Zhang Jun. / "August 2000." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (p. 109-144). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Lam Yuk Lan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 147-167). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 摘要 --- p.iii / ACKNOWLEDGEMENTS --- p.v / PUBLICATIONS --- p.vii / TABLE OF CONTENTS --- p.ix / LIST OF FIGURES --- p.xiii / LIST OF TABLES --- p.xiv / LIST OF ABBREVIATIONS --- p.xvi / Chapter Section 1: --- Background --- p.1 / Chapter Chapter 1: --- Cell-free circulating DNA --- p.1 / DNA and Man --- p.2 / Cell-free Circulating DNA in Plasma and Serum --- p.4 / The Discovery and Early Development --- p.4 / Clinical Implications --- p.5 / Cancers --- p.5 / Prenatal diagnosis --- p.11 / Pregnancy abnormalities --- p.14 / Organ transplantation --- p.15 / Trauma and post-traumatic complications --- p.15 / "Origin, Mechanisms and Characteristics" --- p.16 / Methods of Analysis --- p.22 / Chapter Chapter 2: --- Trauma and Organ Failure --- p.25 / Trauma and Society --- p.25 / The Problem of Organ Failure --- p.26 / Definitions --- p.27 / Pathogenesis --- p.228 / Inflammation --- p.29 / Predictions --- p.30 / Trauma Scoring Systems --- p.31 / Abbreviated Injury Scale --- p.32 / Injury Severity Score --- p.32 / Other scoring systems --- p.33 / Definition of Trauma --- p.33 / Chapter Chapter 3: --- Stroke --- p.35 / The Burden of Stroke --- p.35 / What is a Stroke ？ --- p.36 / The Causes --- p.40 / Pathophysiology --- p.41 / Diagnosis and Tests --- p.42 / Assessments and prognosis --- p.44 / Biochemical Markers --- p.47 / Chapter Chapter 4: --- Aims of the study --- p.48 / Chapter Section 2: --- Materials and Methods --- p.50 / Chapter Chapter 5: --- Methods of analysis on cell-free circulating DNA --- p.51 / Materials --- p.51 / DNA Extraction from the Plasma Samples --- p.51 / Real-time Quantitative PCR --- p.52 / Methods --- p.54 / DNA Extraction from the Plasma Samples --- p.54 / Real-time Quantitative PCR --- p.56 / Principle --- p.56 / The β-globin TaqMan Assay --- p.59 / Calibration of the β-globin TaqMan System --- p.62 / Contamination Control --- p.64 / Chapter Section 3: --- Cell-free circulating DNA after trauma --- p.65 / Chapter Chapter 6: --- Cell-free circulating DNA concentration as a prognostic marker in patients after trauma --- p.66 / Introduction --- p.66 / Methods --- p.68 / Results --- p.71 / Discussion --- p.84 / Chapter Chapter 7: --- Temporal changes of cell-free circulating DNA after trauma --- p.89 / Introduction --- p.89 / Methods --- p.90 / Results --- p.92 / Discussion --- p.106 / Chapter Section 4: --- Cell-free circulating DNA concentration after stroke --- p.109 / Chapter Chapter 8: --- Cell-free circulating DNA concentration in patients with stroke --- p.110 / Introduction --- p.110 / Methods --- p.111 / Results --- p.115 / Discussion --- p.129 / Chapter Chapter 9: --- Daily changes of cell-free circulating DNA concentration after stroke --- p.132 / Introduction --- p.132 / Methods --- p.132 / Results --- p.133 / Discussion --- p.137 / Chapter Section 5: --- Conclusion and future perspectives --- p.139 / Chapter Chapter 10: --- Conclusion and Future Perspectives --- p.140 / Conclusion --- p.140 / Future perspectives --- p.145 / BIBLIOGRAPHY --- p.147 / APPENDIX 1: Goriśةmultiple organ failure score --- p.168 / "APPENDIX 2: Definitions and criteria for ARDS, ALI and MODS" --- p.170 / APPENDIX 3: Computed axial tomography and magnetic resonance imaging --- p.172 / APPENDIX 4: Glasgow Coma Scale --- p.173 / APPENDIX 5: Post-Stroke Modified Rankin Scale --- p.174

In conclusion, the studies in this thesis have provided new information on the molecular nature of circulating DNA. Alteration of the size of circulating DNA is demonstrated in different physiological and pathological conditions. The non-bisulfite-based approach described in this thesis has provided a more sensitive and precise way for the detection and quantification of aberrantly methylated DNA sequences in the circulation. This method has tremendous potential for being applied for noninvasive cancer detection and prenatal diagnosis. / In the second chapter of this thesis, the molecular nature of circulating Epstein-Barr virus (EBV) DNA is studied. Circulating EBV DNA has previously been shown to be a valuable marker for the detection, monitoring and