DNA CHIP PLATFORM: A HIGH-THROUGHPUT GENOTYPING TECHNOLOGY FOR GENETIC DIAGNOSIS AND PHARMACOGENETIC PROFILING LU YI (Bachelor of Science, Wuhan University, PR China) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PAEDIATRICS NATIONAL UNIVERSITY OF SINGAPORE 2005 THIS THESIS IS DEDICATED TO: MY PARENTS MY ELDER BROTHER MY FRIENDS AND COLLEAGUES AND ALL MY MENTORS i ACKNOWLEDGEMENTS First of all, I would like to express my sincere gratitude and appreciation to my principal supervisor, Dr. Yeoh Eng Juh Allen, Associate Professor, Department of Paediatrics, National University of Singapore (NUS), for his invaluable guidance during my studentship in NUS. His encouragement and constructive criticisms have taught me to work independently, think scientifically, and understand the principles to be a researcher. I am deeply appreciative of his advice and guidance over the years. Secondly, I must thank Ms. Kham Kow Yin Shirley, Senior Lab Officer, Department of Paediatrics, NUS. From the first day in this foreign land, “Auntie Shirley”, as she is fondly known to everyone, has always cared for me in my study, my lab work, even my daily life. Her kindness has helped me quickly adapt to this new environment enabling me to devote my time into the research. Her useful advice, as well as her help in all aspects, is truly unforgettable. I also wish to record my sincere appreciation to Associate Professor Quah Thuan Chong, Department of Paediatrics, NUS, and Dr. Heng Chew Kiat, Department of Paediatrics, NUS, for their guidance and expert advice which enabled me to perform my research systematically and their support in the review and revision of my publications. I would also thank my colleagues in the laboratory for their warm friendship and making me part of this research family. Finally, my projects would not have started without financial aid from NUS research scholarship generously provided by National University of Singapore. ii TABLE OF CONTENTS Page Dedication i Acknowledgements ii Table of Contents iii Summary viii List of Tables x List of Figures xii List of Abbreviations xiv Preface Chapter 1: Introduction 1.1 β-thalassemia and β-globin gene 1.1.1 β-thalassemia 1.1.2 Techniques for the genetic diagnosis of β-thalassemias 1.1.2.1 The principle of the minisequencing 10 1.1.2.2 Applications of the minisequencing 12 1.2 Pharmacogenetic analyses in childhood acute lymphoblastic leukaemia 22 1.2.1 Childhood ALL and drugs commonly used in its treatment 23 1.2.2 Xenobiotics-metabolizing genes and their common polymorphisms 26 1.2.2.1 6-Mercaptopurine metabolism 26 1.2.2.2 Folic acid metabolism 29 1.2.2.3 Phase I and phase II enzymes 34 1.2.2.4 Drug transporters 39 iii 1.2.3 Statistical methods commonly used in the study of polymorphismdisease association 41 1.2.3.1 Hardy–Weinberg equilibrium test 42 1.2.3.2 Chi-square (χ2) test 43 1.2.3.3 Z-test 44 1.2.3.4 Fisher’s exact test 45 1.2.3.5 Binary logistic regression 46 1.2.3.6 Linkage disequilibrium (LD) 46 1.2.3.7 Receiver operating characteristic (ROC) curve 48 1.2.3.8 Some important terms in statistical analysis 50 1.3 Aims 53 1.3.1 Screening β-thalassemia mutations using APEX methodology 53 1.3.2 Pharmacogenetic profiling of children with ALL using AsPEX strategy 54 Chapter 2: Materials and Methods 57 2.1 APEX genotyping platform for β-globin and TPMT genes 57 2.1.1 APEX methodology 57 2.1.2 DNA samples 58 2.1.3 PCR amplifications 59 2.1.4 APEX primers 61 2.1.5 Microarray preparation 63 2.1.6 Chip layout 64 2.1.7 Hybridization 64 2.1.8 APEX reactions 65 2.1.9 Fluorescence detection 66 2.1.10 Signal intensity analysis 66 iv 2.1.11 Tests on unbalanced PCR amplification 2.2 AsPEX genotyping strategy for pharmacogenetic profiling 67 67 2.2.1 Study design 67 2.2.2 Genotyping strategy 69 2.2.3 Multiplex PCRs and purification 71 2.2.4 Multiplex single nucleotide AsPEX 74 2.2.5 Chip layout and preparation 76 2.2.6 Hybridization 78 2.2.7 Fluorescence detection and analysis 78 2.2.8 Statistical analyses 79 2.2.9 Sensitivity comparison between APEX and AsPEX 81 2.2.10 Test on potential extension biases of AsPEX primers terminated with different nucleotides 