Cardiovascular Journal of Africa: Vol 27 No 4 (July/August 2016)

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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 27, No 4, July/August 2016 228 AFRICA
Correlation analysis between ApoM gene-promoter
polymorphisms and coronary heart disease
Yao Zhang, Li-Zhu Huang, Qing-Ling Yang, Yan Liu, Xin Zhou
Abstract
Objectives: Apolipoprotein M (ApoM), a 25-kDa plasma
protein belonging to the lipocalin protein family, is predomi-
nantly associated with high-density lipoprotein cholesterol
(HDL-C). Studies have suggested ApoM to be important
for the formation of pre-β-HDL and to increase cholesterol
efflux from macrophage foam cells. The aim of this study was
to explore the association of single-nucleotide polymorphisms
(SNPs) in the ApoM promoter with coronary atherosclerotic
disease (CAD), and the contribution of latent factors.
Methods: ApoM was measured in samples from two separate
case–control studies, of whom 88 patients developed CAD
and 88 were controls. Whole-blood samples from subjects
were genotyped by PCR-restriction fragment length polymor-
phism (PCR-RFLP). Luciferase activities were measured for
HepG2 cells with two SNPs, rs805296 (T-778C) and rs940494
(T-855C), and after interfering with or overexpressing the
predicted transcription factors. The ability of the SNPs to
combine with nucleoproteins was analysed by electophoretic
mobility shift assay (EMSA).
Results: Mean plasma ApoM concentrations in the CAD
and non-CAD groups were 9.58 ± 4.30 and 12.22 ± 6.59 μg/
ml, respectively. Correlation studies of ApoM concentrations
with several analytes showed a marked positive correlation
with HDL-C, fasting plasma glucose and triglyceride levels.
The CC genotype showed lower luciferase activities compared
to the TC and TT genotypes. The ApoM-855 mutant-type
could bind to the AP-2α. Interference and overexpression of
AP-2 increased and decreased luciferase activities of the wild
and mutant types to different degrees.
Conclusion: ApoM may be a biomarker of CAD. ApoM-
855 T→C substitution provides binding sites for AP-2α and
reduces ApoM transcription activity.
Keywords: coronary heart disease, ApoM, SNP, luciferase activity
Submitted 21/3/14, accepted 8/1/16
Cardiovasc J Afr 2016; 27: 228–237
DOI: 10.5830/CVJA-2016-001
Apolipoprotein M (ApoM) is a novel lipocalin superfamily
protein.1,2 Although also found in low-density lipoprotein
(LDL), very low-density lipoprotein and chylomicrons, ApoM
is primarily found in high-density lipoprotein (HDL) where it
binds to sphingosine-1-phosphate (S1P) anchors.3
Recent studies have suggested that ApoM may affect HDL
metabolism, increasing the formation of pre- β-HDL particles.4
ApoM has been shown to protect LDL against Cu++ -induced
oxidation,5 and to contribute to the anti-inflammatory function
of HDL.6 Small circulating HDLs are involved in reverse
cholesterol transport, and ApoM may affect this process by
regulating pre-β-HDL.7 The binding of ApoM to S1P in HDL
particles may also have an antioxidant role.
By affecting the immune and anti-inflammatory functions of
HDL,8,9 ApoMmay reduce atherosclerosis-related inflammation,
preventing the onset and development of atherosclerosis. We
used the online prediction software, TRANSFAC, to predict
transcription factor (TF) binding sites for the normal and
mutated ApoM-855 and ApoM-778 sites, and found ApoM
T-855C provided binding sites for activating protein 2 (AP-2).
Activating protein-2α (AP-2α) was one of the first identified
and studied TFs. 10 The AP-2 gene, encoding a 437-amino acid
protein of ~52 kDa, regulates the transcription of various
genes regulating embryonic development, cell growth and
differentiation.11,12 Vertebrates possess five subtypes of AP-2, α,
β, γ, δ and ε.13,14
The AP-2 protein has been shown to regulate atherosclerosis-
associated genes, including matrix metalloproteinase-2, vascular
endothelial growth factor, ApoE, tryptase and adiponectin
ATP-binding cassette transporter AI (ABCAI). In addition,
AP-2α plays a role in atherosclerosis. It may mediate foam cell
formation in mouse and human atherosclerotic lesions. 15 AP-2α
was found in ApoE -/- mouse lesions within the artery wall, but was
not detected in mouse arteries without atherosclerotic lesions.
Similarly, AP-2α was observed in the human atherosclerotic
aortic wall, mainly within the atherosclerotic plaque.
Recent studies involving the genetics of ApoM have led to
major breakthroughs in metabolic and disease characteristics. In
particular, associations have been found between diabetes and
polymorphisms in the promoter region of the ApoM gene. 16,17
To explore the association of ApoMgene polymorphisms with
coronary heart disease (CHD) in a Chinese Han population, we
performed a population-based case–control study. We examined
whether ApoM promoter polymorphisms could lead to changes
in TF binding, and therefore, changes in promoter activity.
Department of Biochemistry and Molecular Biology,
Wan Nan Medical College, Anhui 241000, China
Yao Zhang, MD,
Clinical Testing and Diagnosis, Experimental Centre of
Bengbu Medical College, Anhui 233000, China
Li-Zhu Huang, MD
Yan Liu, MD
Department of Biochemistry and Molecular Biology, Beng
Bu Medical College, Anhui 233000, China
Qing-Ling Yang, MD
Centre for Gene Diagnosis, Zhongnan Hospital, Wuhan
University, Wuhan, Peoples’ Republic of China
Xin Zhou, PhD