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Background: DNA methylation showed notable potential to act as a diagnostic marker in many cancers. Many studies proposed DNA methylation biomarker in OSCC detection, while most of these studies are limited to specific cohorts or geographical location. However, the generalizability of DNA methylation as a diagnostic marker in oral cancer across different geographical locations is yet to be investigated., Methods: We used genome-wide methylation data from 384 oral cavity cancer and normal tissues from TCGA HNSCC and eastern India. The common differentially methylated CpGs in these two cohorts were used to develop an Elastic-net model that can be used for the diagnosis of OSCC. The model was validated using 812 HNSCC and normal samples from different anatomical sites of oral cavity from seven countries. Droplet Digital PCR of methyl-sensitive restriction enzyme digested DNA (ddMSRE) was used for quantification of methylation and validation of the model with 22 OSCC and 22 contralateral normal samples. Additionally, pyrosequencing was used to validate the model using 46 OSCC and 25 adjacent normal and 21 contralateral normal tissue samples., Results: With ddMSRE, our model showed 91% sensitivity, 100% specificity, and 95% accuracy in classifying OSCC from the contralateral normal tissues. Validation of the model with pyrosequencing also showed 96% sensitivity, 91% specificity, and 93% accuracy for classifying the OSCC from contralateral normal samples, while in case of adjacent normal samples we found similar sensitivity but with 20% specificity, suggesting the presence of early disease methylation signature at the adjacent normal samples. Methylation array data of HNSCC and normal tissues from different geographical locations and different anatomical sites showed comparable sensitivity, specificity, and accuracy in detecting oral cavity cancer with across. Similar results were also observed for different stages of oral cavity cancer., Conclusions: Our model identified crucial genomic regions affected by DNA methylation in OSCC and showed similar accuracy in detecting oral cancer across different geographical locations. The high specificity of this model in classifying contralateral normal samples from the oral cancer compared to the adjacent normal samples suggested applicability of the model in early detection., (© 2024. The Author(s).)

The digital twin (DT) is a concept widely used in industry to create digital replicas of physical objects or systems. The dynamic, bi-directional link between the physical entity and its digital counterpart enables a real-time update of the digital entity. It can predict perturbations related to the physical object's function. The obvious applications of DTs in healthcare and medicine are extremely attractive prospects that have the potential to revolutionize patient diagnosis and treatment. However, challenges including technical obstacles, biological heterogeneity, and ethical considerations make it difficult to achieve the desired goal. Advances in multi-modal deep learning methods, embodied AI agents, and the metaverse may mitigate some difficulties. Here, we discuss the basic concepts underlying DTs, the requirements for implementing DTs in medicine, and their current and potential healthcare uses. We also provide our perspective on five hallmarks for a healthcare DT system to advance research in this field., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

Background: Restraining or slowing ageing hallmarks at the cellular level have been proposed as a route to increased organismal lifespan and healthspan. Consequently, there is great interest in anti-ageing drug discovery. However, this currently requires laborious and lengthy longevity analysis. Here, we present a novel screening readout for the expedited discovery of compounds that restrain ageing of cell populations in vitro and enable extension of in vivo lifespan., Methods: Using Illumina methylation arrays, we monitored DNA methylation changes accompanying long-term passaging of adult primary human cells in culture. This enabled us to develop, test, and validate the CellPopAge Clock, an epigenetic clock with underlying algorithm, unique among existing epigenetic clocks for its design to detect anti-ageing compounds in vitro. Additionally, we measured markers of senescence and performed longevity experiments in vivo in Drosophila, to further validate our approach to discover novel anti-ageing compounds. Finally, we bench mark our epigenetic clock with other available epigenetic clocks to consolidate its usefulness and specialisation for primary cells in culture., Results: We developed a novel epigenetic clock, the CellPopAge Clock, to accurately monitor the age of a population of adult human primary cells. We find that the CellPopAge Clock can detect decelerated passage-based ageing of human primary cells treated with rapamycin or trametinib, well-established longevity drugs. We then utilise the CellPopAge Clock as a screening tool for the identification of compounds which decelerate ageing of cell populations, uncovering novel anti-ageing drugs, torin2 and dactolisib (BEZ-235). We demonstrate that delayed epigenetic ageing in human primary cells treated with anti-ageing compounds is accompanied by a reduction in senescence and ageing biomarkers. Finally, we extend our screening platform in vivo by taking advantage of a specially formulated holidic medium for increased drug bioavailability in Drosophila. We show that the novel anti-ageing drugs, torin2 and dactolisib (BEZ-235), increase longevity in vivo., Conclusions: Our method expands the scope of CpG methylation profiling to accurately and rapidly detecting anti-ageing potential of drugs using human cells in vitro, and in vivo, providing a novel accelerated discovery platform to test sought after anti-ageing compounds and geroprotectors., (© 2024. The Author(s).)

Recently, DNA methylation clocks have been proven to be precise age predictors, and the application of these clocks in cancer tissue has revealed a global age acceleration in a majority of cancer subtypes when compared to normal tissue from the same individual. The polycomb repressor complex 2 plays a pivotal role in the aging process, and its targets have been shown to be enriched in CpG sites that gain methylation with age. This complex is further regulated by the chromatin remodeling complex SWItch/Sucrose Non-Fermentable and its core subunit, notably the tumor suppressor gene SMARCB1, which under physiological conditions inhibits the activity of the polycomb repressor complex 2. Hence, the loss of function of core members of the SWItch/sucrose non-fermentable complex, such as the tumor suppressor gene SMARCB1, results in increased activity of polycomb repressor complex 2 and interferes with the aging process. SMARCB1-deficient neoplasms represent a family of rare tumors, including amongst others malignant rhabdoid tumors, atypical teratoid and rhabdoid tumors, and epithelioid sarcomas. As aging pathways have recently been proposed as therapeutic targets for various cancer types, these tumors represent candidates for testing such treatments. Here, by deriving epigenetic age scores from more than 1000 tumor samples, we identified epigenetic age acceleration as a hallmark feature of epithelioid sarcoma. This observation highlights the potential of targeting aging pathways as an innovative treatment approach for patients with epithelioid sarcoma., (© 2024. The Author(s).)

