Non invasive prenatal testing of haemoglobin barts using fetal DNA from the maternal plasma

NON-INVASIVE PRENATAL TESTING OF HAEMOGLOBIN BART’S USING FETAL DNA FROM THE MATERNAL PLASMA SHERRY HO SZE YEE NATIONAL UNIVERSITY OF SINGAPORE 2008 NON-INVASIVE PRENATAL TESTING OF HAEMOGLOBIN BART’S USING FETAL DNA FROM THE MATERNAL PLASMA SHERRY HO SZE YEE A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF OBSTETRICS AND GYNAECOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2008 ACKNOWLEDGEMENTS I undertook this work at the Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore. Firstly, I would like to thank my supervisors, A/P Mahesh Choolani, A/P Arijit Biswas and Dr Khalil Razvi for their constant support and scientific guidance. I would also like to thank A/P Sinuhe Hahn (University of Basel, Switzerland), Jacquie Keer (LGC Ltd, UK) and their team members for their friendship and guidance in the initial part of this project. I would also like to extend my gratitude towards A/P Evelyn Koay (Molecular Diagnosis Centre, NUH), A/P Samuel Chong (Department of Paediatrics, NUS) and Dr Jerry Chan for their invaluable advice. It has been a great pleasure working with our post-doctoral fellows, Dr Sukumar Ponnusamy and Dr Narasimhan Kothandaraman. My special thanks to my team members, Dr Nuruddin Binte Mohammed, Dr Qin Yan, Zhang Huoming, Zhao Changqing, Fan Yiping, Dr Sonia Baig, Ho Lai Meng and Tan Lay Geok who had made research enjoyable. I am most grateful to all laboratory, administrative, clinical and nursing staff who had been enthusiastic in the recruitment of patients, and patients who participated in this study. I would like to thank my family: my parents, especially my mother, Gek Khim, for her constant faith in me, and my siblings for their love and encouragement. Finally, I would like to thank my husband, Dennis Chong, for always being there for me, and most of all, God, for making all this possible. i TABLE OF CONTENTS ACKNOWLEDGEMENTS TABLE OF CONTENTS SUMMARY LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS Units/ Symbols i ii v vii viii xi xiii CHAPTER 1: INTRODUCTION 1.1 Background 1.2 Prenatal testing 1.2.1 Prenatal screening 1.2.1.1 Screening for chromosomal disorders 1.2.1.2 Screening for single gene disorders 1.2.2 Prenatal diagnosis 1.2.2.1 Invasive procedures of prenatal diagnosis and their risks 1.2.2.2 Laboratory analysis of fetal material 1.3 Non-invasive prenatal diagnosis 1.3.1 Fetal cells in maternal blood 1.3.2 Fetal RNA in maternal blood 1.3.3 Fetal DNA in maternal blood 1.3.3.1 Sources of fetal DNA in maternal blood 1.3.3.2 Sensitivity and reproducibility of fetal DNA detection in maternal blood 1.3.3.3 Preeclampsia - Disease model for quantitative analysis of fetal DNA in maternal blood 1.3.3.4 Thalassaemia - Disease model for qualitative analysis of fetal DNA in maternal blood 1.4 DNA molecular technologies 1.4.1 DNA sequencing 1.4.2 Conventional PCR 1.4.3 Quantitative real-time PCR (QRT-PCR) 1.4.4 Quantitative fluorescence PCR (QF-PCR) 1.4.5 Fluorescence in situ hybridisation (FISH) 1.5 Experimental aims and hypotheses 1.5.1 Hypotheses 1 5 7 10 12 13 16 17 21 23 24 42 63 63 63 65 70 73 78 79 ii CHAPTER 2: MATERIALS AND METHODS 80 2.1 Samples 2.1.1 Research ethics board approval 2.1.2 Amniotic fluid 2.1.3 Trophoblast 2.1.4 Fetal blood 2.1.5 Umbilical cord blood 2.1.6 Adult peripheral blood 2.1.7 Cell lines 2.2 Deoxyribonucleic acid (DNA) analysis 2.2.1 DNA isolation 2.2.1.1 Amniotic fluid samples 2.2.1.2 Trophoblast samples 2.2.1.3 Blood samples - buffy coats and plasma 2.2.1.4 Cell lines 2.2.2 Conventional PCR 2.2.3 Real-time PCR 2.2.4 Quantitative fluorescent (QF)-PCR (singleplex and multiplex) 2.2.4.1 Microsatellite markers 2.2.5 DNA sequencing 2.3 Fluorescence in situ hybridisation (FISH) 2.3.1 FlashFISH 2.4 Statistical analysis 2.4.1 PowerStats V12 2.4.2 SPSS CHAPTER 3: SENSITIVITY AND SPECIFICITY OF REAL-TIME PCR 3.1 Introduction 3.2 Materials and methods 3.3 Results 3.3.1 Qualitative analysis and relative quantitation of APP gene expression 3.3.2 Sensitivities of HBB/APP multiplex QRT-PCR assays 3.3.3 Standard curves 3.4 Discussion CHAPTER 4: DETECTION OF FETAL DNA FROM MATERNAL PLASMA 4.1 Introduction 4.2 Materials and methods 80 80 80 80 81 81 81 81 82 82 82 82 83 83 83 84 87 88 91 92 92 98 98 99 100 100 100 101 101 103 104 105 107 107 107 iii 4.3 Results 4.4 Discussion CHAPTER 5: USE OF MICROSATELLITE MARKERS TO IDENTIFY HB BART'S IN FETAL DNA 5.1 Introduction 5.2 Materials and methods 5.3 Results 5.3.1 Assessment of maternal DNA contamination 5.3.2 Heterozygosity and PIC 5.4 Discussion CHAPTER 6: USE OF MICROSATELLITE MARKERS TO EXCLUDE HB BART'S NON-INVASIVELY USING FETAL DNA FROM