Prenatal diagnosis is an established obstetrics practice in many countries. Currently available methods to obtain fetal materials for a definitive diagnosis involve invasive procedures such as chorionic villus sampling and amniocentesis. Due to the invasive nature of the procedures, confirmatory testing is usually recommended only for pregnant women who are screened as being high risk of bearing a fetus with abnormalities. There has been an urge to develop safer alternatives in obtaining fetal genetic materials for prenatal assessment. Thus, the discovery of circulating fetal DNA in maternal plasma and serum has opened up new possibilities for noninvasive prenatal diagnosis. / The use of genetic markers such as Y chromosomal sequences from a male fetus or paternally-inherited polymorphic markers has been well-described in the literature. However, there is an obvious limitation on the use of these markers because they are gender- and/or polymorphism-dependent. This thesis focuses on the development of a universal fetal marker, namely SERPINB5 (encoding maspin), based on the intrinsic epigenetic differences between the placenta (the major contributor of fetal genetic materials in maternal plasma) and maternal blood cells (postulated to be the predominant source of cell-free DNA in the circulation). Analysis of the methylation profile of the SERPINB5 promoter in the placenta and maternal blood cells, and the development of methods and protocols to detect the differentially methylated SERPINB5 promoter molecules are described. The application of this epigenetic marker in prenatal assessment of the fetal chromosomal aneuploidy is illustrated by an epigenetic allelic ratio (EAR) approach. Basically, the ratio of a single-nucleotide polymorphism (SNP) on the placenta-derived SERPINB5 molecules is shown to be deviated in the maternal plasma from trisomic pregnancies when compared to the euploid ones. Results described in this thesis show the promising potential for the EAR approach for the noninvasive prenatal detection of fetal chromosomal aneuploidies. In addition, a novel approach for methylation analyses on the amplification and detection of restriction enzyme-digested DNA fragments will be discussed. This thesis has essentially described the evolution of a fetal epigenetic marker from basic science to potential clinical application. / Tong, Yu Kwan. / Adviser: Yuk-Ming Dennis Lo. / Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 0953. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 149-172). / 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. / Abstract also in Chinese. / School code: 1307.

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

Lo, Yin Wai Wyatt. / "December 2010." / Thesis (M.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 186-206). / Abstracts in English and Chinese. / ABSTRACT --- p.II / 摘要 --- p.VII / ACKNOWLEDGEMENTS --- p.X / PUBLICATIONS --- p.XI / TABLE OF CONTENTS --- p.XII / LIST OF TABLES --- p.XVIII / LIST OF FIGURES --- p.XXI / LIST OF ABBREVIATIONS --- p.XXIV / Chapter SECTION I: --- BACKGROUND --- p.1 / Chapter CHAPTER 1: --- PRENATALTESTNG --- p.2 / Chapter 1.1. --- THE AIM --- p.2 / Chapter 1.2. --- INVASIVE PRENATAL DIAGNOSIS --- p.4 / Chapter 1.3. --- NONINVASIVE PRENATAL SCREENING --- p.6 / Chapter CHAPTER 2: --- NONINVASIVE PRENATAL DIAGNOSIS --- p.10 / Chapter 2.1. --- CIRCULATING FETAL CELLS IN PRENATAL DIAGNOSIS --- p.10 / Chapter 