Subjects of this nested case-control study were selected from among participants of the Tehran Lipid and Glucose Study (TLGS), a large-scale, community-based, prospective study being performed on sample of residents of district 13 of Tehran, capital of Iran. The first phase of the TLGS was conducted from 1999 to 2001 in 15,005 subjects, aged ≥3 years, and follow-up examinations have been conducted every 3 years (2002–2005; 2006–2008; 2008–2011 and 2011–2014) to identify newly developed diseases. Details of this ongoing cohort study have been published elsewhere [11, 12].
Of 11,001 and 9807 individuals, aged ≥18 years, who participated in baseline and second follow–up surveys respectively, 5280, were excluded because of having MetS at either baseline or the second follow-up survey. In the current study, from among participants who developed MetS in third (n = 918), the fourth (n = 827) or fifth (n = 1050) phases, 1198 cases were randomly selected. After excluding individuals with a history of cardiovascular events, weight loss or gain >5 kg in the last 6 months, pregnancy and lactating, or those taking any CVD/anticoagulant/steroid or hormonal medication, 1158 cases were included in the study. Each case was individually pair matched randomly with a control by age (±5 years) and sex from among those who had not developed ≥1 MetS components at the time that the corresponding case developed MetS. After excluding cases/controls lacking DNA purification in the range of 1.7 < A260/A280 < 2, and those whose reported energy intake divided by the predicted energy intake did not qualify for the ±3 SD range, finally data of 1630 (815 pairs) with MetS and matched controls remained for analysis.
Informed written consents were obtained from all participants. The study protocol was approved by two ethical committees, the ethical committee of the Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran and the medical research ethics committee of JundiShapour University of Medical Sciences, Ahvaz, Iran.
Dietary intake was assessed with the use of a valid and reliable, 168-item semi-quantitative food frequency questionnaire (FFQ) to assess the usual food intake of individuals during the 12 months before the examination. The consumption frequency of each food item on a daily, weekly or monthly basis was converted to daily intakes; portion sizes were then converted to grams, using household measures. Because the Iranian Food Composition Table (FCT) is incomplete, we used the United States Department of Agriculture (USDA) FCT to analyze foods and beverages ; however, the Iranian FCT was used for some national foods and beverages, which are not listed in the USDA FCT . Based on macronutrient composition and using current literature, 25 food groups were categorized [15–17].
Anthropometric assessment was done using a standardized process. Weight was measured to the nearest 100 g, using digital scales while the subjects were minimally clothed and not wearing shoes. Height was measured to the nearest 0.5 cm with a tape measure in a standing position and with shoulders in a normal alignment and without shoes. Waist circumference (WC) was measured to the nearest 0.1 cm, at the umbilical level over light clothing, using an unstretched tape meter without any pressure to body surface. Blood pressure (BP) was measured on the right arm in a sitting position twice, after a 15 min rest in the sitting position and finally, the mean of the two measurements was reported as the participant's BP.
Physical activity level was assessed using the Persian translated modifiable activity questionnaire (MAQ) with high reliability and relatively moderate validity . The frequency and time spent on light, moderate, hard and very hard intensity activities, according to the list of common activities of daily life over the past year were obtained, and the activity data was transformed into metabolic equivalent hours per week (METs/h/wk) [18–20].
Fasting blood samples were taken after 10–12 h of overnight fasting. Fasting plasma glucose (FPG) and triglycerides (TG) were measured by the enzymatic colorimetric method and high density lipoprotein cholesterol (HDL-C) was measured after precipitation of apo-lipoprotein β with phosphotungstic acid. Analyses were performed using Parsazmun kits (Tehran, Iran) and a Selectra 2 autoanalyzer (Vital Scientific, Spankeren, Netherlands).