prognostication of nasopharyngeal carcinoma and several cancers associated with EBV infection. Using DNase digestion and ultracentrifugation analysis, circulating EBV DNA was shown to be free DNA fragments instead of being associated with viral particles. Furthermore, a quantitative system was developed for measuring the size of these EBV DNA molecules and showed that over 80% of the circulating EBV DNA molecules are shorter than 180 bp. / In the subsequent chapters, this DNA size measurement technique has been applied for analyzing the integrity of plasma genomic DNA in cancer patients and pregnant women. Increased plasma DNA integrity was observed in both of these groups of individuals. Moreover, the size of plasma DNA in cancer patients was shown to normalize after successful treatment and the failure of such normalization was shown to be associated with poor prognosis. In pregnant women, in addition to the overall increase in plasma DNA size, the maternal-derived DNA molecules were further shown to be longer than the fetal-derived ones. This observation opens up the possibility for fetal DNA enrichment through size fractionation of maternal plasma DNA. / The discovery of circulating nucleic acids in plasma and serum has led to the development of numerous promising noninvasive diagnostic tests. To date, circulating nucleic acid analysis has been applied to many different areas, including cancer detection, prenatal diagnosis, monitoring of organ transplant recipients and acute medicine. However, despite the extensive investigations on their clinical applications, the information on the molecular characteristics of the circulating nucleic acids is lacking. / The latter part of the thesis describes the principle of a non-bisulfite-based method for the detection of aberrant DNA methylation in plasma/serum. Using this technique, a universal fetal DNA marker has been developed based on an epigenetic approach. The placentally derived hypermethylated RASSF1A sequence has been developed as a gender and polymorphism-independent marker for fetal DNA in maternal plasma. In pregnant women undergoing noninvasive prenatal rhesus D genotyping, false negative cases were successfully identified through the analysis of this new fetal DNA marker. The quantitative analysis of circulating methylated RASSF1A sequences has further been shown to be useful for the detection and prognostication of hepatocellular carcinoma. / Chan, Kwan Chee. / Adviser: Y.M. Dennis. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3307. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 143-162). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Lee Tracy Yuen Han. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 168-187). / Abstracts in English and Chinese. / Abstract --- p.ii / 摘要 --- p.v / Acknowledgements --- p.vi / Table of contents --- p.viii / List of tables --- p.xii / List of figures --- p.xiii / List of abbreviations --- p.xiv / Chapter Chapter 1: --- Prenatal diagnosis --- p.1 / Chapter 1.1 --- Historical overview --- p.1 / Chapter 1.1.1 --- Prenatal diagnosis --- p.1 / Chapter 1.1.2 --- Circulating fetal nucleated cells --- p.2 / Chapter 1.1.3 --- Cell-free fetal DNA in maternal plasma --- p.3 / Chapter 1.2 --- Biological characteristics of circulating DNA --- p.4 / Chapter 1.3 --- Origin of circulating DNA --- p.6 / Chapter 1.4 --- Clinical applications of circulating fetal DNA --- p.7 / Chapter 1.5 --- Quantitative aberrations in circulating fetal DNA --- p.9 / Chapter 1.6 --- Epigenetic approach in detecting circulating fetal DNA --- p.11 / Chapter Chapter 2: --- Epigenetics --- p.14 / Chapter 2.1 --- Historical overview --- p.14 / Chapter 2.2 --- Mechanisms of DNA methylation --- p.15 / Chapter 2.3 --- Roles of DNA methylation --- p.17 / Chapter 2.4 --- Aberrations in DNA methylation --- p.20 / Chapter 2.5 --- Epigenetic diagnostic markers --- p.22 / Chapter 2.6 --- Significance of epigenetic markers in noninvasive prenatal diagnosis --- p.23 / Chapter 2.7 --- Aim of thesis --- p.23 / Chapter Chapter 3: --- Materials and methods --- p.25 / Chapter 3.1 --- Preparation of samples --- p.25 / Chapter 3.1.1 --- Collection of placental tissues --- p.25 / Chapter 3.1.2 --- Preparation of plasma and blood cells --- p.25 / Chapter 3.2 --- Nucleic acid extraction --- p.26 / Chapter 3.2.1 --- DNA extraction from placental tissues --- p.26 / Chapter 3.2.2 --- DNA extraction from plasma --- p.26 / Chapter 3.2.3 --- DNA extraction from blood cells --- p.29 / Chapter 3.3 --- Bisulfite genomic sequencing --- p.30 / Chapter 3.3.1 --- Principles of bisulfite modification --- p.30 / Chapter 3.3.2 --- Primer design for bisulfite sequencing --- p.31 / Chapter 3.3.3 --- Bisulfite genomic sequencing --- p.31 / Chapter 3.3.3.1 --- Bisulfite modification --- p.31 / Chapter 3.3.3.2 --- Bisulfite genomic sequencing --- p.32 / Chapter 3.3.4 --- Data and statistical analysis --- p.38 / Chapter 3.4 --- MALDI-TOF Mass Spectrometry (MS) --- p.39 / Chapter 3.4.1 --- Principle of MALDI-TOF MS and MassEXTEND assay --- p.39 / Chapter 3.4.2 --- Methylation-sensitive restriction enzyme digestion and MassEXTEND assay for PDE9A and H19 --- p.41 / Chapter 3.5 --- Quantitative measurements of nucleic acids --- p.44 / Chapter 3.5.1 --- Principles of real-time quantitative PCR --- p.44 / Chapter 3.5.2 --- Methylation-specific qMSP assay for M- and U-PDE9A --- p.47 / Chapter Chapter 4: --- Systematic screening of 8 CGIs in third trimester pregnancy --- p.49 / Chapter 4.1 --- Introduction --- p.49 / Chapter 4.2 --- Methods --- p.50 / Chapter 4.2.1 --- Subjects --- p.50 / Chapter 4.2.2 --- Experimental design --- p.51 / Chapter 4.3 --- Results --- p.54 / Chapter 4.3.1 --- Methylation profile of 8 CGIs in maternal blood cells and paired placental tissues --- p.54 / Chapter 4.3.1.1 --- Methylation profile of PDE9A in third trimester pregnancy --- p.56 / Chapter 4.3.1.2 --- Methylation profile of PPP1R2P2 in third trimester pregnancy --- p.60 / Chapter 4.3.1.3 --- Methylation profile of LOC115376 in third trimester pregnancy --- p.65 / Chapter 4.3.1.4 --- Methylation profile of AM L1 in third trimester pregnancy --- p.71 / Chapter 4.3.1.5 --- Methylation profile of COL6A2 in third trimester pregnancy --- p.78 / Chapter 4.3.1.6 --- Methylation profile of PRDM15 in third trimester pregnancy --- p.82 / Chapter 4.3.1.7 --- Methylation profile of CG1111 in third trimester pregnancy --- p.86 / Chapter 4.3.1.8 --- Methylation profile of CGI121 in third trimester pregnancy --- p.90 / Chapter 4.3.2 --- Methylation profile of regions upstream and downstream of PDE9A in maternal blood cells and paired placental tissues --- p.94 / Chapter 4.3.2.1 --- Methylation profiles of PDE9A_A and PDE9ÁؤB in third trimester pregnancy --- p.96 / Chapter 4.3.2.2 --- Methylation profile of PDE9ÁؤC in third trimester pregnancy --- p.100 / Chapter 4.4 --- Discussion --- p.104 / Chapter Chapter 5: --- "Methylation analysis of PPP1R2P2, PDE9A, PDE9A B and PDE9ÁؤC in first trimester pregnancy" --- p.108 / Chapter 5.1 --- Introduction --- p.108 / Chapter 5.2 --- Methods --- p.109 / Chapter 5.2.1 --- Subjects --- p.109 / Chapter 5.2.2 --- Experimental design --- p.109 / Chapter 5.3 --- Results --- p.110 / Chapter 5.3.1 --- Methylation profile of PPP1R2P2.Region A in first trimester pregnancy. --- p.110 / Chapter 5.3.2 --- Methylation profile of PDE9A in first trimester pregnancy --- p.115 / Chapter 5.3.3 --- Methylation profile of PDE9A B in first trimester pregnancy --- p.119 / Chapter 5.3.4 --- Methylation profile of PDE9A C in first trimester pregnancy --- p.123 / Chapter 5.4 --- Discussion --- p.127 / Chapter Chapter 6: --- "Methylation analysis of PPP1R2P2, PDE9A and PDE9ÁؤB in Trisomy 21 pregnancy" --- p.129 / Chapter 6.1 --- Introduction --- p.129 / Chapter 6.2 --- Methods --- p.130 / Chapter 6.2.1 --- Subjects --- p.130 / Chapter 6.2.2 --- Experimental design --- p.131 / Chapter 6.3 --- Results --- p.131 / Chapter 6.3.1 --- Methylation profile of PPP1R2P2 in trisomy 21 placental tissues --- p.131 / Chapter 6.3.2 --- Methylation profile of PDE9A in trisomy 21 placental tissues --- p.136 / Chapter 6.3.3 --- Methylation profile of PDE9A B in trisomy 21 placental tissues --- p.140 / Chapter 6.4 --- Discussion --- p.144 / Chapter Chapter 7: --- Investigation of imprinting status of PDE9A --- p.145 / Chapter 7.1 --- Introduction --- p.145 / Chapter 7.2 --- Methods --- p.147 / Chapter 7.2.1 --- Subjects --- p.147 / Chapter 7.2.2 --- Experimental design --- p.147 / Chapter 7.3 --- Results --- p.149 / Chapter 7.3.1 --- SNP detection in enzyme digested placental tissues by H19 assay --- p.149 / Chapter 7.3.2 --- SNP detection in enzyme digested placental tissues by PDE9A assay --- p.151 / Chapter 7.4 --- Discussion --- p.153 / Chapter Chapter 8: --- Detection of U-PDE9A DNA sequences in maternal plasma --- p.155 / Chapter 8.1 --- Introduction --- p.155 / Chapter 8.2 --- Methods --- p.156 / Chapter 8.2.1 --- Subjects --- p.156 / Chapter 8.2.2 --- Experimental design --- p.156 / Chapter 8.3 --- Results --- p.157 / Chapter 8.3.1 --- Detection of U-PDE9A DNA sequences in maternal plasma --- p.157 / Chapter 8.3.2 --- Clearance of U-PDE9A DNA sequences from maternal plasma after delivery --- p.157 / Chapter 8.4 --- Discussion --- p.160 / Chapter Chapter 9: --- Conclusion and future perspectives --- p.162 / Chapter 9.1 --- Methylation profiles of CpG islands on chromosome 21q --- p.162 / Chapter 9.2 --- Investigation of imprinting status of PDE9A --- p.164 / Chapter 9.3 --- Development of a universal epigenetic marker --- p.165 / Chapter 9.4 --- Future perspectives --- p.166 / References --- p.168

Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available via the Internet as viewed 10 July 2008.

1997年，胎兒DNA被首次證實存在於母體血漿中。這一發現促進了無創產前診斷技術的發展。由於孕婦血漿中含大量來自母體的背景DNA，這給針對胎兒特異性DNA序列以外的產前診斷變得有挑戰性。近期開發的大規模平行測序技術把DNA定量精度提升到了一個空前的水平。本團隊已證實這一技術可應用於對胎兒染色體非整倍體和對胎兒全基因組的檢測。由於目前平行測序的費用仍相當昂貴，目標性測序技術可提高目標區域的數據比例從而降低測序成本。 / 在論文第一部分，本人論述了目標性測序在母體血漿DNA應用的可行性。本實驗採用雜交型富集技術對X染色體的外顯子進行富集。我們用平行測序比較了經由和未經富集處理的樣本。對比發現，經富集處理的樣本在目標區域的平均測序深度是未經富集處理樣本的213倍。目標區域的母體和胎兒DNA分子的富集程度相當。經富集處理後，目標區域的胎兒特異性等位基因的檢測率從3.5%提升至95.9%。 / 在論文第二部分，本人論述了目標性測序對胎兒21三體無創產前診斷的應用價值。我們對7，13，18和21號染色體上的單核苷酸多態性位點進行目標性測序。目標性測序數據顯示，在父源性21三體的樣本中，21號染色體上的胎兒特異性等位基因與共有性等位基因的比值上升約2倍。而在母源性21三體的樣本中，這一比值則下降約11%。本人採用電腦模擬實驗探討胎兒DNA濃度，有效等位基因數量和測序深度對檢測準確率的影響。 / 在論文第三部分，本人論述了目標性測序對胎兒單基因疾病無創產前診斷的應用。針對兩個需進行β地中海貧血產前診斷的家庭，我們對其β球蛋白基因進行目標性測序。我們用數字PCR技術推導了父母親β球蛋白基因區域的單倍型。經過相對性單倍型劑量分析，兩個胎兒的β地中海貧血遺傳狀況均得到了正確的推斷。其中一對夫婦位於致病區域的單倍型結構相近。 / 鑒於目標性測序技術可降低測序成本和提高目標序列的通量，其在血漿DNA的應用將有助於平行測序技術在無創性產前診斷、癌症診斷和移植監控等分子診斷學領域的發展。 / The presence of fetal DNA in the cell-free plasma of pregnant women was first reported in 1997. This discovery has facilitated the development of noninvasive prenatal diagnosis. The coexistence in maternal plasma of a minor population of fetal DNA among a major background of maternal DNA has posed challenges for extending noninvasive prenatal diagnostic applications that require analytical information beyond the detection of fetus-specific DNA sequences. The recent availability of massively parallel sequencing has enhanced the precision of DNA quantification to an unprecedented level. Our group has demonstrated the application of massively parallel sequencing in noninvasive prenatal diagnosis of chromosomal aneuploidies, as well as genome-wide fetal whole genome sequencing and mutational profiling. While the current costs of massively parallel sequencing are relatively expensive, targeted massively parallel sequencing may enhance the cost-effectiveness compared with the non-targeted approach because it increases the proportion of informative data from the regions-of-interest. / In the first part of this thesis, I have demonstrated the feasibility of targeted massively parallel sequencing in maternal plasma DNA. In this proof-of-principle study, hybridization-based target enrichment was used to enrich exons on chromosome X. Plasma DNA libraries with and without target enrichment were analyzed by massively parallel sequencing. For the targeted regions, the mean sequencing depth of the enriched samples was 213-fold higher than that of the non-enriched samples. Maternal and fetal DNA molecules were enriched to similar extents within the targeted regions. With target enrichment, the detection rate of fetus-specific alleles within the targeted regions increased from 3.5% to 95.9%. / In the second part of this thesis, I have demonstrated the potential application of targeted massively parallel sequencing of plasma DNA for noninvasive prenatal diagnosis of trisomy 21 using an allelic ratio approach. Targeted sequencing was used to enrich single nucleotide polymorphism loci on chr7, chr13, chr18 and chr21. The targeted sequencing data showed that the ratio between fetus-specific and shared alleles increased by approximately 2-fold on chr21 in a paternally-derived trisomy 21 case, and decreased by approximately 11% on chr21 for maternally-derived trisomy 21 cases. I have also used computer simulation to determine the impact of fractional fetal DNA concentration, number of informative alleles