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Hla Genetic Profile of Malay Sub- Ethnic Groups of Peninsular Malaysia As Determined by Pcr-Ssp and Sbt Methods

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Hla Genetic Profile of Malay Sub- Ethnic Groups of Peninsular Malaysia As Determined by Pcr-Ssp and Sbt Methods HLA GENETIC PROFILE OF MALAY SUB- ETHNIC GROUPS OF PENINSULAR MALAYSIA AS DETERMINED BY PCR-SSP AND SBT METHODS ALLIA BINTI SHAHRIL UNIVERSITI SAINS MALAYSIA 2016 HLA GENETIC PROFILE OF MALAY SUB-ETHNIC GROUPS OF PENINSULAR MALAYSIA AS DETERMINED BY PCR-SSP AND SBT METHODS by ALLIA BINTI SHAHRIL Thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy August 2016 ACKNOWLEDGEMENT In the name of Allah, the Beneficent, the Merciful. I wish to express my sincere gratitude to Universiti Sains Malaysia (USM) Health Campus for providing me the opportunity to continue my studies here. I would also like to acknowledge the Ministry of Higher Education for giving me a scholarship thus enable me to pursue my dream. My upmost gratitude goes to my supervisors, Professor Norazmi Mohd Nor, Dr. Zafarina Zainuddin and Mr. Panneerchelvam for their endless support, guidance and encouragement. Not to forget, Dr. Edinur Hisham Atan for helping me out especially in the process of writing and statistics. I would also like to thank my colleagues, Puan Nur Haslindawaty, En. Nor Waliyuddin, Puan Tasnim, Puan Firdaus and Puan Sharifah Nany for helping me throughout my research work. I would also like to thank the assistant scientific officers Encik Azwan and Puan Rosniah for helping me during my laboratory work. Last but not least, special thanks go to my beloved husband and my parents for their endless support and encouragement. They are my stimuli and without them I won’t be able to be here. I love all of you so much. ii TABLE OF CONTENTS Acknowledgement ii Table of Contents iii List of Tables xi List of Figures xiv List of Abbreviations xviii List of Publications xxi Abstrak xxii Abstract xxiv CHAPTER 1: INTRODUCTION 1 1.1 Background of the Study 1 1.2 Rationale of the Study 5 1.3 Objectives of the Study 6 1.3.1 General Objective 6 1.3.2 Specific Objectives 6 CHAPTER 2: LITERATURE REVIEW 7 2.1 The Malays 7 2.1.1 The Origin of Malay population 7 2.1.2 Malay Sub-ethnic Groups 13 2.1.2(a) Kelantan 14 2.1.2(b) Patani 14 2.1.2(c) Champa 14 2.1.2(d) Kedah of Lembah Bujang 15 2.1.2(e) Acheh 15 iii 2.1.2(f) Mandailing 16 2.1.3 Previous Genetic Studies on the Malay population 16 2.2 Human Leukocyte Antigen (HLA) 20 2.2.1 HLA Classes 21 2.2.2 HLA Function 26 2.2.3 HLA Nomenclature 26 2.2.4 HLA typing methods 29 2.2.4(a) Microlymphocytotoxicity Assay 29 2.2.4(b) Restriction Fragment Length Polymorphism (RFLP) 30 2.2.4(c) Sequence Specific Oligonucleotide Primer (SSOP) 31 2.2.4(d) Sequence Specific Primer (SSP) 32 2.2.4(e) Sequence Based Typing (SBT) 33 2.2.4(f) Next Generation Sequencing (NGS) 34 2.3 HLA and Disease 35 2.4 HLA Application 39 2.4.1 Population Genetics 39 2.4.2 Transplantation 40 2.4.3 Disease Association 41 2.4.4 Forensic Purpose 42 CHAPTER 3: MATERIALS AND METHODS 43 3.1 Samples 43 3.2 Materials 43 3.2.1 Chemicals and consumables 43 3.2.2 Preparations of Solutions 46 iv 3.2.2(a) 70% Ethanol 46 3.2.2(b) 80% Ethanol 46 3.2.2(c) 10X Tris Borate EDTA buffer (10X TBE) 46 3.2.2(d) 0.5X Tris Borate EDTA Buffer (0.5X TBE) 46 3.2.2(e) Orange G Loading Dye 47 3.2.3 Kits 47 3.2.3(a) QIAamp® DNA blood mini kit 47 3.2.3(b) Olerup SSPTM HLA-A-B-C and Olerup SSPTM HLA- 47 DQ-DR SSP Combi Tray kit 3.2.3(c) Secore® A, Secore® B and Secore® DRB1 Locus 47 Sequencing kit 3.3 Methodology 48 3.3.1 DNA Extraction 48 3.3.2 Detection of genomic DNA 49 3.3.3 Quantification of DNA 49 3.3.4 Sequence Specific Primer (SSP) Method 50 3.3.4(a) PCR amplification of HLA-A, -B and –C loci 51 3.3.4(b) PCR amplification of HLA-DRB and -DQB1 53 3.3.4(c) Agarose gel electrophoresis of PCR products 53 3.3.4(d) Allele identification 54 3.3.5 Sequence Based Typing (SBT) Method 54 3.3.5(a) Preparation of HLA-A and -B amplification 55 Reactions 3.3.5(b) Preparation of HLA-DRB1 amplification reactions 56 v 3.3.5(c) Agarose Gel Electrophoresis 56 3.3.5(d) Purification of PCR Amplicons (ExoSAP-ITTM) 57 3.3.5(e) Sequencing Reaction 59 3.3.5(f) Preparation of Sequencing Reactions 59 3.3.5(g) Ethanol Precipitation of Sequencing Reaction 61 Products 3.3.5(h) Electrophoresis of Sequencing Reaction