Balkan Journal of Medical Genetics

ASSOCIATION OF NFKB1, NKX2-5, GATA4 AND RANKL GENE POLYMORPHISMS WITH SPORADIC CONGENITAL HEART DISEASE IN GREEK PATIENTS
Aidinidou L1, Chatzikyriakidou A1, Giannopoulos A2, Karpa V1, Tzimou I2, Aidinidou E3, Fidani L1,*
*Corresponding Author: Professor Liana Fidani, Department of General Biology, Medical School, Aristotle University of Thessaloniki, University Campus, GR-54124, Thessaloniki, Greece. Tel.: +30- 231-099-9165. Fax: +30-231-099-9019. E-mail: sfidani@auth.gr
page: 15

MATERIALS AND METHODS

A total of 43 CHD patients (15 female, 28 male, 5.6± 1.6 average age of years), selected based on echocardiogram evidence of CHD and 100 healthy controls with no history of CHD were included in the present study. Genomic DNA of each individual was extracted from a sample of venous blood by use of modified salting out method. The PCR-RFLP method was performed to genotype the polymorphism of NFKB1 (–94ins/delATTG), NKX2-5, GATA4 and RANKL. The primers were designed using the primer-BLAST program in NCBI (National Center for Biotechnology Information) This tool is publicly available at http://www.ncbi.nlm.nih.gov/tools/primer-blast. A New England Biolabs (NEB)-cutter, version 2.0, is a program available via a web server (http://tools.neb. com/ NEB-cutter), was used to find the restriction enzymes. The primer sequences are shown in Table 1. The amplified 281 bp (deletion allele) or 285 bp (insertion allele) fragments were then digested using PfIMI restriction endonuclease for the –94ins/delATTG polymorphism of NFKB1. The wild type allele D yields a 281 bp band and allele I yielded two fragments of 240 and 45 bp, respectively. The A and G alleles of the NKX2-5 gene polymorphism produced DNA fragments of 264+37 and 213+51+37 bp, respectively, using BpmI restriction endonuclease. For the GATA4 gene polymorphism, the C and T alleles produced DNA fragments of 261 and 174+87 bp, respectively, using the HpyCH4III restriction endonuclease. For the RANKL gene polymorphism, the A and G alleles produced DNA fragments of 149+61 and 210 bp, respectively, using the EcoRV restriction endonuclease. All four single nucleotide polymorphisms (SNPs) were assessed for Hardy-Weinberg equilibrium (HWE) by the χ2 test. Statistical analysis was performed by the Statistical Package for the Social Sciences (SPSS) version 24.0 software (https://www.ibm.com/product/spssstatistics) and Project for Statistical Computing (R) (version 3.6.1) software (https://www.r-project.org/), to identify the differences in variant contributions between the two groups under the six models of genetic association. Haldane Anscombe correction (0.5 is added to all cells of the contingency table) was used when we had cells in the contingency table with zero counts. In addition, the false discovery rate (FDR) method was applied to correct for bias caused by multiple comparisons [14]. The odds ratio (OR) and 95% confidence interval (95% CI) was estimated, and a p value of <0.05 was considered to be statistically significant.

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