1) The document summarizes a student's research project on studying the association between lipid profile, oxidative stress markers, and gene polymorphisms in relation to early onset coronary heart disease. 2) Biochemical analysis found higher levels of triglycerides, cholesterol, LDL, VLDL, glucose, and oxidative stress markers in early coronary heart disease patients compared to controls. 3) Molecular analysis found the CC genotype of the eNOS gene was more prevalent among early coronary heart disease patients compared to controls.

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

2. External Guide: M. Sailaja
Designation:Senior Scientific Assistant
Institute of Genetics O.U.
Internal Guide: Richa Joshi
Designation : Assistant Professor
MJRP University

3. 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.

4. 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.

5. 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

6. 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

7. 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

8. 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

12. 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 :

15. RESULTS:
4.598
1.575
0
1
2
3
4
5
Patients Controls
ConcentrationofNO(µM)
Estimation of NO
Patients
Controls
 BIOCHEMICAL STUDIES
4.348
2.084
0
1
2
3
4
5
Patients Controls
ConcentrationofMDA(µM)
Estimation of MDA
Patients
Controls
9.886
9.3
0
1
2
3
4
5
6
7
8
9
10
Patients Controls
ConcentrationofCalcium(µM)
Estimation of Calcium Levels
Patients
Controls
212.267
83.267
0
50
100
150
200
250
Patients Controls
Concentrationofglucose(µM)
Estimation of Glucose Level
Patients
Controls

16. 27.733
30.733
0
5
10
15
20
25
30
35
Patients Controls
ConcentrationofHDL(µM)
Estimation of HDL
Patients
Controls
110.667
101.8
0
20
40
60
80
100
120
Patients Controls
ConcentrationofLDL(µM)
Estimation of LDL
Patients
Controls
50
26.933
0
10
20
30
40
50
60
Patients Controls
ConcentrationofVLDL(µM)
Estimation of VLDL
Patients
Controls
250.333
118.133
0
50
100
150
200
250
300
Patients Controls
ConcentrationofTriglycerides(µM)
Estimation of Triglycerides
Patients
Controls
LIPID PROFILE

17. 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

18. MPO Genotyping
Genotyping 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

19. 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

20. MPO genotyping frequency

21.  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 PATIENTS
L
PCR – ANALYSIS
387 bp
250 bp
176 bp

22. Genotype Patients Controls
TT 4 9
TC 8 6
CC 3 0
Total 15 15
Statistics of eNOS Genotype:
4
8
3
9
6
0
0
1
2
3
4
5
6
7
8
9
10
TT TC CC
Frequencies
Genotype
Patients
Controls
Table: eNOS genotypes in patients and controls

23. Table: T & C alleles in patients and controls
16
14
24
6
0
5
10
15
20
25
30
T C
Frequencies
Allele
Patients
Controls
Allele Patients Controls Total
T 16 24 40
C 14 6 20

24. CONCLUSION
In 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%).

25.  Manisha Nair and Dorairaj Prabhakaran, Why Do South Asians Have High Risk for
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 Leeder, S, Raymond S, Greenberg H. A Race Against Time: The challenge of
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 Viswanathan Mohan et al, Raj Deepa, Subramaniam Shanthi Rani, GopalPremalatha,
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REFERENCE

26. THANK YOU !!!