Computational protocol: Comparative analysis of inflammatory gene expression levels in metabolic syndrome and coronary artery disease

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Protocol publication

[…] A total of 178 male participants, comprising 57 with symptomatic CAD and 121 age-matched male individuals without CAD, were included in this study. These were enrolled during May 2012 to February 2014, and were randomly selected from Indian Atherosclerosis Research Study (IARS). The IARS is an ongoing epidemiological study with an aim to investigate the genetic factors and biomarkers associated with CAD along with their risk factors in the Asian Indian population. The IARS families were enrolled from Narayana Institute of Cardiac Sciences and other local hospitals/clinics in Bengaluru, in southern India, and the Asian Heart Institute and Research Centre, Mumbai, in Western India. A detailed design of the IARS is published elsewhere. Given a sample size of 178, and considering the effect size to be medium, β/α ratio of 4, with a standard value of 0.20 and 0.05 for β and α, respectively, the power of study was estimated to be 95 per cent (G*Power 3.1.6 tool, (Franz Faul, University of Kiel, Kiel, Germany).Symptomatic individuals with CAD were defined as those with abnormal electrocardiogram (ECG), angiographically confirmed the presence of CAD, having >70 per cent stenosis in any one major epicardial artery or >50 per cent stenosis in two or more arteries, those undergoing percutaneous transluminal coronary angioplasty or bypass graft surgery. This included 63 per cent chronic stable angina patients and 37 per cent myocardial infarction cases. CAD severity was defined based on the number of diseased coronary vessels as one, two or three or more, showing >50 per cent luminal narrowing of the vessels. Control subjects were clinically asymptomatic for CAD, having no known history of CAD or sudden death in the family and normal ECG and were characterized for the presence of MetS. All participants were free from concomitant infection (HIV, hepatitis B surface antigen, hepatitis C virus, cold, cough, fever) and other major illnesses (cancer, liver failure, arthritis, etc.). All participants selected for the study provided informed written consent. The study was approved by the institutional ethics committee.Definition and classification of metabolic syndrome (MetS): The presence of MetS in this study participants was determined based on the modified criteria suitable for Asian Indians, which includes lower cut-offs for waist circumference (WC) ≥90 cm, serum triglyceride (TG) ≥150 mg/dl (1.7 mmol/l), high-density lipoprotein-cholesterol (HDL-C) ≤40 mg/dl (1.03 mmol/l), blood pressure of ≥130/85 mmHg and fasting blood glucose (FBG) level of ≥110 mg/dl (6.1 mmol/l). Individuals having three or more of the five risk components were classified as having MetS. A MetS score of 0-5 was determined based on the number of MetS components present in an individual.The 121 individuals without CAD were characterized for the presence of MetS. Individuals having three or more risk factors were categorized into MetS, those with one or two risk factors into non-MetS group while individuals having no risk factors served as controls. A score of 0 to >4 was assigned based on the number of MetS components present in an individual. Group-wise breakdown of the 121 individuals was as follows: (i) 18 without any risk factors (control group), (ii) 50 with one or two risk factors (non-MetS group), and (iii) 53 with three or more risk factors (MetS group) (). In most groups, abdominal obesity was the most common feature, followed by low HDL-C and high TG levels.Data collection: Detailed demographics, anthropometrics, vital parameters, medical history, medication and pedigree information were recorded for each participant. Presence of T2DM, hypertension and CVD was ascertained based on self-report of physician's diagnosis and/or use of prescription medication along with perusal of their medical records. