Lam, Yuk Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 142-157). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 摘要 --- p.iv / ACKNOWLEDGEMENTS --- p.vi / CONTRIBUTORS --- p.viii / PUBLICATIONS --- p.ix / LIST OF TABLES --- p.x / LIST OF FIGURES --- p.xi / LIST OF ABBREVIATIONS --- p.xiii / TABLE OF CONTENTS --- p.xiv / Chapter SECTION I: --- BACKGROUND --- p.1 / Chapter CHAPTER 1: --- PRENATAL DIAGNOSIS OF FETAL ANEUPLOIDIES --- p.2 / Chapter 1.1 --- The need for prenatal screening and diagnosis --- p.2 / Chapter 1.2 --- Patau Syndrome (Trisomy 13) --- p.2 / Chapter 1.3 --- Current methods for fetal aneuploidy detection --- p.4 / Chapter 1.3.1 --- Routine prenatal screening tests --- p.4 / Chapter 1.3.2 --- Definitive prenatal diagnosis by invasive procedures --- p.7 / Chapter 1.4 --- New approach for noninvasive prenatal diagnosis --- p.11 / Chapter 1.4.1 --- Circulating fetal cells --- p.11 / Chapter 1.4.2 --- Cell-free fetal nucleic acids in maternal circulation --- p.12 / Chapter 1.4.3 --- Diagnostic applications of cell-free fetal nucleic acids in maternal plasma --- p.12 / Chapter CHAPTER 2: --- DEVELOPMENT OF FETAL EPIGENETIC MARKERS IN MATERNAL PLASMA --- p.17 / Chapter 2.1 --- Limitations of fetal DNA markers --- p.17 / Chapter 2.2 --- DNA methylation is an actively-researched area under the field of epigenetics --- p.18 / Chapter 2.3 --- Genome-scale DNA methylation analysis brings new insight into epigenetics --- p.20 / Chapter 2.4 --- The first demonstration of using an epigenetic method for detecting maternally-inherited fetal DNA in maternal plasma --- p.22 / Chapter 2.5 --- The first universal marker for fetal DNA in maternal plasma --- p.24 / Chapter 2.6 --- Discovery of more fetal epigenetic markers --- p.25 / Chapter 2.6.1 --- Methylated fetal epigenetic markers are more desirable --- p.25 / Chapter 2.6.2 --- Discovery of hypermethylated fetal epigenetic markers by studying tumor suppressor genes --- p.26 / Chapter 2.6.3 --- Discovery of hypermethylated fetal epigenetic markers on chromosome 21 --- p.28 / Chapter 2.7 --- Noninvasive detection of fetal aneuploidies using fetal epigenetic markers --- p.29 / Chapter 2.7.1 --- Noninvasive detection of fetal trisomy 18 by the epigenetic allelic ratio (EAR) approach --- p.29 / Chapter 2.7.2 --- Noninvasive detection of fetal trisomy 21 by the epigenetic-genetic (EGG) approach --- p.30 / Chapter 2.8 --- Aim of thesis --- p.32 / Chapter SECTION II: --- MATERIALS AND METHODS --- p.34 / Chapter CHAPTER 3: --- METHODS FOR QUANTITATIVE ANALYSIS OF DNA METHYLATION --- p.35 / Chapter 3.1 --- Subject recruitment and sample collection --- p.35 / Chapter 3.2 --- Sample processing --- p.38 / Chapter 3.3 --- DNA extraction --- p.38 / Chapter 3.3.1 --- Placental tissues --- p.38 / Chapter 3.3.2 --- Maternal blood cells --- p.39 / Chapter 3.3.3 --- Maternal plasma --- p.40 / Chapter 3.4 --- Methylated DNA immunoprecipitation and tiling array analysis (MeDIP-chip) --- p.41 / Chapter 3.4.1 --- Principles --- p.41 / Chapter 3.4.2 --- DNA sample and array processing --- p.43 / Chapter 3.4.2.1 --- DNA preparation and target hybridization --- p.43 / Chapter 3.4.2.2 --- Data analysis --- p.44 / Chapter 3.5 --- DNA methylation analysis on randomly-chosen regions on chromosome / Chapter 3.6 --- Bisulfite conversion --- p.46 / Chapter 3.6.1 --- Principles of bisulfite conversion --- p.46 / Chapter 3.6.2 --- Procedures of bisulfite conversion --- p.46 / Chapter 3.7 --- Quantitative analysis of DNA