Targeted bisulfite sequencing analysis of Alzheimer’s disease candidate genes reveals differential methylation across neurofibrillary tangle burden in the prefrontal cortex.

Abstract

AbstractBackgroundRecent epigenome‐wide association studies (EWAS) identified several loci in specific genes showing robust and reproducible alterations in DNA methylation in Alzheimer’s disease (AD) brain samples. Standardly, assessing methylation in EWAS is done using microarrays, which target a limited number of sites in each gene. Here, we performed targeted bisulfite sequencing of candidate genes associated with AD to determine the exact extent of methylation changes across neurofibrillary tangle burden (NFT) within these loci.MethodPrefrontal cortex brain samples from 58 individuals were grouped by Braak stage (control 0‐II; intermediate III‐IV; AD V‐VI). Following DNA extraction, 30 genomic regions were captured using Agilent SureSelect target baits. After next‐generation bisulfite sequencing, reads were aligned and the methylation status of cytosine residues called using the Bismark program. Differentially methylated positions (DMPs) were analyzed across the three groups. Furthermore, the presence of differentially methylated regions (DMRs), made up of several DMPs was assessed. Methylation levels in genomic features, such as promoters and gene bodies, of the target regions were also compared.ResultMethylation levels were quantified for each group. Linear regression controlled for co‐variation before differences were examined using a one‐way ANOVA, with Tukey’s post‐hoc test. Sites in several genes showed stepwise increases in DNA methylation across the groups. Interestingly, amongst the most robust sites, differences in methylation in the intermediate group when compared to the control and AD samples were observed. DMRs were observed in the groups, as were methylation differences between promotors and gene bodies in several targeted regions.ConclusionThis work provides further evidence that dysregulation of methylation is associated with pathological changes in AD prefrontal cortex. Differential methylation with intermediate NFT pathology, at sites showing no difference between control and AD samples, potentially indicates early pathological changes. As DNA methylation is reversible, this could present potential targets for pharmacological intervention.