81 Chapter 3: Results 82 3.1 APEX genotyping platform for β-globin and TPMT genes 82 3.1.1 PCR amplifications 82 3.1.2 APEX reactions 83 3.1.3 Accuracy validation 86 3.1.4 Analyses of fluorescence intensity 88 3.1.5 Efficacy testing on unbalanced PCR amplification to generate ssDNA 90 3.2 AsPEX genotyping strategy for pharmacogenetic profiling 91 3.2.1 PCR amplifications 91 3.2.2 Multiplex AsPEX reaction 91 3.2.3 Analyses of fluorescence intensity 96 v 3.2.4 Genotype/allele frequencies of polymorphisms in Chinese, Malay and Indian populations 105 3.2.5 Individual polymorphisms and the risk of developing childhood ALL 113 3.2.6 Combined genotypes and the risk of developing childhood ALL 117 3.2.7 Polymorphisms and the risk of ALL relapse 120 3.2.8 Sensitivity comparison between APEX and AsPEX 125 3.2.9 Signal intensity of the AsPEX primer pair with balanced concentrations 126 Chapter 4: Discussion 4.1 Technical issues regarding the chip-based genotyping platforms 128 128 4.1.1 Comparison between APEX and AsPEX 129 4.1.2 Comparisons between APEX/AsPEX and commercial chip-based genotyping platforms 131 4.1.3 Comparisons between APEX/AsPEX and other genotyping techniques 133 4.1.4 Useful tips for the design of APEX/AsPEX DNA chip 135 4.1.5 Analyses of fluorescence intensity 138 4.1.6 Limitations of APEX and AsPEX 139 4.1.7 Future trends in genotyping technology 141 4.2 Polymorphisms in xenobiotics-metabolizing genes and their impact on the risk of developing childhood ALL and risk of ALL relapse 143 4.2.1 Significant differences in allele frequencies among Chinese, Malay and Indian populations 144 4.2.2 The impact of MTHFR C677T, RFC G80A and NQO1 C609T polymophisms on the susceptibility to develop childhood ALL 145 4.2.2.1 MTHFR C677T 146 4.2.2.2 RFC G80A 148 4.2.2.3 NQO1 C609T 150 vi 4.2.2.4 The effects of combined genotypes 153 4.2.3 Factors that may alter the risk of relapse in children with ALL 156 4.2.4 Limitations of the study 158 4.2.5 Current obstacles in pharmacogenetics research of childhood ALL and future directions 160 Bibliography 162 Appendix 187 vii SUMMARY Genetic polymorphisms/mutations not only cause inherited diseases like Mendelian single gene diseases, but may also, in concert, alter the risk of developing certain cancers by defining an at-risk population, or affect a patient’s response to therapy by altering the metabolism of therapeutic drugs or modulate their pharmacokinetics. Therefore, systematical profiling of such polymorphisms/mutations may help to document their prevalence, to understand the complexity of carcinogenesis, and to optimize therapeutic efficacy by tailoring the dosages to an individual who is likely to respond well to the drugs and will not suffer corresponding side effects. However, these involve determining the polymorphisms of many genes in various individuals at different times, making it critical to develop a single platform that is cheap, readily customizable and can interrogate tens of polymorphisms in a single run. β-thalassemia, caused by mutations in the β-globin gene, is the most common inherited disease in the world. It causes decreased production of the β-globin which creates an imbalance in α- and β-globin production resulting in anemia. Population screening and prenatal diagnosis are currently the most effective measures to control this disease. The first project for this thesis was to design a rapid and robust methodology to simultaneously screen multiple mutations causing β-thalassemias. We integrated the outstanding multiplexing capacity of DNA chip technology and high accuracy of single-nucleotide primer extension strategy to establish an Arrayed Primer Extension (APEX) genotyping platform capable of detecting 23 polymorphisms in β-globin gene and polymorphisms in thiopurine Smethyltransferase (TPMT) gene in a single assay. Two hundreds DNA samples with known genotypes were used to validate this strategy. Accuracy of 97.3% and 100% viii for β-globin and TPMT genes, respectively, were achieved. Further analysis on fluorescence intensities enabled us to set cut-off values, 5.0 and 10.0, to determine the genotype quantitatively. Our