MATERNAL PLASMA 6.1 6.2 6.3 6.4 109 119 122 122 122 125 132 132 133 137 Introduction Materials and methods Results Discussion 137 138 139 148 CHAPTER 7: COMMENTARY 151 7.1 Introduction 7.2 Hypotheses 7.3 Findings 7.3.1 Sensitivity and specificity of real-time PCR (QRT-PCR) 7.3.2 Detection of fetal DNA from maternal plasma 7.3.3 Use of microsatellite markers to identify Hb Bart's from fetal DNA 7.3.4 Use of microsatellite markers to exclude Hb Bart's non-invasively using fetal DNA from maternal plasma 7.4 Limitations 7.5 Directions for future research 7.6 Conclusions BIBLIOGRAPHY APPENDICES 151 152 152 152 153 154 154 155 159 160 162 210 iv SUMMARY Alpha thalassaemia is the most common inherited monogenic disorder amongst haemoglobinopathies in Southeast Asia (SEA). Carriers of double alpha-globin gene deletions such as the common -SEA deletion, are at risk of carrying fetuses with the fatal haemoglobin (Hb) Bart’s hydrops fetalis. At-risk couples are offered prenatal diagnosis for subsequent genetic counselling when found to be affected. The risk of procedural-related miscarriages associated with prenatal diagnosis is however, unacceptable to some parents. Non-invasive techniques will allow at-risk couples to undergo prenatal screening with ease. The presence of cell-free fetal DNA in the maternal plasma is an alternative source of fetal genetic material for non-invasive prenatal testing. In principle, any fetal DNA sequence that differs from maternal DNA sequence can be identified in maternal plasma. The low level of fetal DNA in the prevailing maternal DNA is, however, a technical challenge. The aim of this thesis was to develop a highly sensitive and specific analytical system to detect fetal-specific markers in the maternal plasma for noninvasive prenatal testing. I explored the use of polymorphic microsatellite markers that may differ between maternal and paternal alleles. This would allow the differentiation and identification between fetal paternally-inherited alleles from the prevailing maternal alleles in the maternal plasma. Quantitative fluorescence polymerase chain reaction (QF-PCR) technique is used for the amplification, detection and analysis of the small amounts of fetal v DNA with paternally-inherited microsatellite markers in the maternal plasma. This novel strategy was to analyse the specific fetal paternally-inherited microsatellite markers that lie within the breakpoints of the common alpha thalassaemia double gene deletions using QF-PCR. The detection of these fetal paternally-inherited microsatellite markers in the maternal plasma would show that the fetus has inherited the unaffected paternal allele and exclude the fetus of Hb Bart’s. I found that fetal paternally-inherited microsatellite markers can be detected and analysed in 10 out of 30 (33.3%) at-risk (n=3) and unaffected (n=7) maternal plasma samples using QF-PCR. Hb Bart’s was excluded noninvasively with 100% accuracy using cell-free fetal DNA in maternal plasma. As such, more than one-third (37.5%, out of 8) at-risk mothers carrying unaffected fetuses would avoid unnecessary invasive tests that could cause miscarriages. The presence of non-specific stutter peaks that mask paternally-inherited fetal STRs limits the detection rate. In conclusion, I have developed an analytical system for the detection and differentiation of small amounts of fetal paternally-inherited alleles from prevailing maternal alleles in the maternal plasma. The use of sizefractionation and single nucleotide polymorphisms (SNPs) within the deleted regions may enhance the rate of detection. This non-invasive prenatal screening method would allow at-risk mothers carrying unaffected fetuses to avoid unnecessary invasive procedures. Hb Bart’s can therefore, be excluded non-invasively using cell-free fetal DNA in the maternal plasma. vi LIST OF TABLES Table 1-1 Risk factors of preeclampsia and HELLP syndrome 28 Table 1-2 Pathophysiology of thalassaemia 44 Table 1-3 Human haemoglobins and their synonyms 48 Table 1-4 The different forms of beta thalassaemia . 51 Table 1-5 Haematological indices of patients with thalassaemia . 58 Table 1-6 Spectral properties of fluorescent probes. . 66 Table 2-1 Dilution series used for standard curves in QRT-PCR . 87 Table 2-2 Primer sequences for QF-PCR. . 