2. ROLE OF CORONARY HEART DISEASE RISK GENE SCORE IN DIFFERENTIATING BETWEEN PAKISTANI CHD CASES AND CONTROLS
Presenter:
Dr. WAFA MUNIR ANSARI
Department of Chemical Pathology,
Army Medical College,
National University of Sciences and Technology, Islamabad, Pakistan.

3. INTRODUCTION Coronary Artery Disease (CAD) burden:
17.1 million deaths worldwide each year 1
leading cause of mortality in the world by the year
Premature CAD (≤ 45yrs) accounts for:
ALMOST 40% IN PAKISTAN 3
30% IN SOUTH ASIA

4. INTRODUCTION INTRODUCTION Genetic factors and atherosclerosis (GWAS) .
Risk of CAD is being assessed by:
Framingham risk score 4
PROCAM 5
Cumulative gene score and risk prediction.
Gene score risk prediction v/s CRF .

5. OBJECTIVES OBJECTIVES
To investigate the role of a 13 SNP CAD risk gene score in a subjects from Pakistan.
To compare the risk allele frequency between Pakistanis and Caucasians (Northwick Park Heart Study II ).
To determine if the gene score improved risk prediction over & above Conventional Risk Factors (CRFs)

6. HYPOTHESIS
HYPOTHESIS
The risk gene score helps to differentiate between Pakistani CAD cases and controls.
Addition of genetics to CAD risk prediction improves stratification for individuals of South Asian origin.

7. MATERIALS AND METHODS MATERIALS AND METHODS Setting
Centre for Research in Experimental Medicine, Army Medical College, Islamabad, Pakistan.
Cardiovascular Genetics Institute, University College London, UK.
Duration:- 8 months after approval of synopsis.

8. STUDY DESIGN STUDY DESIGN CASE-CONTROL STUDY n=408
CASES (PCAD Patients)
n=203
CONTROLS
(n=205)

9. SUBJECTS
SUBJECTS
450 subjects aged ≤ 45 years with a history of chest pain, scheduled to undergo coronary angiography were selected.
Technique: Non-probability convenience sampling.

10. SUBJECTS INCLUSION CRITERIA EXCLUSION CRITERIA SUBJECTS
Both males and females.
Age ≤45 yrs.
Coronary artery stenosis >70% in at least one coronary vessel (cases n=203).
Angio-negative controls (n=205) were age and sex matched.
EXCLUSION CRITERIA
Neoplastic , hematological , renal, hepatic ,autoimmune or thyroid disease.
Congenital or valvular heart disease.
Taking anti-inflammatory drugs.
Unwilling to give consent.

11. DNA EXTRACTION
-QIAamp DNA Blood Mini kit (Qiagen, USA).
- The 13 SNPs were genotyped in a using KASPar and Taqman assays.

12. STATISTICAL ANALYSIS
STATA v13.1 (StataCorp 2013) and SPSS version 22.0 (IBM Corp 2013).
Case control comparison:
Two-sided t-tests (continuous variables)
χ2 tests (categorical variables).

13. STATISTICAL ANALYSIS
logistic regression for the relationship between quintile of gene score and CHD.
Gene scores = number of risk alleles x the natural log of the odds ratio for each SNP and adding the products together.

14. RESULTS

15. 13 SNPs included in the gene score.
Gene/Locus
Source
Process rs
MIA3
GWAS - rs
9p21 rs
CXCL12 rs
MRAS
rs646776
SORT1
rs662799
APOA5
Candidate Gene
Lipid Metabolism rs
APOB rs
LPA rs rs
PCSK9
rs429358
APOE
rs7412
rs328
LPL

16. Gene/Locus
SNP
Risk Allele
Odds Ratio
MIA3 rs C
1.14
9p21 rs G
1.29
CXCL12 rs
1.17
APOA5
rs662799
1.19
APOB rs A
1.73
LPA rs
1.92 rs
1.70
MRAS rs T
1.15
LPL
rs328
1.25
SORT1+ rs
PCSK9 rs
1.43
APOE
rs429358
1.06
rs7412
0.80

