MTHFR C677T AND A1298C GENOTYPES AND HAPLOTYPES IN SLOVENIAN COUPLES WITH UNEXPLAINED INFERTILITY PROBLEMS AND IN EMBRYONIC TISSUES FROM SPONTANEOUS ABORTIONS
Stangler Herodež Š1,*, Zagradišnik B1, Erjavec Škerget A1, Zagorac A1, Takač I2,3, Vlaisavljević V4, Lokar L5, 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
page: 31

MATERIALS AND METHODS

Probands. The total study sample included 775 probands representing three different groups: couples with UFP, SAET and healthy controls. Couples with UFP (n = 200, 100 females and 100 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 the study enrolment, all probands were childless as a couple. Both female and male probands did not exhibit any other condition known to affect fertility (e.g., azoospermia in males or premature ovarian failure in females). Spontaneously aborted embryonic tissues (SAET) (n = 353) were also sent for routine karyotyping from the Department of Obstetrics and Gynecology, University Clinical Centre Maribor, Maribor, Slovenia. The pregnancies ended between the 6th and 20th week of gestation, with the majority occurring earlier than week 15. Fetal tissues included chorionic villi, placenta, fetal skin and they all originated from at least a second pregnancy loss in each case. Because few fetal samples were from couples participating in the study, both groups were considered unrelated. The healthy controls (n = 222, 111 females, 111 males) were blood donors with a good reproductive history, who were recruited through the Department of Transfusiology, University Medical 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. Cytogenetic Analysis. A routine cytogenetic analysis was performed on metaphase chromosomes from embryonic tissues. Chromosomes were harvested according to standard cytogenetic methods and analyzed by G-bands [G-bands by trypsin using Giemsa (GTG)]. Karyotypes were defined according to ISCN (2009). DNA Extraction and Analysis. Peripheral venous blood was collected in standard collection tubes containing EDTA as the anticoagulant. Genomic DNA was extracted from blood leukocytes with a simple salting-out method [23]. Genomic DNA from embryonic tissues was obtained using a QIAGEN Blood Mini Kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer’s instructions. All DNA samples were screened for the C677T/A1298C gene polymorphisms of MTHFR using an allelespecific polymerase chain reaction (PCR). Two reactions were performed per sample with the QIAGEN Multiplex PCR Kit (Cat. #206143; Qiagen) by amplifying one allele of each polymorphism (two duplex reactions). The mix included 1 × master mix, 1 mM of each primer and 50 ng of genomic DNA. In reaction 1, alleles 677T and 1298A were amplified with MTHFR677T-F (5’- GAA GGT GTC TGC GGG CGT-3’), MTHFRC677TR (5’-AGC AAC GCT GTG CAA GTT CTG-3’), MTHFR1298A-F (5’-AGG AGC TGA CCA GTG AGG A-3’) and MTHFR1298C-R (5’-TTC TCC CTT TGC CAT GTC C-3’). In reaction 2, alleles 677C and 1298C were amplified with MTHFR C677C-F (5’-GAA GGT GTC TGC GGG CGC-3’), MTHFR677T- R, MTHFR1298C-F (5’-AGG AGC TGA CCA GTG AGG C-3’) and MTHFR1298C-R. The annealing temperature was 60°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 allele scoring. Multiplex Ligation-Dependent Probe Amplification. Cytogenetic analysis of all SAET was performed. For those samples where karyotypeing was not possible the multiplex ligation-dependent probe amplification (MLPA) was performed using commercial MLPA kits containing subtelomeric probes (P036, P070; MRC Holland, Amsterdam, The Netherlands), according to the manufacturer’s protocol. The MLPA data analysis was performed as previously reported [24]. Statistical Analyses. For various genotypes and haplotypes, an odds ratio (OR) and 95% confidence interval (95% CI) were calculated. Differences between the tested groups and control groups were assessed by the c2 test and the Fisher’s exact test. Genotypes for the tested groups and the controls were assessed for departures from the Hardy-Weinberg equilibrium (HWE).



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