Subjects:The study was carried on 50 myocardial infarction patients and 50control subjects. Both males and females are included. All patientsand control subjects were Egyptians living in Ismailia city.
All theregulations of the ethics committee were considered. The controlsubjects were judged to be free of coronary heart disease by history,clinical examination and electrocardiography. Acute MI patients werecollected from the cardiovascular coronary care unit at Suez Canal171Œ>aliGa I (Badran et aŒuniversity hospital (CCU) from January 2010 to April 2010.
Thediagnosis of MI was established in the presence of chest pain lasting >20 min combined with ST-segment elevation or pathological Q waveson a surface electrocardiogram. Patients with MI had to show aregional wall motion abnormalities corresponding to theelectrocardiographic infarct localization. A set of questionnaires thatincluded details of medical history and cigarette smoking wascompleted.
BP (blood pressure), weight, height was recorded, andbody mass index (BMI) was calculated. Coronary heart disease riskfactors were recorded for all individuals Diagnosis of diabetesmellitus was based on an actively antidiabetic treatment and/or fastingblood glucose >126 mg/dL on two occasions or 2-h postprandialserum glucose>200 mg/dl or a random plasma glucose >200 mg/dl orin a patient with classic symptoms of hyperglycemia or hyperglycémiecrisis. (American Diabetes Association, 2010).
Diagnosis ofhypertension was based on an elevated systolic (>140 mmHg) and/ordiastolic (>90 mmHg) BP on three occasions and/or the cun-ent use, ofantihypertensive drugs (Chalmers et al, 1999). Dyslipidemia wasdiagnosed if fasting total serum cholesterol > 200 mg/dl, LDLeholesterol> 130 mg/dl, fasting serum triglycérides > 150 mg/dl orHDL-C (<40 mg/dL in men or <50 mg/dL in women) or treated bystatins National Cholesterol Education Program (NCEP), 2002.Patients were considered obese when body-mass index the weight inkilograms divided by the square of the height in meters was >25while could be considered as cigarette smoker when they smoked 10or more cigarettes daily for more than 6 months (Yamada et ai,2002). All the regulations of the ethics committee were considered.Blood sampling:Blood from MI patients was collected within 24 hours of admission.
Four ml of venous blood were collected after an overnight fast, 2 ml invacutainer tubes eontaining EDTA as anticoagulant and 2 ml in plaintubes. The former tubes were used for DNA extraction for subsequentgenetic investigations, and the later one were centrifuged after clottingformation and the obtained serum were used for assessment of lipidprofile and serum glucose then stored at-20°C.Biochemical analysisFasting serum glucose and 2 hours postprandial blood glucose.
Glucose was determined using the standard kit based on the glucose172 -oxidase and peroxidase method according to the method of Trinder,(1969) on Roche Diagnostics Hitachi 912® system (RocheDiagnostics, Indianapolis, IN)Lipid profiIe:-Serum total cholesterol was measured usingRocli/Hitachi cholesterol kit Enzymatic colorimetric determinationaccording to the method of Alian et al. (1974) and Roeschleau et al.(1974). Serum triglycérides were measured using Roch/Hitachitriglycérides kit Enzymatic colorimetric determination according tothe method of Siedel et al. (1993) and Whalfeld et al. (1974). HDLcholesterolwas measured using Roch/Hitachi HDL-cholesterol kit andwas estimated by phosphotungstic acid precipitation followed byenzymatic analysis in supernatant fraction (Lopes-Virella et al.,1977).
LDL-cholesterol was calculated by the FriedewaldFormuIa:LDL cholesterol.= Total cholesterol – HDL cholesterolTriglycerides./5)The formula is generally agreed to be inaccurate in non- fastingpatients and when triglycérides are greater than 400 mg/dL (Mamiemietal., 1995).Extraction of genomic DNAGenomic DNA was extracted from the peripheral blood leucocytes ofEDTA anticoagulant blood according to Miller et al. (1988) usingQIAamp DNA Blood Mini Kit (Cat # 51106; Qiagen, UK). DNAsamples were subjected to DNA quantitation using the NanoDrop®(ND)-IOOO spectrophotometer (NanoDrop Technologies, Inc.Wilmington, USA).
DNA Genotyping of SNPs(rs8089) and(rs 18663 89) TaqMan allelicdiscrimination systems were designed and used for genotyping of theSNPs in THBS2 (rs8089) and THBS4 (rsl866389) (Koch et al.,2008).The sequences of the primers are kept confidential by thecompany, the sequences of probes are shown in Table 1. Presence ofthe fluorogenic dyes FAM (6-carboxy-fluorescein) and VIC(proprietary dye of Applied Biosystems) at the 5′ ends of the probemolecules accomplished allele-specific signaling.
Minor groovebinder groups were conjugated with the 3′ ends of the probeoligonucleotides to facilitate formation of stable duplexes between theprobes and their single-stranded DNA targets. TaqMan reactions wereanalyzed on the ABI Prism 7000 Sequence Detection System (AppliedBiosystems)173(DaHCia I (Badran et aCCStatistical analysisStatistical analysis was carried out using the SPSS version 17. Dataare presented as mean±SD. Differences between the means of the 2continuous variables were evaluated by Student t-test.
The allelicfrequencies and genotype distribution were estimated by genecounting. Differences between non continuous variables, genotypedistribution, alíele frequency, and Hardy-Weinberg equilibrium were^ tested by y2 analysis and fisher’s exact test. The odds ratio (OR) for”^ CAD and their 95% confidence interval (CI) associated with eachmutated alíele was also calculated.
Statistical significance was atP<0.05.