Studies on DNA methylation (DNAm) in Alzheimer's disease (AD) have recently highlighted several genomic loci showing association with disease onset and progression. Here, we conducted an epigenome-wide association study (EWAS) using DNAm profiles in entorhinal cortex (EC) from 149 AD patients and control brains and combined these with two previously published EC datasets by meta-analysis (total n=337). We identified 12 cytosine-phosphate-guanine (CpG) sites showing epigenome-wide significant association with either case-control status or Braak's tau-staging. Four of these CpGs, located in proximity to TENT5A, PALD1, PRF1, and DIRAS1, were not reported previously. Integrating DNAm levels with RNA sequencing-based mRNA expression data generated in the same individuals showed significant DNAm-mRNA correlations for 6 of the 12 significant CpGs. By calculating rates of epigenetic age acceleration using two recently proposed "epigenetic clock" estimators we found a significant association with accelerated epigenetic aging in AD patients vs. controls. In summary, our study represents the hitherto most comprehensive EWAS in AD using EC and highlights several novel differentially methylated loci with potential effects on gene expression.

DNA methylation age acceleration (DNAmAA, derived from an epigenetic clock) and relative leukocyte telomere length (rLTL) are widely accepted biomarkers of aging. Nevertheless, it is still unclear which aspects of aging they represent best. Here we evaluated longitudinal associations between baseline rLTL and DNAmAA (estimated with 7-CpG clock) and functional assessments covering different domains of aging. Additionally, we made use of cross-sectional data on these assessments and examined their association with DNAmAA estimated by five different DNAm age measures. Two-wave longitudinal data was available for 1,083 participants of the Berlin Aging Study II (BASE-II) who were re-examined on average 7.4 years after baseline as part of the GendAge study. Functional outcomes were assessed with Fried’s frailty score, Tinetti mobility test, falls in the past 12 months (yes/no), Finger-floor distance, Mini Mental State Examination (MMSE), Center for Epidemiologic Studies Depression Scale (CES-D), Activities of Daily Living (ADL), Instrumented ADL (IADL) and Mini Nutritional Assessment (MNA). Overall, we found no evidence for an association between the molecular biomarkers measured at baseline, rLTL and DNAmAA (7-CpG clock), and functional assessments assessed at follow-up. Similarly, a cross-sectional analyses of follow-up data did also not show evidence for associations of the various DNAmAA measures (7-CpG clock, Horvath’s clock, Hannum’s clock PhenoAge, and GrimAge) with functional assessments. In conclusion, neither rLTL nor 7-CpG DNAmAA were able to predict impairment in the analyzed assessments over a ~7-year time-course. Similarly, DNAmAA estimated from five epigenetic clocks was not a good cross-sectional marker of health deterioration either.

DNA methylation age acceleration (DNAmAA, derived from an epigenetic clock) and relative leukocyte telomere length (rLTL) are widely accepted biomarkers of aging. Nevertheless, it is still unclear which aspects of aging they represent best. Here we evaluated longitudinal associations between baseline rLTL and DNAmAA (estimated with 7-CpG clock) and functional assessments covering different domains of aging. Additionally, we made use of cross-sectional data on these assessments and examined their association with DNAmAA estimated by five different DNAm age measures. Two-wave longitudinal data was available for 1,083 participants of the Berlin Aging Study II (BASE-II) who were re-examined on average 7.4 years after baseline as part of the GendAge study. Functional outcomes were assessed with Fried's frailty score, Tinetti mobility test, falls in the past 12 months (yes/no), Finger-floor distance, Mini Mental State Examination (MMSE), Center for Epidemiologic Studies Depression Scale (CES-D), Activities of Daily Living (ADL), Instrumented ADL (IADL) and Mini Nutritional Assessment (MNA). Overall, we found no evidence for an association between the molecular biomarkers measured at baseline, rLTL and DNAmAA (7-CpG clock), and functional assessments assessed at follow-up. Similarly, a cross-sectional analyses of follow-up data did also not show evidence for associations of the various DNAmAA measures (7-CpG clock, Horvath's clock, Hannum's clock PhenoAge, and GrimAge) with functional assessments. In conclusion, neither rLTL nor 7-CpG DNAmAA were able to predict impairment in the analyzed assessments over a ~7 year time-course. Similarly, DNAmAA as estimated by five epigenetic clocks was not a good cross-sectional marker of health deterioration either.

It is well documented that some brain regions, such as association cortices, caudate, and hippocampus, are particularly prone to age-related atrophy, but it has been hypothesized that there are individual differences in atrophy profiles. Here, we document heterogeneity in regional-atrophy patterns using latent-profile analysis of 1,482 longitudinal magnetic resonance imaging observations. The results supported a 2-group solution reflecting differences in atrophy rates in cortical regions and hippocampus along with comparable caudate atrophy. The higher-atrophy group had the most marked atrophy in hippocampus and also lower episodic memory, and their normal caudate atrophy rate was accompanied by larger baseline volumes. Our findings support and refine models of heterogeneity in brain aging and suggest distinct mechanisms of atrophy in striatal versus hippocampal-cortical systems.

