Implementation of modern methods to identify DNA methylation marks for episodic memory impairment and Alzheimer’s disease

Alzheimer’s disease (AD) is a progressive neurodegenerative disease and a common cause of dementia worldwide. The pathogenic mechanisms of AD are not completely understood. Both genetic and external factors, like lifestyle and other diseases, can alter AD risk. The impact of external factors may be mediated by epigenetic mechanisms, like DNA methylation. The first clinical symptoms of AD usually manifest as episodic memory impairment (EMI), however, AD can be diagnosed only at a later stage when neurodegeneration is far-progressed. New methods are required to enable early diagnosis and characterization of the disease mechanisms. The main goals of this study were to implement new methods for characterization of disease marks and mechanisms and to identify new DNA methylation marks for EMI and AD with these methods. Reduced Representation Bisulfite Sequencing (RRBS) was first implemented by utilising human embryonic stem cells and by identifying DNA methylation changes in these cells during transformation to abnormal karyotype. RRBS was then utilised to identify blood DNA methylation marks for AD in Finnish disease discordant twin pairs. Such marks were detected in eleven genomic regions and the one in adenosine deaminase RNA specific B2 gene (ADARB2) was validated in Swedish twin cohorts. A new twin sample cohort was collected and genome-wide bisulphite sequencing method was implemented to identify plasma cell-free DNA methylation marks for EMI. No markers were detected. Patient-specific induced pluripotent stem cell lines and a brain organoid model were generated to study the pathogenic mechanisms of AD. In conclusion, the results show that DNA methylation marks associated with AD can be detected not only in the brain but also in blood, however, more research is required to evaluate whether these marks can be utilised in diagnostics. Several methods enabling identification of disease marks were successfully implemented, samples from a new cohort were collected and pluripotent stem cell-based model was established to study AD. These resources will be valuable for future research aiming to identify mechanisms and markers for AD.Alzheimerin tauti (AT) on keskushermoston rappeumaan johtava sairaus, joka aiheuttaa yli puolet maailman dementiatapauksista. Taudin aiheuttajia ei vielä täysin tunneta. Sekä perintötekijät että ulkoiset tekijät, kuten elintavat ja muut sairaudet vaikuttavat AT-riskiin. Ulkoisten tekijöiden vaikutus saattaa johtua muutoksista epigeneettisissä mekanismeissa, kuten DNA:n metylaatiossa. Episodisen muistin heikkeneminen (EMH) aiheuttaa yleensä AT:n ensimmäiset kliiniset oireet, mutta sairaus pystytään diagnosoimaan vasta huomattavasti myöhemmin, jolloin hermo-solutuho on edennyt jo pitkälle. Tarvitaan uusia menetelmiä AT:n mekanismien tutkimiseen sekä sairauden varhaiseen diagnosointiin. Tämän tutkimuksen päätavoitteina oli ottaa käyttöön uusia menetelmiä tautimarkkerien ja -mekanismien tutkimiseen sekä etsiä niiden avulla DNA-metylaatiomarkkereita AT:lle sekä EMH:lle. RRBS (Reduced Representation Bisulfite Sequencing) otettiin ensin käyttöön tutkimalla DNA-metylaatiomuutoksia ihmisen alkion kantasoluissa niiden karyotyyppien muuttuessa epänormaaleiksi. Seuraavaksi RRBS:n avulla tunnistettiin AT:hen liittyviä metylaatiomarkkereita veressä suomalaisilla kaksospareilla, joista toisella on AT ja toisella ei. Markkereita havaittiin 11 genomin alueella. Näistä yksi, joka sijaitsi adenosine deaminase RNA specific B2 -geenissä (ADARB2), validoitiin ruotsalaisessa kaksosaineistossa. Keräsimme näytteitä uuteen kaksosaineistoon, jossa tutkimme EMH:hon liittyviä metylaatiomarkkereita plasman soluvapaassa DNA:ssa. Merkittäviä markkereita ei havaittu. Lopuksi perustimme indusoidut pluripotentit kantasolulinjat sekä aivo-kudosmallin AT:hen liittyvien mekanismien tutkimista varten. Tämän tutkimuksen aikana otettiin käyttöön useita uusimpia genomin ja epigenomin tutkimusmenetelmiä. Keräsimme arvokkaan näyteaineiston, jota voidaan hyödyntää muistin heikentymisen tutkimuksessa. Tutkimuksen tulosten mukaan AT:hen liittyviä DNA-metylaatiomuutoksia on havaittavissa aivokudoksen lisäksi myös veressä. Tarvitaan kuitenkin lisätutkimuksia selvittämään, voidaanko metylaatiomarkkereita hyödyntää AT:n diagnostiikassa

LuxRep: a technical replicate-aware method for bisulfite sequencing data analysis

