A total of 413 randomly selected patients with CRC and 185 control subjects from the Republic of Macedonia were recruited from the University Clinic of Abdominal Surgery and the Institute of Radiotherapy and Oncology in Skopje, between January 2006 and October 2008 (Table 1). Written informed consent was obtained from all participants of the study before the collection of the specimens. A questionnaire was used to elicit detailed information on demographic and clinical variables, prior disease history and family history of cancer. The average age of the patients was 60.27 ± 11.08 years. The control subjects consisted of 94 adults (average age 74.52 ± 10.46) and 91 newborns without any history of malignant disease. Since both control groups showed a similar genotype distribution, the results were integrated in further calculations.

      Genomic DNA was isolated from peripheral blood and tumors using Proteinase K digestion/phenol-chloroform extraction and ethanol precipitation. The MTHFR C677T polymorphism (rs1801133) was genotyped by real-time polymerase chain reaction (PCR) (MxPro 3005P, Stratagene, La Jolla, CA, USA) using the TaqMan SNP genotyping assay according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA).

      Descriptive comparisons [i.e., means, standard deviation (SD), frequencies as percentages] of CRC patients and controls were conducted using a chi-square test for categorical variables and analysis of covariance 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 MTHFR genotype and certain risk factors, stratified analyses were conducted.