Analysis Of DNA Methylation Patterns Research Articles

Resolving the 22q11.2 deletion using CTLR-Seq reveals chromosomal rearrangement mechanisms and individual variance in breakpoints

We developed a generally applicable method, CRISPR/Cas9-targeted long-read sequencing (CTLR-Seq), to resolve, haplotype-specifically, the large and complex regions in the human genome that had been previously impenetrable to sequencing analysis, such as large segmental duplications (SegDups) and their associated genome rearrangements. CTLR-Seq combines in vitro Cas9-mediated cutting of the genome and pulse-field gel electrophoresis to isolate intact large (i.e., up to 2,000 kb) genomic regions that encompass previously unresolvable genomic sequences. These targets are then sequenced (amplification-free) at high on-target coverage using long-read sequencing, allowing for their complete sequence assembly. We applied CTLR-Seq to the SegDup-mediated rearrangements that constitute the boundaries of, and give rise to, the 22q11.2 Deletion Syndrome (22q11DS), the most common human microdeletion disorder. We then performed de novo assembly to resolve, at base-pair resolution, the full sequence rearrangements and exact chromosomal breakpoints of 22q11.2DS (including all common subtypes). Across multiple patients, we found a high degree of variability for both the rearranged SegDup sequences and the exact chromosomal breakpoint locations, which coincide with various transposons within the 22q11.2 SegDups, suggesting that 22q11DS can be driven by transposon-mediated genome recombination. Guided by CTLR-Seq results from two 22q11DS patients, we performed three-dimensional chromosomal folding analysis for the 22q11.2 SegDups from patient-derived neurons and astrocytes and found chromosome interactions anchored within the SegDups to be both cell type-specific and patient-specific. Lastly, we demonstrated that CTLR-Seq enables cell-type specific analysis of DNA methylation patterns within the deletion haplotype of 22q11DS.

ETMR-23. CNS SARCOMAS WITHCIC- ORATXN1-ALTERATIONS — A META-ANALYSIS OF CLINICAL AND MOLECULAR FEATURES IN A LARGE SERIES OF PATIENTS

Abstract BACKGROUND In 2021, the 5th WHO classification of central nervous system (CNS) tumors acknowledged CNS sarcomas with CIC alterations as a distinct tumor type, which may also expand to non-CIC alterations including ATXN1. To tackle the lack of treatment protocols for these highly aggressive tumors, we conducted a meta-analysis to provide an overview of clinical and molecular characteristics, define clinically relevant subtypes, and ultimately advise on treatment guidelines. METHODS We collected molecular and clinical data for >200 patients with CIC- or ATXN1-altered CNS sarcomas by screening our in-house DNA methylation database (581 potential patients initially identified), publication databases (50 patients), and contacting centers worldwide (60 patients). Tumor tissue (FFPE and fresh frozen) for further molecular characterization was obtained from a subset of patients. DNA methylation-based clustering analyses (t-SNE, UMAP, consensus partitioning) were performed, integrating gene fusion details (RNAseq, FISH), copy-number variations, and clinical patient data to identify associations between (epi-)genetic and clinical parameters. RESULTS Most prominent fusion partners for CIC (n=47) and ATXN1 (n=9) were NUTM1 (n=18), LEUTX (n=17) and DUX4 (n=15). Clustering analyses of DNA methylation patterns indicated several epigenetic subtypes associated with different fusion types. Potential clinical implications are still under evaluation. Age distribution of patients at diagnosis in this cohort ranged from two months to 71 years (median=7.5 years). We observed no gender bias. Survival analyses are currently being performed, first results on survival and follow-up studies will be shown at the symposium. CONCLUSION We show first-of-its-kind data describing a large cohort of CNS sarcomas with CIC or ATXN1 alterations, outlining molecular and clinical characteristics, which may facilitate the development of guidelines for optimal treatment of these tumors based on retrospective response data. New molecular insights may also open up an avenue for targeted therapeutic approaches.

