CAG REPEAT NUMBER IN ANDROGEN RECEPTOR GENE AND MALE INFERTILITY
Plaseski T1,2, Noveski P1, Dimitrovski C2, Kocevska B2, Efremov GD1, Plaseska-Karanfilska D1,*
*Corresponding Author: : Dr. Dijana Plaseska-Karanfilska, Macedonian Academy of Sciences and Arts, Research Center for Genetic Engineering and Biotechnology, Av. Krste Misirkov 2, POB 428, 1000 Skopje, R. Macedonia; Tel: +389 2 3235 410 Fax: +389 2 3115 434; E-mail: dijana@manu.edu.mk
page: 19

MATERIALS AND METHODS

Materials. A total of 222 patients (representing 231 X chromosomes) attending the Andrological Outpatient Unit at the Clinic for Endocrinology and Metabolic Disorders, Faculty of Medicine, (Skopje, R. Macedonia?), were enrolled in the study. All patients gave their informed consent to participate in the study. Semen analysis was performed in accordance with the World Health Organization (WHO) guidelines. All patients were routinely screened for the presence of chromosomal aneuploidies of the X and Y chromosomes by quantitative fluorescent polymerase chain reaction (QF-PCR) analysis and for the presence of Y microdeletions by multiplex analysis of six sequence tagged sites (STS) in the three AZF regions. The studied group included 27 patients with a known cause of infertility (Y microdeletions, XXY, XX males and obstructive azoospermia representing 36 X chromosomes). The other 195 patients were grouped according to the results of semen analysis (patients with azoospermia, n = 73; severe oligozoospermia or sperm counts of <5 x 106/mL, n = 51; mild oligozoospermia or sperm counts of >5 x 106/m, n = 31; and normozoospermia with unexplained couple infertility, n = 40). In addition, 152 men who have fathered at least one child by natural conception and whose paternity was proved by DNA analysis served as controls.

Methods. Genomic DNA was isolated from leukocytes using the Proteinase K/SDS digestion-phenol/chloroform extraction-ethanol precipitation method (9). The CAG repeat number was determined by fluorescent PCR amplification of exon 1 of the AR gene using the following primers: direct 5'-(HEX) TCC AGA ATC TGT TCC AGA GCG TGC-3' and reverse 5'-GCT GTG AAG GTT GCT GTT CCT CA-3'. The size of the PCR product was determined by capillary electrophoresis on an ABI PRISMô 310 Genetic Analyzer (Applied BioSystems, Foster City, CA, USA). The number of CAG repeats predicted by the Genescan software (Applied BioSystems) was compared with the actual CAG repeats determined by direct dideoxy terminator cycle sequencing using the Big Dye Terminator Sequencing Kit v1.0 (Applied BioSystems) in male DNA samples with 14, 19, 21, 25 and 29 CAG repeats. Figure 1 shows the Genescan profiles of DNAs with 19, 21, 23 and 26 CAG repeats, while Figure 2 shows part of the DNA sequence of AR gene exon 1 in a DNA sample with 19 CAG repeats.

Statistical Analysis. The frequencies of the CAG repeat alleles were compared between the various groups of infertile/subfertile males and fertile controls using the χ 2 and Fisher's exact tests. Differences in the mean of (CAG)n length between different groups of patients vs. controls were tested by the independent samples t-test. Statistical significance was defined as p <0.05.




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