3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Fragile X Syndrome: A Family Study(1997-10-31) Wessels, Tina-MarieFragile X syndrome is, second to Down syndrome, the commonest form of genetic mental retardation. The aim of this research project was to investigate the impact of having a child with this syndrome on the family relationships. The subjects were 21 mothers and 9 fathers of affected children. The data were collected by means of specially constructed questionnaires in interviews with 19 mothers and 8 fathers and completed by post in three cases. A control group of parents with a normal child, matched for sex and age of the affected child, family size and ethnic groups, was interviewed. The data were computerised and analyzed. The results showed that more experimental parents than controls enjoyed their child’s nature, but disliked the behavioural problems. About half of the experimental parents tended not to reward good behaviour physically. However, although most of the affected children were accepted by their siblings, they had fewer friends and more problems with their peers. Some parents thought that their relationship with their spouse had improved and others thought that it had deteriorated after the affected child’s birth. Most parents in both study groups would request prenatal diagnosis in subsequent pregnancies and significantly more experimental parents than controls would request a termination of pregnancy for an affected fetus. Most parents were satisfied with the health service they received. These results show that family dynamics are disturbed by the presence of a child with FMR. Counsellors and therapists working with these families should be aware of the effects of the syndrome on the familyItem Epigenetic inheritance of aberrant DNA methylation signatures as a consequence of chronic paternal alcohol exposure and the effect on embryonic gene expression in mice(2015) Ismail, AyeshaEpigenetic mechanisms regulate gene expression, a particularly important activity during foetal development. DNA methylation contained within promoter and regulatory intergenic regions influence gene activity. In utero alcohol exposure as a result of maternal consumption during pregnancy has been associated with disruption of foetal DNA methylation and gene expression, leading to neurological dysfunction, growth retardation and facial anomalies. While similar phenotypes in offspring have been associated with chronic preconception paternal alcohol exposure, the mechanisms underlying these effects remain largely unexplored. This study aimed to: (1) validate significant changes in sperm DNA methylation in a list of ten candidate genes in male mice chronically exposed for ten weeks to ethanol (n=10) compared to a calorie-equivalent sucrose solution (n=10); (2) validate significant changes in gene expression in candidate genes in the brain, liver and placenta of E16.5 embryos sired by ethanol (n=24) compared to sucrose (n=24) treated male mice; (3) quantify DNA methylation changes in candidate genes in the three embryonic tissues. (4) Lastly, previously generated microarray data were reanalysed using bioinformatics tools to generate a top ranked candidate differentially expressed gene list that was used to identify and analyse biological functions or pathways significantly over represented among these genes using PANTHER and DAVID. This study was unable to provide validation for most of the significant differences observed in the sperm DNA methylome in the original study, most likely because of the low sperm DNA concentration. Significant methylation differences were however observed at individual CpG sites in three candidate genes (Igf1r, Odc1, Depdc1b) in specific tissues of embryos sired by ethanol-exposed males relative to embryos sired by sucrose-treated males. There was concordance in the direction of altered gene expression between the cases and controls using the microarray and real-time PCR approaches for two genes in the brain (Grm7 and Zfp317), three genes in the liver (Igf1r, Vwf and Depdc1b) and one gene in the placenta vii (Vwf). However, none of the candidate genes selected for validation showed statistically significant changes. This may be a result of the modest fold changes observed in the microarray experiment that as shown in many cases, often do not replicate. The remainder of the genes showed no changes in expression in the test embryos relative to the control. The functional enrichment analysis revealed biological processes that were over represented in the brain and liver indicating that they may be more vulnerable to the effects of alcohol, compared to the placenta. Overall, the study could not provide a statistically significant correlation between methylation changes in the sperm that were inherited by the offspring which subsequently dysregulated gene expression in the embryo. However, as trends toward significance and significant DNA methylation changes were observed in the embryonic tissues, this study supports the idea that preconception paternal alcohol exposure can induce epigenetic alterations in a locus and organ specific manner within offspring.Item Methylation profiling of paternally imprinted loci in male gametes following alcohol exposure(2012) Pitamber, Punita NavnitalFetal Alcohol Syndrome (F AS), the most severe form of Fetal Alcohol Spectrum Disorder (F ASD), has traditionally been associated with maternal alcohol consumption during pregnancy. However, a number of animal studies have shown an association between paternal preconception alcohol consumption and developmental abnormalities in the offspring that resemble the features of F AS. Dysregulation of epigenetic factors (such as DNA methylation) in the presence of alcohol may provide a plausible mechanism by which paternal