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured. BMI was calculated as a ratio of weight in kilogram to height in square metre.Laboratory assay: Venous blood (22 ml) was collected in evacuated tubes after overnight fasting of 10-12 h. Serum, EDTA and citrate plasma samples were separated by centrifugation within two hours of sampling and aliquots were stored at −80°C for biochemical analysis. Plasma levels of total cholesterol (TC), TG, HDL-C and FBG were measured using reagents from Siemens Dimension Flex reagent cartridge (Siemens Healthcare Diagnostics Ltd., UK) and standards from Randox laboratories (Crumlin, UK) on Siemens dimension Xpand plus instrument (Siemens Dade Behring, Liederbach, Germany). Liquichek lipid control was used as an assayed quality control (Bio-Rad, USA) that was run along with every experiment. Low-density lipoprotein-cholesterol (LDL-C) was calculated by applying Friedewald's formula.Selection of candidate genes: The network approach helped identify key inflammatory genes from a large panel of genes involved in the inflammatory process and the direct interactions among the genes in the leukotriene pathway. The 18 genes prioritized for the present study are listed in and include the leukotriene pathway genes, inflammatory genes and key transcription factors.Real-time quantitative PCR (qPCR) assay: Total RNA was isolated from 3 ml of fresh whole blood sample using QIAamp RNA Blood Mini Kit (Qiagen Inc., Germany), treated with DNase I (Fermentas, Canada) and reverse transcribed to cDNA using cDNA archive kit (Applied Biosystems, USA), following the manufacturer's instructions. RNA was quantified by absorbance at 260 nm and the purity was estimated by the ratio A260/A280 nm and A260/A230 nm using NanoDrop 1000 TM (Thermo Fisher Scientific, USA). Quantitative real-time polymerase chain reaction (q-PCR) was performed in duplicates on 7900 HT Fast RT-PCR system (Applied Biosystems) either based on TaqMan or SYBR green chemistry. Arachidonate 5-lipoxygenase (ALOX5), arachidonate 5-lipoxygenase activating protein (ALOX5AP) and leukotriene A4 hydrolase (LTA4H) gene expression was measured using TaqMan chemistry and the primer/probe pairs with the following assay IDs - Hs01095330_m1 (ALOX5), Hs00233463_m1 (ALOX5AP) and Hs1075882_ml (LTA4H), were purchased from Applied Biosystems.The primer pairs used for the SYBR green-based gene expression assay were selected either from PrimerBank or designed using PrimerQuest® program (Integrated DNA Technologies, Coralville, USA) for those genes where the primers were not available in PrimerBank. All primer sequences were further verified using BLAST search. The RT-PCR efficiency of each primer pair was determined by constructing the standard curve with serial dilution of sample. Relative quantitation for the Taqman assay was calculated using RQ Manager v1.2 software (Applied Biosystems) while the relative expression for SYBR green assay was estimated by comparative Ct method. Beta-glucuronidase (GUSB) gene served as the endogenous control. The expression levels were initially normalized to an endogenous control and mRNA abundance in each sample was determined in relation to a reference sample (calibrator). All assays were set up in duplicates. The outlier samples were re-tested and persistent outliers were excluded from further analysis.Statistical analysis: Student's t test and multivariate analysis were used to determine the differences in normalized mRNA expression levels and other quantitative traits between the cases and controls. Normality distribution of mRNA expression levels was assessed with Q-Q plot. Statistical differences for experiments with three or more groups were determined using analysis of variance (ANOVA), and the linearity of association was tested with polynomial contrast for linear trend. Binary logistic regression analysis was used to assess the association of mRNA expression with CAD and to estimate the corresponding odds ratio (OR). Pearson's correlation was performed to evaluate the correlation in gene expression between the genes under study. Age, hypertension, diabetes and WC was considered as potential confounders and appropriately adjusted during analysis. All analyses were performed using SPSS v 17.0 (SPSS Inc., Chicago, IL, USA) statistical software package. Quantitative variables are expressed as mean±standard error of mean, unless stated otherwise. All statistical tests were two-sided, with 95 per cent confidence interval (CI). […]

Pipeline specifications

Software tools G*Power, PrimerQuest
Databases PrimerBank
Applications Miscellaneous, qPCR
Organisms Homo sapiens
Diseases Coronary Artery Disease, Metabolic Diseases
Chemicals Leukotrienes