ScDMV: a zero-one inflated beta mixture model for DNA methylation variability with scBS-seq data.

The utilization of single-cell bisulfite sequencing (scBS-seq) methods allows for precise analysis of DNA methylation patterns at the individual cell level, enabling the identification of rare populations, revealing cell-specific epigenetic changes, and improving differential methylation analysis. Nonetheless, the presence of sparse data and an overabundance of zeros and ones, attributed to limited sequencing depth and coverage, frequently results in reduced precision accuracy during the process of differential methylation detection using scBS-seq. Consequently, there is a pressing demand for an innovative differential methylation analysis approach that effectively tackles these data characteristics and enhances recognition accuracy. We propose a novel beta mixture approach called scDMV for analyzing methylation differences in single-cell bisulfite sequencing data, which effectively handles excess zeros and ones and accommodates low-input sequencing. Our extensive simulation studies demonstrate that the scDMV approach outperforms several alternative methods in terms of sensitivity, precision, and controlling the false positive rate. Moreover, in real data applications, we observe that scDMV exhibits higher precision and sensitivity in identifying differentially methylated regions, even with low-input samples. In addition, scDMV reveals important information for GO enrichment analysis with single-cell whole-genome sequencing data that are often overlooked by other methods. The scDMV method, along with a comprehensive tutorial, can be accessed as an R package on the following GitHub repository: https://github.com/PLX-m/scDMV.

Characterization of Limnospira platensis PCC 9108 R-M and CRISPR-Cas systems

The filamentous cyanobacterium Limnospira platensis, formerly known as Arthrospira platensis or spirulina, is one of the most commercially important species of microalgae. Due to its high nutritional value, pharmacological and industrial applications it is extensively cultivated on a large commercial scale. Despite its widespread use, its precise manipulation is still under development due to the lack of effective genetic protocols. Genetic transformation of Limnospira has been attempted but the methods reported have not been generally reproducible in other laboratories. Knowledge of the transformation defense mechanisms is essential for understanding its physiology and for broadening their applications. With the aim to understand more about the genetic defenses of L. platensis, in this work we have identified the restriction-modification and CRISPR-Cas systems and we have cloned and characterized thirteen methylases. In parallel, we have also characterized the methylome and orphan methyltransferases using genome-wide analysis of DNA methylation patterns and RNA-seq. The identification and characterization of these enzymes will be a valuable resource to know how this strain avoids being genetically manipulated and for further genomics studies.

A qPCR technology for direct quantification of methylation in untreated DNA

DNA methylation is important for gene expression and alterations in DNA methylation are involved in the development and progression of cancer and other major diseases. Analysis of DNA methylation patterns has until now been dependent on either a chemical or an enzymatic pre-treatment, which are both time consuming procedures and potentially biased due to incomplete treatment. We present a qPCR technology, EpiDirect®, that allows for direct PCR quantification of DNA methylations using untreated DNA. EpiDirect® is based on the ability of Intercalating Nucleic Acids (INA®) to differentiate between methylated and unmethylated cytosines in a special primer design. With this technology, we develop an assay to analyze the methylation status of a region of the MGMT promoter used in treatment selection and prognosis of glioblastoma patients. We compare the assay to two bisulfite-relying, methyl-specific PCR assays in a study involving 42 brain tumor FFPE samples, revealing high sensitivity, specificity, and the clinical utility of the method.

Do not be scared of the genome's 5th base-Explaining phenotypic variability and evolutionary dynamics through DNA methylation analysis.

