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Gene Polymorphisms and Coronary Heart Disease
Fang Zheng
Clinic Lab, Zhongnan Hospital,
Wuhan University
Coronary Heart Disease is still the No. 1 killer in the world.
The prevention of CHD is based on the control of
several factors associated with a disease or clinical
condition and suspected to play a pathogenetical role,
defined as risk factors.The risk factors of CHD
included:
Genetics
Environmental
Life style
Emerged CHD Risk Factors
Age
Clotting factors
Sex
Fibrinolytic factors
Hypertension
Hyperhomocysteinaemia
Smoking
Inflammation factors
Diabetes
Endothelium factor
Hyperlipidaemia
Nutrition factor
Obesity
Post-menopausal
status
Genetic factors
But only in 5% of hereditary CHD, the gene
background was clear. In the others, each
genetic factor played a minor role in
occurrence and development of the disease.
Rare mutations (e.g., in the LDLR and
APOE genes) may have a major effect,
whereas genes belonging to normal
polymorphism have only a moderate effect.
But even genes with only a slight effect can
be clinically important in combination with
other genes.
The importance of polymorphism analyses
will increase significantly in the near future.
What’s normal polymorphism?
The occurrence in a population (or among
populations) of several phenotypic forms
associated with alleles of one gene or homologs
of one chromosome. The occurrence together in
the same population of more than one allele or
genetic marker at the same locus with the least
frequent allele or marker occurring more
frequently than can be accounted for by
mutation alone.
Polymorphism and Mutation
They are both single nucleotide polymorphism，SNP.
 polymorphism－－normal phenotype
 mutation
－－disease
polymorphism－－more
 mutation
－－less

Cardiovascular disease is complex as a
consequence of pleiotropy. These included
environmental and genetics factors. Gene
polymorphism played an important role in the
occurrence and development of cardiovascular
disease. And it can be applied on the prediction,
diagnosis, treatment and prognosis.
1. The gene polymorphisms as
independent risk predictors