Asteraceae is the world's richest plant family and is found on all continents, in environments ranging from the coast to the highest mountains. The family shows all growth forms and, as in other angiosperm families, species richness is concentrated in tropical regions. South America has the highest diversity of Asteraceae in the world, yet taxonomic and distributional knowledge gaps remain. This study compiles an updated catalog of Asteraceae native to South America, based on national and regional checklists and ongoing large-scale flora projects. The resulting checklist includes a total of 6,940 species and 564 genera native to South America to date, which represent about a quarter of the family's global diversity. Countries already considered to be megadiverse show the greatest diversity, such as Brazil with 2,095 species, followed by Peru (1,588), Argentina (1,377), and Colombia (1,244), with this diversity mainly focused on the Brazilian Highlands and the Andes. Species endemism also peaks in Brazil, but Sørensen distances reveal the Chilean flora to be eminently different from the rest of the continent. Tribes better represented in the continent are Eupatorieae, Senecioneae and Astereae, also with a remarkably presence of entirely South American subfamilies representing earliest diverging lineages of the Asteraceae, such as Barnadesioideae, Wunderlichioideae, Famatinanthoideae, and Stifftioideae. It is estimated that the discovery and description curves have not yet stabilized, and the number of species is likely to increase by 5 to 10% in the coming years, posing major challenges to continental-scale conservation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Moreira-Muñoz, Monge, Grossi, Ávila, Morales-Fierro, Heiden, Britto, Beck, Nakajima, Salgado, Rodríguez-Cravero and Gutiérrez.)

The cumulative number of stem cell divisions in a tissue, known as mitotic age, is thought to be a major determinant of cancer-risk. Somatic mutational and DNA methylation (DNAm) clocks are promising tools to molecularly track mitotic age, yet their relationship is underexplored and their potential for cancer risk prediction in normal tissues remains to be demonstrated. Here we build and validate an improved pan-tissue DNAm counter of total mitotic age called stemTOC. We demonstrate that stemTOC's mitotic age proxy increases with the tumor cell-of-origin fraction in each of 15 cancer-types, in precancerous lesions, and in normal tissues exposed to major cancer risk factors. Extensive benchmarking against 6 other mitotic counters shows that stemTOC compares favorably, specially in the preinvasive and normal-tissue contexts. By cross-correlating stemTOC to two clock-like somatic mutational signatures, we confirm the mitotic-like nature of only one of these. Our data points towards DNAm as a promising molecular substrate for detecting mitotic-age increases in normal tissues and precancerous lesions, and hence for developing cancer-risk prediction strategies., (© 2024. The Author(s).)

Allogeneic hematopoietic cell transplantation (HCT) is used to treat many blood-based disorders and malignancies, however it can also result in serious adverse events, such as the development of acute graft-versus-host disease (aGVHD). This study aimed to develop a donor-specific epigenetic classifier to reduce incidence of aGVHD by improving donor selection. Genome-wide DNA methylation was assessed in a discovery cohort of 288 HCT donors selected based on recipient aGVHD outcome; this cohort consisted of 144 cases with aGVHD grades III-IV and 144 controls with no aGVHD. We applied a machine learning algorithm to identify CpG sites predictive of aGVHD. Receiver operating characteristic (ROC) curve analysis of these sites resulted in a classifier with an encouraging area under the ROC curve (AUC) of 0.91. To test this classifier, we used an independent validation cohort (n = 288) selected using the same criteria as the discovery cohort. Attempts to validate the classifier failed with the AUC falling to 0.51. These results indicate that donor DNA methylation may not be a suitable predictor of aGVHD in an HCT setting involving unrelated donors, despite the initial promising results in the discovery cohort. Our work highlights the importance of independent validation of machine learning classifiers, particularly when developing classifiers intended for clinical use., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Webster, Ecker, Moghul, Liu, Dhami, Marzi, Paul, Kuxhausen, Lee, Spellman, Wang, Feber, Rakyan, Peggs and Beck.)

The majority of disease-associated variants identified through genome-wide association studies are located outside of protein-coding regions. Prioritizing candidate regulatory variants and gene targets to identify potential biological mechanisms for further functional experiments can be challenging. To address this challenge, we developed FORGEdb ( https://forgedb.cancer.gov/ ; https://forge2.altiusinstitute.org/files/forgedb.html ; and https://doi.org/10.5281/zenodo.10067458 ), a standalone and web-based tool that integrates multiple datasets, delivering information on associated regulatory elements, transcription factor binding sites, and target genes for over 37 million variants. FORGEdb scores provide researchers with a quantitative assessment of the relative importance of each variant for targeted functional experiments., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Aberrant DNA methylation (DNAm) is known to be associated with the aetiology of cancer, including colorectal cancer (CRC). In the past, the availability of open access data has been the main driver of innovative method development and research training. However, this is increasingly being eroded by the move to controlled access, particularly of medical data, including cancer DNAm data. To rejuvenate this valuable tradition, we leveraged DNAm data from 1,845 samples (535 CRC tumours, 522 normal colon tissues adjacent to tumours, 72 colorectal adenomas, and 716 normal colon tissues from healthy individuals) from 14 open access studies deposited in NCBI GEO and ArrayExpress. We calculated each sample's epigenetic age (EA) using eleven epigenetic clock models and derived the corresponding epigenetic age acceleration (EAA). For EA, we observed that most first- and second-generation epigenetic clocks reflect the chronological age in normal tissues adjacent to tumours and healthy individuals [e.g., Horvath ( r = 0.77 and 0.79), Zhang elastic net (EN) ( r = 0.70 and 0.73)] unlike the epigenetic mitotic clocks (EpiTOC, HypoClock, MiAge) ( r < 0.3). For EAA, we used PhenoAge, Wu, and the above mitotic clocks and found them to have distinct distributions in different tissue types, particularly between normal colon tissues adjacent to tumours and cancerous tumours, as well as between normal colon tissues adjacent to tumours and normal colon tissue from healthy individuals. Finally, we harnessed these associations to develop a classifier using elastic net regression (with lasso and ridge regularisations) that predicts CRC diagnosis based on a patient's sex and EAAs calculated from histologically normal controls (i.e., normal colon tissues adjacent to tumours and normal colon tissue from healthy individuals). The classifier demonstrated good diagnostic potential with ROC-AUC = 0.886, which suggests that an EAA-based classifier trained on relevant data could become a tool to support diagnostic/prognostic decisions in CRC for clinical professionals. Our study also reemphasises the importance of open access clinical data for method development and training of young scientists. Obtaining the required approvals for controlled access data would not have been possible in the timeframe of this study., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Widayati, Schneider, Panteleeva, Chernysheva, Hrbkova, Beck, Voloshin and Chervova.)