results show that APEX is a reliable strategy to detect mutations causing β-thalassemia and TPMT deficiency. The second project involved an investigation of the impact of 14 polymorphisms in xenobiotics-metabolizing genes (TPMT, NQO1, MTHFR, GSTP1, CYP1A1, CYP2D6, MDR1 and RFC) on the risk of developing childhood acute lymphoblastic leukaemia (cALL) and the risk of relapse. paediatric cancer. ALL is the most common type of Defective handling of environmental xenobiotics due to polymorphisms in related metabolizing genes is one of the suspected reasons for the development of cALL. In addition, since efficacy of drugs may be similarly altered due to the polymorphic genes involved in drug metabolism, it is hypothesized that these polymorphisms may influence a patient’s treatment response. Instead of using conventional RFLP test which could only detect these polymorphisms individually, we developed another DNA chip-based method by exploiting multiplex allele-specific primer extension (AsPEX) which was able to detect all 14 polymorphisms simultaneously. We screened 225 cALL cases and 334 controls, and found that polymorphisms at NQO1-C609T, MTHFR-C677T and RFC-G80A are associated with a reduced risk of developing cALL in Chinese and/or Malay populations. However, we did not identify the influence of polymorphisms on the risk of relapse. In conclusion, DNA chip platform is a high-throughput and reliable technology for genetic diagnosis and pharmacogenetic profiling. ix Kim YI. Folate and cancer prevention: a new medical application of folate beyond hyperhomocysteinemia and neural tube defects. Nutr Rev. 1999, 57(10): 314-21. Kitada M. Genetic polymorphism of cytochrome P450 enzymes in Asian populations: focus on CYP2D6. Int J Clin Pharmacol Res. 2003, 23(1): 31-5. Koch WH. Technology platforms for pharmacogenomic diagnostic assays. Nat Rev Drug Discov. 2004, 3(9): 749-61. Kracht T, Schrappe M, Strehl S, Reiter A, Elsner HA, Trka J, et al NQO1 C609T polymorphism in distinct entities of paediatric hematologic neoplasms. Haematologica. 2004, 89(12): 1492-7. Krajinovic M, Richer C, Sinnett H, Labuda D, Sinnett D. Genetic polymorphisms of N-acetyltransferases and and gene-gene interaction in the susceptibility to childhood acute lymphoblastic leukaemia. Cancer Epidemiol Biomarkers Prev. 2000, 9(6): 557-62. Krajinovic M, Labuda D, Mathonnet G, Labuda M, Moghrabi A, Champagne J, et al Polymorphisms in genes encoding drugs and xenobiotic metabolising enzymes, DNA repair enzymes, and response to treatment of childhood acute lymphoblastic leukaemia. Clin Cancer Res. 2002a, 8(3):802-10. 172 Krajinovic M, Labuda D, Sinnett D. Glutathione S-transferase P1 genetic polymorphisms and susceptibility to childhood acute lymphoblastic leukaemia. Pharmacogenetics. 2002b, 12(8): 655-8. Krajinovic M, Lamothe S, Labuda D, Lemieux-Blanchard E, Theoret Y, Moghrabi A, et al Role of MTHFR genetic polymorphisms in the susceptibility to childhood acute lymphoblastic leukaemia. Blood. 2004a, 103(1): 252-7. Krajinovic M, Lemieux-Blanchard E, Chiasson S, Primeau M, Costea I, Moghrabi A. Role of polymorphisms in MTHFR and MTHFD1 genes in the outcome of childhood acute lymphoblastic leukaemia. Pharmacogenomics J. 2004b, 4(1): 66-72. Krajinovic M, Sinnett H, Richer C, Labuda D, Sinnett D. Role of NQO1, MPO and CYP2E1 genetic polymorphisms in the susceptibility to childhood acute lymphoblastic leukaemia. Int J Cancer. 2002c, 97(2): 230-6. Krawczak M, Boehringer S, Epplen JT. Correcting for multiple testing in genetic association studies: the legend lives on. Hum Genet. 2001, 109(5): 566-7. Kuppuswamy MN, Hoffmann JW, Kasper CK, Spitzer SG, Groce SL, Bajaj SP. Single nucleotide primer extension to detect genetic diseases: experimental application to hemophilia B (factor IX) and cystic fibrosis genes. Proc Natl Acad Sci U S A. 1991, 88(4): 1143-7. 173 Kurg A, Tonisson N, Georgiou I, Shumaker J, Tollett J, Metspalu A. Arrayed primer extension: solid-phase four-color DNA resequencing and mutation detection technology. Genet Test. 2000, 4(1): 1-7. Kwok PY. SNP genotyping with fluorescence polarization detection. Hum Mutat. 