and sequencing depth on the detection accuracy. / In the third part of this thesis, I have demonstrated the feasibility of targeted massively parallel sequencing of maternal plasma DNA for noninvasive prenatal diagnosis of monogenic diseases. Targeted sequencing was used to enrich the β-globin gene region in two families undergoing prenatal diagnosis for β-thalassemia. Parental haplotypes of the β-globin gene region were deduced via digital polymerase chain reaction. Relative haplotype dosage analysis was performed successfully to determine the β-thalassemic status for the fetuses, including one family in which the parents had similar haplotype structures in the disease-causing region. / Because of its potential to save costs and increase throughput, targeted sequencing may catalyse the translation of massively parallel sequencing based molecular diagnostics into many fields, including noninvasive prenatal diagnosis, cancer diagnostics and transplantation monitoring. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liao, Jiawei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 147-155). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese. / ABSTRACT --- p.i / ACKNOWLEDGEMENTS --- p.vi / PUBLICATIONS --- p.vii / CONTRIBUTORS --- p.viii / TABLE OF CONTENTS --- p.ix / LIST OF TABLES --- p.xiii / LIST OF FIGURES --- p.xiv / LIST OF ABBREVIATIONS --- p.xvi / Chapter SECTION I : --- BACKGROUND --- p.1 / Chapter CHAPTER 1: --- Cell-free fetal DNA and targeted massively parallel sequencing --- p.2 / Chapter 1.1 --- Cell-free fetal DNA in maternal plasma --- p.2 / Chapter 1.2 --- NIPD by qualitative analysis --- p.3 / Chapter 1.2.1. --- Fetal sex assessment --- p.4 / Chapter 1.2.2. --- RHD genotyping --- p.5 / Chapter 1.2.3. --- Other implementations --- p.5 / Chapter 1.3 --- NIPD by quantitative analysis --- p.6 / Chapter 1.3.1. --- NIPD of chromosomal aneuploidies --- p.6 / Chapter 1.3.2. --- NIPD of autosomal recessive monogenic diseases --- p.8 / Chapter 1.4 --- Massively parallel sequencing of maternal plasma --- p.9 / Chapter 1.4.1. --- Massively parallel sequencing --- p.9 / Chapter 1.4.2. --- MPS-based NIPD of chromosomal aneuploidies --- p.11 / Chapter 1.4.3. --- MPS-based prenatal fetal whole-genome scanning --- p.15 / Chapter 1.5 --- Targeted massively parallel sequencing of maternal plasma --- p.19 / Chapter 1.5.1. --- Microdroplet-based PCR --- p.19 / Chapter 1.5.2. --- Molecular inversion probe --- p.20 / Chapter 1.5.3. --- On-array capture --- p.21 / Chapter 1.5.4. --- In-solution capture --- p.21 / Chapter 1.5.5. --- Implementation on plasma DNA --- p.22 / Chapter 1.6 --- Aims of this thesis --- p.29 / Chapter SECTION II : --- Feasibility of targeted MPS in maternal plasma DNA --- p.30 / Chapter CHAPTER 2: --- Targeted MPS of maternal plasma DNA permits efficient and unbiased detection of fetal alleles --- p.31 / Chapter 2.1 --- Introduction --- p.31 / Chapter 2.2 --- Methods --- p.34 / Chapter 2.2.1 --- Study participants and sample collection --- p.34 / Chapter 2.2.2 --- Sample processing and DNA extraction --- p.34 / Chapter 2.2.3 --- DNA quantification --- p.36 / Chapter 2.2.4 --- Microarray genotyping --- p.39 / Chapter 2.2.5 --- Plasma DNA library preparation --- p.39 / Chapter 2.2.6 --- Plasma DNA library validation --- p.40 / Chapter 2.2.7 --- Target enrichment --- p.44 / Chapter 2.2.8 --- Massively parallel sequencing and alignment --- p.45 / Chapter 2.3 --- Results --- p.48 / Chapter 2.3.1 --- Efficiency of target enrichment --- p.48 / Chapter 2.3.2 --- Sequence coverage within the targeted region --- p.53 / Chapter 2.3.3 --- Fetus-specific allele detection --- p.59 / Chapter 2.3.4 --- Fractional fetal DNA concentrations before and after enrichment --- p.63 / Chapter 2.4 --- Discussion --- p.66 / Chapter SECTION III : --- NIPD of trisomy 21 by targeted MPS of maternal plasma DNA --- p.71 / Chapter CHAPTER 3: --- NIPD of fetal trisomy 21 by allelic ratio analysis using targeted MPS of maternal plasma DNA --- p.72 / Chapter 3.1 --- Introduction --- p.72 / Chapter 3.2 --- Methods --- p.74 / Chapter 3.2.1 --- Study participants and sample collection --- p.74 / Chapter 3.2.2 --- Sample processing and DNA extraction --- p.74 / Chapter 3.2.3 --- Targeted MPS of plasma DNA libraries --- p.74 / Chapter 3.2.4 --- F-S ratio calculation --- p.76 / Chapter 3.2.5 --- Microarray genotyping --- p.78 / Chapter 3.2.6 --- Computer simulation --- p.78 / Chapter 3.3 --- Results --- p.80 / Chapter 3.3.1 --- Efficiency of target enrichment --- p.80 / Chapter 3.3.2 --- Estimation of fractional fetal DNA concentrations --- p.83 / Chapter 3.3.3 --- F-S ratio calculation using non-targeted sequencing data --- p.83 / Chapter 3.3.4 --- F-S ratio calculation using targeted sequencing data --- p.85 / Chapter 3.3.5 --- Computer simulation --- p.85 / Chapter 3.4 --- Discussion --- p.90 / Chapter SECTION IV : --- NIPD of monogenic diseases by targeted MPS of maternal plasma DNA --- p.94 / Chapter CHAPTER 4: --- NIPD of monogenic diseases by targeted MPS of maternal plasma: application to Beta-thalassemia --- p.95 / Chapter 4.1 --- Introduction --- p.95 / Chapter 4.2 --- Methods --- p.98 / Chapter 4.2.1 --- Sample collection and DNA extraction --- p.98 / Chapter 4.2.2 --- Microarray-based genotyping --- p.100 / Chapter 4.2.3 --- Targeted MPS of plasma DNA libraries --- p.100 / Chapter 4.2.4 --- Genotyping by Sanger sequencing --- p.103 / Chapter 4.2.5 --- Haplotyping by digital PCR --- p.105 / Chapter 4.2.6 --- RHDO analysis --- p.105 / Chapter 4.3 --- Results --- p.110 / Chapter 4.3.1 --- Effectiveness of targeted sequencing --- p.110 / Chapter 4.3.2 --- Determination of fetal HBB genotype in the first family --- p.112 / Chapter 4.3.3 --- Determination of fetal HBB genotype in the second family --- p.113 / Chapter 4.4 --- Discussion --- p.115 / Chapter SECTION V : --- CONCLUDING REMARKS --- p.120 / Chapter CHAPTER 5: --- Conclusion and future perspectives --- p.121 / Chapter 5.1 --- Targeted MPS in plasma DNA --- p.121 / Chapter 5.2 --- Targeted MPS in NIPD of chromosomal aneuploidies --- p.124 / Chapter 5.3 --- Targeted MPS in NIPD of monogenic diseases --- p.126 / Chapter Appendix I: --- Primer names and sequences for genotyping and haplotyping of βeta-globin gene cluster region --- p.128 / Chapter Appendix II: --- Primers used in PCRs and sequencing for the parents in the first family --- p.132 / Chapter Appendix III: --- Primers used in PCRs and sequencing for the parents in the second family --- p.138 / Chapter Appendix IV: --- RHDO analysis on maternal plasma DNA in the first family --- p.140 / Chapter Appendix V: --- RHDO analysis on maternal plasma DNA in the second family --- p.145 / References --- p.147

Another attempt was made to identify CpG-rich paralogues on chromosome 21 for dosage analysis. Methylation profiles of 14 paralogous CpG-rich clusters were screened by bisulfite genomic sequencing and/or combined bisulfite restriction analysis. One of the paralogue pairs showed similar differential methylation patterns, and three other CpG-rich clusters located on chromosome 21 were hypomethylated in the placentas and completely methylated in the maternal blood cells. Detection methods for these novel epigenetic markers were described and discussed, and potential applications were also suggested. / In the second part of this thesis, a technique called digital PCR was used for detecting and quantifying cell-free fetal DNA in maternal plasma. DNA templates are first diluted to a single molecule level and partitioned to separate compartments before subjecting to polymerase chain reaction amplification. Quantification is then made by counting the number of positive reactions directly. Such a technique has allowed the reliable detection of fetal DNA from a high background of maternal plasma DNA, and allows absolute quantification of fetal DNA without using a calibration curve. As a proof-of-principle project, a non-polymorphism-based approach called digital relative chromosome dosage (RCD) method was developed to detect chromosomal imbalance in trisomic cases. The implementation of digital PCR was further facilitated with the technology of integrated fluidics circuits (IFCs), by which nanolitre volumes of reaction mixtures could be manipulated in a high-throughput manner. Such a microfluidics digital PCR system was evaluated systematically and shown to be highly accurate, precise and sensitive compared to other existing detection platforms. The technology has been applied with the RCD approach for rapid detection of trisomy 21 from amniotic fluid samples and 100% accuracy was attained. With the development of new universal markers and robust detection platforms, it is envisioned that circulating fetal DNA