POLYMORPHISM OF THE IL13 GENE MAY BE ASSOCIATED WITH UTERINE LEIOMYOMAS IN SLOVENIAN WOMEN
Krsteski J, Jurgec S, Pakiž M, But I, Potočnik U,
*Corresponding Author: Professor Uroš Potočnik, Ph.D., Centre for Human Molecular Genetics and Pharmacogenomics, Faculty of Medicine, University of Maribor, Taborska Ulica 8, 2000 Maribor, Slovenia. Tel: +386-2-2345-854, Fax: +386-2- 2345-820, E-mail: uros.potocnik@um.si
page: 51

MATERIALS AND METHODS

Study Subjects. In this study, we recruited 181 ULM patients and 41 women without ULM as a control group at the Department of General Gynecology and Gynecological Urology (University Medical Centre Maribor, Maribor, Slovenia) as described previously [17]. Additionally, 92 subjects were included to represent the general population. Uterine leiomyomas patients were divided into two subgroups: 85 were patients with solitary ULM and 96 were patients with multiple ULM, with a mean age at diagnosis of 46 ± 11 years. Controls were also divided into two subgroups. The first group (healthy control) consisted of 41 women without ULM but diagnosed with pelvic organ prolapse as described previously [17], with a mean age of 60 ± 11 years, and the second group (normal population) consisted of 92 healthy individuals, with a mean age of 43 ± 12 years. Demographic and clinical parameters of ULM patients analyzed in this study have been described previously [17]. Additionally, quantitative measurements of the 17β-estradiol levels in serum were performed using ARCHITECT i2000SR Immunoassay Analyzer (Abbott Laboratories, Abbott Park, IL, USA) according to the manufacturer’s instructions. The study protocol was approved by the Slovenian National Committee for Medical Ethics and the Institutional Review Board (KME 43/10/15). Selection and Genotyping of IL4 (rs2070874), IL4R (rs1801275), IL12B (rs6887695), IL12RB1 (rs11575934) and IL23R (rs7517847) Single Nucleotide Polymorphisms. The rs2070874 (IL4) and rs1801275 (IL4R) SNPs were selected according to their known functional role in IL expression. Single nucleotide polymorphisms rs 11575934 (IL12RB1), rs6887695 (IL12B), rs20541 (IL13) and rs7517847 (IL23R) were previously reported to be associated with other ULM-related immune-mediated diseases, such as inflammatory bowel disease [22], glioblastoma [23], cervical adenocarcinoma and an increased risk of tuberculosis [24]. Genotyping of IL4 (rs2070874), IL4R (rs1801275), IL12B (rs6887695, located ~60 kb upstream of the IL12B coding region), IL12RB1 (rs11575934) and IL23R (rs 7517847) SNPs was performed by polymerase chain reaction (PCR) followed by the restriction fragment length polymorphism (RFLP) technique. The PCR primers and restriction enzymes were selected using the GeneRunner program (Hastings Software Inc., Hastings, NY, USA). A total of 50 ng of genomic DNA was mixed with oligonucleotide primers in a final volume of 10 μL containing 2 mM MgCl2, 0.2 mM dNTP mix, 10 mM Tris-HCl and 0.25 U Taq polymerase (Fermentas, Waltham, MA, USA). The PCR conditions, primer sequences and the concentration of each primer are shown in Table 1. Polymerase chain reaction amplification was performed in a Professional Standard PCR Thermocycler (TProfessional Basic; Biometra GmbH, Göttingen, Germany). The PCR products were digested with a restriction enzyme at 37 °C overnight. The PCR products were electrophoresed on a 2.0% agarose gel stained with ethidium bromide, and visualized on an ultraviolet transilluminator. Restriction enzymes and fragment sizes after digestion are listed in Table 1. Genotyping of the IL13 (rs20541) Single Nucleotide Polymorphism. Genotyping of the IL13 (rs20541) SNP was performed by high resolution melting (HRM) curve analysis following touchdown PCR amplification. Primers were designed using Primer3 (http://simgene.com/ Primer3), and manufactured by Sigma-Aldrich Produktions GmbH, Steinheim, Germany. Primer sequences were as follows: forward 5’-CTG CAA ATA ATG ATG CTT TCG-3’ and reverse 5’-ACC TGC TCT TAC ATT TAA AGA AAC TT-3’. The touchdown PCR amplification was performed on a LightCycler®480 detection system (Roche Applied Science, Mannheim, Germany). Samples were amplified in reactions containing 2 μL DNA (2.5 ng/μL), 3 μL of 2X LightCycler® 480 High Resolution Melting Master Mix (Roche Applied Science), 200 nM of each primer, 0.8 μL of MgCl2 (2 mM final concentration) and RNase-free water in a final reaction volume of 10 μL. The touchdown PCR program was as follows: initial denaturation at 95 °C for 10 min., followed by 45 cycles of 10 seconds at 95 °C, 15 seconds at 65 °C (secondary target temperature 53 °C, with 0.5 °C/step) and 10 seconds at 72 °C. The HRM curve analysis was performed with temperature ranges used for the melting curve generation from 65 °C to 95 °C with 25 signal acquisitions per °C. Statistical Analysis. All statistical analyses were performed with the Statistical Package for the Social Sciences (SPSS), version 23 (IBM Corporation, Armonk, NY, USA). Genetic polymorphisms were expressed as bivariate descriptive parameters, either by each of the two SNP alleles or by either of two genetic models assuming a dominant/recessive influence of the non-ancestral allele on a particular phenotype. Appropriate statistical techniques and methods were used, depending on properties of the measured parameter. Association analyses were done by either the two-sided Fisher’s exact test, risk determination, Mann-Whitney test or Independent Samples t-test. Predictive models for bivariate variables describing phenotype groups were constructed using logistic regression with a stepwise exclusion of parameters (backward: Wald exclusion method with parameter exclusion criteria at p >0.05). Initially included parameters were: all SNPs, adenomyosis, age at diagnosis, family predisposition, menarche, number of pregnancies, parity, body mass index, intake of oral contraceptives, intake of gestagenes, age at first sexual intercourse, and smoking. We did not perform a Bonferroni correction in our statistical analysis to avoid an unnecessary increase of type II errors [25]. Power of statistical association analysis (p) for dominant and recessive genetic models was calculated using G*Power computer software (Heinrich Heine University, Dusseldorf, Germany). The power of the study was calculated post hoc, using the generally accepted standard error rate α = 0.05.



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