2002, 19(4): 315-23. Larson RA, Wang Y, Banerjee M, Wiemels J, Hartford C, Le Beau MM, et al Prevalence of the inactivating 609C-->T polymorphism in the NAD(P)H:quinone oxidoreductase (NQO1) gene in patients with primary and therapy-related myeloid leukaemia. Blood. 1999, 94(2): 803-7. Laverdiere C, Chiasson S, Costea I, Moghrabi A, Krajinovic M. Polymorphism G80A in the reduced folate carrier gene and its relationship to methotrexate plasma levels and outcome of childhood acute lymphoblastic leukaemia. Blood. 2002, 100(10): 3832-4. Lennard L, Lilleyman JS, Van Loon J, Weinshilboum RM. Genetic variation in response to 6-mercaptopurine for childhood acute lymphoblastic leukaemia. Lancet. 1990, 336(8709): 225-9. Lindroos K, Liljedahl U, Raitio M, Syvanen AC. Minisequencing on oligonucleotide microarrays: comparison of immobilisation chemistries. Nucleic Acids Res. 2001, 29(13): E69-9. 174 Locatelli F, Stefano PD. New insights into haematopoietic stem cell transplantation for patients with haemoglobinopathies. Br J Haematol. 2004, 125(1): 3-11. Mahgoub A, Idle JR, Dring LG, Lancaster R, Smith RL. Polymorphic hydroxylation of Debrisoquine in man. Lancet. 1977, 2(8038): 584-6. Marsh S, McLeod HL. Cancer pharmacogenetics. Br J Cancer. 2004, 90(1): 8-11. Marzolini C, Paus E, Buclin T, Kim RB. Polymorphisms in human MDR1 (Pglycoprotein): recent advances and clinical relevance. Clin Pharmacol Ther. 2004, 75(1): 13-33. Masson LF, Sharp L, Cotton SC, Little J. Cytochrome P-450 1A1 gene polymorphisms and risk of breast cancer: a HuGE review. Am J Epidemiol. 2005, 161(10): 901-15. Matsuo K, Suzuki R, Hamajima N, Ogura M, Kagami Y, Taji H, et al Association between polymorphisms of folate- and methionine-metabolising enzymes and susceptibility to malignant lymphoma. Blood. 2001, 97(10): 3205-9. McLeod HL, Krynetski EY, Relling MV, Evans WE. Genetic polymorphism of thiopurine methyltransferase and its clinical relevance for childhood acute lymphoblastic leukaemia. Leukaemia. 2000, 14(4): 567-72. 175 McLeod HL, Siva C. The thiopurine S-methyltransferase gene locus - implications for clinical pharmacogenomics. Pharmacogenomics. 2002, 3(1): 89-98. Mitchell P. Microfluidics--downsizing large-scale biology. Nat Biotechnol. 2001, 19(8): 717-21. Mitra RD, Church GM. In situ localized amplification and contact replication of many individual DNA molecules. Nucleic Acids Res. 1999, 27(24): e34. Mo QH, Zhu H, Li LY, Xu XM. Reliable and high-throughput mutation screening for β-thalassaemia by a single-base extension/fluorescence polarization assay. Genet Test. 2004, 8(3): 257-62. Nei M, Saitou N. In: Kalow W, Goedde HW, Agarwal DP, editors. Ethnic differences in reactions to drugs and xenobiotics. New York, NY: Alan R liss; 1986, pp. 21-37. Ng I, Law HY. Challenges in screening & prevention of thalassaemia in Singapore. Journal of Paediatrics and Child Health 2003, 2(2): 29-38. Nyholt DR. Genetic case-control association studies--correcting for multiple testing. Hum Genet. 2001, 109(5): 564-5. Okada Y, Nakamura K, Wada M, Nakamura T, Tsukamoto N, Nojima Y, et al Genotyping of thiopurine methyltransferase using pyrosequencing. Biol Pharm Bull. 2005, 28(4): 677-81. 176 Old JM, Petrou M, Modell B, Weatherall DJ. Feasibility of antenatal diagnosis of beta thalassaemia by DNA polymorphisms in Asian Indian and Cypriot populations. Br J Haematol. 1984, 57(2): 255-63. Olivieri NF. The beta-thalassaemias. N Engl J Med. 1999, 341(2): 99-109. Pastinen T, Kurg A, Metspalu A, Peltonen L, Syvanen AC. Minisequencing: a specific tool for DNA analysis and diagnostics on oligonucleotide arrays. Genome Res. 1997, 7(6): 606-14. Pastinen T, Perola M, Niini P, Terwilliger J, Salomaa V, Vartiainen E, et al Arraybased multiplex analysis of candidate genes reveals two independent and additive genetic risk factors for myocardial infarction in the Finnish population. Hum Mol Genet. 1998, 7(9): 1453-62. Pastinen T, Raitio M, Lindroos K, Tainola P, Peltonen L, Syvanen AC. A system for specific, high-throughput genotyping by allele-specific primer extension on microarrays. Genome Res. 2000, 10(7): 1031-42. Picciano MF, Stokstad, Gregory JF. Folic acid metabolism in health and disease. New York: Wiley-Liss, Inc 1990, pp. 1-21. Prakash AS, Beall H, Ross D, Gibson NW. Sequence-selective alkylation and crosslinking induced by mitomycin C upon activation by DT-diaphorase. Biochemistry. 