Background and Aims: Understanding diaspore morphology and how much a species invests on dispersal appendages is key for improving our knowledge of dispersal in fragmented habitats. We investigate diaspore morphological traits in high-Andean Compositae and their main abiotic and biotic drivers and test whether they play a role in species distribution patterns across the naturally fragmented high-Andean grasslands., Methods: We collected diaspore trait data for 125 Compositae species across 47 tropical high-Andean summits, focusing on achene length and pappus-to-achene length ratio, with the latter as a proxy of dispersal investment. We analysed the role of abiotic (temperature, elevation and latitude) and biotic factors (phylogenetic signal and differences between tribes) on diaspore traits and whether they are related to distribution patterns across the Andes, using phylogenomics, distribution modelling and community ecology analyses., Key Results: Seventy-five percent of the studied species show small achenes (length <3.3 mm) and 67% have high dispersal investment (pappus length at least two times the achene length). Dispersal investment increases with elevation, possibly to compensate for lower air density, and achene length increases towards the equator, where non-seasonal climate prevails. Diaspore traits show significant phylogenetic signal, and higher dispersal investment is observed in Gnaphalieae, Astereae and Senecioneae, which together represent 72% of our species. High-Andean-restricted species found across the tropical Andes have, on average, the pappus four times longer than the achene, a significantly higher dispersal investment than species present only in the northern Andes or only in the central Andes., Conclusions: Small achenes and high diaspore dispersal investment dominate among high-Andean Compositae, traits typical of mostly three tribes of African origin; but traits are also correlated with the environmental gradients within the high-Andean grasslands. Our results also suggest that diaspore dispersal investment is likely to shape species distribution patterns in naturally fragmented habitats., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Annals of Botany Company.)

We report a personalized tumor-informed technology, Patient-specific pROgnostic and Potential tHErapeutic marker Tracking (PROPHET) using deep sequencing of 50 patient-specific variants to detect molecular residual disease (MRD) with a limit of detection of 0.004%. PROPHET and state-of-the-art fixed-panel assays were applied to 760 plasma samples from 181 prospectively enrolled early stage non-small cell lung cancer patients. PROPHET shows higher sensitivity of 45% at baseline with circulating tumor DNA (ctDNA). It outperforms fixed-panel assays in prognostic analysis and demonstrates a median lead-time of 299 days to radiologically confirmed recurrence. Personalized non-canonical variants account for 98.2% with prognostic effects similar to canonical variants. The proposed tumor-node-metastasis-blood (TNMB) classification surpasses TNM staging for prognostic prediction at the decision point of adjuvant treatment. PROPHET shows potential to evaluate the effect of adjuvant therapy and serve as an arbiter of the equivocal radiological diagnosis. These findings highlight the potential advantages of personalized cancer techniques in MRD detection., Competing Interests: Declaration of interests Z.Z., C.W., X.L., D.P., F.Q., S.W., X.Z., and S.C. are employment and stock/stock option ownership in Burning Rock Biotech. The remaining authors declare no potential conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Epithelioid sarcoma is a rare and aggressive mesenchymal tumour, the genetic hallmark of which is the loss of expression of SMARCB1, a key member of the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodelling complex. Hampered by its rarity, epithelioid sarcoma has received little research attention and therapeutic options for this disease remain limited. SMARCB1-deficient tumours also include malignant rhabdoid tumour, atypical teratoid and rhabdoid tumour, epithelioid malignant peripheral nerve sheath tumour, and poorly differentiated chordoma. Histologically, it can be challenging to distinguish epithelioid sarcoma from malignant rhabdoid tumour and other SMARCB1-deficient tumours, whereas methylation profiling shows that they represent distinct entities and facilitates their classification. Methylation studies on SMARCB1-deficient tumours, although not including epithelioid sarcomas, reported methylation subgroups which resulted in new clinical stratification and therapeutic approaches. In addition, emerging evidence indicates that immunotherapy, including immune checkpoint inhibitors, represents a promising therapeutic strategy for SMARCB1-deficient tumours. Here, we show that some epithelioid sarcomas share methylation patterns of malignant rhabdoid tumours indicating that this could help to distinguish these entities and guide treatment. Using gene expression data, we also showed that the immune environment of epithelioid sarcoma is characterised by a predominance of CD8 + lymphocytes and M2 macrophages. These findings have potential implications for the management of patients with epithelioid sarcoma. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

Background: The feasibility of DNA methylation-based assays in detecting minimal residual disease (MRD) and postoperative monitoring remains unestablished. We aim to investigate the dynamic characteristics of cancer-related methylation signals and the feasibility of methylation-based MRD detection in surgical lung cancer patients., Methods: Matched tumor, tumor-adjacent tissues, and longitudinal blood samples from a cohort (MEDAL) were analyzed by ultra-deep targeted sequencing and bisulfite sequencing. A tumor-informed methylation-based MRD (timMRD) was employed to evaluate the methylation status of each blood sample. Survival analysis was performed in the MEDAL cohort (n = 195) and validated in an independent cohort (DYNAMIC, n = 36)., Results: Tumor-informed methylation status enabled an accurate recurrence risk assessment better than the tumor-naïve methylation approach. Baseline timMRD-scores were positively correlated with tumor burden, invasiveness, and the existence and abundance of somatic mutations. Patients with higher timMRD-scores at postoperative time-points demonstrated significantly shorter disease-free survival in the MEDAL cohort (HR: 3.08, 95% CI: 1.48-6.42; P = 0.002) and the independent DYNAMIC cohort (HR: 2.80, 95% CI: 0.96-8.20; P = 0.041). Multivariable regression analysis identified postoperative timMRD-score as an independent prognostic factor for lung cancer. Compared to tumor-informed somatic mutation status, timMRD-scores yielded better performance in identifying the relapsed patients during postoperative follow-up, including subgroups with lower tumor burden like stage I, and was more accurate among relapsed patients with baseline ctDNA-negative status. Comparing to the average lead time of ctDNA mutation, timMRD-score yielded a negative predictive value of 97.2% at 120 days prior to relapse., Conclusions: The dynamic methylation-based analysis of peripheral blood provides a promising strategy for postoperative cancer surveillance., Trial Registration: This study (MEDAL, MEthylation based Dynamic Analysis for Lung cancer) was registered on ClinicalTrials.gov on 08/05/2018 (NCT03634826). https://clinicaltrials.gov/ct2/show/NCT03634826 ., (© 2023. The Author(s).)

