Thesis (doctoral)--Lund University, 1994. / Added t.p. with thesis statement inserted.

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

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.

Our lab has previously reported the identification of a novel endogenous 19-nor steroid, estradienolone (ED), in pregnant women that strongly bound to sex hormone binding globulin. Estrogen-receptor related receptors (ERRs), which have no known natural ligands, are a family of orphan receptors consisting of 3 isoforms: ERRalpha, ERRbeta and ERRgamma. The ERRs have been shown to actively modulate estrogenic responses, to play an essential role in pregnancy, and are implicated in breast cancer prognosis. My results show that ED acts as an antagonist of the ERRalpha confirming preliminary results obtained by our group. Studies of cellular responses demonstrate that ED has strong anti-mitogenic properties. ED inhibited the growth of both estrogen receptor (ER)-positive (MCF-7) and ER-negative (MDA-MB-231) breast cancer cells in a dose-dependent manner but did not have any effects on the proliferation of the non-cancerous immortalized epithelial breast MCF-10A cells. The finding that ED inhibits proliferation of both ER negative and ER positive breast cancer cells, and regulate ERR transcriptional activity may have important ramifications in breast cancer therapy.

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