1993, 32(21): 5518-25. 177 Pui CH, Boyett JM, Rivera GK, Hancock ML, Sandlund JT, Ribeiro RC, et al Longterm results of Total Therapy studies 11, 12 and 13A for childhood acute lymphoblastic leukaemia at St Jude Children's Research Hospital. Leukaemia. 2000, 14(12): 2286-94. Pui CH. Treatment of acute leukaemias: new directions for clinical research. 2003, Humana Press, Totowa, NJ. Quah TC, Sun L, Chew FT, Yeoh A, Lee BW. Survival of childhood leukaemia in Singapore. Med Pediatr Oncol. 1996, 26(5): 318-24. Rebbeck TR, Spitz M, Wu X. Assessing the function of genetic variants in candidate gene association studies. Nat Rev Genet. 2004, 5(8): 589-97. Relling MV, Hancock ML, Boyett JM, Pui CH, Evans WE. Prognostic importance of 6-mercaptopurine dose intensity in acute lymphoblastic leukaemia. Blood. 1999, 93(9): 2817-23. Relling MV, Yanishevski Y, Nemec J, Evans WE, Boyett JM, Behm FG, et al Etoposide and antimetabolite pharmacology in patients who develop secondary acute myeloid leukaemia. Leukaemia. 1998, 12(3): 346-52. Roses AD. Pharmacogenetics place in modern medical science and practice. Life Sci. 2002, 70(13): 1471-80. 178 Ross D, Kepa JK, Winski SL, Beall HD, Anwar A, Siegel D. NAD(P)H:quinone oxidoreductase (NQO1): chemoprotection, bioactivation, gene regulation and genetic polymorphisms. Chem Biol Interact. 2000, 129(1-2): 77-97. Rubnitz JE, Pui CH. Recent advances in the treatment and understanding of childhood acute lymphoblastic leukaemia. Cancer Treat Rev. 2003, 29(1): 31-44. Rund D, Rachmilewitz E. β-thalassaemia. N Engl J Med. 2005, 353(11): 1135-46. Sahai H, Khurshid A. Statistics in epidemiology: methods, techniques, and applications. 1996, CRC Press, Boca Raton. Sambrook J, Russell DW. Molecular cloning: a laboratory manual, 3rd ed. 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Schaeffeler E, Fischer C, Brockmeier D, Wernet D, Moerike K, Eichelbaum M, et al Comprehensive analysis of thiopurine S-methyltransferase phenotype-genotype correlation in a large population of German Caucasians and identification of novel TPMT variants. Pharmacogenetics. 2004, 14(7): 407-17. Schmiegelow K, Schroder H, Gustafsson G, Kristinsson J, Glomstein A, Salmi T, et al Risk of relapse in childhood acute lymphoblastic leukaemia is related to RBC methotrexate and mercaptopurine metabolites during maintenance chemotherapy. Nordic Society for Paediatric Hematology and Oncology. J Clin Oncol. 1995, 13(2): 345-51. 179 Schulz WA, Krummeck A, Rosinger I, Eickelmann P, Neuhaus C, Ebert T, et al Increased frequency of a null-allele for NAD(P)H: quinone oxidoreductase in patients with urological malignancies. Pharmacogenetics. 1997, 7(3): 235-9. Shather HN. Statistical evaluation of prognostic factors in ALL and treatment results. Med Pediatr Oncol. 1986, 14(3): 158-65. Shchepinov MS, Case-Green SC, Southern EM. Steric factors influencing hybridisation of nucleic acids to oligonucleotide arrays. Nucleic Acids Res. 1997b, 25(6): 1155-61. Shchepinov MS, Udalova IA, Bridgman AJ, Southern EM. Oligonucleotide dendrimers: synthesis and use as polylabelled DNA probes. Nucleic Acids Res. 1997a, 25(22): 4447-54. Shi MM. Enabling large-scale pharmacogenetic studies by high-throughput mutation detection and genotyping technologies. Clin Chem. 2001, 47(2): 164-72. Silverman LB, Declerck L, Gelber RD, Dalton VK, Asselin BL, Barr RD, et al Results of Dana-Farber Cancer Institute Consortium protocols for children with newly diagnosed acute lymphoblastic leukaemia (1981-1995). Leukaemia. 2000, 14(12): 2247-56. Sinnett D, Krajinovic M, Labuda D. Genetic susceptibility to childhood acute lymphoblastic leukaemia. Leuk Lymphoma. 2000, 38(5-6): 447-62. 180 Skibola CF, Smith MT, Kane E, Roman E, Rollinson S, Cartwright RA, et al Polymorphisms in the methylenetetrahydrofolate reductase gene are associated with susceptibility to acute leukaemia in adults. Proc Natl Acad Sci U S A. 1999, 96(22): 12810-5. Smith M, Arthur D, Camitta B, Carroll AJ, Crist W, Gaynon P, et al Uniform approach to risk classification and treatment assignment for children with acute lymphoblastic leukaemia. J Clin Oncol. 