An HphI polymorphism in the E-selectin gene
is associated with premature coronary artery
disease.
ApoE gene polymorphism is related to
coronary heart disease.
E23K polymorphism in KCNJ11 gene has
relationships with coronary heart disease.
Every gene
variants that
contribute to
CHD like
tiny weights
in balance.
1.1 The G98/T polymorphism in E-selectin gene and CHD
E-selectin belongs to a family of structurally related
"selectin' molecules including E-, P- and L-selectin and
participates in the endothelial-leukocyte adhesion .
Experiments using E-and P-selectin-double-knockout
mice suggest that E- and P-selectin together play an
important role in both early and advanced stages of the
atherosclerotic lesion development.
Several polymorphisms in the E-selectin gene have
been identified as new risk factors for the early
atherosclerosis.
The transversion of G98T mutation
abolishes the HphI recognition site.
HphI
NT 92 TTGGGTGAAAAG103
NT 92 TTGGGTTAAAAG103
HphI
bp
Kb
1.35
0.63
332
194
138
0.31
0.19
0.12
The PCR product was digested by HphI and
separated on 2 % agarose gel electrophoresis.
GG genotype:
98
TT genotype:
98
PCR amplification of the genomic DNA,
subcloning and DNA sequencing were carried
out.
Table Frequency of the E-selectin G98T mutation in the
angiographically documented premature CAD and controls (The original
population: all males aged  50 yr.old, all females aged  60 yr. old; the subset: All males
aged  45 yr.old, all females aged  55 yr. old)
N
The populationa
Control
71
CAD
93
Pb
The subsetc:
Control
50
CAD
51
Pb
G-allele (%)
T-allele (%)
Total alleles
128 (90.14)
160 (86.02)
14 (10.93)
26 (13.98)
NS
142
186
90 (90.00)
80 (78.43)
10 (10.00)
22d (21.57)
< 0.05
a: In control, 32 males, 39 females; in CAD, 51 males, 42 females.
b: Chi-square statistical analysis was done using Sigma Stat (ver.2.0, SPSS Inc., Chicago, IL).
c: In control, 21 males, 29 females; in CAD, 28 males, 23 females.
d: Include 18 heterozygotes (GT) and two homozygotes (TT).
100
102
Multivariate Logistic Regression Analysis
variable
Odds Ratio (95%, CI)
G98T
3.58 (1.20-10.67)
=0.022
S128R
4.11 (1.24-13.56)
=0.020
TC
0.99 (0.97-1.07)
NS
TG
1.00 (0.99-1.01)
NS
LDLB
1.02 (0.99-1.05)
=0.071
Smoke, Y/N
5.87 (1.84-18.75)
P
=0.003
1.2 The E23K polymorphism in KCNJ11 gene and CHD
The ATP-sensitive potassium channel (KATP) were complexes of two
subunits, a regulatory sulfonylurea receptor (SUR) and an ATPsensitive and pore-forming inwardly rectifying K+ channel (Kir 6.X).
The Kir 6.X subunits including Kir 6.1 and Kir 6.2 had two
transmembrane domains and form the pore, conferring channel
sensitivity to ATP and other cell metabolites such as ADP.
The G to A mutation in the Kir 6.2, the ATP-sensitive potassium
channel subunit, resulted a Glutamate (E) to Lysine (K) substitution
at codon 23, and the A allele was shown to have a relationship with
high risk to type 2 Diabetes in previous study.
Table. Genotype and Allele Frequencies of the E23K Polymorphism of
Kir6.2 Gene in CHD Patients and Controls
Genotype(%)
Group
GG
GA+AA
Allele(%)
G
A
CHD(n=119)
50(42.0)
69(58.0)
151(63.4)
87(36.6)
Controls(n=101)
29(28.7)
72(71.3)
115(56.9)
87(43.1)
χ2
4.202
1.940
P value
0.040
0.164
OR(95%CI)
1.799 (1.023~3.163)*
1.313 (0.895~1.927) **
*: The frequency of GG genotype was compared with that of GA+AA genotype;
**: The frequency of G allele was compared with A allele.
1.3 The Apolipoprotein (Apo)E gene polymorphism and
CHD
 Apolipoprotein E gene is located on chromosome 19q13.2.
 ApoE plays a critical role in the formation of very low
density lipporotein(VLDL) and chylomicrons. Genetically,
ApoE is polymorphic isoforms of proteins, E2, E3, E4
respectively.
 The ApoE alleles modulate the risk for CHD, cerebral
aheroscerosis and Alzheimer’s disease.
NH2 --------112---------158-----------COOH
2
5’---TGC--------TGC----------3’
Cys
3

HhaI
5’----TGC--------CGC---------3’
Cys
4
Cys
Arg
HhaI HhaI
5’-----CGC-------CGC---------3’
Arg
Arg
There were three alleles and six genotypes.
ApoE PAGE/RFLP pattern
M ε4/4
ε2/2
ε3/3
ε4/3
ε3/2
ε4/2
1.4 The Apolipoprotein (Apo)E and low density lipoprotein
gene polymorphism and CHD
There is a significant relationship between“ + ”allele of low
density lipoprotein gene, ε4 and high total cholesterol, LDL
cholesterol levels. The cholesterol levels of individuals with
LDL-R AvaⅡ(-/-) and NcoI (-/-) genotype were lower than
those with LDL-R AvaⅡ (+/+) and NcoI(+/+) genotypes.
1.5 Fibronectin gene and CHD