The crumbs cell polarity complex plays a crucial role in apical-basal epithelial polarity, cellular adhesion, and morphogenesis. Homozygous variants in human CRB1 result in autosomal recessive Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP), with no established genotype-phenotype correlation. The associated protein complexes have key functions in developmental pathways; however, the underlying disease mechanism remains unclear. Using the oko meduzy m289/m289 (crb2a -/- ) zebrafish, we performed integrative transcriptomic (RNA-seq data) and methylomic [reduced representation bisulphite sequencing (RRBS)] analysis of whole retina to identify dysregulated genes and pathways. Delayed retinal cell specification was identified in both the crb2a -/- zebrafish and CRB1 patient-derived retinal organoids, highlighting the dysfunction of cell cycle modulation and epigenetic transcriptional control. Differential DNA methylation analysis revealed novel hypermethylated pathways involving biological adhesion, Hippo, and transforming growth factor β (TGFβ) signalling. By integrating gene expression with DNA methylation using functional epigenetic modules (FEM), we identified six key modules involving cell cycle control and disturbance of TGFβ, bone morphogenetic protein (BMP), Hippo, and SMAD protein signal transduction pathways, revealing significant interactome hotspots relevant to crb2a function and confirming the epigenetic control of gene regulation in early retinal development, which points to a novel mechanism underlying CRB1-retinopathies. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

Intratumour heterogeneity (ITH) fuels lung cancer evolution, which leads to immune evasion and resistance to therapy 1 . Here, using paired whole-exome and RNA sequencing data, we investigate intratumour transcriptomic diversity in 354 non-small cell lung cancer tumours from 347 out of the first 421 patients prospectively recruited into the TRACERx study 2,3 . Analyses of 947 tumour regions, representing both primary and metastatic disease, alongside 96 tumour-adjacent normal tissue samples implicate the transcriptome as a major source of phenotypic variation. Gene expression levels and ITH relate to patterns of positive and negative selection during tumour evolution. We observe frequent copy number-independent allele-specific expression that is linked to epigenomic dysfunction. Allele-specific expression can also result in genomic-transcriptomic parallel evolution, which converges on cancer gene disruption. We extract signatures of RNA single-base substitutions and link their aetiology to the activity of the RNA-editing enzymes ADAR and APOBEC3A, thereby revealing otherwise undetected ongoing APOBEC activity in tumours. Characterizing the transcriptomes of primary-metastatic tumour pairs, we combine multiple machine-learning approaches that leverage genomic and transcriptomic variables to link metastasis-seeding potential to the evolutionary context of mutations and increased proliferation within primary tumour regions. These results highlight the interplay between the genome and transcriptome in influencing ITH, lung cancer evolution and metastasis., (© 2023. The Author(s).)

We evaluated associations between nine epigenetic age acceleration (EAA) scores and 18 cardiometabolic phenotypes using an Eastern European ageing population cohort richly annotated for a diverse set of phenotypes (subsample, n = 306; aged 45-69 years). This was implemented by splitting the data into groups with positive and negative EAAs. We observed strong association between all EAA scores and sex, suggesting that any analysis of EAAs should be adjusted by sex. We found that some sex-adjusted EAA scores were significantly associated with several phenotypes such as blood levels of gamma-glutamyl transferase and low-density lipoprotein, smoking status, annual alcohol consumption, multiple carotid plaques, and incident coronary heart disease status (not necessarily the same phenotypes for different EAAs). We demonstrated that even after adjusting EAAs for sex, EAA-phenotype associations remain sex-specific, which should be taken into account in any downstream analysis involving EAAs. The obtained results suggest that in some EAA-phenotype associations, negative EAA scores (i.e., epigenetic age below chronological age) indicated more harmful phenotype values, which is counterintuitive. Among all considered epigenetic clocks, GrimAge was significantly associated with more phenotypes than any other EA scores in this Russian sample.

Purpose: Rare disease diagnosis is challenging in medical image-based artificial intelligence due to a natural class imbalance in datasets, leading to biased prediction models. Inherited retinal diseases (IRDs) are a research domain that particularly faces this issue. This study investigates the applicability of synthetic data in improving artificial intelligence-enabled diagnosis of IRDs using generative adversarial networks (GANs)., Design: Diagnostic study of gene-labeled fundus autofluorescence (FAF) IRD images using deep learning., Participants: Moorfields Eye Hospital (MEH) dataset of 15 692 FAF images obtained from 1800 patients with confirmed genetic diagnosis of 1 of 36 IRD genes., Methods: A StyleGAN2 model is trained on the IRD dataset to generate 512 × 512 resolution images. Convolutional neural networks are trained for classification using different synthetically augmented datasets, including real IRD images plus 1800 and 3600 synthetic images, and a fully rebalanced dataset. We also perform an experiment with only synthetic data. All models are compared against a baseline convolutional neural network trained only on real data., Main Outcome Measures: We evaluated synthetic data quality using a Visual Turing Test conducted with 4 ophthalmologists from MEH. Synthetic and real images were compared using feature space visualization, similarity analysis to detect memorized images, and Blind/Referenceless Image Spatial Quality Evaluator (BRISQUE) score for no-reference-based quality evaluation. Convolutional neural network diagnostic performance was determined on a held-out test set using the area under the receiver operating characteristic curve (AUROC) and Cohen's Kappa (κ)., Results: An average true recognition rate of 63% and fake recognition rate of 47% was obtained from the Visual Turing Test. Thus, a considerable proportion of the synthetic images were classified as real by clinical experts. Similarity analysis showed that the synthetic images were not copies of the real images, indicating that copied real images, meaning the GAN was able to generalize. However, BRISQUE score analysis indicated that synthetic images were of significantly lower quality overall than real images ( P < 0.05). Comparing the rebalanced model (RB) with the baseline (R), no significant change in the average AUROC and κ was found (R-AUROC = 0.86[0.85-88], RB-AUROC = 0.88[0.86-0.89], R-k = 0.51[0.49-0.53], and RB-k = 0.52[0.50-0.54]). The synthetic data trained model (S) achieved similar performance as the baseline (S-AUROC = 0.86[0.85-87], S-k = 0.48[0.46-0.50])., Conclusions: Synthetic generation of realistic IRD FAF images is feasible. Synthetic data augmentation does not deliver improvements in classification performance. However, synthetic data alone deliver a similar performance as real data, and hence may be useful as a proxy to real data. Financial Disclosure(s): Proprietary or commercial disclosure may be found after the references., (© 2022 by the American Academy of Ophthalmology. Published by Elsevier Inc.)