in maternal plasma can be applied to an expanding range of clinical applications in the near future. / The first part of my thesis focused on the discovery of new epigenetic markers for fetal DNA detection. Methylation profiles of 7 selected CpG islands on chromosome 21 were revealed by bisulfite sequencing, in the hope of identifying regions with differential methylation patterns between placentas and maternal blood cells. Out of the 14 sub-regions of these CpG islands, five displayed significant difference between the two tissue type and were promising marker candidates. / The presence of circulating fetal DNA in maternal plasma provides a non-invasive source of fetal genetic materials for prenatal diagnosis. Reliance on Y-chromosomal sequences for detecting fetal-specific signals from the background of maternal plasma DNA, however, has restricted the applications to 50% of pregnancies. For a wider extent of diagnostic applications, sex- and polymorphism-independent fetal markers would be necessary. Recently, an epigenetic approach has been adopted to discriminate between the fetal and maternal DNA circulating in maternal plasma. Based on the differential DNA methylation status between the fetus and mother, universal fetal DNA markers have been developed and applied to detect fetal signals in maternal plasma. Identification of more of such markers is important for the development of the field. / Lun, Miu Fan. / Adviser: Yuk Ming Dennis Lo. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3292. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 233-256). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

O objetivo do presente trabalho foi o desenvolvimento de um teste não invasivo para a trissomia do cromossomo 21 através da análise de ácidos nucleicos fetais livres no plasma materno por sequenciamento de última geração realizado no sequenciador automático Ion Torrent. A metodologia proposta para o teste é a análise de SNPs com alta taxa de heterozigosidade na população brasileira localizados em dois genes presentes no cromossomo 21 (PLAC4 e C21orf105), e a detecção da cópia extra do cromossomo 21 é feita pela razão dos alelos desses SNPs, sendo que a razão de 1:1 indica que o feto é normal, e a razão de 2:1 indica que o feto tem uma cópia extra do cromossomo 21. Para a validação da metodologia foram utilizadas 50 amostras de DNA livre extraídas de líquido amniótico, previamente caracterizadas por análise citogenética consideradas como o padrão ouro pois contém apenas material genético fetal abundante. A metodologia foi validada com sucesso nessas amostras, sendo que as 24 amostras de fetos com trissomia foram claramente distinguidas das 26 amostras de fetos normais. A metodologia validada foi aplicada a 44 amostras de DNA livre extraídas de plasma de gestantes (21 amostras de fetos com trissomia do 21 e 23 de fetos normais), porém não foi possível fazer a distinção entre fetos normais e fetos com trissomia do 21, possivelmente devido à variações na fração fetal do DNA livre em relação à fração materna. Como nosso objetivo principal não foi alcançado, Propomos aqui que o teste realizado em líquido amniótico seja utilizado como uma alternativa mais simples, rápida e barata ao cariótipo convencional atualmente utilizado para fazer o diagnóstico da trissomia do cromossomo 21 em amostras coletadas por procedimentos invasivos, enquanto as deficiências do teste não-invasivo pelo plasma materno são aprimoradas / The purpose of this study was to develop a test for trisomy 21 by analyzing cell-free fetal nucleic acids in maternal plasma by next-generation sequencing performed on automated sequencer Ion Torrent. The proposed methodology for the test is based on analysis of SNPs with high heterozygosity rates in Brazilian population, located in two genes present on chromosome 21 (PLAC4 and C21orf105), and the detection of the extra copy of chromosome 21 is made by the allelic-ratio of these SNPs, where 1:1 ratio indicates a normal fetus, and the 2:1 ratio indicates that the fetus has an extra copy of chromosome 21. In order to validate the methodology 50 cell-free DNAs extracted from amniotic fluid were used representing a gold standard since it contains abundant genetic material exclusively from the fetus. The methodology has been successfully validated in these samples, all the 24 samples from fetuses with