Background: DNA methylation is commonly measured using bisulfite sequencing (BS-seq). The quality of a BS-seq library is measured by its bisulfite conversion efficiency. Libraries with low conversion rates are typically excluded from analysis resulting in reduced coverage and increased costs.Results: We have developed a probabilistic method and software, LuxRep, that implements a general linear model and simultaneously accounts for technical replicates (libraries from the same biological sample) from different bisulfite-converted DNA libraries. Using simulations and actual DNA methylation data, we show that including technical replicates with low bisulfite conversion rates generates more accurate estimates of methylation levels and differentially methylated sites. Moreover, using variational inference speeds up computation time necessary for whole genome analysis.Conclusions: In this work we show that taking into account technical replicates (i.e. libraries) of BS-seq data of varying bisulfite conversion rates, with their corresponding experimental parameters, improves methylation level estimation and differential methylation detection.</p

Placental DNA methylation marks are associated with maternal depressive symptoms during early pregnancy

Maternal depressive symptoms during pregnancy are a significant risk factor for adverse developmental and health outcomes of the offspring. The molecular mechanisms mediating the long-term effects of this exposure are not well understood. Previous studies have found association between prenatal exposure to maternal psychological distress and placental DNA methylation of candidate genes, which can influence placental barrier function and development of the fetus. Our objective in this study was to determine epigenome wide association of maternal depressive symptoms in early pregnancy with the placental DNA methylation. For this purpose we examined DNA methylomes of 92 placental samples by using reduced representation bisulfite sequencing. The placental samples were collected after deliveries of 39 girls and 59 boys, whose mothers had Edinburgh Postnatal Depression Score ranging from 0 to 19 at gestational week 14. According to our results maternal depressive symptoms are associated with DNA methylation of 2833 CpG sites, which are particularly over-represented in genic enhancers. The genes overlapping or nearest to these sites are functionally enriched for development of neurons and show expression enrichment in several regions of developing brain. The genomic regions harboring the DNA methylation marks are enriched for single nucleotide polymorphisms associated with mental disease trait class. Potential cellular signaling cascades mediating the effects include inflammatory and hormonal pathways. As a conclusion our results suggest that maternal depressive symptoms during early pregnancy are associated with DNA methylation marks in placenta in genes, which are important for the development and long-term health of the brain. Whether similar marks can be detected in exposed children remains to be elucidated in further studies.</p

Umbilical cord blood DNA methylation in children who later develop type 1 diabetes

Aims/hypothesis Distinct DNA methylation patterns have recently been observed to precede type 1 diabetes in whole blood collected from young children. Our aim was to determine whether perinatal DNA methylation is associated with later progression to type 1 diabetes. Methods Reduced representation bisulphite sequencing (RRBS) analysis was performed on umbilical cord blood samples collected within the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) Study. Children later diagnosed with type 1 diabetes and/or who tested positive for multiple islet autoantibodies (n = 43) were compared with control individuals (n = 79) who remained autoantibody-negative throughout the DIPP follow-up until 15 years of age. Potential confounding factors related to the pregnancy and the mother were included in the analysis. Results No differences in the umbilical cord blood methylation patterns were observed between the cases and controls at a false discovery rate Conclusions/interpretation Based on our results, differences between children who progress to type 1 diabetes and those who remain healthy throughout childhood are not yet present in the perinatal DNA methylome. However, we cannot exclude the possibility that such differences would be found in a larger dataset.Peer reviewe

Peripheral blood DNA methylation differences in twin pairs discordant for Alzheimer's disease

Background Alzheimer's disease results from a neurodegenerative process that starts well before the diagnosis can be made. New prognostic or diagnostic markers enabling early intervention into the disease process would be highly valuable. Environmental and lifestyle factors largely modulate the disease risk and may influence the pathogenesis through epigenetic mechanisms, such as DNA methylation. As environmental and lifestyle factors may affect multiple tissues of the body, we hypothesized that the disease-associated DNA methylation signatures are detectable in the peripheral blood of discordant twin pairs. Results Comparison of 23 disease discordant Finnish twin pairs with reduced representation bisulfite sequencing revealed peripheral blood DNA methylation differences in 11 genomic regions with at least 15.0% median methylation difference and FDR adjusted p valuePeer reviewe

Peripheral blood DNA methylation differences in twin pairs discordant for Alzheimer's disease

Background Alzheimer's disease results from a neurodegenerative process that starts well before the diagnosis can be made. New prognostic or diagnostic markers enabling early intervention into the disease process would be highly valuable. Environmental and lifestyle factors largely modulate the disease risk and may influence the pathogenesis through epigenetic mechanisms, such as DNA methylation. As environmental and lifestyle factors may affect multiple tissues of the body, we hypothesized that the disease-associated DNA methylation signatures are detectable in the peripheral blood of discordant twin pairs. Results Comparison of 23 disease discordant Finnish twin pairs with reduced representation bisulfite sequencing revealed peripheral blood DNA methylation differences in 11 genomic regions with at least 15.0% median methylation difference and FDR adjusted p value Conclusions DNA methylation differences can be detected in the peripheral blood of twin pairs discordant for Alzheimer's disease. These DNA methylation signatures may have value as disease markers and provide insights into the molecular mechanisms of pathogenesis. We found no evidence that the DNA methylation marks would be associated with gene expression in blood. Further studies are needed to elucidate the potential importance of the associated genes in neuronal functions and to validate the prognostic or diagnostic value of the individual marks or marker panels.</p