Genomic DNA was extracted from peripheral blood using standard salting-out method . The selected polymorphism (rs12970134) was studied by Tetra-primer ARMS method. The primers determined through the national center for biotechnology information (NCBI) site . Our T-ARMS assay with different inner allele specific primers produce allele-specific PCR products; forward: ATA CTG ACT CTT ACC AAA CA AAG CAC GAA and reverse: AGC ACC CTT CTG ATA AAT CTT TGT TAG C. Two outer primers produce a PCR product to be used as an internal control for reaction; forward: AGT AAG AGT GAA GAT TTG AGG GAT GGA GA and reverse: TCT CTT CGA GGA GTG TTT GAG TCT GA. For the SNP mentioned, the PCR reaction was optimized in a 12.5 μl total volume containing 1.5 μl DNA template, 6.25 μl master mix containing MgCl2, Smart Taq polymerase (CinnaGene co; Iran), BSA 0.1 % (TaKaRa; Japan), 2 μl primer containing (outers and inners) and 2.75 μl Water.
The PCR amplification was carried out with an initial denaturation at 94 °C for 3 min, denaturation at 95 °C for 30 s (35 cycles), 45 s of annealing at 63.13 °C (35 cycles), 1 min of extension at 72 °C and an additional 10 min of extension at 72 °C at the end of the final cycle. PCR products were resolved by electrophoresis in a 1.8 % agarose gel, this procedure which rendered three bands in heterozygotes (614, 372 and 298 bp) and two bands in homozygotes (mutant allele carrier resulting in 614 and 298 bp, wild carrier allele resulting in 614 and 372 bp). A 614 bp band was always obtained as the control for the success of the amplification. To validate the accuracy of genotype scoring by tetra-primer ARMS-PCR, the three (614, 372 and 298 bp) fragments were directly sequenced.
MetS was defined according to the modified definition of the National Cholesterol Education Program/Adult Treatment panel III (ATP III) , as having three or more of the following criteria: 1) abdominal obesity, WC ≥ 95 cm for both genders, according to the newly-introduced cut-off points for Iranian adults , 2) high TG (≥150 mg/dL) or drug treatment, 3) low HDL-C (<40 mg/dL in men or < 50 mg/dL in women) or drug treatment, 4) high blood pressure (SBP/DBP ≥ 130/85 mmHg) or antihypertensive drug treatment and 5) high FPG (≥110 mg/dL) or drug treatment for elevated glucose.
For the descriptive analysis, a comparison of qualitative and quantitative variables between cases and controls was done, using the student t and Chi square test statistics respectively; TG concentration (a non-symmetric quantitative variable) was log-transformed before the statistical analysis. The genotype and allele frequencies for the analyzed polymorphism were obtained using Power-Marker software. Pearson’s Chi-square statistic was used to calculate the Hardy-Weinberg equilibrium.
Dietary patterns were identified using factor analysis with varimax rotation, based on 25 food groups. Dietary patterns were extracted based on the eigenvalues (>1), scree plot, factor interpretability, and the variance explained (>5 %).
Conditional logistic regression was used to estimate the interactions of SNP with quartiles of dietary factors in relation to MetS after adjustment for baseline BMI. Two likelihood scores were obtained performing this statistical analysis, with and without the interaction terms; P value for interaction was determined by performing the likelihood ratio test.
Conditional logistic regression was used to generate odds ratios (ORs) for MetS for individuals with the carrier or non carrier of risk allele (GG/AG + AA) across quartiles of dietary pattern scores, food groups and nutrients intake (Q1-Q4). The lowest quartile of dietary factors and the homozygote group with major allele were examined as the reference group. Unconditional logistic regression was performed to estimate the interactions of MC4R SNP with quartiles of dietary pattern scores, food groups and nutrients intake in relation to MetS components. All ORs were adjusted for variables proven to be associated with MetS components, including age, gender, educational level, smoking status, physical activity and energy intake. To determine the P value for trend across the quartiles of dietary factors, logistic regression was used, using the median of each quartile of dietary pattern scores as a continuous variable. Data were analyzed using STATA statistical package v.12.0 or SPSS Version 16.0 (Version 16.0; SPSS, Chicago, IL).