A final extension step at 72°C for 2 min was added after the last PCR cycle. After amplication, the PCR products were digested with BsmI, ApaI and TaqI endonucleases. Following restriction endonuclease digestion, genotyping was determined by ethidum bromide-UVB illumination of the fragments separated on gels of 2% agarose. The presence of BsmI, ApaI or TaqI restriction site was defined as the lower-case ‘b’, ‘a’ and ‘t’, respectively, and the absence of the site was defined as the upper-case ‘B’, ‘A’ or ‘T’. The BsmI restriction site resulted in two fragments (645 bp and 177 bp). Digestion with ApaI produced

BIBW2992 chemical structure two fragments of 531 bp and 214 bp when the restriction site was present. Digestion with TaqI resulted in three fragments of approximately 205, 290 bp and 245 bp in the presence of TaqI polymorphic site, and in fragments of 245 and 495 bp in the absence of a TaqI polymorphic site. Continuous data are expressed as mean ± standard deviation, and the categorical data are expressed as number (percentage). Comparisons of differences in the categorical date between groups were performed using the chi-square test. Distributions of continuous variables were analyzed by the Student’s t-test or one-way ANOVA test with least significant difference (LSD) post-hoc correction between groups where

appropriate. Stepwise logistic regression analysis was performed to assess the influence of each factor on the risk of developing HCC. All analyses were carried out using SPSS software version find more 15.0 (SPSS Inc., Chicago, IL). All tests were 2-tailed, and a p-value

of less than 0.05 was considered statistically significant. The basic demographical and clinical features of the patients are shown in Table 1. The mean age of HCC patients was significantly higher than those with cirrhosis, chronic hepatitis and controls (P < 0.001). Patients with HCC had a higher male-to-female ratio than other groups (P = 0.001). There was no significant difference in BMI among these groups. The HCC subjects had lower platelet count compared to those with chronic hepatitis; whereas the platelet Tryptophan synthase count was comparable between cirrhosis and HCC groups. Firstly, HCC patients were compared with a control cohort of 100 healthy volunteers with regard to allelic frequency. The distribution of the alleles of BsmI, ApaI and TaqI was in accordance with the Hardy-Weinberg equilibrium in both individuals of HCC and controls (P > 0.05 for any). Patients with HCC had a higher frequency of ApaI CC genotype (P = 0.027) and bAt[CCA]-haplotype consisting of BsmI C, ApaI C and TaqI A alleles (P = 0.037) as compared to control subjects. For the BsmI and TaqI polymorphisms, no significant associations were found.