Balkan Journal of Medical Genetics

ASSOCIATIONS BETWEEN VASPIN RS2236242 GENE POLYMORPHISM, WALKING TIME AND THE RISK OF METABOLIC SYNDROME
Suliga E1, Kozieł D2, Cieśla E3, Rębak D2, Wawszczak M2,*, Adamus-Białek W2, Naszydłowska E4, Piechowska A2, Głuszek S2
*Corresponding Author: Monika Wawszczak, MSc, Department of Surgery and Surgical Nursery with Genetics and Research Laboratory, Institute of Medical Sciences, Jan Kochanowski University, al. IX Wieków Kielc 19a, 25-516, Kielce, Poland. Tel: +48-413696978. E-mail: mwawszczak@ujk.edu.pl
page: 41

MATERIALS AND METHODS

Study Population. Genomic DNA isolated from 108 participants with MetS and 110 controls of the Polish- Norwegian Study (PONS) project were used in this study. All participants were Caucasians. The study included blood pressure measurements, the analysis of collected fasting-blood samples, anthropometric measurements and a questionnaire interview. Detailed information regarding the project and research procedures were described in a previously published study [9] In 108 participants, MetS was diagnosed on the basis of the International Diabetes Federation criteria [10]. Ethics. The study was approved by the Ethics Committee from the Cancer Centre and Institute of Oncology in Warsaw (data collection) and by the Committee on Bioethics at the Faculty of Health Sciences, Jan Kochanowski University in Kielce, Poland (data analysis) (decision number: 45/2016). Genotyping. Determination of the vaspin rs2236242 polymorphism was conducted by the tetra-primer amplification- refractory mutation system polymerase chain reaction (T-ARMS PCR) method, according to the protocol described by Hashemi et al. [7]. The external primers VasFO (5’-GGA GGC AGA CCA GGC ACT AGA AA-3’) and VasRO (5’-ACC ATC TCT CTG GCT TCA GGC TTC- 3’) were applied for the amplification of 378 bp DNA of vaspin gene fragment. Inter primers were specific to each genotype: Vas FI primer (5’-AAG ACG CCG CTT CTG TGC ACT-3’) for the T allele and Vas RI primer (5’-CAC AGG GAC CCA GGA TAA CTT GCT3’) for the A allele. The commercially available PCR premix (PCR Mix Plus; A&A Biotechnology, Gdynia, Poland) was prepared according to the manufacturer’s instructions. Briefly, 1 μL of each primer (10 pmol/μL), 1 μL of template DNA (~25 ng/μL), 12.5 μL of PCR Pre Mix and made up to 25 μL with DNAse-free water. The procedure of T-ARMS PCR was optimized and final conditions were: denaturation at 95 °C for 3 min., amplification for 30 cycles at 95 °C for 1 min., 60 °C for 1 min., and 72 °C for 1 min., and a final extension at 72 °C for 8 min. After DNA amplification, the gel documentation system (InGenius; Syngene, Cambridge, Cambridgeshire, UK) was used for product visualization. The specific genotypes were characterized, respectively: 174 and 378 bp for homozygous T, 248 and 378 bp for homozygous A, and 174, 248 and 378 bp for heterozygous TA (Figure 1). Meta-Analysis of the Vaspin Genotypes. The metaanalysis of the vaspin rs2236242 polymorphism included the data from Iran [7], Egypt [6,11] and Poland (this study). The distribution of the genotype was examined in MetS volunteers (n = 459) and controls (n = 459). Statistical Analyses. All data were analyzed using the Statistical Package Statistica software (version 13.1) (TIBCO Software Inc., Palo Alto, CA, USA). Mean ± standard deviation (SD) and medians with interquartile ranges (Me; Q1-Q3) for continuous variables and proportions expressed as percentages for categorical variables, were used to describe the baseline characteristic of the group. Differences between MetS participants and the control group were assessed through the χ2 test and the U Mann-Whitney-test. The χ2 test was also used to analyze genotype and allele frequencies and to determine whether the genotype distribution was congruent with Hardy-Weinberg equilibrium expectations. The multivariate logistic regression analyses were used to estimate the odds ratio (OR) and 95% confidence interval (95% CI) for MetS and its five components. The TT was a reference genotype in the analysis. In the second model, the risk for MetS and its components was adjusted for age, gender, walking time (≤ or >60 min./day) and smoking. The choice of confounders in models II and III was based on the scores obtained from the cluster analysis. It was performed on raw values by means of a single linkage method. A distance measurement was the Euclidean distance. In the third model, body mass index (BMI), was added (as a continuous variable). Additionally, in models II and III, interactions between vaspin rs2236242 gene polymorphism and walking, in relation to MetS and its components, were considered. A reference point for walking was time ≤60 min./day. In all analyses, a p value of <0.05 was considered to be statistically significant.

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