The results presented here do not suggest a
positive association between HindIII, TaqI, MspI,
and 3076A/C polymorphisms in FN gene and
CHD. We didn't find any relationship between
four polymorphism in fibronectin gene and CHD.
 But a non difference does not imply a non effect.
 But we found pFN levels in circulation were
decreased significantly in patients with CHD.
Figure. MspI (a), Taq I (b), (3076A/C c), and HindIII (d) digestions of the PCR
product showing different genotypes. The size of each fragment is indicated on the
gel. M1：A 100-bp DNA ladder, MBI Fermentas. M2：A 50-bp DNA ladder, MBI
Fermentas. P: PCR products
Table Genotype and Allele Frequencies of the Polymorphisms of the
FN Gene in CHD Patients and Controls
Genotype
P value
Allele
MspI in intron 26
Phenotype
CC
CD
DD
CHD (n=109)
62 (56.9)
47 (43.1)
0 (0)
Controls (n=123)
75 (61.0)
48 (39.0)
0 (0)
C
D
0.527
171 (78.4) 47 (21.6)
3076A/C in
exon20
Phenotype
AA
AC
CC
CHD (n=109)
96 (88.0)
12 (11.0)
1 (1.0)
Controls (n=123)
108 (87.8)
15 (12.2)
0 (0)
A
C
204 (93.6) 14 (6.4)
0.95
Fig The sequence reports. a: genotype AA for 3076A/C; b:genotype CC for
3076A/C (Eco81I: CC↓TNA↑GG) ; c: genotype CC for MspI﹡(MspI: C↓CG↑G)
﹡allele was designated as C (CCAG), D(CCGG) for the MspI polymorphism
1.6 Interleukin-1β gene and CHD
Interleukin-1β (IL-1 β) belongs to a family of
cytokines including IL-1, IL-4, IL-8, IL-10, and
IL-13.
 IL-1 β plays a dominant role in several immune
reactions
 IL-1β also was found to associate to
atherosclerotic events in vitro and in vivo.

Polymorphism analyze
1
CC
2
CT
3
TT
4 PCR
Comparison of distribution of IL-1β 3954C/T genotypes
between CHD group and control group
group
n
Gene type(%)
CC
CT
TT
Allele frequency
C
T
control
130
0.931
0.069
0.000
0.965
0.035
CHD
78
0.897
0.090
0.013
0.942
0.058
(P>0.05)
The concentrations of C-reactive protein in different
genotype groups
Gene type
n
CRP median
Inter-quartile
range
CC
121
2.05 *
0.67-3.64 *
CT
9
3.98
1.69-6.68
* (P<0.05)


The TaqⅠpolymorphism of IL-1β was
associated with the concentrations of CRP in
normal people
The TaqⅠpolymorphism of IL-1β was not
associated with with CHD
1.7 CRP gene and CHD

CRP, The first acute-phase protein to be described,
its plasma concentration increases during
inflammatory states.

Recently, CRP might have an important role in
the pathogenesis and prediction of CHD.

Polymorphism analysis
Figure 1-1 Determination of the
+1444 C/T polymorphism in the
CRP gene by PCR-RFLP.
Lane M：DNA Marker;
Lane A：PCR product;
Lane B：homozygous CC;
Lane C：heterozygous CT;

Polymorphism analysis
Figure The chromatogram of homozygous CC

Comparison of distribution of +1444 C/T
genotypes and alleles between CHD group and
control group
Group
s
n
CHD
Contro
l
Genotype Distribution(%)
Allele Distribution(%)
CC
CT+TT
C
T
128
114(89.1)
14(10.9)
242(94.5)
14(5.5)
119
107(89.9)
12(10.1)
226(95.0)
12(5.0)
2
0.048
0.045
P
0.827
0.832
OR(95%CI)
0.913(0.404¬2.063) *
0.918(0.416¬2.027) △
*: genotype CC vs CT+TT；△：allele C vs T

The concentrations of C-reactive protein
in different groups
Groups
n
median Inter-quartile range
CHD
128
1.21
0.63～2.58
CC
114
1.21
0.67～2.48
CT+TT
14
1.52
0.51～3.73
Control 119
0.77
0.59～1.22
CC
107
0.75
0.59～1.15
CT+TT
12
1.42
0.66～3.05

The +1444 C/T polymorphism of CRP was
associated with the basal concentrations of CRP in
normal people.