17. Demographic Characteristics of the Pakistani sample set
Trait
CASES (n=203)
Mean ± SD
Controls (n=205)
Age (y) Mean ± SD
40.7±4.23
35.1±7.55
Height (m)
1.69 ± 0.07
1.68 ± 0.07
Weight (kg)
73.9 ± 4.0*
70.6 ± 13.6
BMI (kg/m2)
25.9 ± 4.36*
23.7 ± 5.15
Systolic BP(mm of Hg)
124.7 ± 12.3**
114.4 ± 6.0
Diastolic BP(mm of Hg)
81.4 ± 8.08**
70.6 ± 4.0
Smokers n (%)
93 (57%) *
36 (26%)
Family history PCAD n(%)
48 (30%)**
12(8.7%)
Family history HTN n(%)
51 (31%)**
17 (12.3%)

18. CAD Risk Gene Score in the Pakistani study(n=380) and NPHSII(n=2775)
Cases (n=200)
Controls (n=180)
p-value
13 SNP CAD
Gene Score Mean (SD)
2.44
(0.42)
2.25
(0.35)
0.04*
NPHSII participants
NPHSII
CHD
(n=284)
No CHD
(n=2491)
2.53 (0.45)
2.43 (0.48)
7x10-3 **
p<0.01
p=4.51 x10-6

19. Association between gene score and CHD in the Pakistani samples.

20. RAF Pakistani Controls (n=180)
Comparison of risk allele frequencies between the Pakistani and NPHSII control groups.
Gene/Locus
SNP
RAF Pakistani Controls (n=180)
RAF NPSHII
No CHD (n=284)
p-value
9p21* rs
0.56**
0.48
9.02x10-5
CXCL12* rs
0.73**
0.86
1.71x10-30
APOA5*
rs662799
0.14**
0.06
1.35x10-18
APOB* rs
0.11**
0.18
1.6x10-5
LPA* rs
0.016**
0.02
6.23x10-3 rs
0.012**
0.08
1.34x10-10
MRAS* rs
0.07**
0.16
3.26x10-8
APOE*
rs429358
0.09**
0.15
5.31x10-5

21. COMPARISON OF SNP RISK ALLELE FREQUENCIES BETWEEN PAKISTANI PCAD CASES AND CONTROLS

22. Comparison of risk allele frequencies between the Pathan and Punjabi Controls from the Pakistani study.
Gene/Locus
SNP
RAF Pathan
Controls
(n=80)
RAF Punjabi controls
(n=100)
p-value
9p21 rs
0.67**
0.52
0.009
CXCL12 rs
0.74*
0.68
0.03
APOA5
rs662799
0.10*
0.16
0.02
SORT1 rs
0.75*
0.72
0.04

23. a) Framingham score b) Framingham score + gene score in Pakistanis
AUC for CAD risk:
a) Framingham score b) Framingham score + gene score in Pakistanis b) a)
a)AUC=0.553
95%CI:
b) AUC= 0.598
95%CI=

24. CONCLUSION
The 13 SNP gene score has the potential to improve risk stratification in high risk Pakistani individuals.
The risk score has utility in differentiating between Pakistani PCAD cases and controls.
The RAF were significantly different between South Asians and Caucasians

25. DISCUSSION
RAF of 5 /13 SNPs were significantly different between Pakistani CHD cases and controls.

26. DISCUSSION
Gene score comparison between Pathans & Punjabis.

27. FUTURE PROSPECTIVES
Developing population specific risk gene scores with bigger sample sizes.
In Silico Analysis of the Functional and Structural Consequences of SNPs
Identification of the functional SNP and effect of the SNP on serum levels of these markers in CAD.

28. CLINICAL APPLICATIONS
Risk stratification array.

29. ACKNOWLEGEMENTS
Special Risk stratification array.

30. REFERENCES
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4.Conroy RM, Pyorala K, Fitzgerald AP, Sans S, Menotti A, et al. (2003) Estimation of ten-year risk of fatal cardiovascular disease in Europe: the SCORE project. European Heart Journal 24:
5.Collins GS & Altman DG (2010) An independent and external validation of QRISK2 cardiovascular disease risk score: a prospective open cohort study. BMJ 340: c2442.

31. THANK YOU