methylation --- p.47 / Chapter 3.7.1 --- Bisulfite PCR and genomic sequencing --- p.47 / Chapter 3.7.1.1 --- Primer design for bisulfite polymerase chain reaction (PCR) --- p.47 / Chapter 3.7.1.2 --- Bisulfite PCR --- p.49 / Chapter 3.7.1.3 --- Cloning --- p.50 / Chapter 3.7.1.4 --- Bisulfite genomic sequencing --- p.52 / Chapter 3.7.1.5 --- Data acquisition and interpretation --- p.53 / Chapter 3.7.2 --- EpiTYPER，a mass-spectrometry-based method --- p.54 / Chapter 3.7.2.1 --- Principles of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) --- p.54 / Chapter 3.7.2.2 --- Primer design of the EpiTYPER assay --- p.55 / Chapter 3.7.2.3 --- The EpiTYPER assay and its principle --- p.56 / Chapter 3.8 --- Methylation-sensitive restriction enzyme (MSRE)-mediated real-time quantitative PCR (qPCR) --- p.61 / Chapter 3.9 --- Digital PCR --- p.66 / Chapter 3.9.1 --- Principles of digital PCR --- p.66 / Chapter 3.9.2 --- Poisson distribution --- p.68 / Chapter 3.10 --- Statistical analyses --- p.69 / Chapter SECTION III: --- SYSTEMATIC IDENTIFICATION OF A FETAL DNA METHYLATION MARKER ON CHROMOSOME 13 FOR DETECTION OF FETAL TRISOMY 13 --- p.70 / Chapter CHAPTER 4: --- SYSTEMATIC IDENTIFICATION OF POTENTIAL FETAL EPIGENETIC MARKERS BY MEDIP-CHIP ANALYSIS --- p.71 / Chapter 4.1 --- Systematic discovery of fetal epigenetic markers on chromosome 13 by MeDIP-chip analysis --- p.71 / Chapter 4.2 --- Experimental design --- p.73 / Chapter 4.3 --- Results --- p.76 / Chapter 4.3.1 --- Identification of differentially methylated DNA regions by MeDIP-chip or non-MeDIP-chip approaches followed by EpiTYPER analysis --- p.76 / Chapter 4.3.2 --- Confirmation of differential methylation patterns and exclusion of regions with high inter-individual variations by EpiTYPER analysis --- p.82 / Chapter 4.3.3 --- Confirmation of differential DNA methylation patterns with higher resolution by bisulfite sequencing --- p.85 / Chapter 4.4 --- Discussion --- p.95 / Chapter CHAPTER 5: --- THE APPLICATION OF FETAL EPIGENETIC MARKER ON CHROMSOME 13 FOR DETECTION OF FETAL TRISOMY 13 --- p.98 / Chapter 5.1 --- Identification of a fetal epigenetic marker on chromosome 13 for the detection of fetal trisomy 13 by the epigenetic-genetic (EGG) chromosome dosage approach --- p.98 / Chapter 5.2 --- Experimental design --- p.101 / Chapter 5.3 --- Results --- p.105 / Chapter 5.3.1 --- Optimization of the digestion protocol --- p.105 / Chapter 5.3.2 --- Detection of digestion-resistant EFNB2-3'UTR moleculesin maternal plasma --- p.109 / Chapter 5.3.3 --- Evaluation of the fetal specificity of digestion-resistant EFNB2´ؤ3 'UTR DNA molecules in maternal plasma --- p.111 / Chapter 5.3.4 --- Comparison of EFNB2-3'UTR methylation profiles between the euploid and trisomy 13 placental tissue samples --- p.115 / Chapter 5.3.5 --- Chromosome dosage analysis by the EGG analysis using placental tissue samples --- p.118 / Chapter 5.4 --- Discussion --- p.122 / Chapter SECTION IV: --- CONCLUDING REMARKS --- p.125 / Chapter CHAPTER 6: --- CONCLUSION AND FUTURE PERSPECTIVES --- p.126 / Chapter 6.1 --- Development of fetal epigenetic markers for noninvasive prenatal diagnosis --- p.126 / Chapter 6.2 --- Systematic identification of fetal epigenetic markers on chromosome13 --- p.127 / Chapter 6.3 --- Detection of fetal trisomy 13 by the epigenetic-genetic (EGG) relative chromosome dosage analysis --- p.129 / Chapter 6.4 --- Future perspectives --- p.132 / Appendix I --- p.134 / Appendix II --- p.136 / REFERENCES --- p.142

