Balkan Journal of Medical Genetics

DETECTION OF MUTATIONS IN THE CYP21A2 GENE: GENOTYPE-PHENOTYPE CORRELATION IN SLOVENIAN COUPLES WITH CONCEIVING PROBLEMS
Stangler Herodež Š1,*, Fijavž L2, Zagradišnik B1, Kokalj Vokač N1,2
*Corresponding Author: Dr. Špela Stangler Herodež, Laboratory of Medical Genetics, University Clinical Centre Maribor, Ljubljanska ulica 5, 2000 Maribor, Slovenia. Tel: +386-2-321-27-37. Fax: +386-2-321-27-55. E-mail: spela.sh@ukc-mb.si
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MATERIALS AND METHODS

Probands. Our study was a retrospective study and included 838 probands representing two different groups: couples with UFP and HCs. Couples with UFP (n = 638, 319 females and 319 males), were referred to our laboratory from the Department of Reproductive Medicine and Gynecologic Endocrinology, University Clinical Centre Maribor, Maribor, Slovenia, for routine karyotyping. At the time of enrollment in the study, all probands were childless as a couple and had not undergone CAH diagnosis. Clinical data of interest was extracted from a questionnaire designed for the needs of the study and existing medical records collected between 2005 and 2012. The HCs (n = 200, 100 females and 100 males) were blood donors with an unremarkable reproductive history, who were recruited through the Department of Transfusiology, University Clinical Centre Maribor, Maribor, Slovenia. All samples were from individuals of Caucasian origin who were residents of different geographical areas of Slovenia. Informed consent was obtained from all participating individuals and ethical approval was granted prior to conducting the study. Clinical and Hormonal Investigations. For female probands, the following clinical and biochemical parameters were determined: the concentration of prolactin in serum (nmol/L), the concentration of dihydroepiandrosterone in serum (nmol/L), the concentration of free testosterone in serum (nmol/L), the concentration of testosterone in serum (nmol/L), concentration of Anti-Muller hormone levels in serum (nmol/L), body mass index (BMI), the presence of a thyroid disease, the presence of an autoimmune disease, the presence of PCOS. The follicular phase of the menstrual cycle was determined: the concentration of follicle-stimulating hormone in serum (nmol/L), the concentration of luteinizing hormone in serum (nmol/L), the concentration of 1,2-estradiol in the serum (nmol/L). In the luteal phase of the menstrual cycle, the concentration of progesterone in the serum (nmol/L) was determined. Basic laboratory parameters of female probands are given in Table 1. DNA Extraction and Analysis. Peripheral venous blood was collected in standard collection vacutainers containing EDTA as anticoagulant. Genomic DNA was extracted from blood leukocytes with a simple salting-out method [15]. All DNA samples were screened for the four most common mutations (p.P30L in exon 1, c.290-13A/ C>G in intron 2, p.I172N in exon 4 and p.V281L in exon 7) in the CYP21A2 gene using allele-specific polymerase chain reaction (PCR). Four PCR reactions were performed per sample with the Multiplex PCR Kit (Cat. #206143; Qiagen GmbH, Hilden, Germany). The mix included 1X master mix, 1 μM of each primer and 50 ng of genomic DNA. In reaction 1, the p.P30L mutation in exon 1 was detected with CAH-30LT-P-F (5’-AAG CTC CGG AGC CTC CAC CTA CT-3’) and CAH-I1-A-R1 (5’-AGC ATA GCA AGA ACC CAT CTG TT-3’). In reaction 2, the c.290-13A/C>G mutation in intron 2 was detected with CAH-I2-A2-F (5’-CTA ACT ACA TAT CTG GTG GGG AGA AAG C-3’) and CAH-In2-M-GR (5’-CAG CTT GTC TGC AGG AGG CGC-3’). In reaction 3, the p.I172N mutation in exon 4 was detected with CAH-172NA-F (5’-TCT CTC CTC ACC TGC AGC ATG AA-3’) and CAH-I172N-ARL (5’-TTC ATG TCG TCC TGC CAG AAA AGC AG-3’). In reaction 4, the p.V281L mutation in exon 7 was detected with CAH-I6-A-F (5’-CAG CAC AAG GTG GGG ACT GGA C-3’). The annealing temperature was 63 °C in 30 amplification cycles. Successful PCR amplification was confirmed by electrophoresis on 3.0% agarose gel, stained with SYBR Green I, and photographed for documentation and mutation scoring. Statistical Analyses. For statistical analysis, the Statistical Package for the Social Science (SPSS), version 21 software program (IBM Corporation, Armonk, NY, USA), was used. Comparison between the means was done by non parametric tests. The results were expressed as an arithmetic mean ± standard deviation (SD). The relations between variables were analyzed by the analysis of variance (ANOVA) test. The distribution of the mutations was compared using the χ2 test; p ≤0.05 was considered statistically significant.

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