trisomy 21 were clearly distinguished from 26 samples of normal fetuses. The validated method was applied to 44 cell-free DNA samples extracted from plasma of pregnant women (21 samples from fetuses with trisomy 21 and 23 from normal fetuses), but unfortunately it was not possible to distinguish between normal and trisomy 21 fetuses, possibly due to variations on the fetal fraction of the cell-free DNA in relation to maternal fraction. As our main goal was not achieved, we propose here that the test performed on amniotic fluid sample could be used as a simpler, faster and cheaper alternative test to traditional karyotype, which is used nowadays to make the diagnosis of trisomy 21 in samples collected by invasive procedures. In parallel, minor improvements in the described method may enable its clinical use

O objetivo do presente trabalho foi o desenvolvimento de um teste não invasivo para a trissomia do cromossomo 21 através da análise de ácidos nucleicos fetais livres no plasma materno por sequenciamento de última geração realizado no sequenciador automático Ion Torrent. A metodologia proposta para o teste é a análise de SNPs com alta taxa de heterozigosidade na população brasileira localizados em dois genes presentes no cromossomo 21 (PLAC4 e C21orf105), e a detecção da cópia extra do cromossomo 21 é feita pela razão dos alelos desses SNPs, sendo que a razão de 1:1 indica que o feto é normal, e a razão de 2:1 indica que o feto tem uma cópia extra do cromossomo 21. Para a validação da metodologia foram utilizadas 50 amostras de DNA livre extraídas de líquido amniótico, previamente caracterizadas por análise citogenética consideradas como o padrão ouro pois contém apenas material genético fetal abundante. A metodologia foi validada com sucesso nessas amostras, sendo que as 24 amostras de fetos com trissomia foram claramente distinguidas das 26 amostras de fetos normais. A metodologia validada foi aplicada a 44 amostras de DNA livre extraídas de plasma de gestantes (21 amostras de fetos com trissomia do 21 e 23 de fetos normais), porém não foi possível fazer a distinção entre fetos normais e fetos com trissomia do 21, possivelmente devido à variações na fração fetal do DNA livre em relação à fração materna. Como nosso objetivo principal não foi alcançado, Propomos aqui que o teste realizado em líquido amniótico seja utilizado como uma alternativa mais simples, rápida e barata ao cariótipo convencional atualmente utilizado para fazer o diagnóstico da trissomia do cromossomo 21 em amostras coletadas por procedimentos invasivos, enquanto as deficiências do teste não-invasivo pelo plasma materno são aprimoradas / The purpose of this study was to develop a test for trisomy 21 by analyzing cell-free fetal nucleic acids in maternal plasma by next-generation sequencing performed on automated sequencer Ion Torrent. The proposed methodology for the test is based on analysis of SNPs with high heterozygosity rates in Brazilian population, located in two genes present on chromosome 21 (PLAC4 and C21orf105), and the detection of the extra copy of chromosome 21 is made by the allelic-ratio of these SNPs, where 1:1 ratio indicates a normal fetus, and the 2:1 ratio indicates that the fetus has an extra copy of chromosome 21. In order to validate the methodology 50 cell-free DNAs extracted from amniotic fluid were used representing a gold standard since it contains abundant genetic material exclusively from the fetus. The methodology has been successfully validated in these samples, all the 24 samples from fetuses with trisomy 21 were clearly distinguished from 26 samples of normal fetuses. The validated method was applied to 44 cell-free DNA samples extracted from plasma of pregnant women (21 samples from fetuses with trisomy 21 and 23 from normal fetuses), but unfortunately it was not possible to distinguish between normal and trisomy 21 fetuses, possibly due to variations on the fetal fraction of the cell-free DNA in relation to maternal fraction. As our main goal was not achieved, we propose here that the test performed on amniotic fluid sample could be used as a simpler, faster and cheaper alternative test to traditional karyotype, which is used nowadays to make the diagnosis of trisomy 21 in samples collected by invasive procedures. In parallel, minor improvements in the described method may enable its clinical use