Errata slip inserted at back / Bibliography: leaves 159-192 / vi, 142, [75] leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Summary: Primary object ... was to construct a pulsed field gel electrophoresis (PFGE) derived long range restriction map of this region by physically linking adjacent DNA probes to common high molecular weight genomic DNA fragments / Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 1994

Thesis (doctoral)--Göteborg University, 2005. / Includes bibliographical references (p. 438-449) and index.

Thesis (doctoral)--Göteborg University, 2005. / Includes bibliographical references (p. 438-449) and index.

Thesis (PhD (Psychiatry))--Stellenbosch University, 2008. / BACKGROUND: Schizophrenia is a heterogeneous illness resulting from complex gene-environment interplay. The majority of molecular genetic work done has involved Caucasian populations, with studies in these and Asian populations showing 2-32% of sufferers to have chromosomal aberrations. So far the discovery of a specific susceptibility mechanism or gene still eludes us, but the use of endophenotypes is advocated as a useful tool in this search. No cytogenetic studies of this nature have been reported in any African schizophrenia population. AIM: The aim of the study was to combine genotypic and phenotypic data, collected in a homogenous population in a structured manner, with the hope of characterising an endophenotype that could be used for more accurate identification of individuals with possible chromosomal abnormalities. METHODOLOGY: A structured clinical interview was conducted on 112 Xhosa schizophrenia patients. (Diagnostic Interview for Genetic Studies, including Schedules for the Assessment of Negative and Positive Symptoms.) Blood samples (karyotyping and/or FISH analysis) as well as urine samples (drug screening) were obtained and nine head and facial measurements were performed. Descriptive statistics were compiled with reference to demographic, clinical and morphological variables. Comparisons between mean differences for these variables were made.

