A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand,
Johannesburg, in fulfillment of the requirements for the degree in Master of Science (Medicine) in
the Division of Human Genetics / Epigenetic 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. / MT2016
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/21283 |
| Date | January 2015 |
| Creators | Ismail, Ayesha |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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