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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 27, No 4, July/August 2016
236
AFRICA
Discussion
The results of this study confirm the previously identified
18,19
association between CHD and SNPs T-855C and T-778C in
the promoter region of the ApoM gene. Luciferase activity
associated with the -855 T
→
C substitution was significantly less
than that of the promoter with -855 TT. With software predictive
analysis we found the possible reason for this finding was that
the -855 T
→
C substitution permitted the TFs AP-2
α
and Sp1 to
bind to the promoter.
When HepG2 cells were transfected with the ApoM promoter
containing the -855 T
→
C substitution, AP-2
α
combined with
the ApoM-855 area, thereby decreasing promoter activity. These
findings confirm that changes in the activation of the ApoM
promoter region may induce variations in the ApoM plasma
concentration.
Our results suggest that C alleles at the ApoM promoter
-855 and -778 were associated with increased CHD risk. In
our population-based case–control study, we enrolled 88 CHD
patients (63 males, mean age: 60.80
±
9.27 years) and 88
unrelated individuals (53 males, mean age: 58.18
±
10.43 years)
as a control group. The CHD group was divided into ACS
and SAP groups, and the plasma levels of TG, TC, HDL-C,
FPG and LDL-C were evaluated. Genomic DNA from whole
blood of these subjects was subjected to PCR amplification and
restriction enzyme digestion to determine genotype with regard
to the ApoM T-855C and T-778C polymorphisms.
CHD patients had higher TG (1.97
±
1.28 mmol/l;
p
=
0.000)
and FPG levels (6.40
±
2.40 mmol/l;
p
=
0.000), and lower HDL-C
levels (1.05
±
0.25 mmol/l;
p
=
0.000) than non-CHD patients.
The allelic frequencies were in Hardy–Weinberg equilibrium.
After adjustment for age, gender and serum glucose level,
multiple logistic regression analysis showed that, compared to
the wild-type TT genotype of the two SNPs, carriers of the C
allele had an increased risk of CHD, with an odds ratio (OR)
of 1.819, 95% confidence interval (CI) of 1.142–2.898, and
p
=
0.012 (T-855C: OR
=
3.206, 95% CI
=
1.139–2.204,
p
=
0.037; T-778C: OR
=
3.290, 95% CI
=
1.487–7.280,
p
=
0.004).
Luciferase activities of the promoter constructs with CC were
significantly lower than those of the constructs with TC and TT.
To detect whether different alleles of the ApoM proximal
promoter region may affect the expression of target genes,
thereby affecting the metabolism of ApoM, we constructed
different genotypes of the promoter reporter gene to examine
how mutations in the ApoM proximal promoter would affect
promoter activity. The presence of a C allele at -855 or -778 bp
of the ApoM promoter region may lead to lower ApoM levels
and allow prediction of disease severity in the patient. However,
due to the small number of cases analysed, more clinical data are
needed for this conclusion to be confirmed.
We investigated whether the DNA sequence of ApoM from
-844 to -869 bp was involved in transcriptional regulation of
the ApoM gene. Using EMSA experiments, we showed that the
mutant allele (-855C) could bind with nuclear proteins, whereas
the wild-type allele (-855T) could not. Competitive inhibition
experiments showed that the combination was due to specific
binding by the TF AP-2
α
.
To explore the role of AP-2
α
in ApoM promoter activity,
we examined the luciferase activities of the wild-type and
mutant-type alleles after interference of AP-2
α
. Whereas AP-2
α
interference increased the luciferase activities of the treated cells,
the wild-type was elevated to a lesser extent than the mutant-type.
There were other AP-2
α
binding sites in addition to the -855 site.
These results suggest that AP-2
α
may be a negative regulatory
factor of ApoM. The increased luciferase activity of the mutant
type with Apo-2
α
interference compared to the wild-type may
indicate that the mutant had more binding sites for AP-2
α
, or
that the mutated -855 site can bind with AP-2
α
.
Multiple epidemiological studies have shown that serum
HDL levels are negatively correlated with the risk of early
CHD.
20
Generally, clinical CAD is divided into two major
types, ACS and SAP. Patients with ACS had significantly lower
ApoM levels, probably due to the fact that ApoM is a major
apolipoprotein of HDL.
It has been confirmed that ApoM is required for pre-
β
-
HDL formation and cholesterol efflux to HDL, and that it
protects against atherosclerosis.
4
ApoM increased formation of
pre-
β
-HDL particles and had a profoundly protective effect on
atherosclerotic lesion formation in hypercholesterolaemic Ldlr
-/-
mice.
4
Atherosclerotic lesion areas in aortic roots and the thoracic
aorta were reduced in Ldlr
-/-
mice infected with Ad-ApoM.
4
The unstable lesion (also vulnerable plaque, the formation
being mainly due to dyslipidaemia) is the basic pathological
aetiological factor of ACS. Therefore the presence of an
enlarged unstable lesion may be because the decreased serum
ApoM level prohibited the formation of sufficient amounts of
mature, functional HDL to promote the mobilisation of cellular
cholesterol
in vivo
.
21
The serum glucose level was different between CHD patients
and normal controls, and serum ApoM and serum glucose levels
were negatively correlated (both
p
<
0.05). Very low ApoM levels
increase the risk of atherosclerosis.
22
Therefore the serum ApoM
level may be a valuable marker for identifying high-risk groups.
TFs are the most important regulators of protein expression
by genes and the most important factors to influence ApoM
expression. Gene promoter regulation may underlie the low
ApoM levels in CHD patients. Recent studies have shown that
several TFs participate in the regulation of ApoM expression,
such as HNF-1
α
, liver receptor homolog-1 (LRH-1), forkhead
box A2 (Foxa2),
23,24
liver X-activated receptor (LXR),
25,26
leptin,
27
interleukin-1 (IL-1),
28
transforming growth factor (TGF) and
epidermal growth factor (EGF).
9
Our study has some limitations. Although our results confirm
findings on the effect of the T-778C polymorphism on CHD,
our analysis of the association of the ApoM promoter region
SNPs with CHD was limited to a single locus. Such single-locus
associations may be different in different populations. We found
that the ApoM plasma concentration was decreased in CHD
patients, the rs805296 and rs9404941 SNPs were associated
with CHD occurrence and severity, and the rs9404941 SNP
was associated with plasma TC and TG changes. However, the
small sample size of this study limits its statistical power, and the
results should be replicated in studies with larger sample sizes
to avoid false positives. Expression of the ApoM protein and its
relationship with diseases need to be further studied and discussed.
Conclusion
ApoM may be a biomarker of CAD. ApoM-855 T
→
C
substitution provides binding sites for AP-2
α
and reduces ApoM
transcription activity.