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... consisting of Telugas and Tamils from southeastern India and a minority of Sikhs and Pathans from north India and accounts for 7.9% of the Singapore population The immigrant Chinese and Indians... ethnic Chinese were not different from that in the ethnic Indians, in spite of a much lower CAD incidence in the ethnic Chinese (57) Because of Singapore's multiracial population structure and differential... 76.7% of the resident Singaporean population The Malay population account for 13.9% of the Singapore population (52) Moreover, the Indian migrants in Singapore were from the Indian subcontinent, with
ASSOCIATION OF CETP AND ADTRP WITH CORONARY ARTERY DISEASE IN MULTI-ETHNIC SINGAPOREAN POPULATION NAEIMEH TAYEBI A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF PEDIATRICS NATIONAL UNIVERSITY OF SINGAPORE 2012 Declaration I hereby declare that the thesis is my original work and it has been written by me in its entirety I have duly acknowledged all the sources of information which have been used in the thesis This thesis has also not been submitted for any degree in any university previously Naeimeh Tayebi December 2012 أ Acknowledgements I would like to thank: A/Prof.Heng and A/Prof.Liu, who have been my great supervisor and cosupervisor respectively, their love of learning and insistence on perfection has been great sources of inspiration I am thankful to them for lending their trust, attention, patience and support during this research and preparation of this thesis; this would not have been possible without their help I wish to thank from Lye Hui Jen, Huang Ning and June-Mui Goh for teaching the techniques in molecular biology to me, and Ke Tingjing, for helping me with the experiments I wish to thank from my GIS friends: Foo Jia Nee, Low Hui Qi, Astrid Irwanto, Herty Liany, Zhao Wanting, Rajkumar Dorajoo, and Li Jingmei, who taught me statistical analysis I wish to thank from National University of Singapore for scholarship and all of my good friends in Singapore, for their support and encouragement Naeimeh Tayebi December 2012 ب Table of Contents Title page Declaration page I Acknowledgments II Summary List of Tables List of Figures List of Abbreviations Chapter 1: Introduction 1.1 Definition of Coronary Artery Disease (CAD) 1.2 Prevalence of CAD 1.3 Pathogenesis of CAD 10 1.4 Conventional risk factors of CAD 11 1.4.1 Hyperlipidemia 12 1.4.2 Age and hypertension 13 1.4.3 Cigarette smoking 13 1.4.4 Diabetes mellitus 14 1.4.5 Coagulation factors 14 1.5 Genetic factors 1.5.1 Various genetic epidemiological approaches 1.6 Background and aims of present study 14 15 19 ت Chapter 2: Methods 22 2.1 Record of demographic information 23 2.2 Blood sampling 23 2.3 Measurement of blood lipids 24 2.4 Extraction of genomic DNA 25 2.5 Optimization 26 2.6 Sanger sequencing 27 2.7 Statistical analysis 28 Chapter 3: Cholesteryl Ester Transfer Protein (CETP) rs708272 and rs1800775 polymorphisms 31 3.1 Introduction 32 3.2 Study subjects 35 3.3 Genotyping and Sequencing 36 3.4 Results 40 3.4.1 Demographics of the Singaporean subjects 40 3.4.2 Frequency distribution of rs708272 and rs1800775 promoter polymorphisms in the three ethnic groups of Singapore 3.4.3 Linkage disequilibrium between rs708272 and rs1800775 sites in the three ethnic groups of Singapore 3.4.4 Impact of CETP polymorphisms on plasma lipid profile 41 3.4.5 Gene environmental interaction 44 42 42 3.5 Discussion 57 3.6 Conclusion 61 ث Chapter 4: The androgen-dependent TFPI regulating protein (ADTRP) polymorphisms 62 4.1 Introduction 63 4.2 Study subjects 64 4.3 Genotyping 65 4.4 Results 68 4.4.1 Demographics of the Singaporean subjects 68 4.4.2 Association of rs6903956 with CAD in the three ethnic groups of Singapore 4.4.3 Association between genotypic of rs6903956 