THE ASSOCIATION BETWEEN SERUM LDL-CHOLESTEROL AND GENETIC VARIATION IN CHROMOSOMAL LOCUS

1P13.3 AMONG CORONARY ARTERY DISEASE PATIENTS IN QATAR

Presented by/Huda Ibrahim

Idil Souleman Wais

Supervisors/ Dr. Nasser Rizk

Dr. Ayman El Menyar

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Outline

Introduction Development of Atherosclerosis Risk Factors Aim of our Study Methodology Results Discussion Limitations and Further Study Acknowledgements References

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Introduction

Atherosclerosis is the most common cause for coronary artery, carotid artery and peripheral arterial diseases.

Atherosclerosis alone is not fatal, but the rupture of the atherosclerotic plaque contributes to the development of acute coronary syndrome which is considered as a life-threatening disease.

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Introduction

Cardiovascular risk factors along with the presence of inflammatory factors may result in endothelial dysfunction and vascular inflammation.

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Development of Atherosclerosis

Once the endothelium becomes defective, development of atherosclerotic lesions will be initiated

Endothelial cells start to disappear during the formation of lesions and platelets adhere to the exposed subendothelial tissue

Plasma molecules and lipoproteins extravasate through the defective endothelium into the subendothelial space depending on their size and concentration

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Development of Atherosclerosis

Atherogenic lipoproteins are oxidized into a form which is cytotoxic, proinflammatory, chemotaxic, and proatherogenic

This mechanism is responsible for LDL modification and the production of small dense LDL

Oxidation of LDL is promoted by a potent oxidant called nitro oxide

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Development of Atherosclerosis

The retention of lipoproteins initiates the release of cytokines and chemokines which are necessary for leukocyte recruitment.

Circulating pro-inflammatory cytokines interact with platelet derived inflammatory and prothrombotic agents and activate specific signalling pathways, initiating:

1. cells adhesion 2. apoptosis 3. increased permeability of the endothelium

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Development of Atherosclerosis

Epithelial cells present in the defective endothelium express adhesion molecules such as selectin, intracellular adhesion molecule

These molecules act as receptors for glycoconjugates and integrins present in monocytes and T lymphocytes which contribute to the plaque formation

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Risk Factors

Risk factors for the development of atherosclerosis include hypercholesterolemia, diabetes, smoking, hypertension, and age

The most important risk factor is elevated cholesterol level which induces endothelial dysfunction

Several studies indicated that familial hypercholesterolemia is linked with increased risk of developing atherosclerosis.

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Risk Factors

Impaired glycemic homeostasis has a direct influence in the formation and propagation of atherosclerotic plaque.

Smoking has been associated with atherosclerosis and mainly in older adults.

It also increases fibrinogen which mediates platelets activation and increases the risk of heart disease

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Risk Factors

Hypertension increases the expression of mediators such as leukocytes adhesion molecules, chemokines and other mediators

These results have been confirmed by using blood-induced pressure reduction treatment that leads to a decrease in the level of the pro-inflammatory markers

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Risk Factors

Also studies demonstrated that there is close relationship between aging and the clinical manifestation of atherosclerosis

Aged subjects show increased susceptibility to atherogenic stimuli and arterial wall undergo changes that result in the progression of severe lesions

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LDL-cholesterol

Is the major cholesterol carriers in circulation and their physiological function is to transport cholesterol to the cells.

LDL is frequently associated with patients of high-risk characteristics: elevated triglycerides low HDL Diabetes insulin resistance obesity

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Muendlein ,A. et al Performed genotyping of variants rs 599839,rs646776

and rs4970834 on chromosome 1 p13.3 in a large cohort of 1610 consecutive patients undergoing coronary angiography.

They found comparing to the common allele , the rare alleles of variants rs 599839,rs646776 and rs4970834 where significantly associated with decreased serum LDL-C (P=0.003)

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P. Linsel-Nitschke et al Analyzed the association of rs599839 with LDL-C in 6605

individuals across a wide age spectrum and with CAD

Found each copy of the G-allele of rs599839 associated with a decrease of serum LDL-C (p = 2.6×10 11) Moreover, each copy of −the G-allele associated with a 9% decrease of CAD risk (90% CI 4–14%)

Rs599839 associates with decreased LDL-C and a lower risk of CAD.

