CORONARY ARTERY DISEASE

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BY: NAME:ABHIJEET SUBHASH GHADGE ROLL NO : 10647 BRANCH : B.TECH BIOTECHNOLOGY 4 th Year MAHATMA JYOTI RAO PHOOLE UNIVERSITY

Transcript of CORONARY ARTERY DISEASE

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BY:

NAME:ABHIJEET SUBHASH GHADGE

ROLL NO : 10647

BRANCH : B.TECH BIOTECHNOLOGY

4th Year

MAHATMA JYOTI RAO PHOOLE UNIVERSITY

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External Guide: M. SailajaDesignation:Senior Scientific Assistant

Institute of Genetics O.U.Internal Guide: Richa JoshiDesignation : Assistant Professor

MJRP University

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AIM & OBJECTIVES

AIM:

To study the association of lipid profile and oxidative stress markers in relation to gene

polymorphisms in early onset of coronary heart disease compared to healthy persons (controls).

OBJECTIVES:

Biochemical studies

Estimation of Nitric Oxide in Plasma

Estimation of Lipid Peroxidation

Estimation of Calcium level in serum

Estimation of Glucose level in serum

Lipid profile (HDL, LDL, VLDL, Triglycerides)

Molecular studies

Isolation of DNA

Quantification of DNA by Agarose Gel Electrophoresis

Polymerase Chain Reaction to amplify genes.

RFLP of the eNOS gene.

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Coronary artery disease (also called CAD) is

the most common type of heart disease. It is also

the leading cause of death for both men and

women in our country.

It occurs when fatty deposits called plaque build

up inside the coronary arteries. The coronary

arteries wrap around

the heart and supply it with blood and oxygen.

When plaque builds up, it narrows the arteries

and reduces the amount of blood that gets to

your heart.

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What causes CAD?Research shows that the exact etiology of CAD is unknown. However, numerous contributing risk factors have been identified. It starts when certain factors damage the inner layers of the coronary arteries. It is classified as modifiable & non-modifiable.

Non- modifiable

-Age

-Sex

-Family History

-Ethnic background

Modifiable

-Smoking

-High amounts of certain fats and cholesterol in the blood

-Physical activity.-Stress (release of Catecholamine)-High amounts of sugar in the blood due to

insulin resistance or diabetes

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Coronary Artery Disease

Coronary artery disease (CAD) is the most common form of cardiovascular (heart)

disease. It occurs when the coronary arteries that supply oxygen and nutrient-rich blood to

heart become blocked over time due to the buildup of fat, cholesterol, and other

substances.

• Atherosclerosis

• Myocardial Infarction

• Angina

• Symptoms

• Risk factors

• Diagnosis

• Treatment

• Prevalence

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WORK DESIGN

WHOLE BLOOD (5-6ml)

MOLECULAR

ANALYSIS (2ml)

BIOCHEMICAL

ANALYSIS (3ml)

GENE POLYMORPHISMS

Of eNOS & MPO.

LIPID

PROFILE

SUBJECTS

CAD =25 &

Controls =25

OXIDATIVE STRESS

MARKERS

(NO, MDA LEVELS)

CALCIUM &

GLUCOSE

LEVELS

DEMOGRAPHIC

STUDIES

INFORMED

CONSENT

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Blood Sample Collection

•5 ml of venous blood is collected for biochemical

and molecular analysis.

•Serum is separated from blood, centrifuged at

1500rpm for 15 minutes and stored at -20°C until

further use.

DNA is extracted from leukocytes of EDTA

blood samples

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PCR – first described in mid 1980’s, Mullis

Nobel prize in 1993.

An in vitro method for the enzymatic

synthesis of specific DNA sequences.

• Template DNA

• Oligonucleotide primers

• dNTP’s

• Thermostable DNA pol

• MgCl2• Buffer (usually supplied as 10X

Requirements :

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RESULTS:

4.598

1.575

0

1

2

3

4

5

Patients Controls

Con

cen

tra

tion

of

NO

M)

Estimation of NO

Patients

Controls

BIOCHEMICAL STUDIES

4.348

2.084

0

1

2

3

4

5

Patients Controls

Co

nce

ntr

ati

on

of

MD

A (

µM

) Estimation of MDA

Patients

Controls

9.8869.3

0

1

2

3

4

5

6

7

8

9

10

Patients Controls

Con

cen

trati

on

of

Calc

ium

M)

Estimation of Calcium Levels

Patients

Controls

212.267

83.267

0

50

100

150

200

250

Patients Controls

Con

cen

trati

on

of

glu

cose

M)

Estimation of Glucose Level

Patients

Controls

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27.73330.733

0

5

10

15

20

25

30

35

Patients Controls

Con

cen

trati

on

of

HD

L (

µM

)