90 Table 3-1 Mean and median of delta cycle threshold, ΔCT, between HBB and APP amplifications (HBB-APP) in normal and DS samples .101 Table 4-1 Table showing fetal DNA (measured by SRY amplifications) and total DNA (measured by HBB amplifications) concentrations in maternal plasma of P1 and P2 .110 Table 4-2 Non-invasive identification of fetal gender using fetal DNA from the maternal plasma. .115 Table 4-3 Table shows the comparisons of cell-free total (HBB) and fetal (SRY) DNA concentrations in maternal plasma obtained from normal pregnancies (n=13) and hypertensive pregnancies (n=2, P1 with HELLP syndrome and P2 with severe PE) 117 Table 5-1 Microsatellite markers (16PTEL05 and 16PTEL06) analysis results and molecular analysis genotypes .127 Table 5-2 Table showing STR analysis results of the 100 amniotic fluid samples 133 Table 6-1 Table showing the genotypes of spiked alpha thalassaemia samples in 1:50 dilution where X is the target DNA and Y is the diluent DNA. 139 Table 6-2 Table showing the results from (a) maternal plasma study, (b) fetal DNA analysis by QF-PCR, and (c) genotypes of 30 couples and their fetuses .145 vii LIST OF FIGURES Figure 1-1 Normal (A) and abnormal (B) placentation . 25 Figure 1-2 Two stages of pregnancies leading to preeclampsia 25 Figure 1-3 Global distribution of haemoglobinopathies. 43 Figure 1-4 Characteristics of haemoglobin in alpha and beta thalassaemias where red symbols indicate deficient globin synthesis. 45 Figure 1-5 Schematic representation of haemoglobin molecule 47 Figure 1-6 Structural genes on chromosomes 16 and 11 47 Figure 1-7 Ontogeny of globin chain synthesis in humans. . 48 Figure 1-8 Mechanisms in the pathophysiology of beta thalassaemia. . 55 Figure 1-9 Alpha thalassaemia deletions throughout the alpha globin gene cluster. . 56 Figure 1-10 QRT-PCR chemistry. . 66 Figure 1-11 Locations (in red) of APP and HBB on chromosomes 21 and 11 respectively 68 Figure 1-12 Amplification curves of real-time PCR 69 Figure 1-13 Standard curve of a QRT-PCR reaction . 70 Figure 1-14 QF-PCR chemistry. 71 Figure 1-15 Short tandem repeats . 72 Figure 1-16 Electropherograms of STRs (D21S1411, D21S11) in trisomy 21 (A) and normal (B) DNA samples isolated from amniocytes. . 73 Figure 1-17 Hybridisation of fluorescence probes onto target sequences . 74 Figure 1-18 FISH of fetal cells. 75 Figure 1-19 Basics of a fluorescent microscope 77 Figure 1-20 Separation of fluorescence emission. 77 Figure 2-1 Microsatellite markers (16PTEL05, 16PTEL06) are located within the breakpoint region of -SEA while control microsatellite viii Tjoa, M., C. Oudejans, J. van Vugt, M. Blankenstein & I. van Wijk. (2004) Markers for presymptomatic prediction of preeclampsia and intrauterine growth restriction. Hypertens Pregnancy, 23: 171-89. Tjoa, M., T. Cindrova-Davies, O. Spasic-Boskovic, D. Bianchi, G. Burton. (2006) Trophoblastic oxidative stress and the release of cell-free fetoplacental DNA. Am J Path, 169: 400-4. Tolson, D., N. Nicholson. (1998) Sequencing RNA by a combination of exonuclease digestion and uridine specific chemical cleavage using MALDITOF. Nucleic Acids Res, 26: 446-451. Tong, Y., C. Ding, R. Chiu, A. Gerovassili, S. Chim, T. Leung, T. Leung, T. Lau, K. Nicolaides & Y. Lo. (2006) Noninvasive prenatal detection of fetal trisomy 18 by epigenetic allelic ratio analysis in maternal plasma: Theoretical and empirical considerations. Clin Chem, 52: 2194-202. Tong, Y. & Y. Lo. (2006) Diagnostic developments involving cell-free (circulating) nucleic acids. Clin Chim Acta, 363: 187-96. Tongsong, T., C. Wanapirak, S. Sirichotiyakul & P. Chanprapaph. (2004) Sonographic markers of hemoglobin Bart disease at midpregnancy. J Ultrasound Med, 23: 49-55. Tongsong, T., C. Wanapirak, S. Sirichotiyakul, W. Piyamongkol & P. Chanprapaph. (1999) Fetal sonographic cardiothoracic ratio at midpregnancy as a predictor of Hb Bart disease. J Ultrasound Med, 18: 807-11. 202 Tsang, J. & Y. Lo. (2007) Circulating nucleic acids in plasma/serum. Pathology, 39: 197-207. Tse, D., P. Anderson, S. Goldbard, A. Gown, C. Hawes, A. Donnenfeld. (1994) Characterization of trophoblast-reactive monoclonal antibodies by flow cytometry and their application for fetal cell isolation. Ann N Y Acad Sci, 731: 162-169. Tsui, N., S. Chim, R. Chiu, T. Lau, E. Ng, T. Leung, Y. Tong, K. Chan & Y. Lo. (2004) Systematic micro-array based identification of placental mRNA in maternal plasma: towards non-invasive prenatal gene expression profiling. J Med Genet, 41: 461-7. Tsui, N., E. Ng & Y. Lo. (2002) Stability of endogenous and added RNA in blood specimens, serum, and plasma. Clin Chem, 48: 1647-53. Tungwiwat, W., G. Fucharoen, S. Fucharoen, T. Ratanasiri, K. Sanchaisuriya, N. Sae-Ung. (2007) Application of maternal plasma DNA analysis for noninvasive prenatal diagnosis of Hb E-beta-thalassemia. Transl Res, 150: 319-25. Uitto, J., E. Pfendner & L. Jackson. (2003) Probing the fetal genome: progress in non-invasive prenatal diagnosis. Trends Mol Med, 9: 339-43. Umansky, S. & L. Tomei. (2006) Transrenal DNA testing: progress and perspectives. Expert Rev Mol Diagn, 6: 153-63. Vamos, E. & I. Liebaers. (1984) Prenatal diagnosis of inborn error of metabolism. Present status and new approaches. Enzyme, 32: 47-55. 