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Aim of Study

Investigate the previously described association of SNPs rs599839, rs646776, and rs4970834 with both serum LDL cholesterol and risk of CAD among IHD patients.

The human genetic research of this area could widen our understanding of the pathophysiology of these CVD among Qatari and non Qatari subjects.

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Experiment Design

Three SNPs located on chromosome 1p13.3 were genotyped in subjects. rs599839 (A/G) TAGGAGCAGGATC[A/G]ACTTCCAGATATACAGAGAATAT

AA

rs6467 76(A/G) TATTTGGGAGCAGTGTCATGGACAT[A/G]GGCAGAGGGAC

AGGCTTATCAGCCA rs4970834 (C/T) CAGCCATCCCACTCCCCACTTACTGA[C/T]CTCTCTGTTCC

CTGCCTAGTCCTAC

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Approvals

Hamad Medical Corporation, Medical Research Center- May 2010

Undergraduate Research Experience Program (UREP), Seventh cycle- March 2010.

UREP 07 -091 – 3- 020.

Qatar University Institutional Review Board (QU-IRB), Request for Ethical Approval.

University Chemical and Biohazard Safety Committee.

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Methodology

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Sample Collection

10 ml of peripheral blood was collected from each participating subject. 5 ml was used for DNA extraction and 5 ml was used for determination of plasma glucose, lipids, insulin and biochemical and cardiac markers

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DNA extracted from whole blood samples of using the QiaAmp DNA blood Mini Kit from Qiagen Cat with

RBC lysis solution

Measuring DNA concentration by using nanodrop Spectrophotometer.

All SNPs were genotyped.Polymorphisms of these SNPs carried out by using TaqMan MGB probe by means of an ABI 7500 [Applied Biosystems, Foster City, CA).

Statistical analysis:

Data are expressed as mean ± SD, median and inter-quartile range [25% - 75%] for non distributed continuous data and percentage for categorical data .

All statistical analysis were performed using SPSS program for Windows

Differences between continuous variables were evaluated by two student’s t-tests. Non-parametric Mann-Whitney and 2-independent samples t-tests were accordingly for analysis.

Genotype distributions and allele frequencies between the study groups were compared by constructing 2X2 contingency tables and X2 or Fischer exact test.

Odds ratio, (ORs) and 95% confidence interval (CI) and corresponding P values were analyzed by logistic regression analysis.

Pairwise linkage disequilibrium coefficients (D’ and r2) were estimated and plotted using the (SVS 7) software

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Measurments

1. Height 2. Weight3. waist circumference4. systolic and diastolic blood pressures5. BMI

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Results

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Variables Group (A) Group (B) P- value

Age 56 (46 – 64) 5 (49 – 61) 0.98

Male

Female

22 (84.62%)

4 (15.83%)

16 (100%)

0

-

Qatari

Non-Qatari

8 (30.8%)

18 (69.2%)

4 (25%)

12 (75%)

-

BMI

Overweight

Obese

27.32 (23.31 – 31.17)

9 (34.6%)

6 (37.5%)

26.96 (24.19 – 32.45)

6 (23.1%)

5 (31.25%)

0.794

0.15

SBP

DBP

HTN

129.19 (18.57)

75.33 (8.66)

7 (26.9%)

134.56 (15.94)

74.88 (8.57)

6 (37.5%)

0.361

0.874

0.07

DM 12 (46.2%) 8 (50%) 0.81

Glucose (Mm)

7.3 (5.88 – 14.45) 6.6 (5.53 – 9.60) 0.509

Table:1

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LDL

(<3.37 mmol/L)

3.17 (0.90) 3.44 (1.01) 0.406

HDL

(>0.9 mmol/L)

1.03 (0.23) 1.08 (0.24) 0.447

TC

(<5.2 mmol/L)

4.89 (0.94) 5.11 (1.40) 0.593

Dyslipdemia 12 (46.2%) 7 (43.75%) 0.58

TG(<2.20 mmol/L) 1.55 (1.06 – 2.18) 1.32 (1.14 – 1.92) 0.637

Insulin (mmol/L) 25.46 (10.12 – 41.83) 4.20 (8.78 – 32.13) 0.484

Troponin

(<0.4 ng/mL)

0.35 (0.02 – 2.13) 2.72 (0.37 – 8.24) 0.030

CK-MB

(0-3 ng/mL)

