Epigenetic modification at imprinted loci following alcohol exposure during prenatal development
Date
2016-10-19
Authors
Masemola, Matshane Lydia
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Abstract
Fetal alcohol syndrome (FAS) is a devastating developmental disorder resulting from alcohol exposure during fetal development. It is a considerable public health problem worldwide, but in several communities in South Africa, specifically in the Western and Northern Cape, it has an exceptionally high prevalence of 68.0 – 89.2 per 1000 children of school going age. FAS is a developmental disorder characterised by facial dysmorphic features, growth retardation and central nervous system abnormalities. Twin concordance studies and animal models suggest that there are genetic and epigenetic susceptibility factors for developing FAS. Imprinted genes are known to play an important role in growth and development and most of them are located in imprinted clusters. The IGF2/H19, DLK1/MEG3 (GTL2), CDKNIC/ KCNQ1OT1 and PEG3 imprinted loci play a critical role in fetal development. Each of these imprinted loci contain several imprinted genes that are reciprocally imprinted, and their differential expression is controlled by differentially methylated regions (DMR) referred to as imprinting control regions (ICRs). The ICR for IGF2/H19 is called H19 ICR and for DLK1/MEG3 (GTL2) is IG-DMR and they are both marked with DNA methylation on their paternal allele. KvDMR1 and PEG3 DMR are ICRs for CDKNIC/KCNQ1OT1 and PEG3 imprinted loci respectively and they are marked with methylation on their maternal allele. DNA methylation at CpG dinucleotides is an epigenetic modification that is important in regulating gene expression during embryogenesis. It is proposed that alcohol-associated alterations in fetal DNA methylation at the four ICRs may contribute to developmental abnormalities seen in FAS and which persist into adulthood.
The aim of the study was to examine the effect of maternal alcohol consumption during pregnancy on DNA methylation profiles at specific ICRs (H19 ICR, IG-DMR, KvDMR1 and PEG3 DMR) between FAS offspring and unaffected controls. The participants were FAS children and controls from the Western and Northern Cape Province. DNA samples extracted from blood and buccal tissues were bisulphite modified and the ICRs were amplified by PCR. The pyrosequencing method was used to derive a quantitative estimate of methylation at selected CpG dinucleotides. Analyses were done for H19 ICR (6 CpG sites; 50 controls and 73 cases); KvDMR1 (7 CpG sites; 55 controls and 86 cases); IG-DMR (10 CpG sites; 56 controls and 84 cases) and PEG3 DMR (7 CpG sites; 50 controls and 79 cases).
Age and gender are reported confounders in DNA methylation studies and their effects were investigated in the present study. In this study age was shown to influence methylation at three of the four loci investigated, IG-DMR, KvDMR1 and PEG3 DMR. The effect of gender on methylation was shown to be significant at only one locus, PEG3 DMR. After adjusting for gender and age, there was a significant difference in methylation (CpG specific and locus averaged) at KvDMR1 and PEG3 DMR but not at the H19 ICR, with only a small effect on average methylation (0.84% lower in cases; p=0.035) at IG-DMR. The two maternally imprinted loci, KvDMR1 and PEG3 DMR, showed significantly lower locus averaged methylation in the FAS cases (1.49%; p<0.001 and 7.09%; p=0.001, respectively). Hypomethylation at the KvDMR1 was unexpected since reduced methylation at KvDMR1 has been associated with Beckwith Wiedemann Syndrome, an overgrowth syndrome. The largest effect was observed at the PEG3 DMR, which regulates the paternal PEG3 gene expression in the brain, but we are yet to understand its impact on the FAS phenotype. This study provides supportive evidence for the role of epigenetic modulation as a mechanism for the teratogenic effect of alcohol by altering the methylation profiles of ICRs of imprinted loci in a locus-specific manner.
Description
A thesis submitted to the Faculty of Health Sciences, University of the
Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of
Doctor of Philosophy
February 2016