Umbilical cord blood DNA methylation in children who later develop type 1 diabetes

Aims/hypothesis: Distinct DNA methylation patterns have recently been observed to precede type 1 diabetes in whole blood collected from young children. Our aim was to determine whether perinatal DNA methylation is associated with later progression to type 1 diabetes.Methods: Reduced representation bisulphite sequencing (RRBS) analysis was performed on umbilical cord blood samples collected within the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) Study. Children later diagnosed with type 1 diabetes and/or who tested positive for multiple islet autoantibodies (n = 43) were compared with control individuals (n = 79) who remained autoantibody-negative throughout the DIPP follow-up until 15 years of age. Potential confounding factors related to the pregnancy and the mother were included in the analysis.Results: No differences in the umbilical cord blood methylation patterns were observed between the cases and controls at a false discovery rate Conclusions/interpretation: Based on our results, differences between children who progress to type 1 diabetes and those who remain healthy throughout childhood are not yet present in the perinatal DNA methylome. However, we cannot exclude the possibility that such differences would be found in a larger dataset.</p

Analysis of Alzheimer's Disease Associated DNA Methylation Changes in Peripheral Blood with Reduced Representation Bisulphite Sequencing

Siirretty Doriast

LuxRep

Funding Information: We acknowledge the computational resources provided by the Aalto Science-IT project and the Finnish Functional Genomics Centre and Biocenter Finland. Funding Information: This work was supported by the Academy of Finland (292660, 311584, 335436). The funding body played no role in the design of the study, the collection, analysis, interpretation of data, or in writing the manuscript. Publisher Copyright: © 2022, The Author(s).Background: DNA methylation is commonly measured using bisulfite sequencing (BS-seq). The quality of a BS-seq library is measured by its bisulfite conversion efficiency. Libraries with low conversion rates are typically excluded from analysis resulting in reduced coverage and increased costs. Results: We have developed a probabilistic method and software, LuxRep, that implements a general linear model and simultaneously accounts for technical replicates (libraries from the same biological sample) from different bisulfite-converted DNA libraries. Using simulations and actual DNA methylation data, we show that including technical replicates with low bisulfite conversion rates generates more accurate estimates of methylation levels and differentially methylated sites. Moreover, using variational inference speeds up computation time necessary for whole genome analysis. Conclusions: In this work we show that taking into account technical replicates (i.e. libraries) of BS-seq data of varying bisulfite conversion rates, with their corresponding experimental parameters, improves methylation level estimation and differential methylation detection.Peer reviewe

From maternal glucocorticoid and thyroid hormones to epigenetic regulation of offspring gene expression : An experimental study in a wild bird species

Offspring phenotype at birth is determined by its genotype and the prenatal environment including exposure to maternal hormones. Variation in both maternal glucocorticoids and thyroid hormones can affect offspring phenotype, but the underlying molecular mechanisms, especially those contributing to long-lasting effects, remain unclear. Epigenetic changes (such as DNA methylation) have been postulated as mediators of long-lasting effects of early-life environment. In this study, we determined the effects of elevated prenatal glucocorticoid and thyroid hormones on handling stress response (breath rate) as well as DNA methylation and gene expression of glucocorticoid receptor (GR) and thyroid hormone receptor (THR) in great tits (Parus major). Eggs were injected before incubation onset with corticosterone (the main avian glucocorticoid) and/or thyroid hormones (thyroxine and triiodothyronine) to simulate variation in maternal hormone deposition. Breath rate during handling and gene expression of GR and THR were evaluated 14 days after hatching. Methylation status of GR and THR genes was analyzed from the longitudinal blood cells sampled 7 and 14 days after hatching, as well as the following autumn. Elevated prenatal corticosterone level significantly increased the breath rate during handling, indicating an enhanced metabolic stress response. Prenatal corticosterone manipulation had CpG-site-specific effects on DNA methylation at the GR putative promoter region, while it did not significantly affect GR gene expression. GR expression was negatively associated with earlier hatching date and chick size. THR methylation or expression did not exhibit any significant relationship with the hormonal treatments or the examined covariates, suggesting that TH signaling may be more robust due to its crucial role in development. This study provides some support to the hypothesis suggesting that maternal corticosterone may influence offspring metabolic stress response via epigenetic alterations, yet their possible adaptive role in optimizing offspring phenotype to the prevailing conditions, context-dependency, and the underlying molecular interplay needs further research.peerReviewe