11.0 (3.46 – 34.86) 68.0 (5.58 – 266.23) 0.08

On anti-diabetic

treatment

10 (38.5%) 10 (62.5%) 0.15

On lipidemic treatment 5 (19.2%) 6(37.5%) 0.22

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Genotype distribution

---------------------------------------------

Genotype Group A Group B

lesion <20% lesion >20%

n, (%) n, (%)

P value

rs59839

AA

AG

GG

0 (0.0)

22.0 (88.0)

3.0 (12.0)

1 (6.25)

12.0 (75.0)

3.0 (18.75)

0. 34

rs646776

AA

AG

GG

10.0 (41.7)

0.0 (0.0)

14.0 (58.3)

8.0 (53.3)

0.0 (0.0)

7.0 (46.7)

0.76

rs4970984

CC

CT

TT

0.0 (0.0)

25.0 (100)

0.0 (0.0)

0.0 (0.0)

16 (0.0)

0.0 (0.0)

0.21

Table:2

Results for rs599839

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Risk factor Standard Error [95% CI] OR P- value

Age 0.07 1.04 (0.91-1.20) 0.517

BMI 0.17 0.77 (0.55-1.07) 0.115

Hypertension 1.44 10.6 (0.61-171.06) 0.10

Risk factor Standard Error [95% CI] OR P- value

Diabetes 1.61 6.49 (0.28-151.81) 0.24

LDL 0.79 0.27(0.02-3.61) 0.33

Dyslipdemia 1.32 0.62(1.32-2.93) 0.54

Results for rs646776

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Risk factor Standard Error Odds Ratio

[95% CI]

P- value

Age 0.04 1.08(0.99-1.19) 0.09

BMI 0.06 1.00(0.90-1.12) 0.94

Hypertension 0.84 0.39(0.08-2.06) 0.27

Risk factor Standard Error Odds Ratio

[95% CI]

P- value

Diabetes 0.82 1.85(0.37-9.27) 0.44

LDL 0.41 1.40(0.63-3.11) 0.40

Dyslipdemia 0.82 4.24(0.83-21.49) 0.08

Discussion

We examined the association between LDL cholesterol and three genetic variations in chromosomal locus 1p13.3 and CAD patients presented with single lesion or more that one lesion among Qatari and non Qatari individual

This study showed that G allele of rs599839 and rs646776 were not statistically associated with CAD risk factor LDL-C [p=0.33,OR=0.27(0.02-3.61) , P=0.4 OR=1.40(0.63-3.11)] in both groups, respectively

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Discussion

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In Qatar

<20% lesion A= 0.44

>20% lesion A=0.43

Caucasian African American

Chinese Japanese

0.32 (G)  0.13 (A)  0.03 (G)  0.08 (G) 

Caucasian African American

Chinese Japanese

0.28(G)  0.26(G)  0.01(G 0.06 (G) 

Caucasian African American

Chinese Japanese

0.28 (T)  0.33 (T)  0.02 (T)  0.04 (T) 

Minor Allele Frequency – rs646776(A/G)

In Qatar

<20% lesion A= 0.42

>20% lesion A=0.53

Minor Allele Frequency – rs4970984 - (C/T)

In Qatar

<20% lesion T= 0.5

>20% lesion T=0.5

Linkage disequilibrium [LD] of rs599839, rs646776, and rs4970834

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BIOM

Pairwise squared correlation coefficient [r2] were 0.029 between rs599839 rs646776 r2=0.0 between rs599839 and rs4970834 r2=4.95973e-32between rs646776 and rs4970834

Conclusion

no statistically significant association between the variants rs599839 A>G, rs646776 A>G and rs4970834 C>T and LDL-C and CVD risks among study subjects.

weak LD was observed between the rs599839 A>G, rs646776 A>G and rs4970834 C>T.

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Limitations

Small sample size Delay HMC approval Limited equipment ( RT PCR)

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Further Study

Further studies with large sample size are needed and other SNPs could be included to explore the association between genotype and phenotype of CVD among Qatari populations and gulf areas.

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Acknowledgment

Special thanks to Dr. Nasser Rizk Dr. Ayman El Menyar (HMC)/ cardiology

department Dr. Murlitharan and Dr. Hamda Al Naemi (Biology

Dept. QU) Mrs. Mozah Al Khinji

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