Epigenetic processes have taken center stage for the investigation of many biological processes, and epigenetic modifications have shown to influence phenotype, morphology and behavioural traits such as stress resistance by affecting gene regulation and expression without altering the underlying genomic sequence. The multiple molecular layers of epigenetics synergistically construct the cell type-specific gene regulatory networks, characterized by a high degree of plasticity and redundancy to create cell-type-specific morphology and function. DNA methylation occurring on the 5' carbon of cytosines in different genomic sequence contexts is the most studied epigenetic modification. DNA methylation has been shown to provide a molecular record of the exposure to a large variety of environmental factors, which might be persistent through the entire lifetime of an organism and even be passed onto the offspring. Animals might display altered phenotypes mediated by epigenetic modifications depending on the developmental stage or the environmental conditions as well as during evolution. Therefore, the analysis of DNA methylation patterns might allow deciphering previous exposures, explaining ecologically relevant phenotypic diversity and predicting evolutionary trajectories enabling accelerated adaption to changing environmental conditions. Despite the explanatory potential of DNA methylation integrating genetic and environmental factors to shape phenotypic variation and contribute significantly to evolutionary dynamics, studies of DNA methylation are still scarce in the field of ecology. This might be at least partly due to the complexity of DNA methylation analysis and the interpretation of the acquired data. In the current issue of Molecular Ecology Resources, Laine and colleagues (Molecular Ecology Resources, 2022) provide a detailed summary of guidelines and valuable recommendations for researchers in the field of ecology to avoid common pitfalls and perform interpretable genome-wide DNA methylation analyses.

An Integrative Analysis of DNA Methylation Pattern in Myotonic Dystrophy Type 1 Samples Reveals a Distinct DNA Methylation Profile between Tissues and a Novel Muscle-Associated Epigenetic Dysregulation.

Myotonic dystrophy type 1 (DM1) is a progressive, non-treatable, multi-systemic disorder. To investigate the contribution of epigenetics to the complexity of DM1, we compared DNA methylation profiles of four annotated CpG islands (CpGis) in the DMPK locus and neighbouring genes, in distinct DM1 tissues and derived cells, representing six DM1 subtypes, by bisulphite sequencing. In blood, we found no differences in CpGi 74, 43 and 36 in DNA methylation profile. In contrast, a CTCF1 DNA methylation gradient was found with 100% methylation in congenital cases, 50% in childhood cases and 13% in juvenile cases. CTCF1 methylation correlated to disease severity and CTG expansion size. Notably, 50% of CTCF1 methylated cases showed methylation in the CTCF2 regions. Additionally, methylation was associated with maternal transmission. Interestingly, the evaluation of seven families showed that unmethylated mothers passed on an expansion of the CTG repeat, whereas the methylated mothers transmitted a contraction. The analysis of patient-derived cells showed that DNA methylation profiles were highly preserved, validating their use as faithful DM1 cellular models. Importantly, the comparison of DNA methylation levels of distinct DM1 tissues revealed a novel muscle-specific epigenetic signature with methylation of the CTCF1 region accompanied by demethylation of CpGi 43, a region containing an alternative DMPK promoter, which may decrease the canonical promoter activity. Altogether, our results showed a distinct DNA methylation profile across DM1 tissues and uncovered a novel and dual epigenetic signature in DM1 muscle samples, providing novel insights into the epigenetic changes associated with DM1.

A Longitudinal and Transancestral Analysis of DNA Methylation Patterns in Systemic Lupus Erythematosus

A Longitudinal and Transancestral Analysis of DNA Methylation Patterns in Systemic Lupus Erythematosus

Whole-Genome DNA Methylation Dynamics of Sheep Preimplantation Embryo Investigated by Single-Cell DNA Methylome Sequencing.