Targeted bisulfite sequencing (TBS) has become the method of choice for the cost-effective, targeted analysis of the human methylome at base-pair resolution. In this study, we benchmarked five commercially available TBS platforms-three hybridization capture-based (Agilent, Roche and Illumina) and two reduced-representation-based (Diagenode and NuGen)-across 11 samples. Two samples were also compared with whole-genome DNA methylation sequencing with the Illumina and Oxford Nanopore platforms. We assessed workflow complexity, on/off-target performance, coverage, accuracy and reproducibility. Although all platforms produced robust and reproducible data, major differences in the number and identity of the CpG sites covered make it difficult to compare datasets generated on different platforms. To overcome this limitation, we applied imputation and show that it improves interoperability from an average of 10.35% (0.8 million) to 97% (7.6 million) common CpG sites. Our study provides guidance on which TBS platform to use for different methylome features and offers an imputation-based harmonization solution that allows comparative, integrative analysis., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Over the past decade, bioethicists, legal scholars and social scientists have started to investigate the potential implications of epigenetic research and technologies on medicine and society. There is growing literature discussing the most promising opportunities, as well as arising ethical, legal and social issues (ELSI). This paper explores the views of epigenetic researchers about some of these discussions. From January to March 2020, we conducted an online survey of 189 epigenetic researchers working in 31 countries. We questioned them about the scope of their field, opportunities in different areas of specialization, and ELSI in the conduct of research and knowledge translation. We also assessed their level of concern regarding four emerging non-medical applications of epigenetic testing-i.e., in life insurance, forensics, immigration and direct-to-consumer testing. Although there was strong agreement on DNA methylation, histone modifications, 3D structure of chromatin and nucleosomes being integral elements of the field, there was considerable disagreement on transcription factors, RNA interference, RNA splicing and prions. The most prevalent ELSI experienced or witnessed by respondents were in obtaining timely access to epigenetic data in existing databases, and in the communication of epigenetic findings by the media. They expressed high levels of concern regarding non-medical applications of epigenetics, echoing cautionary appraisals in the social sciences and humanities literature., (© 2022. The Author(s).)

Reliably documenting plant diversity is necessary to protect and sustainably benefit from it. At the heart of this documentation lie species concepts and the practical methods used to delimit taxa. Here, we apply a total-evidence, iterative methodology to delimit and document species in the South American genus Victoria (Nymphaeaceae). The systematics of Victoria has thus far been poorly characterized due to difficulty in attributing species identities to biological collections. This research gap stems from an absence of type material and biological collections, also the confused diagnosis of V. cruziana . With the goal of improving systematic knowledge of the genus, we compiled information from historical records, horticulture and geography and assembled a morphological dataset using citizen science and specimens from herbaria and living collections. Finally, we generated genomic data from a subset of these specimens. Morphological and geographical observations suggest four putative species, three of which are supported by nuclear population genomic and plastid phylogenomic inferences. We propose these three confirmed entities as robust species, where two correspond to the currently recognized V. amazonica and V. cruziana , the third being new to science, which we describe, diagnose and name here as V. boliviana Magdalena and L. T. Sm. Importantly, we identify new morphological and molecular characters which serve to distinguish the species and underpin their delimitations. Our study demonstrates how combining different types of character data into a heuristic, total-evidence approach can enhance the reliability with which biological diversity of morphologically challenging groups can be identified, documented and further studied., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Smith, Magdalena, Przelomska, Pérez-Escobar, Melgar-Gómez, Beck, Negrão, Mian, Leitch, Dodsworth, Maurin, Ribero-Guardia, Salazar, Gutierrez-Sibauty, Antonelli and Monro.)

In this article, we propose a system design and implementation for output-sensitive reconstruction, transmission and rendering of 3D video avatars in distributed virtual environments. In our immersive telepresence system, users are captured by multiple RGBD sensors connected to a server that performs geometry reconstruction based on viewing feedback from remote telepresence parties. This feedback and reconstruction loop enables visibility-aware level-of-detail reconstruction of video avatars regarding geometry and texture data, and considers individual and groups of collocated users. Our evaluation reveals that our approach leads to a significant reduction of reconstruction times, network bandwidth requirements and round-trip times as well as rendering costs in many situations.

Aim: Osteosarcoma (OS) is the most common primary bone tumour in children and adolescents. Circulating free (cfDNA) and circulating tumour DNA (ctDNA) are promising biomarkers for disease surveillance and prognostication in several cancer types; however, few such studies are reported for OS. The purpose of this study was to discover and validate methylation-based biomarkers to detect plasma ctDNA in patients with OS and explore their utility as prognostic markers., Methods: Candidate CpG markers were selected through analysis of methylation array data for OS, non-OS tumours and germline samples. Candidates were validated in two independent OS datasets (n = 162, n = 107) and the four top-performing markers were selected. Methylation-specific digital droplet PCR (ddPCR) assays were designed and experimentally validated in OS tumour samples (n = 20) and control plasma samples. Finally, ddPCR assays were applied to pre-operative plasma and where available post-operative plasma from 72 patients with OS, and findings correlated with outcome., Results: Custom ddPCR assays detected ctDNA in 69% and 40% of pre-operative plasma samples (n = 72), based on thresholds of one or two positive markers respectively. ctDNA was detected in 5/17 (29%) post-operative plasma samples from patients, which in four cases were associated with or preceded disease relapse. Both pre-operative cfDNA levels and ctDNA detection independently correlated with overall survival (p = 0.0015 and p = 0.0096, respectively)., Conclusion: Our findings illustrate the potential of mutation-independent methylation-based ctDNA assays for OS. This study lays the foundation for multi-institutional collaborative studies to explore the utility of plasma-derived biomarkers in the management of OS., Competing Interests: Conflict of interest statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2022. Published by Elsevier Ltd. All rights reserved.)