Human response to ionising radiation (IR) shows a wide variation. This is most clearly seen in the radiation-response of cells as measured by frequencies of chromosomal aberrations. Different frequencies of IR-induced aberrations can be conveniently observed in phytohaemagglutin-stimulated peripheral blood T-lymphocytes from both normal individuals and sporadic cancer cases, in either metaphase chromosomes or as micronuclei in the following cell cycle. Metaphase cells show frequent chromatid breaks, defined as chromatid discontinuities or terminal deletions, if irradiated in the G 2 -phase of the cell cycle. It has been shown that the frequency of chromatid breaks in cells from approximately 40% of sporadic breast cancer patients, are significantly higher than in groups of normal individuals. This suggests that elevated radiation-induced chromatid break frequency may be linked with susceptibility to breast cancer. It is known that chromatid breaks are initiated by a double strand break (DSB), but it appears that the two are linked only indirectly as repair kinetics for DSBs and chromatid breaks do not match. Therefore, the underlying causes of the wide variation in frequencies of chromatid breaks in irradiated T-lymphocytes from different normal individuals and from sporadic breast cancer cases are still unclear but it is unlikely to be linked directly to DSB rejoining. My research has focused on the mechanism through which chromatid breaks are formed from initial DSBs. The lack of a direct association suggested that a signalling process might be involved, connecting the initial DSB and resulting chromatid break. The signal model, suggested that the initial DSB is located within a chromatin loop that leads to an intra- or interchromatid rearrangement resulting in incomplete mis-joining of chromatin ends during the decatenation of chromatids during G 2 . It was therefore proposed that topoisomerase II alpha (topo IIα) might be involved, mainly because of its ability to incise DNA and its role in sister chromatid decatenation. During my PhD research I have used a strategy of altering topo II activity or expression and studying whether this alters IR-induced chromatid break frequency. The first approach involved cell lines that varied in topo IIα expression. The frequency of IR-induced chromatid breaks was found to correlate positively with topo IIα expression level, as measured in three different cell lines by immunoblotting, i.e. two cell lines with lower topo IIα expression exhibited lower chromatid break frequency. Topo II activity in these three cell lines was also estimated indirectly by the ability of a topo IIα poison to activate the G 2 /M checkpoint, and this related well with topo IIα expression. A second approach involved ‘knocking down’ topo IIα protein expression by silencing RNA (siRNA). Lowered topo IIα expression was confirmed by immunoblotting and polymerase chain reaction. SiRNA-lowered topo IIα expression correlated with a decreased IR-induced chromatid break frequency. In a third series of experiments cells were treated with ICRF-193, a topo IIα catalytic inhibitor. It was shown that inhibition of topo IIα also significantly reduced IR-induced chromatid breaks. I also showed that lowered chromatid break frequency was not due to cells with high chromatid break frequencies being blocked in G 2 as the mitotic index was not altered significantly in cells with lowered topo IIα expression or activity. These experiments show that topo IIα is involved in IR-induced chromatid break formation. The final experiments reported here attempted to show how topo II might be recruited in the process of forming IR-induced chromatid breaks. Hydrogen peroxide was used as a source of reactive oxygen species (reported to poison topo IIα) and it was shown that topo IIα under these conditions is involved in the entanglement of metaphase chromosomes and formation of chromatin ‘dots’ as well as chromatid breaks. Experiments using atomic force microscopy attempted to confirm these dots as excised chromatin loops. The possible role of topo IIα in both radiation- and hydrogen peroxide-induced primary DNA damage was also tested. It was shown that topo IIα does not affect radiation-induced DSBs, even though it does affect chromatid break frequency. Also, topo IIα does not affect hydrogen peroxide-induced DNA damage at low doses. The results support the idea that topo IIα is involved in the conversion of DSBs to chromatid breaks after both irradiation and treatment with hydrogen peroxide at a low concentrations. I have demonstrated that topo IIα is involved in forming IR-induced chromatid breaks, most likely by converting the initial DSBs into chromosomal aberrations as suggested by the signal model.

Thesis (doctoral)--Göteborg University, 2005. / Includes bibliographical references (p. 438-449) and index.