203 Van der Schoot, C., A. Soussan, J. Koelewijn, G. Bonsel, L. Paget-Christiaens, M. de Haas. (2006) Non-invasive antenatal RHD typing. Transfus Clin Biol, 13: 53-57. Van der Schoot, C., S. Hahn, L. Chitty. (2008) Non-invasive prenatal diagnosis and determination of fetal Rh status. Semin Fetal Neonatal Med, 13: 63-68. van Wijk, I., A. de Hoon, R. Jurhawan, M. Tjoa, S. Griffioen, M. Mulders, J. van Vugt & C. Oudejans. (2000) Detection of apoptotic fetal cells in plasma of pregnant women. Clin Chem, 46: 729-31. Verma, L., F. Macdonald, P. Leedham, M. McConachie, S. Dhanjal & M. Hultén. (1998) Rapid and simple prenatal DNA diagnosis of Down's syndrome. Lancet, 352: 9-12. Vichinsky, E. (2005) Changing patterns of thalassemia worldwide. Ann N Y Acad Sci, 1054: 18-24. Viguera, E., D. Canceill, S. Ehrlich. (2001) In vitro replication slippage by DNA polymerases from thermophilic organisms. J Mol Biol, 312: 323-33. Wachtel, S., S. Elias, J. Price, G. Wachtel, O. Phillips, L. Shulman, C. Meyers, J. Simpson, M. Dockter. (1991) Fetal cells in the maternal circulation: isolation by multiparameter flow cytometry and confirmation by polymerase chain reaction. Hum Reprod, 6: 1466-9. Wachtel, S., D. Sammons, M. Manley, G. Wachtel, G. Twitty, J. Utermohlen, O. Phillips, L. Shulman, D. Taron, U. Muller, P. Koeppen, T. Ruffalo, K. Addis, 204 R. Porreco, J. Murata-Collins, N. Parker, L. McGavran. (1996) Fetal cells in maternal blood: recovery by charge flow separation. Hum Genet, 98: 162-6. Wachtel, S., D. Sammons, G. Twitty, J. Utermohlen, E. Tolley, O. Phillips, L. Shulman. (1998) Charge flow separation: quantification of nucleated red blood cells in maternal blood during pregnancy. Prenat Diagn, 18: 455-63. Wald, N., A. Kennard, A. Hackshaw & A. McGuire. (1998) Antenatal screening for Down's syndrome. Health Technol Assess, 2; i-iv: 1-112. Wald, N. & J. Morris. (2003) A new approach to antenatal screening for Fragile X syndrome. Prenat Diagn, 23: 345-51. Walknowska, J., F. Conte & M. Grumbach. (1969) Practical and theoretical implications of fetal-maternal lymphocyte transfer. Lancet, 1: 1119-22. Walsh, P., N. Fildes, R. Reynolds. (1996) Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vWA. Nucleic Acids Res, 24: 2807-12. Wapner, R. (2005) Invasive prenatal diagnostic techniques. Semin Perinatol, 29: 401-4. Wataganara, T., E. LeShane, A. Farina, G. Messerlian, T. Lee, J. Canick & D. Bianchi. (2003) Maternal serum cell-free fetal DNA levels are increased in cases of trisomy 13 but not trisomy 18. Hum Genet, 112: 204-8. 205 Wataganara, T., A. Chen, E. LeShane, L. Sullivan, L. Borgatta, D. Bianchi, K. Johnson. (2005) Cell-free fetal DNA levels in maternal plasma after elective first trimester termination of pregnancy. Fertil Steril, 81: 638-44. Watanabe, A., A. Sekizawa, A. Taguchi, H. Saito, T. Yanaihara, M. Shimazu, I. Matsuda. (1998) Prenatal diagnosis of ornithine transcarbamylase deficiency by using a single nucleated erythrocyte from maternal blood. Hum Genet, 102: 611-5. Wauters, J., E. Assche, A. Antsaklis, J. Tepperberg, S. Sharp, M. Kilpatrick, T. Tafas, P. Tsipouras. (2007) Fully automated FISH examination of amniotic fluid cells. Prenat Diagn, 27: 951-5. Weatherall, D. (2001) Phenotype-genotype relationships in monogenic disease: lessons from the thalassaemias. Nat Rev Genet, 2: 245-55. Webster, W., A. Lipson & P. Brown-Woodman. (1987) Uterine trauma and limb defects. Teratology, 35: 253-60. Weinman, J., M. Johnston. (1988) Stressful medical procedures: analysis of the effects of the psychological intervention and of the stressfulness of the procedures. In Maes S (ed.). Topics in Health Psychology Chichester, J. Wiley: 205-18. Wessman, M., K. Ylinen, S. Knuutila. (1992) Fetal granulocytes in maternal venous blood detected by in situ hybridization. Prenat Diagn, 12: 993-1000. 206 Wisniewski, K., A. Dalton, C. McLachlan, G. Wen, H. Wisniewski. (1985) Alzheimer’s disease in Down’s syndrome: clinicopathologic studies. Neurology, 35: 957-61. Witters, I., K. Devriendt, E. Legius, G. Matthijs, D. Van Schoubroeck, F. Van Assche & J. Fryns. (2002) Rapid prenatal diagnosis of trisomy 21 in 5049 consecutive uncultured amniotic fluid samples by fluorescence in situ hybridisation (FISH). Prenat Diagn, 22: 29-33. Wolf, J. (1996) Liver disease in pregnancy. Med Clin North Am, 80: 1167-87. Wong, B. & Y. Lo. (2003) Cell-free DNA and RNA in plasma as new tools for molecular diagnostics. Expert Rev Mol Diagn, 3: 785-97. Wu, T., D. Zhang, J. Chia, K. Tsao, C. Sun & J. Wu. (2002) Cell-free DNA: measurement in various