alcohol consumption could result in offspring affected with features of F AS. Imprinted genes are expressed in a parentof- origin manner due to DNA methylation at distinct differentially methylated regions (DMRs) and are essential for normal embryonic development. There are only two known paternally methylated DMRs in humans, with an additional one described in mice - associated with Rasgrfl. The first aim of this study was to determine whether the human RASGRFl gene contains a DMR and whether this DMR is paternally methylated. In order to assess the imprint status of RASGRF 1, a number of computational assessments were done to identify key features of imprinted loci. Pyrosequencing analysis was used to assess the methylation status of various CpO islands surrounding RASGRFi in peripheral blood and sperm DNA samples. The RASGRF i-associated CpO regions were not found to exhibit differential methylation in a parent-of-origin manner. The second aIm of the study was to examine the effect of paternal alcohol consumption on the methylation status of the IG-DMR locus in male gametes and to detennine whether alcohol is correlated with methylation in a dose-dependant manner. Methylation assessment was done using the quantitative pyrosequencing technology. While an overall reduction in methylation was noted in males who consumed alcohol after adjusting for confounding variables, the amount of alcohol consumed did not correlate with overall methylation. When analyzed by individual CpG sites, alcohol consumption was found to correlate preferentially with demethylation at CpG 3 while alcohol-dosage preferentially correlated with demethylation at CpG 7. Age was significantly correlated with an increase in the overall methylation at JG-DMR and at individual sites within JG-DMR. In conclusion, these findings support the hypothesis that paternal preconception alcohol consumption can lead to hypomethylation of nonnally hypennethylated DMRs of specific imprinted genes in human spenn. This in tum could have significant implications with regard to the regulation of developmentally significant genes in the zygote and fetus, resulting in developmental, behavioral and neurocognitive disorders.Item Transgenerational inheritance of DNA methylation alterations at the H19 imprinting control region following maternal ethanol exposure in mice(2013) Ungerer, MichelleFoetal Alcohol Syndrome (FAS) is characterised by growth retardation, craniofacial dysmorphology and neurodevelopmental deficits. Whilst, not all alcohol exposed offspring display alcohol-related developmental anomalies, the percentage of affected offspring is greatly underestimated. Common behavioural disorders, such as ADHD and anxiety, are likely to be linked to the transgenerational effects of in utero alcohol exposure. Epigenetics has been highlighted as a potential mechanism in the aetiology of alcohol teratogenesis due to alcohol’s disruptive effects on the folate pathway, and subsequently DNA methylation. The imprinted H19/Igf2 domain is critical in foetal growth and development. The locus is regulated by the methylation-sensitive CTCF binding protein which binds to the H19 imprinting control region (ICR) upstream of the H19 locus. CTCF binding allows for the reciprocal expression of H19 and Igf2 in an allele-specific parent of origin manner. Due to the monoallelic expression of imprinted genes, DNA methylation changes within their control regions can lead to altered gene expression and possibly disease. Furthermore, if these alterations occur in the germline, disease states or susceptibility to disease may be transmittable to future generations. A mouse model was used to investigate the potential transgenerational effects of F0 chronic maternal ethanol exposure on parturition, growth, locomotor activity and anxiety. Furthermore, the transgenerational inheritance of H19 ICR DNA methylation was investigated and its possible contribution to the aforementioned phenotypes was determined. Phenotypic analysis revealed significantly reduced F1 fertility following alcohol exposure (P = 0.003) but no other significant perturbations in parturition. Although not significant at all generations, alcohol’s effects on growth and behaviour were apparent. DNA was extracted from tail biopsies, bisulfite modified and the CTCF1 and CTCF2 regions of the H19 ICR amplified. DNA methylation quantification via Pyrosequencing revealed significantly reduced mean methylation profiles at CTCF1 and CTCF2 within the F1 EtOH exposed group (P = 0.021), with CpG sites 1, 2, 4 and 6 of CTCF1 and CpG sites 1, 2, 3 (P = 0.021) and 5 (P = 0.043) of CTCF2 displaying statistically significant differences. In contrast, the EtOH group of the F2 generation showed an increase in CTCF1 mean methylation that trended towards significance (P = 0.083) suggesting a potential recovery or compensatory mechanism within the epigenetic machinery. The F3 generation EtOH exposed group displayed decreased CTCF1 mean methylation levels (P = 0.083). The F2 and F3 generations showed no significant difference in CTCF2 methylation levels between treatment groups. The significant change in CTCF1 methylation at the F1 generation and the trend towards significance in the F2 and F3 generations indicated potential transgenerational inheritance of altered H19 ICR DNA methylation. Correlations between DNA methylation at the H19 CTCF1 and CTCF2 binding sites with growth rate and behaviour measures revealed no significant relationships. This dissertation supports the involvement of epigenetic mechanisms in alcohol teratogenesis. In addition it contributes to the growing field of transgenerational epigenetic inheritance, with implications for the treatment of those with Foetal Alcohol Syndrome and/or Foetal Alcohol Spectrum Disorders and their progeny.