Balkan Journal of Medical Genetics

POLYMORPHISM OF THE ADRB2 rs1042713 GENE IS NOT ASSOCIATED WITH SPONTANEOUS PRETERM BIRTH: ANALYSES IN A SLOVENIAN SAMPLE AND META ANALYSIS
Peterlin A1, Maver A1, Jan Z2, Lovrecic L1, Tul N2, Peterlin B1
*Corresponding Author: Professor Borut Peterlin, Clinical Institute of Medical Genetics, Division of Obstetrics and Gynecology, University Medical Centre Ljubljana, Šlajmerjeva 3, 1000 Ljubljana, Slovenia. Tel/Fax: +386-1-5401-137. E -mail: borut.peterlin@guest.arnes.si
page: 35

MATERIALS AND METHODS

Case-Control Association Study in the Slovenian Population. Participants of the study all signed a written informed consent. The Republic of Slovenia National Medical Ethics Committee approved the study. Definition of SPTB and Inclusion Criteria for the SPTB Cases. We included healthy mothers with singleton pregnancies who delivered after a spontaneous onset of labor (SPTB) before completed 37 weeks’ gestation. Gestational age was determined by the last menstrual period and confirmed by an ultrasound examination in the first trimester. Cases with known risk factors for SPTB (e.g., diabetes, hypertension, kidney disease, autoimmune conditions, infections, uterine malformations and complications during pregnancy) or neonates born with congenital anomalies or evidence of infection were excluded. All analyzed subjects were of Caucasian origin. Additional information on maternal characteristics is shown in Table 1. Study Sample in the Case-Control Association Analyses. We conducted a case-control study including 98 female patients with SPTB and 135 female controls who gave birth at the Division of Obstetrics and Gynecology, University Medical Centre in Ljubljana, Slovenia. Controls were age-matched healthy mothers who delivered after an uncomplicated pregnancy after 37 weeks and delivered a neonate with appropriate-for-gestational-age birth weight (Table 1). Genotype Analyses. Genomic DNA was isolated from peripheral blood leukocytes using standard procedures. Real-time polymerase chain reaction (PCR) method performed on a 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using KASPar SNP genotyping chemistry carried out genotyping of the single nucleotide polymorphism (SNP). The PCR reaction mix of 8 μL final volume consisted of 3 μL of DNA sample, 4 μL of reaction mix 2X, 0.11 μL assay mix and 0.89 μL H2O. The protocol for PCR amplification was as follows: initial denaturation step at 94 °C for 15 min., then 10 cycles of denaturation at 94 °C for 20 seconds, followed by 5 seconds at 57 °C or 61 °C, 10 seconds at 72 °C, 10 seconds at 94 °C, 20 seconds at 57 °C or 61 °C, and final extension at 72 °C for 40 seconds. The allelic discrimination analysis was performed using SDS Software Version 1.2 (Applied Biosystems). Genotype assignment was performed and interpreted independently by two investigators. Statistical Analyses. We analyzed the significance of associations between allelic and genotype frequencies and disease status using the χ2 test. Odds ratios (ORs) and their respective 95% confidence intervals (95% CIs), were calculated to compare allelic and genotype distribution in patients and controls. To provide an additional quality step of the genotyping process we calculated the χ2 goodnessof- fit tests for deviation of genotype distribution from those predicted by Hardy-Weinberg equilibrium. The investigated associations were regarded as significant when they reached p ≤0.05. The R statistical language (version 3.0) was used to perform the analyses. To calculate the power of the study DSS Researcher’s Toolkit (https://www.dssresearch.com/Knowledge Center/ toolkitcalculators/statisticalpowercalculators.aspx) was utilized. Calculations showed that our power to detect a significant result in the presence of the actual genotype relative risk equal to at least 2.0 was 85.8% when taking into account the sample size, the significance threshold of 0.05, and the risk genotype frequency of 15%. Meta Analyses. A literature search to find potential eligible studies of the association between ADRB2 rs1042713 and SPTB was conducted in PubMed (National Center for Biotechnology Information, January 1966-December 2016), Scopus (December 2016), Google Scholar (December 2016), and HugeNavigator (December 2016). We limited our search to articles in the English language. Keywords searched included: (ADRB2 or β-2-adrenergic receptor gene or polymorphisms) AND (preterm birth or preterm labor). The AND operator was used to create various combinations of selected terms. Studies were selected and reviewed by two independent authors who reached a consensus on all of the items. Study Selection and Data Extraction. We included human studies meeting following criteria: 1) a genotype of ADRB2 rs1042713 and 2) case-control study in which genotyping was carried out for the group of SPTB cases and control group; 3), SPTB defined as <37 weeks’ gestation; 4) control group defined as women who gave birth after 37 weeks’ gestation. For each study included in the meta analysis, we extracted authors, year of publication, study population geographic origins, number of SPTB cases and controls, SPTB definition, an occurrence of preterm premature rupture of membranes (PPROM), inclusion criteria for control women, and genotype count for SPTB cases and controls. We classified subjects into three genotypes: AA, GA and GG. Then pooled effect was calculated for the dominant genetic model (GA+GG vs. AA) and recessive genetic model (AA+GA vs. GG) in the ADRB2 rs1042713 polymorphism. Cochrane’s Q and I2 tests were used to assess heterogeneity between the studies, with the null hypothesis that there is no difference in findings of primary studies. Heterogeneity was considered significant when p <0.1 for Cochrane’s Q statistics. Random effect model (der Simonian-Laird) was applied upon the detection of heterogeneity; otherwise, fixed effect model (Maentel- Haenszel) was used. The random effect model takes into account diversity of included studies due to intra-study sampling errors and inter-study variances, while the fixed effect model assumes that the observed variations between studies are caused by chance alone. Publication bias was assessed by Funnel plot. The asymmetry of the Funnel plot was analyzed with the Egger’s test. The analysis was carried out with the R statistical language (version 3.0).

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