carcinomas and establishment of normal reference range. Clin Chim Acta, 321: 77-87. Zhong, X., M. Bürk, C. Troeger, L. Jackson, W. Holzgreve & S. Hahn. (2000a) Fetal DNA in maternal plasma is elevated in pregnancies with aneuploid fetuses. Prenat Diagn, 20: 795-8. Zhong, X., W. Holzgreve & S. Hahn. (2000b) Detection of fetal Rhesus D and sex using fetal DNA from maternal plasma by multiplex polymerase chain reaction. BJOG, 107: 766-9. Zhong, X., W. Holzgreve, J. Li, K. Aydinli & S. Hahn. (2000c) High levels of fetal erythroblasts and fetal extracellular DNA in the peripheral blood of a 207 pregnant woman with idiopathic polyhydramnios: case report. Prenat Diagn, 20: 838-41. Zhong, X., D. Hahn, C. Troeger, A. Klemm, G. Stein, P. Thomson, W. Holzgreve & S. Hahn. (2001a) Cell-free DNA in urine: a marker for kidney graft rejection, but not for prenatal diagnosis? Ann N Y Acad Sci, 945: 250-7. Zhong, X., W. Holzgreve & S. Hahn. (2001b) Circulatory fetal and maternal DNA in pregnancies at risk and those affected by preeclampsia. Ann N Y Acad Sci, 945: 138-40. Zhong, X., W. Holzgreve & S. Hahn. (2001c) Risk free simultaneous prenatal identification of fetal Rhesus D status and sex by multiplex real-time PCR using cell free fetal DNA in maternal plasma. Swiss Med Wkly, 131: 70-4. Zhong, X., H. Laivuori, J. Livingston, O. Ylikorkala, B. Sibai, W. Holzgreve & S. Hahn. (2001d) Elevation of both maternal and fetal extracellular circulating deoxyribonucleic acid concentrations in the plasma of pregnant women with preeclampsia. Am J Obstet Gynecol, 184: 414-9. Zhong, X., W. Holzgreve & S. Hahn. (2002) The levels of circulatory cell free fetal DNA in maternal plasma are elevated prior to the onset of preeclampsia. Hypertens Pregnancy, 21: 77-83. Zhong, X., Y. Wang, S. Chen, X. Pan, N. Zhu, C. Hahn, B. Huppertz, W. Holzgreve & S. Hahn. (2004) Circulating fetal DNA in maternal plasma is increased in pregnancies at high altitude and is further enhanced by preeclampsia. Clin Chem, 50: 2403-5. 208 Zhong, X., S. Gebhardt, R. Hillermann, K. Tofa, W. Holzgreve & S. Hahn. (2005a) Circulatory nucleosome levels are significantly increased in early and late-onset preeclampsia. Prenat Diagn, 25: 700-3. Zhong, X., S. Gebhardt, R. Hillermann, K. Tofa, W. Holzgreve & S. Hahn. (2005b) Parallel assessment of circulatory fetal DNA and corticotropinreleasing hormone mRNA in early- and late-onset preeclampsia. Clin Chem, 51: 1730-3. Zimmermann, B., W. Holzgreve, F. Wenzel & S. Hahn. (2002) Novel real-time quantitative PCR test for trisomy 21. Clin Chem, 48: 362-3. Zimmermann, B., A. El-Sheikhah, K. Nicolaides, W. Holzgreve, S. Hahn. (2005) Optimized real-time quantitative PCR measurement of male fetal DNA in maternal plasma. Clin Chem, 51: 1598-1604. 209 APPENDICES Publications/ Conference Proceedings 1. SSY Ho, LL Su, A Biswas, M Choolani. Elevation of fetal DNA in the maternal plasma of patients with pregnancy complications: case studies on severe eclampsia and HELLP syndrome. Manuscript in preparation. 2. Ho SS, Chong SS, Koay ES, Chan YH, Sukumar S, Chiu LL, Wang W, Roy A, Rauff M, Su LL, Biswas A, Choolani M. Microsatellite markers within --SEA breakpoints for prenatal diagnosis of HbBarts hydrops fetalis. Clin Chem 2007;53:173-9. 3. M Choolani, SSY Ho, K Razvi, S Ponnusamy, S Baig, NM Fisk, A Biswas. FastFISH: technique for ultrarapid fluorescence in situ hybridization on uncultured amniocytes yielding results within h of amniocentesis. Mol Hum Reprod 2007;13(6):355-9. 4. SSY Ho, JFV Ho, WY Chua, KH Chew, A Loganath, CG Lee, SY Ng, A Biswas, M Choolani, SS Chong, YS Chong. Lack of association of the Asp298 variant of the endothelial nitric oxide synthase gene with preeclampsia in a Malay population. Singapore Journal of Obstetrics and Gynecology 2005;36:26-30. 5. Guo CF, Haider Kh H, Ye L, Jiang SJ, Law PK, Wong P, Shim SNW, Choolani M, Ho SSY, Sim KWE. Human skeletal myoblast are immunoprivilaged and survive following xenotransplantation in the rat infarcted heart. 17th Annual Scientific Meeting (SCS). Singapore, 2627 Mar 2005 - Shortlist for Young Investigator Award. 210 6. CF Guo, KH Haider, L Ye, SJ Jiang, PK Law, P Wong, SNW Shim, M Choolani, SSY Ho, KWE Sim. Cyclosporine treatment enhances cell survival after human myoblast transplantation into rat infarcted heart. ISMICS: 8th Annual Scientific Meeting, New York, USA, 1-4 June 2005. 7. CF Guo, KH Haider, L Ye, SJ Jiang, PK Law, P Wong, SNW Shim, M Choolani, SSY Ho, KWE Sim. Xenotransplanted human skeletal myoblast for the infarcted heart repair. ESH-EBMT-EUROCHORD Euroconference on Stem Cell Research, Cascais, Portugal, 15-17 April 2005 – Awarded with European Commission’s Marie Curie Actions Scholarship. 