We performed a genome-wide, semi-unbiased quantitative comparison of frontal cortex DNA methylation from age- and gender-matched LOAD cases and controls. The CpG site most strongly associated with case status was located in the promoter region of TMEM59
, a gene recently implicated in AD pathogenesis [42
]. This gene is involved in the post-translational modification of AβPP
and causes retention of AβPP
in the Golgi apparatus [42
]. The magnitude of methylation difference at this site between cases and controls was very modest (7.3% difference using the Illumina array), but the direction of association was confirmed using an alternate method of DNA methylation detection (2.7% difference using pyrosequencing). In an expanded population including a higher number of younger cases and controls, an interaction between age and case status was detected. Thus, age modifies the association between disease status and methylation. There was not a primary association between case status and methylation when the younger population was included. In the original sample of LOAD cases and controls, TMEM59
DNA methylation levels corresponded to functional changes in TMEM59
gene expression. LOAD cases had lower methylation and higher expression of TMEM59
than control samples. No differences in the level of the full length TMEM59
protein were observed between cases and controls; however a smaller protein that bound the TMEM59
antibody was significantly higher in controls. This TMEM59
protein size pattern is consistent with the TMEM59
control protein lysate. The shorter protein may represent a proteolytic product of the full length protein. The shorter protein is approximately 17 kDa, which could also be consistent with translation of an alternative RNA transcript beginning at exon 5 of the TMEM59
gene. The observed differences in protein expression levels are consistent with epigenetic regulation. Further molecular research is needed to better understand the gene expression and protein regulation and potential role of DNA methylation at this site.
Well-studied genes known to be involved in AD pathogenesis or identified through GWAS for genetic association with LOAD were largely not associated with disease-specific DNA methylation differences in this study. A notable exception was PSEN1
, which was modestly hypomethylated in LOAD cases. Consistent with previous human postmortem tissue studies, PSEN1
showed very low levels of methylation in our population [43
]. Here, LOAD cases had reduced DNA methylation that was associated with increased PSEN1
gene expression, suggesting the DNA methylation change may be functional at this site. Studies in mice and neuroblastoma cell lines demonstrate that PSEN1
gene expression is regulated by DNA methylation at specific promoter CpG sites and this regulation depends on B vitamin availability [44
]. The correlation between DNA methylation and gene expression in our study support the cell line and mouse model findings. The Illumina HumanMethylation27 BeadArray platform used in this study also allowed for discovery of novel gene associations with AD. For example, methylation change was observed within the DMR for the imprinted gene, DIRAS3
. Genomic imprinting in AD is a potential mechanism to explain epidemiological parent-of-origin inheritance observations [45
Greater than 900 genes (6% of genes featured on the array) were differentially methylated by case status after adjusting for age and gender. Overall, the disease related methylation effect size (2.9%) was relatively modest, and the global methylation distributions of AD cases and controls were similar. Together these findings suggest that DNA methylation may play a role in LOAD and the individual effects at each CpG site may be subtle. The magnitude and absolute number of DNA methylation changes observed in this study are consistent with previous reports in the literature performed on candidate gene subsets. In a case-control study of prefrontal cortex DNA methylation of twelve genes, only two genes were associated with AD status and the differences in methylation were less than 10% [47
Gene set enrichment analysis revealed key patterns in the identified set of disease associated CpG sites. First, gene ontology analysis showed hypermethylation of genes involved in transcription and DNA replication, while membrane transporters were hypomethylated. Second, hypermethylation was enriched at genes containing POU3F2
binding motifs. POU3F2
is a transcription factor critical in central nervous system development that regulates Nestin gene expression, a protein important for radial axon growth [48
]. Third, positional analysis showed hypomethylation with case status at 19q13 and hypermethylation at 19p13, cytogenic band regions genetically linked with AD [5
LOAD cases and control sample groups were similar with respect length of time in storage, but LOAD cases had shorter PMI then control samples (p = 0.0004). During PMI, samples may be exposed to damaging lower pH conditions and higher temperatures where enzymes may be active. PMI was not a significant confounder at the TMEM59 site, however, PMI may be a factor at other specific locations throughout the DNA methylome.
This study measured genome-wide DNA methylation differences between LOAD case and control subjects aged 69 to 95. Across this relatively short age range, DNA methylation was associated with age at over 2,400 CpG sites, representing more than 8% of the sites on the BeadArray. Both hyper- and hypomethylation was observed. Previous studies have observed global hypomethylation with age in the brain [49
], but gene-specific studies of aging and methylation have noted varying patterns [43
]. These results further support age as an important covariate to consider in statistical models of DNA methylation in late life.
Age is a major factor in epigenetic change in the brain [17
], potentially confounding or modifying disease specific associations. In a study of cerebral cortex DNA from gestation to 104 years of age, eight of fifty loci showed increases in methylation through late life and two sites presented changes suggestive of an acceleration of age-related change in a subset of samples with LOAD [43
]. Additional evidence supports increased age-dependent epigenetic drift with disease. In LOAD prefrontal cortex samples representing a 30 year age range, an age-specific epigenetic drift was more prominent among cases compared to controls. The average methylation in promoters of MTHFR
increased by 6.8%, while control samples decreased with age by 10.6% [47
Cultured cells can potentially have very different epigenetic profiles than primary cells as an artifact of growth in culture [50
], and thus use of primary human frontal cortex tissue is a strength of this study. DNA methylation is brain region specific and greater differences have been seen between the cerebellum and cortex regions than by gender, age, PMI, race, or cause of death [18
]. This study consistently used frontal cortex tissue because of its role in advanced AD. As with many studies of epigenomic, transcriptomic, or proteomic changes in the human brain, the tissue samples represented populations of mixed cell types, an important limitation, which may have resulted in attenuated effects. Epigenomes are cell type specific [15
], and brain cell types have different roles in AD [52
]. The AD brain has an active changing cell population including neuronal loss and glial activation [53
] that may in part be responsible for the observed results. DNA methylation data, however, was not enriched in inflammatory mediators, which would have supported changes due to gliosis. This study considered brain region specific methylation and as epigenomic platforms require lower input DNA amounts, future research may be able to also consider cell type specific changes.
Results from large DNA methylome and transcriptome maps of the human brain suggest that intragenic CpG sites rather than promoter CpG islands may better correlate with transcription [15
]. The genome-wide sites included on the Illumina Infinium HumanMethylation27 BeadArray are more likely to be located within promoter region CpG islands. Important methylation events located elsewhere throughout the genome would be missed using this platform and may be included in future research utilizing alternative technologies.
These results must be interpreted with caution because this study had a small sample size relative to the large number of CpG site comparisons and the magnitude of observed methylation differences between LOAD cases and controls was moderate. The results from the array were technically validated at the top CpG site, but it is not clear whether this observation will be consistent across populations. While a small study, we identified modest DNA methylation differences as a potential event in LOAD.