1996, 14(1): 18-24. Stanulla M, Schaeffeler E, Flohr T, Cario G, Schrauder A, Zimmermann M, et al Thiopurine methyltransferase (TPMT) genotype and early treatment response to mercaptopurine in childhood acute lymphoblastic leukaemia. JAMA. 2005, 293(12): 1485-9. Stanulla M, Schrappe M, Brechlin AM, Zimmermann M, Welte K. Polymorphisms within glutathione S-transferase genes (GSTM1, GSTT1, GSTP1) and risk of relapse in childhood B-cell precursor acute lymphoblastic leukaemia: a case-control study. Blood. 2000, 95(4): 1222-8. Sutcharitchan P, Saiki R, Fucharoen S, Winichagoon P, Erlich H, Embury SH. Reverse dot-blot detection of Thai beta-thalassaemia mutations. Br J Haematol. 1995, 90(4): 809-16. 181 Syvanen AC, Sajantila A, Lukka M. Identification of individuals by analysis of biallelic DNA markers, using PCR and solid-phase minisequencing. Am J Hum Genet. 1993, 52(1): 46-59. Syvanen AC. Accessing genetic variation: genotyping single nucleotide polymorphisms. Nat Rev Genet. 2001, 2(12): 930-42. Syvanen AC. Detection of point mutations in human genes by the solid-phase minisequencing method. Clin Chim Acta. 1994, 226(2): 225-36. Syvanen AC. From gels to chips: "minisequencing" primer extension for analysis of point mutations and single nucleotide polymorphisms. Hum Mutat. 1999, 13(1): 1-10. Taton TA, Mirkin CA, Letsinger RL. Scanometric DNA array detection with nanoparticle probes. Science. 2000, 289(5485): 1757-60. Taub JW, Matherly LH, Ravindranath Y, Kaspers GJ, Rots MG, Zantwijk CH. Polymorphisms in methylenetetrahydrofolate reductase and methotrexate sensitivity in childhood acute lymphoblastic leukaemia. Leukaemia. 2002, 16(4): 764-5. Tillib SV, Strizhkov BN, Mirzabekov AD. Integration of multiple PCR amplifications and DNA mutation analyses by using oligonucleotide microchip. Anal Biochem. 2001, 292(1): 155-60. 182 Timan IS, Aulia D, Atmakusma D, Sudoyo A, Windiastuti E, Kosasih A. Some haematological problems in Indonesia. Int J Hematol. 2002, 76 Suppl 1: 286-90. Traver RD, Siegel D, Beall HD, Phillips RM, Gibson NW, Franklin WA, et al Characterization of a polymorphism in NAD(P)H: quinone oxidoreductase (DTdiaphorase). Br J Cancer. 1997, 75(1): 69-75. Tully G, Sullivan KM, Nixon P, Stones RE, Gill P. Rapid detection of mitochondrial sequence polymorphisms using multiplex solid-phase fluorescent minisequencing. Genomics. 1996, 34(1): 107-13. van der Put NM, Gabreels F, Stevens EM, Smeitink JA, Trijbels FJ, Eskes TK, et al A second common mutation in the methylenetetrahydrofolate reductase gene: an additional risk factor for neural-tube defects? Am J Hum Genet. 1998, 62(5): 1044-51. Wang W, Kham SK, Yeo GH, Quah TC, Chong SS. Multiplex minisequencing screen for common Southeast Asian and Indian beta-thalassaemia mutations. Clin Chem. 2003, 49(2): 209-18. Weatherall DJ, Clegg JB. The thalassaemia Syndromes, 4th edn. 2001, Blackwell Scientific Publications, Oxford. Weinshilboum R. Inheritance and drug response. N Engl J Med. 2003, 348(6): 529-37. 183 Weisberg I, Tran P, Christensen B, Sibani S, Rozen R. A second genetic polymorphism in methylenetetrahydrofolate reductase (MTHFR) associated with decreased enzyme activity. Mol Genet Metab. 1998, 64(3): 169-72. Weisberg IS, Jacques PF, Selhub J, Bostom AG, Chen Z, Curtis Ellison R, et al The 1298A-->C polymorphism in methylenetetrahydrofolate reductase (MTHFR): in vitro expression and association with homocysteine. Atherosclerosis. 2001, 156(2): 409-15. Westin L, Xu X, Miller C, Wang L, Edman CF, Nerenberg M. Anchored multiplex amplification on a microelectronic chip array. Nat Biotechnol. 