Rielly, Nicholas Jude
(2020)
Assessing DNA methylation epialleles for human cognitive dysfunction and brain disease.
PhD thesis, University of Nottingham.
Abstract
In 2016, neurological disorders were the leading cause of disability adjusted life years (number of years lost to illness or disability) and the second leading cause of death. Extensive research has been performed on associations between genetic variants and risk for neurodegenerative diseases. Now, epigenomic processes such as DNA methylation, the addition of a methyl group to the 5th carbon of cytosine (5mC) and its oxidised derivatives (5hmC, 5fC, and 5CaC), are being increasingly studied. DNA methylation contributes to changes in cognition and ageing and has been associated with several neurodegenerative diseases such as Parkinson’s disease and Alzheimer’s disease. However, little research has focused the effect methylation at these sites may have on the regulation of gene expression.
In this study 5mC, 5hmC, and 5fC/CaC DNA methylation levels were measured at the complementary antisense CpG of the BDNF Val66Met rs6265 variant, CpG sites at APOE rs7412 and rs429358 risk variants which determine APOE ɛ isoforms (ɛ2 - ɛ4), and DNMT3L rs7354779 (R278G) which are known risk variants for cognitive diseases. Methylation was quantified through bisulphite, oxibisulphite, and methylation assisted bisulphite sequencing and compared across multiple tissues, brain regions and across mild cognitive impairment, Alzheimer’s disease, dementia with Lewy bodies, and Parkinson’s disease as well as compared with sense and antisense BDNF transcript expression.
Analysis of methylation at the antisense BDNF Val66Met CpG (AS-rs6265) showed significantly higher levels of 5mC compared to 5hmC methylation in C homozygotes (p = 1×10-3). Tissue as well as tissue and genotype had significant associations with 5mC and 5hmC methylation, respectively, in the Imperial samples. Whole brain and hippocampus tissues, as well as Parkinson’s disease and Alzheimer’s disease analyses of C homozygotes all showed patterns where 5mC was higher than 5hmC levels. 5fC/CaC methylation was observed in all tissues and disease states. Samples from individuals with mild cognitive impairment or from the cerebellum showed higher (but non-significant) levels of AS-rs6265 5fC/CaC than 5mC methylation, and AS-rs6265 5fC/CaC methylation was bimodal in distribution.
BDNF sense mRNA transcript expression correlated strongly and significantly with BDNF antisense transcript AS-204 expression across all samples while an alternative antisense transcript (AS-201) showed a negative correlation with both transcripts suggesting that AS-201 may be acting as a cis-regulatory element for BDNF sense mRNA. Expression analysis indicated significantly higher expression of BDNF antisense AS-201 transcript and lower expression of BDNF sense mRNA and BDNF antisense AS-204 in dementia with Lewy bodies samples compared to control samples (p = 2.4×10-2). BDNF mRNA showed a significant decrease in expression with AS-rs6265 5mC methylation in dementia with Lewy bodies (p = 3×10-2) and mild cognitive impairment samples (p = 0.04) and 5hmC levels were found to significantly correlate with decreased AS-201 transcript abundance in cerebellum (p = 2×10-3) and significantly correlate with increased mRNA abundance in C homozygotes (p = 0.03), respectively.
5mC methylation was non-significantly higher than 5hmC methylation across both APOE ɛ2-ɛ4 rs7412 and rs429358 variant sites and also observed in Parkinson’s disease, dementia with Lewy bodies, and Alzheimer’s disease samples at the rs7412 variant site. 5fC/CaC was observationally higher than 5mC in cerebellum and dementia with Lewy bodies samples at the rs429358 epiallele. When analysing by ɛ isoform, 5hmC methylation showed evidence of allele specific methylation and 5hmC methylation was highest in ɛ3/ɛ4 isoform individuals, which may suggest that methylation is being regulated and could influence disease risk.
This study indicates that methylation at risk variant sites shows brain region and disease specific methylation patterns and may, through cis-acting regulatory mechanisms, spatially and temporally regulate expression of proteins in disease. Furthermore, it provides new mechanistic insight into antisense strand methylation and ratios of 5mC to 5hmC in ASM.
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