2.2. --- CIRCULATING FETAL NUCLEIC ACIDS IN PRENATAL DIAGNOSIS --- p.12 / Chapter 2.3.1. --- Biology of circulating fetal DNA . --- p.14 / Chapter 2.3.2. --- Clinical applications of circulating fetal DNA --- p.15 / Chapter 2.3.2.1. --- Qualitative analysis of fetal DNA in maternal plasma --- p.16 / Chapter 2.3.2.2. --- Quantitative analysis of fetal DNA in maternal plasma --- p.17 / Chapter 2.4. --- CIRCULATING FETAL RNA IN MATERNAL PLASMA --- p.20 / Chapter 2.4.1. --- Biology of circulating fetal RNA --- p.20 / Chapter 2.4.2. --- Clinical applications of circulating fetal RNA --- p.22 / Chapter 2.4.2.1. --- Quantitative analysis of fetal RNA in maternal plasma --- p.22 / Chapter CHAPTER 3: --- TECHNICAL CHALLENGES IN ANALYZING CIRCULATING FETAL NUCLEIC ACIDS --- p.26 / Chapter 3.1. --- INTRODUCTION --- p.26 / Chapter 3.2. --- "PREANALYTICAL ISSUES IN MATERNAL PLASMA NUCLEIC ACID ANALYSE"";" --- p.28 / Chapter 3.2.1. --- Low abundance of cell-free fetal nucleic acids in maternal plasma --- p.28 / Chapter 3.2.2. --- High level of maternal background in maternal plasma --- p.30 / Chapter 3.3. --- ANALYTICAL ISSUES IN MATERNAL PLASMA NUCLEIC ACID ANALYS --- p.IS / Chapter 3.3.1. --- Imprecise measurement of fetal nucleic acid quantity --- p.33 / Chapter 3.3.2. --- Coexistence of fetal nucleic acids and maternal nucleic acid background in maternal plasma --- p.36 / Chapter 3.4. --- AIMS OF THIS THESIS --- p.41 / Chapter SECTION II: --- MATERIALS AND METHODS --- p.42 / Chapter CHAPTER 4: --- QUANTITATIVE AND QUALITATIVE ANALYSIS OF NUCLEIC ACIDS --- p.43 / Chapter 4.1. --- SAMPLE COLLECTION AND PROCESSING --- p.43 / Chapter 4.1.1. --- Preparation of plasma and blood cells --- p.43 / Chapter 4.1.2. --- Preparation of placental tissues --- p.44 / Chapter 4.2. --- NUCLEIC ACID EXTRACTION --- p.45 / Chapter 4.2.1. --- Extraction of total RNA --- p.45 / Chapter 4.2.1.1. --- Plasma samples --- p.45 / Chapter 4.2.1.2. --- Placental tissue samples --- p.46 / Chapter 4.2.2. --- Extraction of genomic DNA --- p.48 / Chapter 4.2.2.1. --- Plasma samples --- p.48 / Chapter 4.2.2.2. --- Blood cell samples --- p.48 / Chapter 4.3. --- CONVENTIONAL REAL-TIME PCR ANALYSIS OF NUCLEIC ACIDS --- p.50 / Chapter 4.3.1. --- Principles of real-time polymerase chain reaction --- p.50 / Chapter 4.3.2. --- Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) --- p.52 / Chapter 4.3.3. --- Quantitative polymerase chain reaction (qPCR) --- p.53 / Chapter 4.4. --- DIGITAL REAL-TIME PCR ANALYSIS OF NUCLEIC ACIDS --- p.55 / Chapter 4.4.1. --- Principles of digital PCR (dPCR) --- p.55 / Chapter 4.4.2. --- 384-reaction well dPCR v --- p.56 / Chapter 4.4.3. --- Microfluidics dPCR --- p.57 / Chapter 4.5. --- MATRIX-ASSISTED LASER DESORPTIONIONIZATION/TIME-OF-FLIGHT MASS SPECTROMETRY (MALDI-TOF MS) ANALYSIS OF NUCLEIC ACIDS --- p.59 / Chapter 4.5.1. --- Principles of MALDI-TOF MS --- p.59 / Chapter 4.5.2. --- DNA genotyping analysis by MassArray Homogenous MassExtend (hME) assay --- p.60 / Chapter 4.6. --- CLONING AND DNA SEQUENCING --- p.63 / Chapter SECTION III: --- IMPROVEMENTS ON MATERNAL PLASMA ANALYSIS OF CIRCULATING RNA --- p.65 / Chapter CHAPTER 5: --- ENRICHMENT OF PLACENTA EXPRESSED MRNA MARKERS BY WHOLE TRANSCRIPTOME PREAMPLIFICATION --- p.66 / Chapter 5.1. --- INTRODUCTION --- p.66 / Chapter 5.2. --- MATERIALS AND METHODS --- p.69 / Chapter 5.2.1. --- Study design --- p.69 / Chapter 5.2.2. --- Subjects and sample collection --- p.71 / Chapter 5.2.3. --- RNA extraction and sample dilution --- p.71 / Chapter 5.2.4. --- Preamplification --- p.73 / Chapter 5.2.5. --- qPCR analysis --- p.74 / Chapter 5.3. --- RESULTS --- p.83 / Chapter 5.3.1. --- Comparison of mRNA expression profiles in placental tissues with and without preamplification --- p.83 / Chapter 5.3.1.1. --- Undiluted placental tissue RNA --- p.84 / Chapter 5.3.1.2. --- Diluted placental tissue RNA --- p.88 / Chapter 5.3.2. --- The effect of RNA input on the degree of amplification --- p.92 / Chapter 5.3.2.1. --- Correlation between RNA input and RNA output --- p.94 / Chapter 5.3.2.2. --- Correlation between RNA input and output/input ratio --- p.96 / Chapter 5.3.3. --- Preamplification of maternal plasma RNA --- p.98 / Chapter 5.3.3.1. --- Concentrations of placenta expressed mRNA in third trimester maternal plasma --- p.98 / Chapter 5.3.3.2. --- Concentrations of placenta expressed mRNA in first trimester maternal plasma --- p.100 / Chapter 5.4. --- DISCUSSION --- p.102 / Chapter SECTION IV: --- IMPROVEMENTS ON MATERNAL PLASMA ANALYSIS OF CIRCULATING DNA --- p.105 / Chapter CHAPTER 6: --- ACCURATE GENE DOSAGE ANALYSIS BY MULTIPLEX QPCR --- p.106 / Chapter 6.1. --- INTRODUCTION --- p.106 / Chapter 6.2. --- MATERIALS AND METHODS --- p.110 / Chapter 6.2.1. --- Study design --- p.110 / Chapter 6.2.2. --- Subjects and sample collection --- p.112 / Chapter 6.2.3. --- DNA extraction and sample dilution --- p.113 / Chapter 6.2.4. --- qPCR analysis --- p.113 / Chapter 6.2.4.1. --- Monoplex assays --- p.114 / Chapter 6.2.4.2. --- Multiplex assays --- p.114 / Chapter 6.2.5. --- Microfluidics dPCR assay --- p.122 / Chapter 6.2.6. --- Gene Dosage Comparison --- p.122 / Chapter 6.2.6.1. --- In adult male samples --- p.123 / Chapter 6.2.6.2. --- In maternal plasma samples --- p.123 / Chapter 6.3. --- RESULTS --- p.125 / Chapter 6.3.1. --- The influence of using the same and different sets of primers for amplifying different chromosomal loci --- p.125 / Chapter 6.3.2. --- Effects of using monoplex and multiplex real-time PCR formulations --- p.130 / Chapter 6.3.3. --- Effects of incorporating calibration curves for template quantification in conventional qPCR --- p.135 / Chapter 6.4. --- DISCUSSION --- p.140 / Chapter CHAPTER 7: --- DPCR DETECTION OF PATERNALLY INHERITED POINT MUTATIONS --- p.144 / Chapter 7.1. --- INTRODUCTION --- p.144 / Chapter 7.2. --- MATERIALS AND METHODS --- p.153 / Chapter 7.2.1. --- Study design --- p.153 / Chapter 7.2.2. --- Subjects and sample collection --- p.157 / Chapter 7.2.3. --- DNA extraction and sample preparation --- p.158 / Chapter 7.2.4. --- MassArray hME assays --- p.159 / Chapter 7.2.5. --- dPCR assay --- p.159 / Chapter 7.3. --- RESULTS --- p.161 / Chapter 7.3.1. --- Validation of the digital HbE assay --- p.161 / Chapter 7.3.2. --- Determination of the minimum fetal DNA amount required for digital PCR detection --- p.165 / Chapter 7.3.3. --- Detection of paternally inherited fetal HbE mutation in maternal plasma --- p.172 / Chapter 7.4. --- DISCUSSION --- p.175 / Chapter SECTION V: --- CONCLUDING REMARKS --- p.180 / Chapter CHAPTER 8: --- CONCLUSION AND FUTURE PERSPECTIVES --- p.181 / Chapter 8.1. --- IMPROVEMENTS ON QUANTITATIVE AND QUALITATIVE ANALYSIS OF FETAL NUCLEIC ACIDS IN MATERNAL PLASMA --- p.181 / Chapter 8.2. --- PERSPECTIVES FOR FUTURE WORK --- p.184 / REFERENCES --- p.186

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.