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.