CARDIOVASCULAR JOURNAL OF AFRICA • Volume 25, No 1, January/February 2014
10
AFRICA
Methods
A total of 33 patients with CSF (mean age 55.8
±
10.3 years) and
48 controls (mean age 53.9
±
11.8 years) with normal coronary
arteries were enrolled in this study. Coronary angiography
was performed in the cardiology clinic between January 2010
and June 2012 on patients who had an indication for elective
coronary angiography due to ischaemia detected on a treadmill
test and/or myocardial perfusion scintigraphy.
A complete history, findings of the physical examination, risk
factors for atherosclerotic heart disease and medications were
recorded. Patients who had been treated with antihypertensive
drugs or those whose baseline blood pressure exceeded 140/90
mmHg were diagnosed with hypertension (HT). Diabetes
mellitus (DM) was defined as fasting blood glucose levels
>
126
mg/dl or the use of anti-diabetic medication. Hyperlipidaemia
was defined as a total cholesterol level
>
200 mg/dl and/or
low-density cholesterol level
>
160 mg/dl. Patients with known
atherosclerotic disease, visualised coronary artery plaque in
coronary angiography, peripheral artery disease, malignancy,
renal and hepatic insufficiency, and chronic inflammatory
disease were excluded from the study. All subjects agreed to
participate in the research and the consent of the local ethics
committee was obtained.
Coronary angiography was performed with a femoral
approach using Judkins catheters and the contrast agent
iopramide (Ultravist-370, Bayer Schering Pharma, Germany)
with angiographic equipment (GE Medical Systems, Innova
2100, USA). The thrombolysis and myocardial infarction (TIMI)
frame rate was 30 frames per second (fps) and angiograms were
recorded on a compact disc in DICOM format. Coronary blood
flow was measured quantitatively using TIMI frame count,
which was determined for each major coronary artery of each
subject included in the study, according to the method first
described by Gibson
et al
.
23
The left anterior descending coronary artery (LAD) is usually
longer than the other major coronary arteries and for that reason
the TIMI frame count of this vessel is often higher. Therefore,
to obtain the corrected TIMI frame count of the LAD, the TIMI
frame count was divided by 1.7.
23
TIMI frame counts in the LAD and left circumflex (LCx)
arteries were assessed in the right anterior oblique projection,
and the right coronary artery (RCA) in the left anterior oblique
projection. The mean TIMI frame count for each subject was
calculated by adding the TIMI frame counts for the LAD/1.7,
LCx and RCA and then dividing the value obtained by 3. The
corrected cut-off values due to the length of normal visualisation
of the coronary arteries were 36.2
±
2.6 frames for the LAD, 22.2
±
4.1 frames for the LCx, and 20.4
±
3 frames for the RCA. Any
values obtained above these thresholds were considered CSF.
Peripheral blood samples from CSF patients and healthy
controls were used for genotyping for point mutations of
PAI-1,
MTHFR
and
ACE
genes and are compared in the results. Three
thrombophilic marker genes, plasminogen activator inhibitor-1
(
PAI-1
, rs1799889); two polymorphic regions for
MTHRF
(C677T, rs1801133 and A1298C, rs1801131), and
ACE
I/D
(rs1799983) genes were analysed in the results.
Genotyping
Peripheral blood samples containing EDTA were collected from
the patients and volunteer controls after a 12-hour overnight fast.
All routine biochemical tests were carried out on an autoanalyser
with the Cobas 6 000 Integra (Roche Diagnostics, IN, USA)
auto-analyser device using the chemiluminescence method.
Venous blood was collected in 6-ml EDTA tubes for isolation
of the genomic DNA and stored at –20°C. A total of 81 DNA
samples from patients with CSF and controls were genotyped by
real-time polymerase chain reaction (PCR) analysis.
The total genomic DNA was extracted by the MagnaPure
Compact (Roche) and Invitek kit extraction techniques
(Invitek
®
; Invisorb spin blood, Berlin, Germany). Target genes
were amplified by real-time PCR, LightCycler 2.0 methods
(Roche) for the CSF cohort and healthy controls. Briefly,
LightCycler FastStart DNA Master HybProbes, master mix
(water, PCR-grade, MgCl
2
, stock solution, primer mix, HtbProbe
mix) and template DNA from patients and controls were used for
real-time amplification for each target gene.
The amplification protocol for
MTHFR
677C
>
T consisted
of a denaturation step of 10 minutes at 95°C. The amplification
conditions for 45 cycles were: denaturation at 95°C for five
seconds, annealing at 55°C for 10 seconds, extension at 72°C
for 15 seconds, melting curve step with denaturation at 95°C for
20 seconds, annealing at 40°C for 20 seconds, melting at 85°C
for two seconds and the cooling step at 40°C for 30 seconds. A
software program (LightCycler 2.0, Roche) was used for detection
of the mutated (channel 640 at 54.5°C) and wild genotype
(channel 640 at 63°C) profiles for target 677 C
>
T SNP analysis.
The amplification protocol for
MTHFR
1298A
>
C consisted
of a denaturation step of 10 minutes at 95°C. The amplification
conditions for 40 cycles were: denaturation at 95°C for five
seconds, annealing at 62°C for 10 seconds, extension at 72°C
for six seconds, melting curve step with denaturation at 72°C
for 30 seconds, annealing at 95°C for 20 seconds, melting
at 40°C for one second and the cooling step at 40°C for 30
seconds. A software program (LightCycler 2.0, Roche) was used
for detection of the mutated (channel 640 at 59°C) and wild
genotype (channel 640 at 65°C) profiles for target 1298A
>
C
SNP analysis.
The amplification protocol for
PAI
-1 5G/4G consisted of
a denaturation step of 10 minutes at 95°C. The amplification
conditions for 40 cycles were: denaturation at 95°C for three
seconds, annealing at 60°C for 10 seconds, extension at 72°C
for 13 seconds, melting curve step with denaturation at 95°C for
30 seconds, annealing at 40°C for one minute, melting at 85°C
for two seconds and the cooling step at 40°C for 30 seconds.
A software program (LightCycler 2.0, Roche) was used for
detection of the mutated (4G) (channel 640 at 54°C) and wild
genotype (5G) (channel 640 at 61°C) profiles for target
PAI
-1
5G/4G analysis.
The amplification protocol for
ACE I/D
consisted of a
denaturation step of 10 minutes at 95°C. The amplification
conditions for 45 cycles were: denaturation at 95°C for three
seconds, annealing at 60°C for 10 seconds, extension at 72°C
for 10 seconds, melting curve step with denaturation at 95°C for
30 seconds, annealing at 40°C for one minute, melting at 85°C
for 10 seconds and the cooling step at 40°C for 30 seconds.
A software program (LightCycler 2.0, Roche) was used for
detection of the mutated (D, del) (channel 640 at 85°C) and wild
genotype (I, Ins) (channel 640 at 93°C) profiles for target
ACE
I/D
analysis.
