Subjects. A total of 331 randomly selected patients with CRC and 101 control subjects from the Republic of Macedonia were studied (Table 1). The average age of the patients was 61.25 ± 11.52 years, and 109 of them were less than 60 years old at diagnosis (average 51.13 ± 8.05 years). The tumors of 32 patients (18 of whom were younger than 60 years at diagnosis) showed MSI. The control subjects consisted of 101 individuals (average age 74.52 ± 10.46) without any history of malignant disease. Samples from the control subjects have been used previously for analysis of numerous genetic markers and were shown to be in Hardy-Weinberg equilibrium [19,20].

Detection of the G870A Polymorphism. Genomic DNA was isolated from peripheral blood (EDTA was used as anticoagulant) and tumors using Proteinase K digestion/ phenol-chloroform extraction and ethanol precipitation. The G870A polymorphism was detected by polymerase chain reactionrestriction fragment length polymorphism (PCRRFLP) analysis using the following primers: 5’-TAC TAC CGC CTC ACA CGC TTC C-3’ and 5’-TTG GCA CCA GCC TCG GCA TTT C-3’. The 135 bp PCR fragment was cleaved with the HpaII restriction enzyme and the digestion products were separated by electrophoresis on a 10% non denaturing polyacrylamide gel (Figure 1).

Table 1. General characteristics of the colorectal cancer patients and controls.

Statistical Analysis. Descriptive comparisons [i.e., means, standard deviation (SD), frequencies as percentages] of cases and controls were conducted using a chi-square test for categorical variables and analysis of co variance for continuous variables. Logistic regression was used to calculate odds ratios (ORs) and corresponding 95% confidential intervals (95% CI). To examine separate and combined effects of the CCND1 genotype and certain risk factors, stratified analyses were conducted.