2000, 18(2): 199-204. Whetstine JR, Gifford AJ, Witt T, Liu XY, Flatley RM, Norris M, et al Single nucleotide polymorphisms in the human reduced folate carrier: characterization of a high-frequency G/A variant at position 80 and transport properties of the His(27) and Arg(27) carriers. Clin Cancer Res. 2001, 7(11): 3416-22. Wiemels JL, Pagnamenta A, Taylor GM, Eden OB, Alexander FE, Greaves MF. A lack of a functional NAD(P)H:quinone oxidoreductase allele is selectively associated with paediatric leukaemias that have MLL fusions. United Kingdom Childhood Cancer Study Investigators. Cancer Res. 1999, 59(16): 4095-9. Wiemels JL, Smith RN, Taylor GM, Eden OB, Alexander FE, Greaves MF, et al Methylenetetrahydrofolate reductase (MTHFR) polymorphisms and risk of molecularly defined subtypes of childhood acute leukaemia. Proc Natl Acad Sci U S A. 2001, 98(7): 4004-9. 184 Wiencke JK, Spitz MR, McMillan A, Kelsey KT. Lung cancer in Mexican-Americans and African-Americans is associated with the wild-type genotype of the NAD(P)H: quinone oxidoreductase polymorphism. Cancer Epidemiol Biomarkers Prev. 1997, 6(2): 87-92. Winner EJ, Prough RA, Brennan MD. Human NAD(P)H:quinone oxidoreductase induction in human hepatoma cells after exposure to industrial acrylates, phenolics, and metals. Drug Metab Dispos. 1997, 25(2): 175-81. Wu G, Hua L, Zhu J, Mo QH, Xu XM. Rapid, accurate genotyping of betathalassaemia mutations using a novel multiplex primer extension/denaturing highperformance liquid chromatography assay. Br J Haematol. 2003, 122(2): 311-6. Yates CR, Krynetski EY, Loennechen T, Fessing MY, Tai HL, Pui CH, et al Molecular diagnosis of thiopurine S-methyltransferase deficiency: genetic basis for azathioprine and mercaptopurine intolerance. Ann Intern Med. 1997, 126(8): 608-14. Yeoh EJ, Ross ME, Shurtleff SA, Williams WK, Patel D, Mahfouz R, et al Classification, subtype discovery, and prediction of outcome in paediatric acute lymphoblastic leukaemia by gene expression profiling. Cancer Cell. 2002, 1(2): 13343. Yip SP, Pun SF, Leung KH, Lee SY. Rapid, simultaneous genotyping of five common Southeast Asian beta-thalassaemia mutations by multiplex minisequencing and denaturing HPLC. Clin Chem. 2003, 49(10): 1656-9. 185 Zammatteo N, Jeanmart L, Hamels S, Courtois S, Louette P, Hevesi L, et al Comparison between different strategies of covalent attachment of DNA to glass surfaces to build DNA microarrays. Anal Biochem. 2000, 280(1): 143-50. Zielinska E, Zubowska M, Bodalski J. Polymorphism within the glutathione Stransferase P1 gene is associated with increased susceptibility to childhood malignant diseases. Pediatr Blood Cancer. 2004, 43(5): 552-9. 186 Appendix  Lu Y, Kham KY, Foo TC, Ariffin H, Quah TC, Yeoh EJ. Genotyping of eight polymorphic genes encoding drug-metabolising enzymes and transporters using a customised oligonucleotide array. Anal Biochem. 2007, 360(1): 105-13.  Lu Y. Genetic polymorphisms associated with the risk of developing childhood acute lymphoblastic leukaemia: a study of Chinese and Malay populations in Southeast Asia. (oral presentation on the 5th Bi-annual Symposium on Childhood Leukaemia, April 30th – May 2nd 2006, NH Hotel Leeuwenhorst, Noordwijkerhout, The Netherlands)  Lu Y, Kham KY, Tan PL, Quah TC, Heng CK, Yeoh EJ. Arrayed Primer Extension (APEX): a robust and reliable genotyping platform for the diagnosis of single gene disorders: β-thalassaemia and thiopurine methyltransferase (TPMT) deficiency. Genet Test. 2005, 9(3): 212-9.  Lu Y. Arrayed Primer Extension (APEX): a solid-phase four-color DNA minisequencing to detect the mutations on the human beta-globin gene and thiopurine methyltransferase (TPMT) gene. (poster presentation, the 3rd Prize of “Best Poster Award” on the 1st Bilateral Symposium on Advances in Molecular Biotechnology and Biomedicine, May 23rd – 24th 2002, Clinical Research Centre, National University of Singapore, Singapore) 187 [...]