 TNF-a (cid:1)1031 T/C FO 59-gctgtggggagaacaaaaggataa-39 RO 59-ggtccccatactcgacttccata-39 FI 59-gaagcaaaggagaagctgagaacaT-39 RI 59-tccagaccctgacttttcctgcG-39 IL-1b (cid:1)511 C/T FO 59-atctggcattgatctggttcatcc-39 RO 59-cttaactttaggaatcttcccactt-39 FI 59-cctgcaattgacagagagctacC-39 RI 59-cttgggtgctgttctctgccgcA-39 IL-6 (cid:1)634 C/G FO 59-acctggagacgccttgaagtaact-39 RO 59-aaaccaaagatgttctgaactgagt-39 FI 59-gccaggcagtctacaacaggcC-39 RI 59-gtgttctggctctccctgtgtgC-39 IL-18 (cid:1)607 A/C F 59-tctccccaagcttactttctgttg-39 R 59-tgcaagccacacggatacc-39 FAM 59-atcattagaattttatTtaataat-39 VIC 59-atcattagaattttatCtaataat-39 TGF-b þ869 C/T F 59-ttccctcgaggccctcctacc-39 MspAII R 59-gccgcagcttggacaggatct-39 MMP-3 (cid:1)1171 5A/6A FO 59-ctcaaagtgctaggattacagacat-39 RO 59-attatctatcaggctttcctctaaact-39 FI 59-cctttgatggggggaaaaac-39 RI 59-aatcaggacaagacatggtttttt-39 MMP-1 (cid:1)1607 1G/2G F 59-acatgttatgccacttagatgaggaaa-39 R 59-gcgtcaagactgatatcttactcataaacaata-39 FAM 59-ttgagataagtcatatCtttcta-39 VIC 59-ttgagataagtcatatCCtttcta-39

Each PCR was carried out in a total volume of 10 ll containing template DNA at 80 ng/ll, 10 pmol primers, 200 lM deoxyribonucleotide triphosphates (dNTPs), 20 mM MgCl , 20 mM Tris-HCl (pH 8), 100 mM KCl, and 1 U Taq 2 polymerase (TaKaRa Ex Taq; TAKARA, Tokyo, Japan). The solution was overlaid with 10 ll of mineral oil. Standard conditions of the RoboCycler (Gene Amp PCR System 9600; Perkin Elmer, Tokyo, Japan) used in this study were as follows: initial denaturation at 958C for 2 minutes, followed by 30 cycles of 30-second denatura- tion (958C), 20-second annealing (608C), and 30-second extension (728C). PCR products were separated by electro- phoresis on a 2% agarose gel, and subsequently stained with ethidium bromide.

TaqMan assay of PCR products was performed according to the manufacturer’s protocol. Reaction components for a single 5-ll reaction (for each well of a 384-well plate) were: template DNA at 5 ng/ll, 2.5 ll of TaqMan universal PCR master mix, 0.125 ll of 40 X assay mix, and 2.375 ll of dH O. Specific forward/reverse PCR primers and Taq- 2 Man minor groove binding DNA oligonucleotide (MGB) probes for MMP-1 and IL-18 SNPs were designed with Primer Express version 1.5 software (Applied Biosystems, Foster City, CA), and were custom-synthesized by Applied Biosystems. Sequences of primers for IL-10 (cid:1)592 A/C are not shown in Table 1 because they were obtained as commercial Assays-on-Demand (Applied Biosystems). Re- action mixtures were loaded into 384-well plates and placed in an ABI Prism 7900HT Sequence Detection System (Applied Biosystems). PCR amplifications were performed as follows: initial denaturation at 958C for 10 minutes, followed by 40 cycles of PCR with a denaturation at 958C for 15 seconds and one step annealing and extension for 1 minute at 608C.

Notes: *Primers for tetra-primer amplification refractory mutation system (ARM) polymerase chain reaction (PCR): FO ¼ forward outer primer; RO ¼ reverse outer primer; FI ¼ forward inner primer; RI ¼ reverse inner primer. Primers for Taqman assay: F ¼ forward primer; R ¼ reverse primer; FAM ¼ reporter1 probe; VIC¼reporter 2 probe. IL¼interleukin; TNF¼tumor necrosis factor; TGF ¼ transforming growth factor; MMP ¼ matrix metalloproteinase.

zRestriction enzymes for PCR-restriction fragment length polymorphism (RFLP) analysis.

Statistical Analysis

64–100 years) from January 2001 through October 2004. Patients with malignancy or with severe organ failure were not enrolled in this study. Participants were drawn from Yamaguchi Prefecture and the surrounding prefectures (assuming similar environmental and social factors), and all were native Japanese. The experimental protocol was approved by the Institutional Ethics Committee of the Yamaguchi University School of Medicine.

SNPs tested in this study did not show departure from Hardy–Weinberg equilibrium when samples used for this study (n ¼ 500) were examined before analysis (p . .05). Association of the genotypes with age and gender was evaluated by the chi-square test. Multivariate logistic regression analysis was then carried out to obtain odds ratios (OR) and 95% confidence intervals (CI). Presence or absence of a given SNP genotype was used as a dependent variable, and age and gender were used as independent variables. A p value of ,.05 was considered statistically significant. The StatView statistical software system (ver- sion 5; SAS Institute Inc., Cary, NC) was used for analyses.

Genotyping

Using a conventional NaI method, we extracted genomic DNA from peripheral blood anti-coagulated with EDTA- 2Na (20). For genotyping of IL-10 (cid:1)819 T/C, TNF-a (cid:1)1031 T/C, IL-1b (cid:1)511 C/T, IL-6 (cid:1)634 C/G, and MMP-3 (cid:1)1171 5A/6A, tetra-primer amplification refractory mutation sys- tem (ARMC) polymerase chain reaction (PCR) was performed with validation by PCR-restriction fragment length polymorphism (RFLP) as described previously

R ESULTS

We genotyped eight SNPs including IL-10 (cid:1)819 T/C, TNF-a (cid:1)1031 T/C, IL-1b (cid:1)511 C/T, IL-6 (cid:1)634 C/G, IL-18 (cid:1)607 A/C, TGF-b þ869 C/T, MMP-1 (cid:1)1607 1G/2G, and MMP-3 (cid:1)1171 5A/6A, and evaluated the relation of age or

Table 2. Comparison of Genotype Distribution of Eight SNPs Between Higher and Lower Age Groups