BMC Cancer 2007, 7:187 http://www.biomedcentral.com/1471-2407/7/187 Forward primer, 5'-TGCCACTTAGATGAGGAAATTG- TAGT-3' and reverse primer, 5'-ACACTTTCCTCCCCTTAT- GGATTC-3', TaqMan® MGB probes, FAM for 1G, 5'- ATAATTAGAAAGATATGACTTATC-3' and VIC for 2G, 5'- ATAATTAGAAAGGATATGACTTAT-3'; used for the MMP- 3 SNP were: Forward primer, 5'-ACATCACTGCCAC- CACTCTGTT-3' and reverse primer, 5'-GGCACCT- GGCCTAAAGACATT-3', TaqMan® MGB probes, FAM for 5A, 5'-AAGACATGGTTTTTC-3' and VIC for 6A, 5'-AGA- CATGGTTTTTTC-3'. Statistical analysis The Chi-square test was used to examine the differences in genotype distribution of the MMP-1 and MMP-3 promot- ers between the OSCC cases and control groups, and to estimate correlation or synergistic effects of MMP-1 geno- types with regard to clinical consequences as well as envi- ronmental factors among the OSCC cases. A p-value of < 0.05 was considered as statistically significant. Univariate analysis was performed by Fisher's exact test (two-sided) on gender, carrier state of MMP-1 1G allele (1G+ or 1G-) and 2G allele (2G+ or 2G-) between the OSCC and con- trol groups. In the consideration of latent interrelations between factors, the effects of MMP-1 2G allele, age and gender were estimated by a multivariate logistic regression model. For each parameter, the OSCC risk was accounted by Odds Ratios (OR) and 95% Confidence Intervals (95% CI). All of the statistical analyses were performed using the SPSS 11.5J software package (SPSS Japan Inc., Tokyo, Japan). Results Genotype distributions of MMP-1 and MMP-3 promoter polymorphisms in OSCC cases and control groups The genotype distributions of the promoter SNPs in MMP-1 and MMP-3 genes did not indicate a departure from the Hardy-Weinberg equilibrium when they were examined separately in the OSCC (n = 170) and control (n = 164) groups, or in all the samples combined (n = 334) (χ2 < 3.84, p > 0.05). The frequency of 1G/2G or 2G/ 2G promoter genotypes having the 2G allele, which is associated with a high MMP-1 expression level, was signif- icantly higher in the OSCC group, and the frequency of Table 1: MMP-1and MMP-3 genotype distribution in OSCC cases and controls MMP-1 2G/2G 1G/2G 1G/1G MMP-3 5A/5A 5A/6A 6A/6A OSCC cases (n = 170) 77 (45.3) 79 (46.5) 14 (8.2) 3 (1.8) 50 (29.4) 117 (68.8) Controls (n = 164) 64 (39.0) 71 (43.3) 29 (17.7) 8 (4.9) 54 (32.9) 102 (62.2) p 0.034 0.188 The data were analyzed by the χ2 test. the 1G/1G homozygote was lower than that of the control group (p = 0.034, Table 1). On the other hand, no differ- ence in MMP-3 genotype distribution (5A/5A, 5A/6A, and 6A/6A) was detected between the OSCC case and control groups. Association between MMP-1 promoter genotypes and various clinical parameters MMP-1 promoter genotypes of OSCC cases were stratified by clinical parameters including gender, age (Table 2), T category, lymph-node metastasis (N category), tumor location in the oral cavity, status of alcohol intake and smoking (Table 3). A significant association was detected only between the age and MMP-1 genotype, which sug- gests accumulation of older age patients with the 1G allele, or young age patients with the 2G allele. Figure 1 shows the age distribution pattern of subjects with MMP- 1 promoter genotypes in OSCC groups, demonstrating a remarkable reduction of the 1G/1G and 1G/2G genotype distributions in OSCC cases less than 45 years and 35 years old. Receiver operating characteristic (ROC) curve consistently exhibited that the 45 years of age could be an optimal cutoff value of the age at which to stratify the young OSCC from the older bracket in following analyses. It was of considerable interest to notice that the MMP-1 promoter genotype has a remarkable difference in distri- bution between subjects over and under 45 years of age. A Table 2: Age and gender distribution in relation to alleles in cases and controls Age* Gender# males females 2G/2G OSCC cases 1G/2G 1G/1G 54.8 ± 15.1 57.5 ± 13.2 60.1 ± 10.9 46 (59.7%) 31 (40.3%) 53 (67.1%) 26 (32.9%) 8 (57.1%) 6 (42.9%) p 0.277 0.570 Controls 2G/2G 1G/2G 1G/1G 52.2 ± 14.8 52.3 ± 14.5 48.1 ± 14.2 38 (59.4%) 26 (40.6%) 46 (64.8%) 25 (35.2%) 20 (69%) 9 (31%) p 0.387 0.640 Data are expressed as mean ± SD or number of subjects (%). * One-way ANOVA test. # χ2 test. Page 3 of 9 (page number not for citation purposes)