DNA methylation age (DNAm age, epigenetic clock) is a novel and promising biomarker of aging. It is calculated from the methylation fraction of specific cytosine phosphate guanine sites (CpG sites) of genomic DNA. Several groups have proposed epigenetic clock algorithms and these differ mostly regarding the number and location of the CpG sites considered and the method used to assess the methylation status. Most epigenetic clocks are based on a large number of CpGs, e.g. as measured by DNAm microarrays. We have recently evaluated an epigenetic clock based on the methylation fraction of seven CpGs that were determined by methylation-sensitive single nucleotide primer extension (MS-SNuPE). This method is more cost-effective when compared to array-based technologies as only a few CpGs need to be examined. However, there is only little data on the correspondence in epigenetic age estimation using the 7-CpG clock and other algorithms. To bridge this gap, in this study we measured the 7-CpG DNAm age using two methods, via MS-SNuPE and via the MethylationEPIC array, in a sample of 1,058 participants of the Berlin Aging Study II (BASE-II), assessed as part of the GendAge study. On average, participants were 75.6 years old (SD: 3.7, age range: 64.9–90.0, 52.6% female). Agreement between methods was assessed by Bland-Altman plots. DNAm age was highly correlated between methods (Pearson’s r = 0.9) and Bland-Altman plots showed a difference of 3.1 years. DNAm age by the 7-CpG formula was 71.2 years (SD: 6.9 years, SNuPE) and 68.1 years (SD: 6.4 years, EPIC array). The mean of difference in methylation fraction between methods for the seven individual CpG sites was between 0.7 and 13 percent. To allow direct conversion of DNAm age obtained from both methods we developed an adjustment formula with a randomly selected training set of 529 participants using linear regression. After conversion of the Illumina data in a second and independent validation set, the adjusted DNAm age was 71.44 years (SD: 6.1 years, n = 529). In summary, we found the results of DNAm clocks to be highly comparable. Furthermore, we developed an adjustment formula that allows for direct conversion of DNAm age estimates between methods and enables one singular clock to be used in studies that employ either the Illumina or the SNuPE method.

Epigenome-wide association studies (EWAS) assessing the link between DNA methylation (DNAm) and phenotypes related to structural brain measures, cognitive function, and neurodegenerative diseases are becoming increasingly more popular. Due to the inaccessibility of brain tissue in humans, several studies use peripheral tissues such as blood, buccal swabs, and saliva as surrogates. To aid the functional interpretation of EWAS findings in such settings, there is a need to assess the correlation of DNAm variability across tissues in the same individuals. In this study, we performed a correlation analysis between DNAm data of a total of n = 120 matched post-mortem buccal and prefrontal cortex samples. We identified nearly 25,000 (3% of approximately 730,000) cytosine-phosphate-guanine (CpG) sites showing significant (false discovery rate q < 0.05) correlations between buccal and PFC samples. Correlated CpG sites showed a preponderance to being located in promoter regions and showed a significant enrichment of being determined by genetic factors, i.e. methylation quantitative trait loci (mQTL), based on buccal and dorsolateral prefrontal cortex mQTL databases. Our novel buccal–brain DNAm correlation map will provide a valuable resource for future EWAS using buccal samples for studying DNAm effects on phenotypes relating to the brain. All correlation results are made freely available to the public online.

Adverse effects of low vitamin D level on mortality and morbidity are controversially discussed. Especially older people are at risk for vitamin D deficiency and therefore exposed to its potentially harmful consequences. A way of measuring differences in the biological age is through DNA methylation age (DNAm age) and its deviation from chronological age, DNAm age acceleration (DNAmAA). We previously reported on an association between vitamin D deficiency and higher 7-CpG DNAmAA in participants of the Berlin Aging Study II (BASE-II). In this study, we employ a quasi-interventional study design to assess the relationship between DNAmAA of five epigenetic clocks and vitamin D supplementation. Longitudinal data were available for 1,036 participants of BASE-II that were reexamined on average 7.4 years later in the GendAge study (mean age at follow-up: 75.6 years, SD = 3.8 years, age range: 64.9–94.1 years, 51.9% female). DNAmAA was estimated with the 7-CpG clock, Horvath’s clock, Hannum’s clock, PhenoAge, and GrimAge. Methylation data were obtained through methylation-sensitive single nucleotide primer extension (MS-SNuPE) or Illumina’s Infinium “MethylationEPIC” array. Vitamin D–deficient participants who chose to start vitamin D supplementation after baseline examination showed a 2.6-year lower 7-CpG DNAmAA (p = 0.011) and 1.3-year lower Horvath DNAmAA (p = 0.042) compared to untreated and vitamin D–deficient participants. DNAmAA did not statistically differ between participants with successfully treated vitamin D deficiency and healthy controls (p > 0.16). Therefore, we conclude that intake of vitamin D supplement is associated with lower DNAmAA in participants with vitamin D deficiency.

Adverse effects of psychological stress on physical and mental health, especially in older age, are well documented. How perceived stress relates to the epigenetic clock measure, DNA methylation age acceleration (DNAmAA), is less well understood and existing studies reported inconsistent results. DNAmAA was estimated from five epigenetic clocks (7-CpG, Horvath's, Hannum's, PhenoAge and GrimAge DNAmAA). Cohen's Perceived Stress Scale (PSS) was used as marker of psychological stress. We analyzed data from 1,100 Berlin Aging Study II (BASE-II) participants assessed as part of the GendAge study (mean age = 75.6 years, SD = 3.8 years, 52.1% women). In a first step, we replicated well-established associations of perceived stress with morbidity, frailty, and symptoms of depression in the BASE-II cohort studied here. In a second step, we did not find any statistically significant association of perceived stress with any of the five epigenetic clocks in multiple linear regression analyses that adjusted for covariates. Although the body of literature suggests an association between higher DNAmAA and stress or trauma during early childhood, the current study found no evidence for an association of perception of stress with DNAmAA in older people. We discuss possible reasons for the lack of associations and highlight directions for future research.