Abstract
BackgroundAlzheimer’s disease is a progressive neurodegenerative disorder that is hypothesized to involve epigenetic dysfunction. Previous studies of DNA modifications in Alzheimer’s disease have been unable to distinguish between DNA methylation and DNA hydroxymethylation. DNA hydroxymethylation has been shown to be enriched in the human brain, although its role in Alzheimer’s disease has not yet been fully explored. Here, we utilize oxidative bisulfite conversion, in conjunction with the Illumina Infinium Human Methylation 450K microarray, to identify neuropathology-associated differential DNA methylation and DNA hydroxymethylation in the entorhinal cortex.ResultsWe identified one experiment-wide significant differentially methylated position residing in the WNT5B gene. Next, we investigated pathology-associated regions consisting of multiple adjacent loci. We identified one significant differentially hydroxymethylated region consisting of four probes spanning 104 bases in the FBXL16 gene. We also identified two significant differentially methylated regions: one consisting of two probes in a 93 base-pair region in the ANK1 gene and the other consisting of six probes in a 99-base pair region in the ARID5B gene. We also highlighted three regions that show alterations in unmodified cytosine: two probes in a 39-base pair region of ALLC, two probes in a 69-base pair region in JAG2, and the same six probes in ARID5B that were differentially methylated. Finally, we replicated significant ANK1 disease-associated hypermethylation and hypohydroxymethylation patterns across eight CpG sites in an extended 118-base pair region in an independent cohort using oxidative-bisulfite pyrosequencing.ConclusionsOur study represents the first epigenome-wide association study of both DNA methylation and hydroxymethylation in Alzheimer’s disease entorhinal cortex. We demonstrate that previous estimates of DNA hypermethylation in ANK1 in Alzheimer’s disease were underestimates as it is confounded by hypohydroxymethylation.
Highlights
Alzheimer’s disease is a progressive neurodegenerative disorder that is hypothesized to involve epigenetic dysfunction
To assess the reproducibility of our results, we first compared the effect size of the 100 top-ranked Braak-associated differentially modified positions identified in our BS data generated in the “discovery cohort,” with BS data previously published on an independent set of samples from the Entorhinal cortex (EC) in Alzheimer’s disease (AD) (“validation cohort 1”) [7]
Our findings indicate that previous studies of DNA methylation in Ankyrin 1 (ANK1) have underestimated increments in 5mC in disease due to confounding by 5hmC and could suggest the loss of active DNA demethylation of ANK1 in AD, as both 5hmC and unmodified cytosine (uC) are lower in disease
Summary
Alzheimer’s disease is a progressive neurodegenerative disorder that is hypothesized to involve epigenetic dysfunction. DNA hydroxymethylation has been shown to be enriched in the human brain, its role in Alzheimer’s disease has not yet been fully explored. The amyloid precursor protein (APP) is normally processed by α- and γ-secretase (non-amyloidogenic pathway), which forms a soluble, non-toxic Aβ fragment called P3. In AD, APP is cleaved by β- and γ-secretase (amyloidogenic pathway) resulting in the formation of a larger Aβ species, which aggregates to form the characteristic plaques associated with the disease. Mendelian inheritance of mutations in the APP gene and the PSEN1 and PSEN2 genes, which encode subunits of the γ-secretase enzyme, have been demonstrated in early-onset familial AD cases, these only account for ~ 5% of disease incidence [3]. The majority of AD cases are sporadic, occur late in life, and have, as yet, no defined etiology, with common single nucleotide polymorphisms (SNPs) accounting for only a third of disease risk [4]
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