The early stages of mammalian embryonic development involve the participation and cooperation of numerous complex processes, including nutritional, genetic, and epigenetic mechanisms. However, in embryos cultured in vitro, a developmental block occurs that affects embryo development and the efficiency of culture. Although the block period is reported to involve the transcriptional repression of maternal genes and transcriptional activation of zygotic genes, how epigenetic factors regulate developmental block is still unclear. In this study, we systematically analyzed whole-genome methylation levels during five stages of sheep oocyte and preimplantation embryo development using single-cell level whole genome bisulphite sequencing (SC-WGBS) technology. Then, we examined several million CpG sites in individual cells at each evaluated developmental stage to identify the methylation changes that take place during the development of sheep preimplantation embryos. Our results showed that two strong waves of methylation changes occurred, namely, demethylation at the 8-cell to 16-cell stage and methylation at the 16-cell to 32-cell stage. Analysis of DNA methylation patterns in different functional regions revealed a stable hypermethylation status in 3′UTRs and gene bodies; however, significant differences were observed in intergenic and promoter regions at different developmental stages. Changes in methylation at different stages of preimplantation embryo development were also compared to investigate the molecular mechanisms involved in sheep embryo development at the methylation level. In conclusion, we report a detailed analysis of the DNA methylation dynamics during the development of sheep preimplantation embryos. Our results provide an explanation for the complex regulatory mechanisms underlying the embryo developmental block based on changes in DNA methylation levels.

PLZF Acetylation Levels Regulate NKT Cell Differentiation.

The transcription factor promyelocytic leukemia zinc finger (PLZF) is encoded by the BTB domain-containing 16 (Zbtb16) gene. Its repressor function regulates specific transcriptional programs. During the development of invariant NKT cells, PLZF is expressed and directs their effector program, but the detailed mechanisms underlying PLZF regulation of multistage NKT cell developmental program are not well understood. This study investigated the role of acetylation-induced PLZF activation on NKT cell development by analyzing mice expressing a mutant form of PLZF mimicking constitutive acetylation (PLZFON) mice. NKT populations in PLZFON mice were reduced in proportion and numbers of cells, and the cells present were blocked at the transition from developmental stage 1 to stage 2. NKT cell subset differentiation was also altered, with T-bet+ NKT1 and RORγt+ NKT17 subsets dramatically reduced and the emergence of a T-bet-RORγt- NKT cell subset with features of cells in early developmental stages rather than mature NKT2 cells. Preliminary analysis of DNA methylation patterns suggested that activated PLZF acts on the DNA methylation signature to regulate NKT cells' entry into the early stages of development while repressing maturation. In wild-type NKT cells, deacetylation of PLZF is possible, allowing subsequent NKT cell differentiation. Interestingly, development of other innate lymphoid and myeloid cells that are dependent on PLZF for their generation is not altered in PLZFON mice, highlighting lineage-specific regulation. Overall, we propose that specific epigenetic control of PLZF through acetylation levels is required to regulate normal NKT cell differentiation.

Exploring epigenetic biomarkers of universal specificities and commonalities among pan-cancer cohorts in The Cancer Genome Atlas.

Aim: To explore the mechanism of cancer by employing a comprehensive analysis of DNA methylationpatterns and variations among pan-cancer cohorts. Materials&methods: This research focused on the discovery of universally specific or common biomarkers by mathematical statistics and machine learning methods in The Cancer Genome Atlas. Results: We found 138 differently methylated CpGs (DMCs)with a common methylation trend and eight common differently methylated regionsin different cancer cohorts. Additionally, we found 99 DMCs to distinguish 32 different cancer cohorts in random forestanalysis because of the specificity mechanism, but each DMC still had high instability. Conclusion: Our results could facilitate the development of biomarkers that are universally specific and common features across pan-cancer cohorts.