Indiana University-Purdue University Indianapolis (IUPUI) / Down syndrome (DS) is the most common aneuploidy to affect humans and occurs in approximately 1 of 750 live births. Individuals with DS present with a wide range of clinical phenotypes. Common craniofacial phenotypic expressions include a small mandible, protruding tongue, and a flattened nasal bridge. These traits may affect the feeding, breathing, and swallowing of individuals with DS. Because some complications may go unnoticed for longer periods of time, we hypothesize that significant cardiac and GI defects may be indicative of feeding and airway difficulties. In order to better understand the secondary phenotypes resulting from DS, we have implemented a retrospective chart review of 137 infants between zero and six months of age who were evaluated through the Down Syndrome Program at Riley Hospital for Children from August 2005 to August 2008. Data regarding cardiac, gastrointestinal, endocrine, airway, auditory, and feeding abnormalities have been collected and incedences and comorbidites of these traits has been examined. Comprehensive results indicate cardiac abnormalities occur in 80% of infants, 60% experience gastrointestinal complications, feeding difficulties occur in 46%, and airway complications occur in 38% of infants. Infants with DS were found to be breastfed less over time, with an increase in tube feeds. Notably, we have found all infants with videofluoroscopic evaluations had some type of dysphagia. The presence of gastrointestinal abnormalities closely correlate with the need for tube feeds, and the comorbidity between GI anomalies and muscle tone appear to indicate the likelihood of feeding difficulties and need for altered feeding strategies. Comorbidities between feeding difficulties were nearly significant with cardiac defects and significant with GI abnormalities. Identification of such associations will help healthcare providers determine the best course of treatment and recommended feeding methodology for infants with DS. In order to utilize an in vitro model to study the craniofacial dysmorphologies seen in individuals with DS, cranial neural crest cells (NC) have been cultured. With these, we have begun to investigate the mechanisms behind a smaller trisomic mandibular precursor as compared to the euploid. With this in vitro model, we will be able to test proliferation, migration, and senescence of NC in a culture system.

Shared characteristics and predictors of psychological distress are beginning to be identified in research on women seeking genetic testing for BRCA1 and BRCA2 gene mutations. This study further explored patterns of psychological distress for 51 community women waiting to receive such genetic test results. There was no significant relationship between psychological distress and family cancer history, personal cancer history, social support networks, and family environment. Women in this sample tended to rely more on females and relatives for support than males and friends. Social support satisfaction was not related to gender or number of relatives providing support. Thirty-four of the 36 women classified on the family environment type were from Personal Growth-Oriented families. Comparisons with normal and distressed family means revealed increased cohesion and expressiveness with decreased conflict, indicative of supportive family environments. Limitations and implications are discussed.

Indiana University-Purdue University Indianapolis (IUPUI) / Down syndrome (DS), a genetic disorder that affects ~1 in 700 live births, is caused by trisomy of human chromosome 21 (Hsa21). Individuals with DS are affected by a wide spectrum of phenotypes which vary in severity and penetrance. However, cognitive and skeletal impairments can be commonly observed in all individuals with DS. To study these phenotypes, we utilized the Ts65Dn mouse model that carries three copies of approximately half the gene orthologs found on Hsa21 and exhibit similar phenotypes as observed in humans with DS. Individuals with DS and Ts65Dn mice have deficits in bone mineral density (BMD), bone architecture, bone strength, learning and memory. Over-expression of DYRK1A, a serine-threonine kinase encoded on Hsa21, has been linked to deficiencies in DS bone homeostasis and cognition. Epigallocatechin-3-gallate (EGCG), an aromatic polyphenol found in high concentrations in green tea, is a selective inhibitor of DYRK1A activity. Normalization of DYRK1A activity by EGCG therefore may have the potential to ameliorate skeletal and cognitive deficits. We hypothesized that supplements containing EGCG obtained from health food stores/ online vendors will not be as effective as EGCG from a chemical company in correcting bone deficits associated with DS. Our results suggest that EGCG improves the bone mineral density of trisomic femurs significantly better than the supplements while the EGCgNOW supplement from NOW FOODS improves trabecular and cortical bone structure. The results from HPLC analysis of supplements showed the presence of other catechins in EGCgNOW and degradation analysis revealed the rapid degradation of supplements. Therefore we hypothesize that the presence of EGCG degradation products and other green tea catechins in supplements may play a role in the differential skeletal effects we observed. We further hypothesized that a three week treatment of adolescent mice with EGCG will lead to an improvement in the learning and memory deficits that are observed in trisomic animals in comparison to control mice. However, our results indicate that three weeks of low-dose EGCG treatment during adolescence is insufficient to improve hippocampal dependent learning and memory deficits of Ts65Dn mice. The possibility remains that a higher dose of EGCG that begins at three weeks but lasts throughout the behavioral test period may result in improvement in learning and memory deficit of Ts65Dn mice.