Currently, most advances in site-specific epigenetic editing for human use are concentrated in basic research, yet, there is considerable interest to translate this technology beyond the bench. This review highlights recent developments with epigenetic editing technology in comparison with the canonical CRISPR-Cas genome editing, as well as the epistemic and ethical considerations with preemptive translation of epigenetic editing into clinical or commercial use in humans. Key considerations in safety, equity, and access to epigenetic editing are highlighted, with a spotlight on the ethical, legal, and social issues of this technology in the context of global health equity.

Bulk-tissue DNA methylomes represent an average over many different cell types, hampering our understanding of cell-type-specific contributions to disease development. As single-cell methylomics is not scalable to large cohorts of individuals, cost-effective computational solutions are needed, yet current methods are limited to tissues such as blood. Here we leverage the high-resolution nature of tissue-specific single-cell RNA-sequencing datasets to construct a DNA methylation atlas defined for 13 solid tissue types and 40 cell types. We comprehensively validate this atlas in independent bulk and single-nucleus DNA methylation datasets. We demonstrate that it correctly predicts the cell of origin of diverse cancer types and discovers new prognostic associations in olfactory neuroblastoma and stage 2 melanoma. In brain, the atlas predicts a neuronal origin for schizophrenia, with neuron-specific differential DNA methylation enriched for corresponding genome-wide association study risk loci. In summary, the DNA methylation atlas enables the decomposition of 13 different human tissue types at a high cellular resolution, paving the way for an improved interpretation of epigenetic data., (© 2022. The Author(s).)

Background: Early detection of esophageal cancer is critical to improve survival. Whilst studies have identified biomarkers, their interpretation and validity is often confounded by cell-type heterogeneity., Results: Here we applied systems-epigenomic and cell-type deconvolution algorithms to a discovery set encompassing RNA-Seq and DNA methylation data from esophageal adenocarcinoma (EAC) patients and matched normal-adjacent tissue, in order to identify robust biomarkers, free from the confounding effect posed by cell-type heterogeneity. We identify 12 gene-modules that are epigenetically deregulated in EAC, and are able to validate all 12 modules in 4 independent EAC cohorts. We demonstrate that the epigenetic deregulation is present in the epithelial compartment of EAC-tissue. Using single-cell RNA-Seq data we show that one of these modules, a proto-cadherin module centered around CTNND2, is inactivated in Barrett's Esophagus, a precursor lesion to EAC. By measuring DNA methylation in saliva from EAC cases and controls, we identify a chemokine module centered around CCL20, whose methylation patterns in saliva correlate with EAC status., Conclusions: Given our observations that a CCL20 chemokine network is overactivated in EAC tissue and saliva from EAC patients, and that in independent studies CCL20 has been found to be overactivated in EAC tissue infected with the bacterium F. nucleatum, a bacterium that normally inhabits the oral cavity, our results highlight the possibility of using DNAm measurements in saliva as a proxy for changes occurring in the esophageal epithelium. Both the CTNND2/CCL20 modules represent novel promising network biomarkers for EAC that merit further investigation., (© 2022. The Author(s).)

We investigated the relationship between 'epigenetic age' (EA) derived from DNA methylation (DNAm) and myocardial infarction (MI)/acute coronary syndrome (ACS). A random population sample was examined in 2003/2005 ( n = 9360, 45-69, the HAPIEE project) and followed up for 15 years. From this cohort, incident MI/ACS (cases, n = 129) and age- and sex-stratified controls ( n = 177) were selected for a nested case-control study. Baseline EA (Horvath's, Hannum's, PhenoAge, Skin and Blood) and the differences between EA and chronological age (CA) were calculated (ΔAHr, ΔAHn, ΔAPh, ΔASB). EAs by Horvath's, Hannum's and Skin and Blood were close to CA (median absolute difference, MAD, of 1.08, -1.91 and -2.03 years); PhenoAge had MAD of -9.29 years vs. CA. The adjusted odds ratios (ORs) of MI/ACS per 1-year increments of ΔAHr, ΔAHn, ΔASB and ΔAPh were 1.01 (95% CI 0.95-1.07), 1.01 (95% CI 0.95-1.08), 1.02 (95% CI 0.97-1.06) and 1.01 (0.93-1.09), respectively. When classified into tertiles, only the highest tertile of ΔAPh showed a suggestion of increased risk of MI/ACS with OR 2.09 (1.11-3.94) independent of age and 1.84 (0.99-3.52) in the age- and sex-adjusted model. Metabolic modulation may be the likely mechanism of this association. In conclusion, this case-control study nested in a prospective population-based cohort did not find strong associations between accelerated epigenetic age markers and risk of MI/ACS. Larger cohort studies are needed to re-examine this important research question.

