Subjects: postprandial serum glucose>200 mg/dl or a random plasma

The study was carried on 50 myocardial infarction patients and 50
control subjects. Both males and females are included. All patients
and control subjects were Egyptians living in Ismailia city. All the
regulations of the ethics committee were considered. The control
subjects were judged to be free of coronary heart disease by history,
clinical examination and electrocardiography. Acute MI patients were
collected from the cardiovascular coronary care unit at Suez Canal
Œ>aliGa I (Badran et aŒ
university hospital (CCU) from January 2010 to April 2010. The
diagnosis of MI was established in the presence of chest pain lasting >
20 min combined with ST-segment elevation or pathological Q waves
on a surface electrocardiogram. Patients with MI had to show a
regional wall motion abnormalities corresponding to the
electrocardiographic infarct localization. A set of questionnaires that
included details of medical history and cigarette smoking was
completed. BP (blood pressure), weight, height was recorded, and
body mass index (BMI) was calculated. Coronary heart disease risk
factors were recorded for all individuals Diagnosis of diabetes
mellitus was based on an actively antidiabetic treatment and/or fasting
blood glucose >126 mg/dL on two occasions or 2-h postprandial
serum glucose>200 mg/dl or a random plasma glucose >200 mg/dl or
in a patient with classic symptoms of hyperglycemia or hyperglycémie
crisis. (American Diabetes Association, 2010). Diagnosis of
hypertension was based on an elevated systolic (>140 mmHg) and/or
diastolic (>90 mmHg) BP on three occasions and/or the cun-ent use, of
antihypertensive drugs (Chalmers et al, 1999). Dyslipidemia was
diagnosed if fasting total serum cholesterol > 200 mg/dl, LDLeholesterol
> 130 mg/dl, fasting serum triglycérides > 150 mg/dl or
HDL-C (<40 mg/dL in men or <50 mg/dL in women) or treated by statins National Cholesterol Education Program (NCEP), 2002. Patients were considered obese when body-mass index the weight in kilograms divided by the square of the height in meters was >25
while could be considered as cigarette smoker when they smoked 10
or 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 in
vacutainer tubes eontaining EDTA as anticoagulant and 2 ml in plain
tubes. The former tubes were used for DNA extraction for subsequent
genetic investigations, and the later one were centrifuged after clotting
formation and the obtained serum were used for assessment of lipid
profile and serum glucose then stored at
Biochemical analysis
Fasting serum glucose and 2 hours postprandial blood glucose.
Glucose was determined using the standard kit based on the glucose
172 –
oxidase and peroxidase method according to the method of Trinder,
(1969) on Roche Diagnostics Hitachi 912® system (Roche
Diagnostics, Indianapolis, IN)
Lipid profiIe:-Serum total cholesterol was measured using
Rocli/Hitachi cholesterol kit Enzymatic colorimetric determination
according to the method of Alian et al. (1974) and Roeschleau et al.
(1974). Serum triglycérides were measured using Roch/Hitachi
triglycérides kit Enzymatic colorimetric determination according to
the method of Siedel et al. (1993) and Whalfeld et al. (1974). HDLcholesterol
was measured using Roch/Hitachi HDL-cholesterol kit and
was estimated by phosphotungstic acid precipitation followed by
enzymatic analysis in supernatant fraction (Lopes-Virella et al.,
1977). LDL-cholesterol was calculated by the Friedewald
FormuIa:LDL cholesterol.= Total cholesterol – HDL cholesterolTriglycerides./5)
The formula is generally agreed to be inaccurate in non- fasting
patients and when triglycérides are greater than 400 mg/dL (Mamiemi
etal., 1995).
Extraction of genomic DNA
Genomic DNA was extracted from the peripheral blood leucocytes of
EDTA anticoagulant blood according to Miller et al. (1988) using
QIAamp DNA Blood Mini Kit (Cat # 51106; Qiagen, UK). DNA
samples 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 allelic
discrimination systems were designed and used for genotyping of the
SNPs in THBS2 (rs8089) and THBS4 (rsl866389) (Koch et al.,
2008).The sequences of the primers are kept confidential by the
company, the sequences of probes are shown in Table 1. Presence of
the fluorogenic dyes FAM (6-carboxy-fluorescein) and VIC
(proprietary dye of Applied Biosystems) at the 5′ ends of the probe
molecules accomplished allele-specific signaling. Minor groove
binder groups were conjugated with the 3′ ends of the probe
oligonucleotides to facilitate formation of stable duplexes between the
probes and their single-stranded DNA targets. TaqMan reactions were
analyzed on the ABI Prism 7000 Sequence Detection System (Applied
(DaHCia I (Badran et aCC
Statistical analysis
Statistical analysis was carried out using the SPSS version 17. Data
are presented as mean±SD. Differences between the means of the 2
continuous variables were evaluated by Student t-test. The allelic
frequencies and genotype distribution were estimated by gene
counting. Differences between non continuous variables, genotype
distribution, 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 each
mutated alíele was also calculated. Statistical significance was at