8. Su LL, Choolani M, Ho SSY, Ponnusamy S, Narasimhan K, Razvi K, Chia D, Biswas A. Ultrarapid prenatal diagnosis by amnioPCR and amnioFISH: routine testing for trisomy 21, but targeted testing for trisomies 13 and 18. First International Scientific Meeting of the International Society of Ultrasound in Obstetrics and Gynaecology (ISUOG), Singapore, 21-25 March 2004 (Abstract). 9. Ho SSY, Donoghue KO, Choolani M. Fetal cells in maternal blood: State of the art for non-invasive prenatal diagnosis. Ann Acad Med Sing 2003; 32(5):5597-603. Presentations with abstracts 1. SSY Ho, W Wang, LL Chiu, SS Chong, ESC Koay, M Rauff, LL Su, A Biswas, M Choolani. Non-invasive prenatal exclusion of alpha thalassaemia double gene deletions using fetal DNA from maternal plasma. Oral presentation, 1st Obstetrics and Gynaecology Research Fair 2007, Singapore. 211 2. SSY Ho, W Wang, LL Chiu, SS Chong, ESC Koay, M Rauff, LL Su, A Biswas, M Choolani. Development of non-invasive prenatal exclusion of alpha thalassaemia using fetal DNA from maternal plasma. Oral presentation, 6th Singapore Congress in Obstetrics & Gynaecology, 21-25 March, 2007. 3. SSY Ho, SS Chong, ESC Koay, M Rauff, LL Su, A Biswas, M Choolani. Quantitative fluorescence-PCR of microsatellite markers on chromosome X: towards non-invasive prenatal exclusion of sex-linked disorders using fetal DNA from maternal plasma. First runner-up (Poster) in 6th Singapore Congress in Obstetrics & Gynaecology, 2125 March, 2007. 4. S Baig, SSY Ho, L Gole, BL Ng, N Kothandaraman, ESC Koay, LL Chiu, A Biswas, M Choolani. Quantitative fluorescence-polymerase chain reaction (QF-PCR) of uncultured amniocytes: increased stringency in the prenatal diagnostic criteria of chromosomal aneuploidies. 6th Singapore Congress in Obstetrics & Gynaecology, 21-25 March, 2007. 5. S Baig, SSY Ho, L Gole, S Ponnusamy, LL Su, K Razvi, A Biswas, M Choolani. One-day fluorescence in situ hybridisation diagnosis of common chromosomal aneuploidy disorders using uncultured amniocytes. 6th Singapore Congress in Obstetrics & Gynaecology, 2125 March, 2007. 6. AP Mahyuddin, HM Zhang, S Ponnusamy, SSY Ho, CQ Zhao, A Biswas, A Venkat, M Rauff, M Choolani. Noninvasive prenatal diagnosis: search for potential cell surface marker for the enrichment of 212 fetal erythroblasts from the maternal blood. 6th Singapore Congress in Obstetrics & Gynaecology, 21-25 March, 2007. 7. HM Zhang, QF Lin, S Ponnusamy, N Kothandaraman, TK Lim, SSY Ho, CQ Zhao, M Rauff, A Venkat, SK Hon, MCM Chung, SB Joshi, M Choolani. Proteomics strategy for identification of membrane proteins from limited clinical samples. 6th Singapore Congress in Obstetrics & Gynaecology, 21-25 March, 2007. 8. HM Zhang, QS Lin, S Ponnusamy, N Kothandaraman, TK Lim, SSY Ho, M Rauff, A Venkat, SK Hon, MCM Chung, SB Joshi, M Choolani. Identification of unique membrane protein(s) of human primitive erythroblasts using 2-D LC MALDI TOF/TOF mass spectrometry. 6th Singapore Congress in Obstetrics & Gynaecology, 21-25 March, 2007. 9. M Choolani, SSY Ho, SS Chong, ESC Koay, S Ponnusamy, L Chui, W Wen, M Rauff, LL Su, A Biswas. Non-invasive prenatal screening for haemoglobin Bart’s hydrops fetalis. Poster presentation. XVIII FIGO, 5th – 10th Nov 2006, Kuala Lumpur, Malaysia. 10. M Choolani, LL Su, SSY Ho, S Ponnusamy, N Kothandaraman, A Biswas. Rapid and cost-effective model for prenatal diagnosis. Oral presentation. XVIII FIGO, 5th – 10th Nov 2006, Kuala Lumpur, Malaysia. 11. MA Choolani, SSY Ho, K Razvi, S Ponnusamy, NB Mohammed, HM Zhang, M Rauff, LL Su, L Gole, A Biswas, N Fisk. FastFISH: improved technique for fluorescence in situ hybridization (FISH) on uncultured amniotic fluid cells that allows reporting within two hours of amniocentesis. IFMSS 25th Annual Meeting, 11th – 15th June 2006, Hawaii, USA. 213 12. SSY Ho, SS Chong, ESC Koay, S Ponnusamy, LL Chui, W Wang, A Roy, M Rauff, LL Su, A Biswas, M Choolani. Fetal DNA from the maternal plasma: noninvasive prenatal exclusion of haemoglobin Bart’s hydrops (--SEA/--SEA). ASHG 56th Annual Meeting, 9-13 October, 2006, New Orleans, Louisiana, USA. 13. SSY Ho, SS Chong, ESC Koay, M Rauff, LL Su, A Biswas, MA Choolani. Non-invasive prenatal exclusion of haemoglobin (Hb) Bart’s hydrops (--SEA/--SEA) using cell-free fetal DNA from the maternal plasma. Poster presentation. NHG ASC 2006; 30th September – 1st October 2006, Singapore. 14. MA Choolani, SSY Ho, K Razvi, S Ponnusamy, N Fisk, A Biswas, Rapid molecular testing in prenatal diagnosis group. FastFISH: new technique of fluorescence in situ hybridization (FISH) on uncultured amniocytes release results within two hours of amniocentesis. Poster presentation. NHG ASC 2006; 30th September – 1st October 2006, Singapore. 