Background: Genome-wide association study (GWAS) single nucleotide polymorphisms (SNPs) are known to preferentially co-locate to active regulatory elements in tissues and cell types relevant to disease aetiology. Further characterisation of associated cell type-specific regulation can broaden our understanding of how GWAS signals may contribute to disease risk., Results: To gain insight into potential functional mechanisms underlying GWAS associations, we developed FORGE2 ( https://forge2.altiusinstitute.org/ ), which is an updated version of the FORGE web tool. FORGE2 uses an expanded atlas of cell type-specific regulatory element annotations, including DNase I hotspots, five histone mark categories and 15 hidden Markov model (HMM) chromatin states, to identify tissue- and cell type-specific signals. An analysis of 3,604 GWAS from the NHGRI-EBI GWAS catalogue yielded at least one significant disease/trait-tissue association for 2,057 GWAS, including > 400 associations specific to epigenomic marks in immune tissues and cell types, > 30 associations specific to heart tissue, and > 60 associations specific to brain tissue, highlighting the key potential of tissue- and cell type-specific regulatory elements. Importantly, we demonstrate that FORGE2 analysis can separate previously observed accessible chromatin enrichments into different chromatin states, such as enhancers or active transcription start sites, providing a greater understanding of underlying regulatory mechanisms. Interestingly, tissue-specific enrichments for repressive chromatin states and histone marks were also detected, suggesting a role for tissue-specific repressed regions in GWAS-mediated disease aetiology., Conclusion: In summary, we demonstrate that FORGE2 has the potential to uncover previously unreported disease-tissue associations and identify new candidate mechanisms. FORGE2 is a transparent, user-friendly web tool for the integrative analysis of loci discovered from GWAS., (© 2021. The Author(s).)

Rationale: Airway macrophages (AMs) are key regulators of the lung environment and are implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a fatal respiratory disease with no cure. However, knowledge about the epigenetics of AMs in IPF is limited. Objectives: To assess the role of epigenetic regulation of AMs during lung fibrosis. Methods: We undertook DNA methylation (DNAm) profiling by using Illumina EPIC (850k) arrays in sorted AMs from healthy donors ( n  = 14) and donors with IPF ( n  = 30). Cell-type deconvolution was performed by using reference myeloid-cell DNA methylomes. Measurements and Main Results: Our analysis revealed that epigenetic heterogeneity was a key characteristic of IPF AMs. DNAm "clock" analysis indicated that epigenetic alterations in IPF AMs were not associated with accelerated aging. In differential DNAm analysis, we identified numerous differentially methylated positions ( n  = 11) and differentially methylated regions ( n  = 49) between healthy and IPF AMs, respectively. Differentially methylated positions and differentially methylated regions encompassed genes involved in lipid (LPCAT1 [lysophosphatidylcholine acyltransferase 1]) and glucose (PFKFB3 [6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3]) metabolism, and importantly, the DNAm status was associated with disease severity in IPF. Conclusions: Collectively, our data identify that changes in the epigenome are associated with the development and function of AMs in the IPF lung.

Comparison of intratumor genetic heterogeneity in cancer at diagnosis and relapse suggests that chemotherapy induces bottleneck selection of subclonal genotypes. However, evolutionary events subsequent to chemotherapy could also explain changes in clonal dominance seen at relapse. We, therefore, investigated the mechanisms of selection in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) during induction chemotherapy where maximal cytoreduction occurs. To distinguish stochastic versus deterministic events, individual leukemias were transplanted into multiple xenografts and chemotherapy administered. Analyses of the immediate post-treatment leukemic residuum at single-cell resolution revealed that chemotherapy has little impact on genetic heterogeneity. Rather, it acts on extensive, previously unappreciated, transcriptional and epigenetic heterogeneity in BCP-ALL, dramatically reducing the spectrum of cell states represented, leaving a genetically polyclonal but phenotypically uniform population with hallmark signatures relating to developmental stage, cell cycle and metabolism. Hence, canalization of cell state accounts for a significant component of bottleneck selection during induction chemotherapy., Competing Interests: Competing Interest M.L was an employee of Fluidigm Corporation at the time of the study.

There is substantial variation in people's responses to adversity, with a considerable proportion of individuals displaying psychological resilience. Epigenetic mechanisms are hypothesised to be one molecular pathway of how adverse and traumatic events can become biologically embedded and contribute to individual differences in resilience. However, not much is known regarding the role of epigenetics in the development of psychological resilience. In this Review, we propose a new conceptual model for the different functions of epigenetic mechanisms in psychological resilience. The model considers the initial establishment of the epigenome, epigenetic modification due to adverse and protective environments, the role of protective factors in counteracting adverse influences, and genetic moderation of environmentally induced epigenetic modifications. After reviewing empirical evidence for the various components of the model, we identify research that should be prioritised and discuss practical implications of the proposed model for epigenetic research on resilience., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Diagnosing bone and soft tissue neoplasms remains challenging because of the large number of subtypes, many of which lack diagnostic biomarkers. DNA methylation profiles have proven to be a reliable basis for the classification of brain tumours and, following this success, a DNA methylation-based sarcoma classification tool from the Deutsches Krebsforschungszentrum (DKFZ) in Heidelberg has been developed. In this study, we assessed the performance of their classifier on DNA methylation profiles of an independent data set of 986 bone and soft tissue tumours and controls. We found that the 'DKFZ Sarcoma Classifier' was able to produce a diagnostic prediction for 55% of the 986 samples, with 83% of these predictions concordant with the histological diagnosis. On limiting the validation to the 820 cases with histological diagnoses for which the DKFZ Classifier was trained, 61% of cases received a prediction, and the histological diagnosis was concordant with the predicted methylation class in 88% of these cases, findings comparable to those reported in the DKFZ Classifier paper. The classifier performed best when diagnosing mesenchymal chondrosarcomas (CHSs, 88% sensitivity), chordomas (85% sensitivity), and fibrous dysplasia (83% sensitivity). Amongst the subtypes least often classified correctly were clear cell CHSs (14% sensitivity), malignant peripheral nerve sheath tumours (27% sensitivity), and pleomorphic liposarcomas (29% sensitivity). The classifier predictions resulted in revision of the histological diagnosis in six of our cases. We observed that, although a higher tumour purity resulted in a greater likelihood of a prediction being made, it did not correlate with classifier accuracy. Our results show that the DKFZ Classifier represents a powerful research tool for exploring the pathogenesis of sarcoma; with refinement, it has the potential to be a valuable diagnostic tool., (© 2021 The Authors. The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland & John Wiley & Sons, Ltd.)