and plasma lipid profile 69 70 4.5 Discussion 76 4.6 Limitation and future direction 79 4.7 Conclusion 80 Chapter 5: Concluding remarks 81 Bibliography 85 Appendices 99 ج Summary Singapore’s multi-ethnic population structure and different prevalence and mortality due to CAD in the ethnic groups provide an excellent opportunity to discover further ethological factors CAD In the study leading to this thesis, two polymorphisms in the Cholesteryl Ester Transfer Protein (CETP), which were results of a candidate gene approach and the polymorphism of androgendependent tissue factor pathway inhibitor (TFPI) regulating protein (ADTRP), that was a result of validation of GWAS, were investigated in relation to CAD and plasma lipid levels among the Chinese, Malay and Indian ethnic groups in Singapore The genotypic distribution of all the three polymorphisms was in line with a population at Hardy Weinberg equilibrium (HWE) The study of two polymorphisms (rs708272 and rs1800775) within the CETP genes included 662 CAD cases with angiographically confirmed CAD (CAD+) and 927 consecutive individuals from the general population This group is labeled as CAD- since those with a history of heart disorders or with ECG abnormalities were excluded For evaluation of allelic and genotypic association of rs708272 and rs1800775 with CAD, subjects with dyslipidemia, hypertension and diabetes were excluded and this subset of CAD- group was labeled as healthy control group For ADTRP gene (rs6903956), 645 CAD+ and 755 CAD- have been selected We then tested the association of these Single Nucleotide Polymorphisms (SNPs) with CAD and lipid profiles The B2 frequency of rs708272 was significantly lower in the Malays than the Indians and Chinese in the healthy control A similar significant trend was observed for the A allele of rs1800775 polymorphism of CETP Significantly lower B2 and A allele frequencies were observed in the Chinese cases compared to healthy controls The absence of the B2 allele was associated with CAD with an odds ratio (OR) of 2.0 (95% CI 1.2 to 3.4) after adjustment for the confounding effects of age, cigarette smoking, BMI and gender The B2 allele of rs708272 and A allele of rs1800775 were significantly associated with higher plasma HDL-C levels in the male Chinese CAD- after adjusting for the significant confounding effects of age, BMI and smoking The B2 allele had similar HDL-C elevating effects in the Indian women However, in the combined population, rs708272 showed only highly significant association with plasma HDL-C The association of rs6903956 within ADTRP gene was also investigated with CAD in the multi-ethnic Singaporean population The risk allele A of rs6903956 was associated significantly only in the Chinese cases with an OR of 2.03 (95% CI 1.04-3.96, p-value = 0.037) when analyzed by each ethnic group separately In a meta-analysis, rs6903956 showed highly significant association with CAD both before (observed p =1.39×10-4; OR= 1.66; 95% CI 1.28-2.15) and after adjustment (p-value = 4.63×10-3; OR =1.86; 95% CI 1.212.87) for conventional risk factors of age, gender, BMI, smoking status and ethnicity No significant association was observed between rs6903956 genotypes and lipid profiles in the Chinese, Malays and Indians, suggesting that the association of this SNP with CAD is not mediated through plasma lipids The limitations of two studies are small sample groups especially for the Malay and Indian populations Therefore, further analysis in larger numbers of cases and controls will help to confirm these results List of Tables 1.5.1 The 36 loci associated with CAD/MI at Genome-Wide Levels of statistical significance 18 2.6 Components of cycle sequencing reaction 27 3.1 Previous studies of polymorphisms in CETP gene 34 3.4.1 Demographics of the Singaporean subjects 46 3.4.2 Genotype