15. SSY Ho, SS Chong, ESC Koay, S Ponnusamy, LL Chiu, W Wang, A Roy, M Rauff, LL Su, A Biswas, MA Choolani. Prenatal diagnosis of haemoglobin (Hb) Bart’s hydrops fetalis by novel analysis of microsatellite markers within the breakpoints of the –SEA deletion in alpha thalassaemia. Poster presentation in International Society for Prenatal Diagnosis (ISPD): 13th International Conference for Prenatal Diagnosis and Therapy, Kyoto, Japan, 28 – 31 May, 2006. 16. Su LL, Biswas A, Ho SSY, Chan YH, Ponnusamy S, Narasimhan K, Choolani M. Targeted rapid prenatal diagnostic tests: A cost-effective 214 model. – Awarded Best Oral Presentation in 15th Malaysian Congress of Obstetrics and Gynaecology, Penang, Malaysia, 2-5 June, 2005. 17. SSY Ho, W Wang, SS Chong, ESC Koay, M Rauff, A Biswas, MA Choolani. Novel prenatal diagnosis of HbBart’s hydrops fetalis by microsatellite analysis within the breakpoints. Ann Acad Med Sing 2005; 34(9):S216. – NUS Clinical Science Award Finalist in Combined Scientific Meeting (CSM) 2005, 4-6 November 2005. 18. SSY Ho, LL Su, WK Chan, SE Chua, RPL Lim, A Biswas, M Choolani. Ultrarapid Down syndrome detection using real-time multiplex PCR of amniotic fluid in prenatal diagnosis. 8th NUS-NUH Annual Scientific Meeting, Singapore, 7-8 October 2004 (Abstract/ Poster Presentation). 19. SSY Ho, Z Damayanti, WY Chua, BL Ng, CM Peh, A Biswas, M Choolani. Foetal gender determination from maternal plasma using real-time PCR: potentials for non-invasive prenatal diagnosis. 8th NUSNUH Annual Scientific Meeting, Singapore, 7-8 October 2004 (Abstract/ Poster Presentation). 20. SSY Ho, WY Chua, A Loganath, CK Heng, M Choolani, SS Chong, YS Chong. Lack of association of the missense Glu298Asp variant of the endothelial nitric oxide synthase gene with preeclampsia in a Malay population. Ann Acad Med Sing 2004;33(5):S132 (1179/SL) – Abstract in National Healthcare Group Annual Scientific Congress (NHG ASC) 2004, 9-10 October 2004. 21. SSY Ho, LL Su, WK Chan, SE Chua, RPL Lim, A Biswas, M Choolani. Ultrarapid prenatal detection of Down syndrome using real-time multiplex polymerase chain reaction (PCR) in amniotic fluid. Ann Acad 215 Med Sing 2004;33(5):S91-2. National Healthcare Group Annual Scientific Congress (NHG ASC) 2004, 9-10 October 2004. Special Poster Presentation Finalist – Awarded Best Poster Presentation for Surgery/ Obstetrics & Gynaecology/ Dentistry/ Ophthalmology (Laboratory Based). 22. LL Su, A Biswas, SSY Ho, S Ponnusamy, N Kothandaraman, D Chia, M Choolani. Rapid prenatal diagnosis by amnioPCR and amnioFISH: Routine testing for Down’s syndrome (Trisomy 21) and sex chromosome trisomies, but targeted testing for Edward’s (Trisomy 18) and Patau’s syndromes 2004;33(5):S53-4. National (Trisomy 13). Healthcare Ann Group Acad Annual Med Sing Scientific Congress (NHG ASC) 2004, 9-10 October 2004. Best Oral Presentation Award Finalist – Surgery/ Obstetrics & Gynaecology/ Dentistry/ Ophthalmology (Clinical Based) – Ethicon Surgical Book Prize (Clinical). 23. SSY Ho, Z Damayanti, WY Chua, BL Ng, CM Peh, A Biswas, M Choolani. Non-invasive prenatal diagnosis of fetal gender using realtime polymerase chain reaction amplification of SRY in maternal plasma. Ann Acad Med Sing 2004;33(5):S61-2. National Healthcare Group Annual Scientific Congress (NHG ASC) 2004, 9-10 October 2004. Best Oral Presentation Award Finalist – Surgery/ Obstetrics & Gynaecology/ Dentistry/ Ophthalmology (Laboratory Based) – Ethicon Surgical Book Prize (Laboratory). 216 Prizes 1. SSY Ho, SS Chong, ESC Koay, M Rauff, LL Su, A Biswas, M Choolani. Quantitative fluorescence-PCR of microsatellite markers on chromosome X: towards non-invasive prenatal exclusion of sex-linked disorders using fetal DNA from maternal plasma. First runner-up (Poster) in 6th Singapore Congress in Obstetrics & Gynaecology, 2125 March, 2007. 2. SSY Ho, SS Chong, ESC Koay, S Ponnusamy, LL Chiu, W Wang, A Roy, M Rauff, LL Su, A Biswas, MA Choolani. Non-invasive prenatal exclusion of haemoglobin Bart’s hydrops fetalis (--SEA/--SEA) using fetal DNA from maternal plasma. Awarded Young Scientist Award in International Society for Prenatal Diagnosis (ISPD): 13th International Conference for Prenatal Diagnosis and Therapy, Kyoto, Japan, 28 – 31 May, 2006. 3. SSY Ho, LL Su, WK Chan, SE Chua, RPL Lim, A Biswas, M Choolani. Ultrarapid prenatal detection of Down syndrome using real-time multiplex polymerase chain reaction (PCR) in amniotic fluid. Ann Acad Med Sing 2004;33(5):S91-2. National Healthcare Group Annual Scientific Congress (NHG ASC) 2004, 9-10 October 2004. Special Poster Presentation Finalist – Awarded Best Poster Presentation for Surgery/ Obstetrics & Gynaecology/ Dentistry/ Ophthalmology (Laboratory Based). 