DNA methylation epitypes highlight underlying developmental and disease pathways in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a molecularly complex disease characterized by heterogeneous tumor genetic profiles and involving numerous pathogenic mechanisms and pathways. Integration of molecular data types across multiple patient cohorts may advance current genetic approaches for improved subclassification and understanding of the biology of the disease. Here, we analyzed genome-wide DNA methylation in 649 AML patients using Illumina arrays and identified a configuration of 13 subtypes (termed “epitypes”) using unbiased clustering. Integration of genetic data revealed that most epitypes were associated with a certain recurrent mutation (or combination) in a majority of patients, yet other epitypes were largely independent. Epitypes showed developmental blockage at discrete stages of myeloid differentiation, revealing epitypes that retain arrested hematopoietic stem-cell-like phenotypes. Detailed analyses of DNA methylation patterns identified unique patterns of aberrant hyper- and hypomethylation among epitypes, with variable involvement of transcription factors influencing promoter, enhancer, and repressed regions. Patients in epitypes with stem-cell-like methylation features showed inferior overall survival along with up-regulated stem cell gene expression signatures. We further identified a DNA methylation signature involving STAT motifs associated with FLT3-ITD mutations. Finally, DNA methylation signatures were stable at relapse for the large majority of patients, and rare epitype switching accompanied loss of the dominant epitype mutations and reversion to stem-cell-like methylation patterns. These results show that DNA methylation-based classification integrates important molecular features of AML to reveal the diverse pathogenic and biological aspects of the disease.

Cytokine-like protein 1–induced survival of monocytes suggests a combined strategy targeting MCL1 and MAPK in CMML

AbstractMouse models of chronic myeloid malignancies suggest that targeting mature cells of the malignant clone disrupts feedback loops that promote disease expansion. Here, we show that in chronic myelomonocytic leukemia (CMML), monocytes that accumulate in the peripheral blood show a decreased propensity to die by apoptosis. BH3 profiling demonstrates their addiction to myeloid cell leukemia-1 (MCL1), which can be targeted with the small molecule inhibitor S63845. RNA sequencing and DNA methylation pattern analysis both point to the implication of the mitogen-activated protein kinase (MAPK) pathway in the resistance of CMML monocytes to death and reveal an autocrine pathway in which the secreted cytokine-like protein 1 (CYTL1) promotes extracellular signal-regulated kinase (ERK) activation through C-C chemokine receptor type 2 (CCR2). Combined MAPK and MCL1 inhibition restores apoptosis of monocytes from patients with CMML and reduces the expansion of patient-derived xenografts in mice. These results show that the combined inhibition of MCL1 and MAPK is a promising approach to slow down CMML progression by inducing leukemic monocyte apoptosis.

DNA methylation studies in cattle.

Investigation of the role of epigenetics in cattle breeding is gaining importance. DNA methylation represents an epigenetic modification which is essential for genomic stability and maintenance of development. Recently, DNA methylation research in cattle has intensified. The studies focus on the definition of methylomes in various organs and tissues in relation to the expression of genes underlying economically important traits, and explore methylome changes under developmental, environmental, disease, and diet influences. The investigations further characterize the methylation patterns of gametes in connection with their quality, and study methylome alterations in the developing naturally or assisted produced zygotes, embryos, and fetuses, considering their viability. A wide array of technologies developed for accurate and precise analysis of DNA methylation patterns is employed for both single-gene and genome-wide studies. Overall, the research is directed towards the identification of single methylation markers or their combinations which may be useful in the selection and breeding of animals to ensure cattle improvement.

Temporal genome-wide DNA methylation signature of post-smolt Pacific salmon challenged with Piscirickettsia salmonis

ABSTRACT Piscirickettsiosis is the most important bacterial disease in the Chilean salmon industry, which has sorted several efforts to its control, generating enormous economic losses. Epigenetic alterations, such as DNA methylation, can play a relevant role in the modulation of the metazoans response to pathogens. Bacterial disease may activate global and local immune responses generating intricate responses with significant biological impact in the host. However, it is scarcely understood how bacterial infections influence fish epigenetic alterations. In the present study, we utilized Pacific salmon and Piscirickettsiosis as model, to gain understanding into the dynamics of DNA methylation among fish-bacterial infection interactions. A genome-wide analysis of DNA methylation patterns in female spleen tissue of Pacific salmon was achieved by reduced representation bisulphite sequencing from a time course design. We determined 2,251, 1,918, and 2,516 differentially methylated regions DMRs among infected and control Pacific salmon in 1 dpi, 5 dpi, and 15 dpi, respectively. The mean methylation difference per DMR among control and infected groups was of ~35%, with an oscillatory pattern of hypo, hyper, and hypomethylation across the disease. DMCs, among the control and infected group, showed that they were statistically enriched in intergenic regions and depleted in exons. Functional annotation of the DMR genes demonstrated three KEGG principal categories, associated directly with the host response to pathogens infections. Our results provide the first evidence of epigenetic variation in fish provoked by bacterial infection and demonstrate that this variation can be modulated across the disease.