and allele frequencies of the rs708272 and rs1800775 promoter polymorphisms in the three ethnic groups of Singapore 48 3.4.2a The P-values between different Genotypes of the rs708272 and rs1800775 promoter polymorphisms in the three ethnic groups of Singapore before and after adjustment for age 49 Linkage disequilibrium coefficients between the rs708272 and rs1800775 sites in the three ethnic groups of Singapore for CAD cases and CAD- 50 3.4.3 3.4.4a Genotypic lipid levels (Mean SD) of the rs708272 polymorphism in the Chinese, Malays and Indian CAD- men 51 3.4.4b Genotypic lipid levels (Mean SD) of the rs708272 polymorphism in the Chinese, Malays and Indian CAD- women 52 3.4.4c Genotypic lipid levels (Mean SD) of the rs1800775 promoter polymorphism in the Chinese, Malays and Indian CAD- men 3.4.4d Genotypic lipid levels (Mean SD) of the rs1800775 promoter polymorphism in the Chinese, Malays and Indian CAD- women 53 Clinical and 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Non-smoker [ ] Smoking: _ years No of sticks/day: Ex-smoker: _ years No of sticks/day: when stopped: Fasting status: Fasting [ ] Non-fasting [ ] Time of last meal: Personal Medical History: CAD [ ] HTN [ ] Diabetes [ ] Any Other: Family History: Diabetic HTN CAD Any other Father Mother Sibs Others Medication: Duration When stopped Lipid lowering drugs B-Blockers Anticoagulants Aspirin Hormones Anti-Diabetics Any other Coronary Grading (No of vessels with >50% obstruction): History of MI: Number of episodes: _ Operation: Year: _ Number of vessels: 99 Appendices APPENDIX HEALTHY SUBJECT DATA FORM Name: Date: Address: Registration No: Telephone No: I/C or Passport (Foreigner) No: Sex: Height: cm B/P: Systolic mmHg Diastolic mmHg Age: Weight: kg Hb: Race: Chinese [ ] Malay [ ] Indian [ ] Others: Educational Level: Occupation: Smoking status: Smoker [ ] Ex-smoker [ ] Non-smoker [ ] Smoking: _ years No of sticks/day: Ex-smoker: _ years No of sticks/day: when stopped: Fasting status: Fasting [ ] Non-fasting [ ] Time of last meal: Personal Medical History: CAD [ ] HTN [ ] Diabetes [ ] Any Other: Family History: Diabetic HTN CAD Any other Father Mother Sibs Others 100 Appendices APPENDIX LIST OF CHEMICALS AND REAGENTS USED Chemicals and reagents Restriction enzymes: AseI, Taqα1 Manufacturer New England Biolabs Inc., Massachusetts, USA Buffer for RFLP New England Biolabs Inc., Massachusetts, USA Exonuclease I ( 20U/µL) New England Biolabs Inc., Massachusetts, USA Shrimp Alkaline Phosphatase(SAP) (1U/µL) Roche Diagnostic Systems, Basel, Switzerland HPLC purified oligonucleotides (primers) for PCR - BST Scientific, 1st Base Pte Ltd, Singapore Deoxynucleotide triphosphate (dNTP) Epicentre Technologies, Wisconsin, USA Taq DNA polymerase, PCR buffer Qiagen, Germany EDTA, Ethanol, Sodium acetate, trisodium citrate Merck, Darmstadt, Germany Proteinase K, Sodium dodecyl sulphate (SDS), phosphotungstic acid/magnesium chloride Sigma Chemical Company, St Louis, Missouri, USA CHOL assay kit for TC and HDLC, TRIG assay kit for Triglyceride Roche Diagnostic Systems, Basel, Switzerland Chloroform, Phenol Roche Diagnostic Systems, Basel, Switzerland BigDye Applied Biosystems, USA 5X sequencing buffer Edge BioSystems, Gaithersburg Hi-Di Formamide Applied Biosystems, USA 101 Appendices APPENDIX LIST OF INSTRUMENTS AND APPARATUS USED Ultracentrifuge Instruments Manufacturer Sorvall RC-5B, Du Pont Instruments, Connect USA High-speed centrifuge KS-5000P Kubota Corporation, Tokyo, Japan PCR amplifier DNA Thermal Cycler, Perkin Elmer Cetus, Boston, USA Electrophoresis power pack ECPS 3000/150.Pharmacia Fine Chemicals,Uppsala, Sweden Horizontal electrophoresis set Electro-4 gel tank Hybaid Ltd,USA Blood tube rotator SB1, Stuart Scientific Co Ltd, Sterling, United Kingdom ABI PRISM® 3100 Genetic Analyzer Applied Biosystems, USA 102