Products 61 on a Capillary Sequencer 3.3.5(i) Data Analysis 63 3.3.5(j) Resolving Ambiguous HLA alleles 63 3.3.6 Statistical Analysis 64 3.3.6(a) Allele frequency 64 3.3.6(b) Haplotype frequency 64 3.3.6(c) Exact test of Hardy Weinberg Equilibrium (HWE) 66 3.3.6(d) Likelihood ratio test of linkage disequilibrium (LD) 66 3.3.6(e) Ewens-Watterson neutrality test 66 3.3.6(f) Exact test of population differentiation 67 3.3.6(g) Principal coordinate analysis 67 CHAPTER 4: POPULATION PROFILES OF MALAY SUB –ETHNIC 68 GROUPS BASED ON HLA SEQUENCE SPECIFIC PRIMERS (SSP) 4.1 DNA Extraction 68 4.2 DNA amplification by PCR-SSP HLA typing 68 4.3 Statistical Analysis 74 4.3.1 Hardy-Weinberg Equilibrium (HWE) 74 vi 4.3.2 Ewens-Watterson neutrality test 74 4.3.3 Linkage Disequilibrium (LD) 74 4.3.4 Allele group frequency 78 4.3.5 Haplotype frequency 84 4.3.6 Exact test of population differentiation 86 4.3.7 Principal Coordinate Analysis (PCO) 94 4.4 Discussion 98 CHAPTER 5: POPULATION PROFILES OF MALAY SUB –ETHNIC 111 GROUPS BASED ON HLA SEQUENCE BASED TYPING (SBT) 5.1 Amplification of HLA loci using HLA-A, -B and -DRB1 locus 111 specific primers 5.2 3130xl Capillary Electrophoresis 111 5.3 Assignment of HLA alleles using uTYPE® v5.0 SBT software 112 5.4 Statistical Analyses 114 5.4.1 Allele group frequency 114 5.4.2 Haplotype frequency for 2 and 3 loci 120 5.4.3 Hardy-Weinberg Equilibrium (HWE) 120 5.4.4 Exact test of population differentiation 124 5.4.5 Ewens-Watterson Neutrality Tests 124 5.4.6 Linkage Disequilibrium (LD) 124 5.4.7 Principle Coordinate Analysis (PCO) 127 5.5 Discussion 129 CHAPTER 6: GENERAL DISCUSSION 134 CHAPTER 7: CONCLUSION 139 vii REFERENCES 140 APPENDICES 152 Appendix 1: Ethical Approval 153 Appendix 2: Consent form and questionnaire 155 Appendix 3: Sequence chromatogram for HLA-A locus 160 Appendix 3.1: HLA-A locus for Exon 2 forward strand 160 Appendix 3.2: HLA-A locus for Exon 2 reverse strand 161 Appendix 3.3: HLA-A locus for Exon 3 forward strand 162 Appendix 3.4: HLA-A locus for Exon 3 reverse strand 163 Appendix 3.5: HLA-A locus for Exon 4 forward strand 164 Appendix 3.6: HLA-A locus for Exon 4 reverse strand 165 Appendix 4: Sequence chromatogram for HLA-B locus 166 Appendix 4.1: HLA-B locus for Exon 2 forward strand 166 Appendix 4.2: HLA-B locus for Exon 2 reverse strand 167 Appendix 4.3: HLA-B locus for Exon 3 forward strand 168 Appendix 4.4: HLA-B locus for Exon 3 reverse strand 169 Appendix 4.5: HLA-B locus for Exon 4 forward strand 170 Appendix 4.6: HLA-B locus for Exon 4 reverse strand 171 Appendix 5: Sequence chromatogram for HLA-DRB1 locus 172 Appendix 5.1: HLA-DRB1 locus for Exon 2 forward strand 172 Appendix 5.2: HLA-DRB1 locus for Exon 2 reverse strand 173 Appendix 5.3: HLA-DRB1 locus for TG codon 86 reverse 174 Strand Appendix 6: A sequence chromatogram analyzed using uTYPE® v5.0 175 viii SBT software. The result is shown in the upper left column. Appendix 7: List of presentations 176 Appendix 7.1: Poster presented in International Symposium on 176 Forensic Science and Environmental Health 2009, organized by the Department of Chemistry Malaysia in Putra World Trade Centre (PWTC), 9-10th November 2009. Appendix 7.2: Poster Presentation, 2nd International Forensic 177 Science Symposium, 14-16 November 2011, Le Meridien Hotel, Kuala Lumpur. Appendix 8: Images during sampling 178 Appendix 9: HLA allele groups in Kelantan individuals (N=60) using 179 HLA-SSP method. Appendix 10: HLA allele groups in Champa individuals (N=42) using 181 HLA- SSP method. Appendix 11: HLA allele groups in Patani individuals (N=30) using 183 HLA- SSP method. Appendix 12: HLA allele groups in Aceh individuals (N=30) using HLA- 184 SSP method. Appendix 13: HLA allele groups in Kedah individuals (N=30) using 185 HLA-SSP method. Appendix 14: HLA allele groups in Mandailing individuals (N=30) using 186 HLA- SSP method. Appendix 15: HLA genotype in Kelantan individuals (N=30) using 187 ix HLA-SBT method. Appendix 16: HLA genotype in Champa individuals (N=30) using HLA- 188 SBT method. Appendix 17: HLA genotype in Patani individuals (N=30) using HLA- 189 SBT method. Appendix 18: HLA genotype in Mandailing individuals (N=30) using 190 HLA-SBT method. x LIST OF TABLES Page Table 2.1 HLA nomenclature 28 Table 2.2 List of HLA genes that confer susceptibility and 36 protection against various autoimmune diseases.
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