217 [...]... amount of fetal DNA in maternal plasma (Koide et al., 2005) The technical challenges in quantifying these low concentrations of cell-free fetal DNA in maternal urine seem unsuitable for noninvasive prenatal diagnosis (Majer et al., 2007) The rapid clearance of fetal DNA from the maternal circulation within 2-hour postpartum had been reported (Lo et al., 1999c) This means that DNA analysis of the maternal. .. to detect unique fetal DNA sequences from the cellular components of the blood of pregnant women in the 1990s The scarcity of these cells in maternal blood makes accurate quantitation and genetic diagnosis difficult The true prevalence of fetal cells in maternal blood is unknown The number of fetal cells in the maternal circulation was estimated to be 1 to 2 fetal cells per ml of maternal blood (Bianchi... number of fetal cells Current strategies for prenatal diagnosis on fetal cells in the maternal blood face technical challenges in the enrichment of fetal cells from the maternal blood, identification of enriched cells and precise analytical methods of these rare fetal cells for accurate diagnosis The limited types of fetal- specific cells in maternal blood, 1 as well as the low frequency of fetal cells... maternal blood (Bianchi et al., 1997) The rarity of fetal cells in 13 the maternal blood and the limited types of specific fetal cell markers by which it can be distinguished and isolated from the maternal cells are drawbacks of using fetal cells for non- invasive prenatal diagnosis (Bischoff et al., 2002; Sekizawa et al., 2007) Other approaches to recover fetal cells from maternal blood include density gradient... al., 1997; Lo et al., 1998b) These findings suggested that the fetal genetic material could be useful for the non- invasive 17 prenatal diagnosis of the fetus The detection of fetal DNA sequences is dependent on the sensitivity of the assay and the amount of target fetal sequences Using Y-chromosome-specific PCR, fetal DNA can be detected in maternal blood in as early as 4 weeks’ gestation (amenorrhoea,... who reject invasive prenatal diagnosis due to the risk of fetal loss or morbidity (Chitty, 1998) Non- invasive and accurate prenatal diagnosis that does not carry any risk of procedural-related fetal loss is therefore desirable to at-risk pregnant women The presence of intact fetal cells and cell-free fetal DNA circulating in the maternal blood forms the basis for non- invasive access to fetal genetic... maternal plasma will not be complicated by the persistence of fetal DNA from a prior gestation (Costa et al., 2001; Bianchi and Lo, 2001; Jackson, 2003; Benachi et al., 2003) 1.3.3.1 Sources of fetal DNA in maternal blood The source of fetal DNA in maternal plasma is currently unknown The placenta, fetal haematopoietic cells, and the fetus itself have all been considered as sources of fetal DNA (Bianchi,... 1.3.3 Fetal DNA in maternal blood In contrast to fetal cells in maternal blood, fetal DNA can be readily detected in maternal plasma and serum (Bianchi and Lo 2001; Lo and Poon 2003; Birch et al., 2005; Galbiati et al., 2005) In 1997, Lo et al demonstrated the presence of fetal DNA in the plasma and serum from healthy pregnant women (Lo et al., 1997) The amount of fetal DNA was estimated to be 3-6% of. .. et al., 2001) In normal pregnancies, fetal DNA concentrations increased from 10.1 18 to 130.5 copies per 0.5 ml maternal plasma in 20 women carrying a male fetus from the 1st to 3rd trimester During the last 8 weeks of gestation, there is a sharp increase of fetal DNA maternal plasma (Lo et al., 1998b) that might be related to the gradual breakdown of the maternal -fetal interface/placental barrier (Bianchi,... plasma, thus allowing non- invasive prenatal testing of paternallyinherited genetic diseases The aim of this thesis was to explore the use of polymorphic microsatellite markers that differ in sequences between paternaland maternal- inherited fetal alleles that may allow discrimination and differentiation between maternal and fetal DNA in the maternal plasma Fetal alleles consist of one maternal- inherited allele . NON-INVASIVE PRENATAL TESTING OF HAEMOGLOBIN BART’S USING FETAL DNA FROM THE MATERNAL PLASMA SHERRY HO SZE YEE A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF. differs from maternal DNA sequence can be identified in maternal plasma. The low level of fetal DNA in the prevailing maternal DNA is, however, a technical challenge. The aim of this thesis. concentrations in maternal plasma of P1 and P2 110 Table 4-2 Non-invasive identification of fetal gender using fetal DNA from the maternal plasma. 115 Table 4-3 Table shows the comparisons of cell-free