The +1444 C/T polymorphism of CRP was not
associated with CHD.
1.8 Conclusion for the study on polymorphisms as
predictors of CHD

We found the polymorphisms of E-selectin, ApoE
gene and KCNJ11 gene were related to CHD
disease.
 The polymorphisms in LDL-R gene and ApoE
gene effected the level of lipid.
 The polymorphisms of IL-1βand CRP genes
influenced the CRP baseline.
 We didn't find any association between
polymorphism in fibronectin gene and CHD.
2.Gene polymorphism and prognosis




Patients with an acute myocardial infarction are of high risk to
develop ischemia –induced ventricular arrhythmias, leading to
sudden cardiac death in about one third of all AMI patients.
The individual susceptibility to ischemia-induced arrhythmias
may be modified by polymorphisms in genes encoding ion
channels.
A.Jeron studied the Kir6.2 gene. Opening of the KATP channel
during ischemia results in action potential shortening in various
studies and may therefore influence the outcome of AMI patients.
However they didn’t find any significant influence of Kir6.2 gene
polymorphism on the risk of SCD in patients with CHD.But they
identified two novel missense mutations in a highly conserved
region of the Kir6.2 gene.
3. Gene polymorphism and
pharmacogenomics
From: Johnson J. A. TRENDS in Genetics 2003 Vol.19
The new pharmacogenetics uses powerful
experimental and data-handling techniques in
DNA analysis to discover and assemble a
comprehensive list of the variations within the
human genome – specifically, SNPs – and then
defines complex genetic profiles of these SNPs
that predict the use of new or existing
therapeutic agents with maximal efficacy and
minimal toxicity.

A genetic test for certain single nucleotide
polymorphisms(SNP) will predict you that:
• You should suffer a severe adverse reaction to it.
• You are expected to shown an excellent response to
a different medication with little chance of side
effects.

This is the promise of pharmacogenetics—
The optimization of drug therapy based
on the individual genetic profile.
For Example:
 The HMG-CoA reductase is the rate-limited
enzyme in the biosynthesis of cholesterol.
 Statins, the HMG-CoA reductase inhibitors, are
widely designed to reduce de novo cholesterol
biosynthesis.
 The efficacy and toxicity of Statins are different
in different individuals.
 Recently, investigation in the relationship of statins
effects and gene polymorphisms related to the lipoprotein
metabolism has been developed.
 These genes included the apolipoprotein E gene, hepatic
lipase gene, lipoprotein lipase gene and CETP gene.
 In 2000, Siest G et al used different HMG-CoA
reductase inhibitors to detect the relationship of apo E
polymorphisms and LDL-C level. It showed the best
effects to reduce LDL-C inε2 carriers.
 Theε4 carriers has elevated level of plasma cholesterol.
And the affinity of LDL particles to LDL receptor was
increased, the activity of HMG-CoA reductase was
decreased. The effect of statins inε4 carriers was poor
may be due to the low base level of HMG-CoA
reductase activity.
 In 2003, Carlquist et al had concluded that the Taq1B
polymorphism in the CETP gene is associated with CETP
activity, HDL concentration, atherosclerosis progression,
and response to statins.
 Their findings suggested, for the first time, the potential of
CETP Taq1B genotyping to enable more effective,
pharmacogenetically directed therapy.
From: Johnson J. A. TRENDS in Genetics 2003 Vol.19
No.11:660-666
In a word,
Identification of all gene variants that contribute to
CHD appears possible; this may considerably
improve our understanding of the aetiology and
mechanisms of this disease.
Furthermore, simultaneous analysis of several
predisposing alleles may help to identify high-risk
individuals and assess prognosis.
And the treatment of CHD could be guided by
screening certain SNP using pharamcogenetic
methods.
…….
From: Pfost D R et al. TIBTECH, 2000 (Vol. 18):334-338
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