DNA Methylomes and Epigenetic Age Acceleration Associations with Poor Metabolic Control in T1D.

Type 1 diabetes (T1D) is an autoimmune disease that leads to insulin deficiency and hyperglycemia. Little is known about how this metabolic dysfunction, which substantially alters the internal environment, forces cells to adapt through epigenetic mechanisms. Consequently, the purpose of this work was to study what changes occur in the epigenome of T1D patients after the onset of disease and in the context of poor metabolic control. We performed a genome-wide analysis of DNA methylation patterns in blood samples from 18 T1D patients with varying levels of metabolic control. We identified T1D-associated DNA methylation differences on more than 100 genes when compared with healthy controls. Interestingly, only T1D patients displaying poor glycemic control showed epigenetic age acceleration compared to healthy controls. The epigenetic alterations identified in this work make a valuable contribution to improving our understanding of T1D and to ensuring the appropriate management of the disease in relation to maintaining healthy aging.

A longitudinal and transancestral analysis of DNA methylation patterns and disease activity in lupus patients.

Epigenetic dysregulation is implicated in the pathogenesis of lupus. We performed a longitudinal analysis to assess changes in DNA methylation in lupus neutrophils over 4 years of follow-up and across disease activity levels using 229 patient samples. We demonstrate that DNA methylation profiles in lupus are partly determined by ancestry-associated genetic variations and are highly stable over time. DNA methylation levels in 2 CpG sites correlated significantly with changes in lupus disease activity. Progressive demethylation in SNX18 was observed with increasing disease activity in African American patients. Importantly, demethylation of a CpG site located within GALNT18 was associated with the development of active lupus nephritis. Differentially methylated genes between African American and European American lupus patients include type I IFN–response genes such as IRF7 and IFI44, and genes related to the NF-κB pathway. TREML4, which plays a vital role in TLR signaling, was hypomethylated in African American patients and demonstrated a strong cis–methylation quantitative trait loci (cis-meQTL) effect among 8855 cis-meQTL associations identified in our study.

Linking epigenetic signature and metabolic phenotype in IDH mutant and IDH wildtype diffuse glioma.

Changes in metabolism are known to contribute to tumour phenotypes. If and how metabolic alterations in brain tumours contribute to patient outcome is still poorly understood. Epigenetics impact metabolism and mitochondrial function. The aim of this study is a characterisation of metabolic features in molecular subgroups of isocitrate dehydrogenase mutant (IDHmut) and isocitrate dehydrogenase wildtype (IDHwt) gliomas. We employed DNA methylation pattern analyses with a special focus on metabolic genes, large-scale metabolism panel immunohistochemistry (IHC), qPCR-based determination of mitochondrial DNA copy number and immune cell content using IHC and deconvolution of DNA methylation data. We analysed molecularly characterised gliomas (n=57) for in depth DNA methylation, a cohort of primary and recurrent gliomas (n=22) for mitochondrial copy number and validated these results in a large glioma cohort (n=293). Finally, we investigated the potential of metabolic markers in Bevacizumab (Bev)-treated gliomas (n=29). DNA methylation patterns of metabolic genes successfully distinguished the molecular subtypes of IDHmut and IDHwt gliomas. Promoter methylation of lactate dehydrogenase A negatively correlated with protein expression and was associated with IDHmut gliomas. Mitochondrial DNA copy number was increased in IDHmut tumours and did not change in recurrent tumours. Hierarchical clustering based on metabolism panel IHC revealed distinct subclasses of IDHmut and IDHwt gliomas with an impact on patient outcome. Further quantification of these markers allowed for the prediction of survival under anti-angiogenic therapy. A mitochondrial signature was associated with increased survival in all analyses, which could indicate tumour subgroups with specific metabolic vulnerabilities.