... that is also produced in reduced amounts By itself, whether in β-thalassaemia mutation as a compound heterozygote – HbE-βthalassaemia – a moderately severe anaemia, in between that of thalassaemia minor and major, afflicts the patient This is termed thalassaemia intermedia Southeast Asia lies in the malaria belt where for thousands of years, malaria is the major cause of death and morbidity As thalassaemia... the carrier has mild microcytosis but otherwise asymptomatic It has been postulated that carriers of β-thalassaemia minor has less severe malaria infections and this provides an evolutionary advantage to the carrier in places of high prevalence of malaria Unfortunately, in the homozygous state – β-thalassaemia major – the patient suffers from severe anaemia, is transfusion dependent for life and will... years of age in the absence of proper ironchelation therapy Although medical therapies for β-thalassaemia major, like chronic blood transfusion with concomitant iron chelation, are currently available and able to prolong the life span of β-thalassaemia major patients, poor patient compliance and high costs limit their roles In the United Kingdom, the estimated cost of managing a β-thalassaemia major... uncommon (Cao, et al., 1994) 5 At the phenotypic level, β-thalassaemia can be classified into 2 clinical syndromes: βthalassaemia trait, characterised by asymptomatic microcytosis, results from the inheritance of one mutant β-globin gene; and thalassaemia major (β0- or β+thalassaemia), which usually result from homozygosity or compound heterozygosity for a mutant β-globin allele β-thalassaemia major patients... Singapore to scrutinise every pregnant women’s mean corpuscular volume when they present for antenatal check up For all women who were found to have microcytosis, a thalassaemia screen and test for iron deficiency were carried out If the mother was found to be a thalassaemia carrier, the father would also be screened for thalassaemia carriage When both parents were found to be thalassaemia carriers, antenatal... thalassaemia disorders confers protection against severe malaria, the evolutionary Darwinian pressure results in a very high frequency of thalassaemia gene carriage and other haemoglobinopathies like HbE in the region (Weatherall, et al., 2001) In Thailand, it has been estimated by the World Bank that, over the next 30 years, approximately 100,000 new cases of HbE-βthalassaemia alone will be added to... research areas in our laboratory for many years and a large number of samples with known mutations of the β-globin chain are available Another important group of SNPs are those that alter the normal functions of enzymes involved in metabolisms of chemical xenobiotics such as environmental carcinogens and medications These SNPs are of particular interest to cancer epidemiological studies or pharmacogenetic. .. assay formats, a feasible solution is to design the minisequencing assay on the solid phase, for example, on the DNA chip platform Solid-phase assay formats In solid-phase assay formats, oligonucleotide reactants, either templates or primers/probes, are immobilised onto a certain solid support, such as a glass microscopic slide or 384-well microtitre plate, to form a DNA chip, or synonymously, a microarray... – are divided into 2 major groups: thalassaemia and structural haemoglobinopathies Thalassaemia results from decreased production of either α- and β-globin chains, causing an imbalance in the ratio α- and β- globin chains in the red cells Unlike structural haemoglobinopathies for example sickle haemoglobin (HbS), where the haemoglobin produced is abnormal, in thalassaemias, the globin gene is normal... thalassaemia carriers, antenatal diagnosis using initially amniocentesis and subsequently chorionic villus sampling, were carried out to determine if the fetus had thalassaemia major As βthalassaemia is an autosomal recessive condition, there is a 1 in 4 chance that the fetus 8 is affected If the fetus is found to be β-thalassaemia major, termination of pregnancy can be offered On a national scale, in order to . conclusion, DNA chip platform is a high - throughput and reliable technology for genetic diagnosis and pharmacoge netic profiling. x LIST OF TABLES Page Table 1.1 Features of traditional geno typing. postulated that carriers of β - thalassaemia minor has less severe malaria infections and this provide s an evolutionary advantage to the carrier in places of high prevalence of malaria . Unfortunately,. gene th at causes β - thalassaemia syndromes. This was one of the research area s in our laboratory for many years and a large number of s amples with known mutations of the β - globin chain are available . Another