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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 27, No 4, July/August 2016
214
AFRICA
Methods
A total of 287 subjects were recruited into the
SIRT1 rs1467568
study (104 CAD patients, 99 age-, gender- and race-matched
controls, and 84 age- and gender-matched black controls)
following institutional ethical approval (BE067/14). The inclusion
criteria for CAD patients were: Indian ancestry and unrelated
adult males aged
<
45 years, and stable CAD confirmed on
angiography. The exclusion criteria for controls included an acute
coronary syndrome/revascularisation procedure in the preceding
three months, chronic renal or liver disease, malignancy and
known inflammatory or infectious disease.
Blood samples were obtained following an overnight fast. A
full pathology report of clinical markers was assessed by routine
laboratory testing at the Global Clinical and Viral Laboratory
(Durban, South Africa), a South African national accreditation
system (SANAS) certified laboratory. The following parameters
were tested: haematology (Roche Sysmex 1800XT), chemistry
(Beckman Coulter DXC600), endocrinology and high-sensitivity
C-reactive protein (hsCRP) (Siemens Centaur XP) and serology
(BDBiosciences FACS Calibur), as per international standards to
obtain levels of total cholesterol, HDL-C, LDL-C, triglycerides,
fasting glucose, two-hour glucose, fasting insulin, glycosylated
haemoglobin, sodium, potassium, bicarbonate, chloride, urea,
creatinine, glomerular filtration rate, CD4, CD8, CD45 and CD3
count. The physical measurements of weight, height, abdominal
circumference, waist circumference and patient history were
conducted by the cardiologist (Dr S Khan).
Genomic DNA was extracted from the whole blood sample
of each patient and control, according to the method described
by Sambrook
et al
.
11
Cells were transferred to 600-
μ
l lysis buffer
[0.5 % sodium dodecyl sulphate (SDS), 150 mM NaCl, 10 mM
ethylenediaminetetra-acetic acid (EDTA), 10 mM Tris–HCl (pH
8.0)]. To this, RNase A (100
μ
g/ml; DNase-free) was added to the
solution and incubated at 37°C for one hour. Proteinase K (200
μ
g/ml) was then added and incubated for three hours at 50°C.
Protein contaminants were then precipitated by adding 5 mM
0.1%potassiumacetate before centrifugation at 5 000
×
g for 15min.
Supernatants containing genomic DNA were transferred to fresh
tubes and extracted with 100% isopropanol on ice, and thereafter
washed with 70% ethanol. DNA samples were dissolved in 10 mM
Tris and 0.1 mM EDTA (pH 7.4, 4°C). DNA concentration was
determined using the Nanodrop 2000 spectrophotometer, and all
samples were standardised to a concentration of 10 ng/
μ
l.
Following the manufacturer’s protocol, TaqMan
®
SNP
predesigned genotyping assay (Life Technologies, Cat #4351379)
was used to genotype all subjects for both SNPs. The TaqMan
®
genotyping assay contains two primers for amplifying the
sequence of interest and two TaqMan
®
minor groove-binding
(MGB) probes for detecting alleles. The presence of two probe
pairs in each reaction allows genotyping of the two possible
variant alleles at the SNP site in a DNA target sequence.
The genotyping assay determines the presence or absence of a
SNP based on the change in fluorescence of the dyes associated
with the probes. The TaqMan
®
MGB probes consist of target-
specific oligonucleotides with a reporter dye at the 5
′
end of
each probe: one VIC
®
-labelled probe to detect allele 1 sequence
(A-allele in the case of
rs1467568
and
rs7895833
) and one
FAM
™
-labelled probe to detect allele 2 sequence (G-allele in the
case of
rs1467568
and
rs7895833
). A fluorescence signal for both
dyes indicates heterozygous for allele 1–allele 2 (AG).
A final reaction mixture consisted of 40
×
TaqMan
®
predesigned genotyping assay, 2
×
TaqMan
®
genotyping master
mix, nuclease-free water, and a 10-ng genomic DNA template.
The experiment was done using the Applied Biosystems
®
ViiA
™
7 Real-Time PCR system.
Statistical analysis
The Hardy–Weinberg equilibrium was used to test for deviation
of allele/genotype frequency. All other statistical analyses were
performed with Graphpad prism software (version 5.0). Allele
and genotype frequencies were calculated using the Fisher’s
exact and chi-squared tests, respectively. The comparison
of biochemical measures between the wild type and variant
genotypes was done with a non-parametric
t
-test. Results are
expressed as mean
±
standard error. A
p
-value less than 0.05 was
considered statistically significant.
Results
SIRT1 rs1467568
The genotype distribution complied with the Hardy–Weinberg
equilibrium in the CADpatients and Indian controls (chi-squared
p
=
0.233 and
p
=
0.941, respectively), but not in the black control
group (chi-squared
p
<
0.05).
No significant difference was observed in the distribution
of the
SIRT1 rs1467568
alleles between the CAD patients and
Indian controls (41 vs 40% respectively,
p
=
0.9196, OR
=
1.040,
95% CI: 0.6998–1.545). The Indian controls presented with a
higher frequency of the variant allele compared to the black
controls (40 vs 18.5%, respectively,
p
<
0.0001, OR
=
2.996,
95% CI: 1.850–4.853). The variant allele was found at a higher
frequency in the total Indian group compared to the total black
population (41 vs 18.5%, respectively,
p
<
0.0001, OR
=
3.057,
95% CI: 1.974–4.733) (Table 1).
SIRT1 rs7895833
The genotype distribution complied with the Hardy–Weinberg
equilibrium in the CAD patients, Indian controls and black
controls (chi-squared
p
=
0.970,
p
=
1.000 and
p
=
0.164,
respectively).
No significant difference was observed in the distribution of
the
SIRT1 rs7895833
alleles between CAD patients and Indian
controls (40.5 vs 41%, respectively,
p
=
0.9188, OR
=
0.9629, 95%
CI: 0.6457–1.436). The Indian controls presented with a higher
frequency of the variant allele compared to the black controls
(41 vs 22% respectively,
p
<
0.0001, OR
=
2.513, 95% CI: 1.578–
4.004). The variant allele was found at a higher frequency in the
total Indian group compared to the total black population (41 vs
22% respectively,
p
<
0.0001, OR
=
2.466, 95% CI: 1.620–3.755)
(Table 1).
Phulukdaree and co-workers reported biochemical measures
of CAD patients and healthy controls in 2012.
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As expected,
in our study, CAD patients presented with more conventional
risk factors, such as higher body mass index (BMI), higher
total and LDL cholesterol and triglyceride levels, and a higher
prevalence of type 2 diabetes mellitus than the control groups.
No association between the SIRT1 SNPs and biochemical
measures were found in the CAD patients (Table 2), Indian
controls (Table 3) and black controls (Table 4).