Acupuncture alleviates chronic pain and comorbid conditions in a mouse model of neuropathic pain: the involvement of DNA methylation in the prefrontal cortex.

Chronic pain reduces life quality and is an important clinical problem associated with emotional and cognitive dysfunction. Epigenetic regulation of DNA methylation is involved in the induction of abnormal behaviors and pathological gene expression. We examined whether acupuncture can restore epigenetic changes caused by chronic pain, and identified the underlying mechanisms in neuropathic pain mice. Acupuncture treatment for 6 months (3 days/week) improved mechanical/cold allodynia and the emotional/cognitive dysfunction caused by left partial sciatic nerve ligation (PSNL)-induced neuropathic pain. The effects of acupuncture were associated with global DNA methylation recovery in the prefrontal cortex (PFC). Analysis of DNA methylation patterns in PFC indicated that 1364 overlapping genes among 4442 and 4416 methylated genes in the PSNL vs sham and PSNL vs acupuncture points groups, respectively, were highly associated with the DNA methylation process. Acupuncture restored the reduced expression of 5-methylcytosine, methyl-cytosine-phospho-guanine binding protein 2, and DNA methyltransferase family enzymes induced by PSNL in PFC. Methylation levels of Nr4a1 and Chkb associated with mitochondrial dysfunction were decreased in PFC of the PSNL mice, and increased by acupuncture. By contrast, high expression of Nr4a1 and Chkb mRNA in PSNL mice decreased after acupuncture. We also found that acupuncture inhibited the expression of Ras pathway-related genes such as Rasgrp1 and Rassf1. Finally, the expression of Nr4a1, Rasgrp1, Rassf1, and Chkb mRNA increased in the neuronal cells treated with Mecp2 small interfering RNA. These results suggest that acupuncture can relieve chronic pain-induced comorbid conditions by altering DNA methylation of Nr4a1, Rasgrp1, Rassf1, and Chkb in the PFC.

Postmortem age estimation via DNA methylation analysis in buccal swabs from corpses in different stages of decomposition\u2014a \u201cproof of principle\u201d study

Age estimation based on the analysis of DNA methylation patterns has become a focus of forensic research within the past few years. However, there is little data available regarding postmortem DNA methylation analysis yet, and literature mainly encompasses analysis of blood from corpses without any signs of decomposition. It is not entirely clear yet which other types of specimen are suitable for postmortem epigenetic age estimation, and if advanced decomposition may affect methylation patterns of CpG sites. In living persons, buccal swabs are an easily accessible source of DNA for epigenetic age estimation. In this work, the applicability of this approach (buccal swabs as source of DNA) under different postmortem conditions was tested. Methylation levels of PDE4C were investigated in buccal swab samples collected from 73 corpses (0–90 years old; mean: 51.2) in different stages of decomposition. Moreover, buccal swab samples from 142 living individuals (0–89 years old; mean 41.2) were analysed. As expected, methylation levels exhibited a high correlation with age in living individuals (training set: r2 = 0.87, validation set: r2 = 0.85). This was also the case in postmortem samples (r2 = 0.90), independent of the state of decomposition. Only in advanced putrified cases with extremely low DNA amounts, epigenetic age estimation was not possible. In conclusion, buccal swabs are a suitable and easy to collect source for DNA methylation analysis as long as sufficient amounts of DNA are present.