Genome-wide gene expression analysis in black South African women who develop gestational diabetes mellitus
Date
2017
Authors
Hobbs, Angela Wendy
Hobbs, Angela Wendy
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Abstract
Gestational diabetes mellitus (GDM) is characterized by high blood glucose levels that first develop during pregnancy. GDM has been linked with many adverse short and long term health outcomes for the developing foetus as well as for the mother. The Developmental Origins of Health and Disease (DOHaD) concept suggests that in the presence of adverse stimuli, the foetus will adapt, through epigenetic mechanisms, to ensure its immediate survival. For this reason, epigenetic modifications are emerging as mediators linking early environmental exposures during pregnancy with programmed changes in gene expression that alter offspring growth and development. The objective of this research study was to explore the role of altered gene expression and methylation in the development of GDM and determine whether these alterations are inherited by the exposed foetus.
Transcriptome sequencing was performed on mRNA extracted from blood samples collected from six women with GDM and from six controls; as well as from exposed (N=6) and unexposed placenta (N=6). Genes that displayed significant (p<0.005) differential expression (log2 fold change >2 and <-2) between cases and controls were identified from the blood (N=60) and placenta (N=56) datasets. Gene ontology and enrichment was performed using DAVID and PANTHER with the aim to narrow down the candidate gene lists.
The ten most likely candidate genes for differential gene expression from the blood dataset were G6PD, DCXR, TKT, ALDOA, PGLS, KCNQ1, C14orf80, KCNQ1, SLC25A22 and GSK3A. Gene enrichment revealed that five of these significantly under-expressed genes (G6PD, DCXR, TKT, ALDOA and PGLS) encode enzymes in the pentose phosphate pathway (PPP). In the placental dataset the top ten candidate genes were CXCR1, CXCR2, G6PD, TKT, IGFBP-1, IGFBP-2, IGFBP-6, GGT3P, MMP12 and GLT1D1. The direction and fold change of differential expression of all twenty genes were validated using TaqMan qPCR probes. Of these twenty genes, the five most promising biological candidates (G6PD, TKT, IGFBP-1, IGFBP-2 and IGFBP-6) were identified and the level of promoter region methylation was assessed using EpiTech Methyl II PCR Assays. The level of methylation in the promoter region of G6PD in both blood and placenta
tissue was found to be significantly higher (p=1.90 x 10-5 and p=1.2 x 10-11 respectively) in the case groups, correlating with decreased mRNA expression levels. There was a significant negative correlation between G6PD mRNA expression in the blood and placenta with the level of maternal glucose at fasting (p=0.006 and p=0.001, respectively), 1-hr (p=0.016 and p=0.007, respectively) and 2-hr post OG (p=0.045 in placenta). We observed a significant positive correlation between G6PD promoter region methylation in both blood and placental tissues with maternal glucose levels at fasting (p=0.023 and p=0.001, respectively) and at 1-hr post OG (p=0.001 and p=0.004, respectively). IGFBP-1 was found to be significantly under-expressed in exposed placental tissue and hypermethylated (p=1.1 x 10-6) at the promoter region when compared to unexposed samples. There was a significant negative correlation between the expression of IGFBP-1 mRNA in the blood and placenta with foetal birth weight (p=0.005 and p=0.017, respectively).
Our results suggest that high glucose levels, an important characteristic of GDM, result in the disturbance of the pentose phosphate pathway, a pathway linked closely to glycolysis, and the IGF-axis, which is important in foetal growth and development. In GDM there is suppression of G6PD mRNA expression in both the blood and placental tissue which influences the pentose phosphate pathway. We hypothesize that this is mediated through an epigenetic mechanism since it is correlated with increased methylation of the G6PD promoter region. Down regulation of G6PD would suppress the PPP and reduce the levels of NADPH production, which may in turn lead to an increase in oxidative stress and an adverse outcome in the mother and foetus. With regard to the IGF-axis, our results demonstrated that IGFBP-1 and IGFBP-2 mRNA expression in the placenta may be inhibited due to the presence of high glucose and insulin levels and this decrease in mRNA expression is likely implicated in the abnormal foetal growth which is often associated with GDM.
This study has provided novel insights into gene expression and DNA methylation changes in the blood of women with GDM and the placenta of their female offspring that involve genes in the PPP and the IGF-axis.
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
December 2017.