Balkan Journal of Medical Genetics

CAG REPEAT NUMBER IN THE ANDROGEN RECEPTOR GENE AND PROSTATE CANCER
Madjunkova S, Eftimov A, Georgiev V, Petrovski D, Dimovski AJ, Plaseska- Karanfi lska D,
*Corresponding Author: Professor Dr. Dijana Plaseska-Karanfi lska, Macedonian Academy of Sciences and Arts, Research Center for Genetic Engineering and Biotechnology “Georgi D. Efremov”, Av. Krste Misirkov 2, POB 428, 1000 Skopje, Republic of Macedonia; Tel.: +389-2-3235-410; Fax: +389-2-3115- 434; E-mail: dijana@manu.edu.mk
page: 31

MATERIALS AND METHODS

patients with benign prostatic hyperplasia (BPH) and 152 males from the general Macedonian population for this study. Informed consent was obtained from all and the study was approved by the Ethic Committees of the Macedonian Academy of Sciences and Arts and Faculty of Pharmacy, Skopje, Republic of Macedonia. Prostate cancer patients and those with BPH were recruited from the Department of Urology, Medical Faculty, Skopje, Republic of Macedonia and were referred to the Pharmacogenetic Laboratory, Faculty of Pharmacy, Skopje, Republic of Macedonia. Only those with histopathologically-confi rmed diagnoses were included. The mean ages of the PC and BPH patients were 68.19  7.36 and 69.72 .55 years, respectively. Males from the general population were selected at the Research Center for Genetic Engineering and Biotechnology “Georgi D. Efremov,” Macedonian Academy of Sciences and Arts, Skopje, Republic of Macedonia to produce a control sample that was age-matched to the samples of PC and BPH patients. Methods. Genomic DNA was extracted from EDTA whole blood following a standard phenol/ chloroform method. The CAG repeat number was determined by fl uorescent polymerase chain reaction (PCR) amplifi cation of exon 1 of AR gene. Approximately 50–100 ng genomic DNA was subjected to 35 cycles of PCR amplifi cation using fl uorescently-labeled forward primer 5’-(HEX) TCC AGA ATC TGT TCC AGA GCG TGC-3’ and unlabeled reverse prime r5’-GCT GTG AAG GTT GCT GTT CCT CA-3’. The PCR amplifi cation was performed as follows: 45 seconds at 94C, 30 seconds at 62C and 1 min. at 72C. The size of the PCR product was determined by capillary electrophoresis (CE) on an ABI PRISM™ 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The number of CAG repeats predicted by the Genescan software v.6 (Applied Biosystems) was compared with the actual CAG repeats determined by direct dideoxy terminator cycle sequencing using the BigDye Terminator Sequencing Kit v1.0 (Applied Biosystems) in male DNA samples as described previously [26]. Statistical Analyses. The frequency of the CAG repeat alleles was compared between the studied groups using the 2 and Fisher’s exact tests. Values were expressed as mean  standard deviation (SD). Differences in the mean of (CAG)n length between different groups of patients (PC, BPH) vs. controls were tested by the independent samples t-test using SPSS 14.0 (SPSS Chicago, IL, USA), after checking for normal distribution. The association of different CAG repeat length was tested at different cutoff points: <19, <20, <21, <22 and >22. Statistical signifi cance was defi ned as p <0.05.

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