G870A POLYMORPHISM IN THE CYCLIN D1 GENE IN COLORECTAL CANCER
Stefanovska A-M1, Josifovski T2, Panovski M2, Jasar D3, Zografski G3, Popevska Z4, Efremov GD1, Dimovski AJ1,*
*Corresponding Author: : Dr. Aleksandar J. Dimovski, Macedonian Academy of Sciences and Arts, Research Center for Genetic Engineering and Biotechnology, Av. Krste Misirkov 2, POB 428, 1000 Skopje, Republic of Macedonia; Tel: +3892-120253; Fax: +3892-115434; E-mail: aleks@manu.edu.mk *Current address: Faculty of Pharmacy, Vodnjanska 17, 1000 Skopje, Republic of Macedonia; Tel: +3892 119694; FAX: + 3892 123054; E-mail: adimovski@baba.ff.ukim.edu.mk
page: 27

MATERIALS AND METHODS

Subjects. One hundred and sixty-seven randomly selected patients with colorectal cancer, and 173 control subjects from the Republic of Macedonia, were included in the study. The control group of patients was previously studied for the presence of microsatellite instability (MSI) in their tumors. The average age of the patients was 62.1  10.8 years, and 61 of them were less than 60 years old at diagnosis (average age 51.0  8.2 years). The tumors of 18 patients (nine of whom were younger than 60 years of age at diagnosis) showed MSI, as defined by the National Cancer Institute (Bethesda, MD, USA) guidelines [16]. The group of control subjects consisted of 72 newborns and 101 aged individuals (average age 76.2  10.8 years) without any history of malignant disease. Samples from both groups of control subjects have been used previously for analysis of numerous genetic markers and were shown to be in Hardy-Weinberg equilibrium.

      Detection of the G870A Polymorphism. The G870A polymorphism was detected by polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) analysis using the primers described by Kong at al. [14]. The 135 bp PCR fragment was cut with the HpaII restriction enzyme and the digested products were separated by electrophoresis on a 13.3% non denaturing polyacrylamide gel that allowed for easy distinction of the three CCND1 genotypes (Fig. 1).

 

 

Figure 1. PCR-RFLP typing of CCND1 genotypes. Lanes 1, 2 and 4: GG homozygotes; lane 3: GA heterozygote; lane 5: AA homozygote




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