Estimation of HDL

Patients

Controls

110.667101.8

0

20

40

60

80

100

120

Patients Controls

Con

cen

trati

on

of

LD

L (

µM

)

Estimation of LDL

Patients

Controls

50

26.933

0

10

20

30

40

50

60

Patients Controls

Con

cen

tra

tio

n o

f V

LD

L (

µM

)

Estimation of VLDL

Patients

Controls

250.333

118.133

0

50

100

150

200

250

300

Patients Controls

Con

cen

trati

on

of

Tri

gly

ceri

des

M)

Estimation of Triglycerides

Patients

Controls

LIPID PROFILE

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DNA was isolated from 2ml of blood using Salting out method (TKM) both from

patients and control group. Concentration of DNA was quantified by 1% agarose gel

electrophoresis. All the samples are showing good concentration of DNA.

ISOLATION OF GENOMIC DNA

Lane 1 2 3 4 5 6 7 8

MOLECULAR STUDIES

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MPO GenotypingGenotyping of MPO G/A, polymorphism was performed using polymerase chain reaction followed by digestion with restriction enzymes AciI

350bp

Lane 7– ladder Lane 1-6 -Patient Samples

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168bp

289bp

121bp

1 2 3 4 5 6 L7

Restriction enzyme analysis of MPO GENE for Aci I enzyme

Lane 1&5-Heterozygous GA (289+168+12 bp1)

Lane2, 3,4-GG Homozygous (168,121bp)

Lane 7-DNA size ladder

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MPO genotyping frequency

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eNOS Genotyping:

Genotyping of eNOs T/C, polymorphism was performed using allele-specific

polymerase chain reaction (PCR).

Lane C1 C2 C3 C4 C5 P1 P2 P3 P4 P5

CONTROLS PATIENTSL

PCR –ANALYSIS

387 bp

250 bp

176 bp

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Genotype Patients Controls

TT 4 9

TC 8 6

CC 3 0

Total 15 15

Statistics of eNOS Genotype:

4

8

3

9

6

00

1

2

3

4

5

6

7

8

9

10

TT TC CC

Fre

qu

enci

es

Genotype

Patients

Controls

Table: eNOS genotypes in patients and controls

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Table: T & C alleles in patients and controls

1614

24

6

0

5

10

15

20

25

30

T C

Fre

qu

enci

es

Allele

Patients

Controls

Allele Patients Controls Total

T 16 24 40

C 14 6 20

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CONCLUSIONIn the present study, increased concentrations of triglycerides, cholesterol, LDL and VLDL were

observed in patients when compared with controls (250.334 vs 118.133 μM; 187.8 vs 139.933 μM; 110.667

vs 101.8 μM; 50 vs 26.933 μM respectively).

Estimation of glucose levels were found to be increased in patients when compared to controls

(212.267 vs 83.267 μM). Most of the patients were found to be diabetic which is the risk factor for CAD.

Serum calcium levels were not found significantly in CAD patients when compared to controls

(9.886 vs 9.3 μM). Most of the CAD patients were found to be hypocalcaemia which is also a risk factor for

CAD.

The results on the mean MDA and nitric oxide levels in Myocardial Infarction patients were

found to be significantly high when compared to controls (4.348 vs 2.084 μM; 4.598 vs 1.575μM

respectively). Elevated levels of MDA and nitrite/nitrate in MI patients cause oxidative stress which further

leads to endothelial damage and pathogenesis of the disease.

Polymorphisms of eNOS gene is significantly associated with the presence of CAD. The results

showed excess of homozygosity for the CC variant among CAD patients as against controls (20% vs 0%)

and heterozygosity for the TC variant among CAD patients as against control group (53.4% vs 40%).

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Manisha Nair and Dorairaj Prabhakaran, Why Do South Asians Have High Risk for

CAD? GLOBAL HEART, VOL. 7, NO. 4, 2012: 307 – 314.

Ghaffar A, Reddy KS, Singhi M. Burden of non-communicable diseases in South Asia.

BMJ 2004; 328:807–10.

Leeder, S, Raymond S, Greenberg H. A Race Against Time: The challenge of

cardiovascular disease in developing economies. 2004. Columbia University. New York

City, New York.

Gupta R, Misra A, Vikram NK, et al. Younger age of escalation of cardiovascular risk

factors in Asian Indian subjects. BMC cardiovascular disorders. 2009; 9-28.

Patel V, Chatterji S, Chisholm D, et al. Chronic diseases and injuries in India. Lancet

2011; 377:413–28.

Viswanathan Mohan et al, Raj Deepa, Subramaniam Shanthi Rani, GopalPremalatha,

Prevalence of Coronary Artery Disease and Its Relationship to Lipids in a Selected

Population in South India, JACC Vol. 38, No. 3, 2001: 682–7.

REFERENCE

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THANK YOU !!!