Methylation profiling of paternally imprinted loci in male gametes following alcohol exposure
Date
2012
Authors
Pitamber, Punita Navnital
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Abstract
Fetal 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.
Description
A dissertation submitted to the Faculty of Health Sciences, University of the
Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Master
of Science in Medicine