In this cross-sectional study atotal of 453 genetically unrelated adult subjects of both genders, ranging from18 to 60 years of age, were enrolled from our previousstudy in which the relationship of twoLXR? SNPs with obesity was investigated on individuals who were randomlyselected from participants of a cohort studyof metabolic syndrome and cardiovascular disease riskfactors performed in Mashhad as a second largest city in Iran (15). The worldhealth organization-body mass index (WHO-BMI) was calculated as a weight to height ratio (kg/m2), anda BMI of 18.5-24.99, 25–29.99 and ?30 kg/m2were taken as cut-off values defining normal weight, overweight and obesity,respectively.
With this in mind, all subjects were classified into threegroups: 209 subjects as overweight, 168 subjects as obese, and 76 healthyindividuals as normal weight. Before participatingvoluntarily in research work, informed writtenconsent was obtained from all participants. Afterthat, a completephysical assessment and anthropometric measurements were performed by trainedhealth workers. Blood samples were drowned after a fasting period, and data on age, sex, geographical birthplace, ethnicity, past medical andfamily history, and dietary habits werecollected. Allsubjects with a history of Stroke, MI (Myocardial infarction),endocrinological abnormalities, diabetes mellitus, alcohol consumption, heart,liver and/or renal disease, or those who were underhigh blood pressure medication and lipid orglucose lowering treatment were excluded from the study. This research project was approved by the Ethical Committee of the Shahid BeheshtiUniversity of Medical Science (SBUMS: Research Project No.
648). 2.2. Anthropometric andBiochemical measurementsAnthropometric and clinical measurements were measured according tointernational standard procedures as described indetails in our previous studies (11, 15).In brief, all parameters including height, weight, waist circumference(WC), hip circumference (HC), waist/hip ratio, systolic blood pressure (SBP)and diastolic blood pressure (DBP), total cholesterol (TC), high- densitylipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C),triglycerides (TG), fasting blood glucose (FBG) and serum C-reactive protein(CRP) were measured. 2.3. GenotypingGenomic DNA was isolated fromthe peripheral blood sample with FlexiGene DNA Kit (Qiagen) in accordance with the manufacturer’sinstructions.
Genotyping of singlenucleotide polymorphisms (SNPs) was performed by allelic discriminationassays using TaqMan probes (C_16059177_10; AppliedBiosystems, USA). Allelic discrimination was performedon Applied Biosystems 7500 real-time PCR System. The following conditionswere used for the polymerase chain reaction: initial denaturation step at 95°C for 10 minutes, and 40 cycles of 92°C for 15 seconds and 60°Cfor 1 minute. Approximately 10% of the samples were regenotyped to ensure reproducibilityand accuracy.
2.4. Statistical analysisAll statistical analysis was performed using SPSS v.
16.0 (SPSS Inc., Ill., USA). The descriptive statisticswere determined for all variables and are presentedas mean (SD) for the normally distributed variables (or as the median (IQR) fornon-normal variables). The chi-squareor Fisher’s exact tests were applied to evaluate differences in allele and genotypedistribution among the examined groups. It was also used to assess any deviation from theHardy-Weinberg equilibrium (HWE) for the study SNP. Independent-samplest-test was used to compare the means of demographicand clinical variables between independent groups.
Analysesof variance (ANOVA) followed by theBonferroni posthoc test, was performed tocompare anthropometric traits for differencesbetween genotype groups. Using data from our previous reportregarding the other common variant (rs17373080) ofLXR? gene (16), haplotypefrequencies for two sites and any potential relationship with excess weight wereestimated using the SNPStats program (17). Logistic regression analyses were used to calculate theodds ratios (ORs) with 95% con?dence interval (CI) ofeach related parameter, both in crude or adjusted for age and gender.All the analyses were two-sided and statistical significance was set at p-value of < 0.05.