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.

Genetics

Environmental Life style

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:

Age Clotting factors

Sex Fibrinolytic factors

Hypertension Hyperhomocysteinaemia

Smoking Inflammation factors

Diabetes Endothelium factor

Hyperlipidaemia Nutrition factor

Obesity

Post-menopausal status

Genetic factors

Emerged CHD Risk 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 poly-morphism ， 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.

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.

1.1 The G98/T polymorphism in E-selectin gene and CHD

The transversion of G98T mutation abolishes the HphI recognition site.

NT 92 TTGGGTGAAAAG103

NT 92 TTGGGTTAAAAG103

HphI

HphI

bp

332

194138

Kb1.350.63

0.310.190.12

The PCR product was digested by HphI and separated on 2 % agarose gel electrophoresis.

PCR amplification of the genomic DNA, subcloning and DNA sequencing were carried out.

98 98

GG genotype: TT genotype:

 

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)

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).

N G-allele (%) T-allele (%) Total allelesThe populationa Control 71 128 (90.14) 14 (10.93) 142 CAD 93 160 (86.02) 26 (13.98) 186 P b NSThe subsetc:Control 50 90 (90.00) 10 (10.00) 100 CAD 51 80 (78.43) 22d (21.57) 102 Pb < 0.05

variable Odds Ratio (95%, CI) P 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) =0.003

Multivariate Logistic Regression Analysis

The ATP-sensitive potassium channel (KATP) were complexes of two s

ubunits, a regulatory sulfonylurea receptor (SUR) and an ATP-sensitive 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.

1.2 The E23K polymorphism in KCNJ11 gene and CHD

Group

Genotype(%) Allele(%)

GG GA+AA G A

CHD(n=119)

Controls(n=101)

χ2

P value

OR(95%CI)

50(42.0) 69(58.0)

29(28.7) 72(71.3)

4.202

0.040

1.799 (1.023~3.163)*

151(63.4) 87(36.6)

115(56.9) 87(43.1)

1.940

0.164

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.

Table. Genotype and Allele Frequencies of the E23K Polymorphism of Kir6.2 Gene in CHD Patients and Controls

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 densi

ty 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.

There were three alleles and six genotypes.

NH2 --------112---------158-----------COOH

2 5’---TGC--------TGC----------3’

Cys Cys

3 5’----TGC--------CGC---------3’

Cys Arg

4 5’-----CGC-------CGC---------3’

Arg Arg

HhaI

HhaIHhaI

ApoE PAGE/RFLP pattern

M ε4/4ε3/3

ε2/2ε3/2

ε4/3ε4/2

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.

1.4 The Apolipoprotein (Apo)E and low density lipoprotein gene polymorphism 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 decre

ased significantly in patients with CHD.

1.5 Fibronectin gene and 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 Allele P value

MspI in intron 26

Phenotype CC CD DD C D 0.527

CHD (n=109) 62 (56.9) 47 (43.1) 0 (0) 171 (78.4) 47 (21.6)

Controls (n=123) 75 (61.0) 48 (39.0) 0 (0)

3076A/C in exon20

Phenotype AA AC CC A C 0.95

CHD (n=109) 96 (88.0) 12 (11.0) 1 (1.0) 204 (93.6) 14 (6.4)

Controls (n=123) 108 (87.8) 15 (12.2) 0 (0)

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(%) Allele frequency

CC CT TT 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 medianInter-quartile

range

CC 121 2.05 * 0.67-3.64 *

CT 9 3.98 1.69-6.68

* (P<0.05)

The Taq polymorphism of IL-1β was Ⅰassociated with the concentrations of CRP in normal people

The Taq polymorphism of IL-1β was not Ⅰassociated with with 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.

1.7 CRP gene and 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

Groups

n

Genotype Distribution(%) Allele Distribution(%)

CC CT+TT C T

CHD 128 114(89.1) 14(10.9) 242(94.5) 14(5.5)

Control

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.

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.

2.Gene polymorphism and prognosis

From: Johnson J. A. TRENDS in Genetics 2003 Vol.19 No.11:660-666

3. Gene polymorphism and pharmacogenomics

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.

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.

For Example:

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

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.

In a word,

…….

From: Pfost D R et al. TIBTECH, 2000 (Vol. 18):334-338

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