Chronic kidney disease (CKD) related cardiovascular disease (CVD) is characterized by vascular remodelling with well-established structural and functional changes in the vascular wall such as arterial stiffness, matrix deposition, and calcification. These phenotypic changes resemble pathology seen in ageing, and are likely to be mediated by sustained alterations in gene expression, which may be caused by epigenetic changes such as tissue-specific DNA methylation. We aimed to investigate tissue specific changes in DNA methylation that occur in CKD-related CVD. Genome-wide DNA methylation changes were examined in bisulphite converted genomic DNA isolated from the vascular media of CKD and healthy arteries. Methylation-specific PCR was used to validate the array data, and the association between DNA methylation and gene and protein expression was examined. The DNA methylation age was compared to the chronological age in both cases and controls. Three hundred and nineteen differentially methylated regions (DMR) were identified spread across the genome. Pathway analysis revealed that DMRs associated with genes were involved in embryonic and vascular development, and signalling pathways such as TGFβ and FGF. Expression of top differentially methylated gene HOXA5 showed a significant negative correlation with DNA methylation. Interestingly, DNA methylation age and chronological age were highly correlated, but there was no evidence of accelerated age-related DNA methylation in the arteries of CKD patients. In conclusion, we demonstrated that differential DNA methylation in the arterial tissue of CKD patients represents a potential mediator of arterial pathology and may be used to uncover novel pathways in the genesis of CKD-associated complications.

Background: DNA methylation (DNAm) is associated with gene regulation and estimated glomerular filtration rate (eGFR), a measure of kidney function. Decreased eGFR is more common among US Hispanics and African Americans. The causes for this are poorly understood. We aimed to identify trans-ethnic and ethnic-specific differentially methylated positions (DMPs) associated with eGFR using an agnostic, genome-wide approach., Methods: The study included up to 5428 participants from multi-ethnic studies for discovery and 8109 participants for replication. We tested the associations between whole blood DNAm and eGFR using beta values from Illumina 450K or EPIC arrays. Ethnicity-stratified analyses were performed using linear mixed models adjusting for age, sex, smoking, and study-specific and technical variables. Summary results were meta-analyzed within and across ethnicities. Findings were assessed using integrative epigenomics methods and pathway analyses., Results: We identified 93 DMPs associated with eGFR at an FDR of 0.05 and replicated 13 and 1 DMPs across independent samples in trans-ethnic and African American meta-analyses, respectively. The study also validated 6 previously published DMPs. Identified DMPs showed significant overlap enrichment with DNase I hypersensitive sites in kidney tissue, sites associated with the expression of proximal genes, and transcription factor motifs and pathways associated with kidney tissue and kidney development., Conclusions: We uncovered trans-ethnic and ethnic-specific DMPs associated with eGFR, including DMPs enriched in regulatory elements in kidney tissue and pathways related to kidney development. These findings shed light on epigenetic mechanisms associated with kidney function, bridging the gap between population-specific eGFR-associated DNAm and tissue-specific regulatory context.

Glioblastoma multiforme (GBM) is an aggressive brain tumor for which current immunotherapy approaches have been unsuccessful. Here, we explore the mechanisms underlying immune evasion in GBM. By serially transplanting GBM stem cells (GSCs) into immunocompetent hosts, we uncover an acquired capability of GSCs to escape immune clearance by establishing an enhanced immunosuppressive tumor microenvironment. Mechanistically, this is not elicited via genetic selection of tumor subclones, but through an epigenetic immunoediting process wherein stable transcriptional and epigenetic changes in GSCs are enforced following immune attack. These changes launch a myeloid-affiliated transcriptional program, which leads to increased recruitment of tumor-associated macrophages. Furthermore, we identify similar epigenetic and transcriptional signatures in human mesenchymal subtype GSCs. We conclude that epigenetic immunoediting may drive an acquired immune evasion program in the most aggressive mesenchymal GBM subtype by reshaping the tumor immune microenvironment., Competing Interests: Declaration of interests S.M.P is a co-founder and shareholder of Cellinta., a biotechnology start-up that is developing cancer therapeutics, including for glioblastoma, and acts as an advisor to the company. J.W.P is a co-founder and shareholder of Macomics. S.A.Q. is co-founder and shareholder and Chief Scientific Officer for Achilles Therapeutics. The other authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

In recent years, there has been a significant increase in whole genome sequencing data of individual genomes produced by research projects as well as direct to consumer service providers. While many of these sources provide their users with an interpretation of the data, there is a lack of free, open tools for generating reports exploring the data in an easy to understand manner. GenomeChronicler was developed as part of the Personal Genome Project UK (PGP-UK) to address this need. PGP-UK provides genomic, transcriptomic, epigenomic and self-reported phenotypic data under an open-access model with full ethical approval. As a result, the reports generated by GenomeChronicler are intended for research purposes only and include information relating to potentially beneficial and potentially harmful variants, but without clinical curation. GenomeChronicler can be used with data from whole genome or whole exome sequencing, producing a genome report containing information on variant statistics, ancestry and known associated phenotypic traits. Example reports are available from the PGP-UK data page (personalgenomes.org.uk/data). The objective of this method is to leverage existing resources to find known phenotypes associated with the genotypes detected in each sample. The provided trait data is based primarily upon information available in SNPedia, but also collates data from ClinVar, GETevidence, and gnomAD to provide additional details on potential health implications, presence of genotype in other PGP participants and population frequency of each genotype. The analysis can be run in a self-contained environment without requiring internet access, making it a good choice for cases where privacy is essential or desired: any third party project can embed GenomeChronicler within their off-line safe-haven environments. GenomeChronicler can be run for one sample at a time, or in parallel making use of the Nextflow workflow manager. The source code is available from GitHub (https://github.com/PGP-UK/GenomeChronicler), container recipes are available for Docker and Singularity, as well as a pre-built container from SingularityHub (https://singularity-hub.org/collections/3664) enabling easy deployment in a variety of settings. Users without access to computational resources to run GenomeChronicler can access the software from the Lifebit CloudOS platform (https://lifebit.ai/cloudos) enabling the production of reports and variant calls from raw sequencing data in a scalable fashion., (Copyright © 2020 Guerra-Assunção, Conde, Moghul, Webster, Ecker, Chervova, Chatzipantsiou, Prieto, Beck and Herrero.)

Cell type heterogeneity presents a challenge to the interpretation of epigenome data, compounded by the difficulty in generating reliable single-cell DNA methylomes for large numbers of cells and samples. We present EPISCORE, a computational algorithm that performs virtual microdissection of bulk tissue DNA methylation data at single cell-type resolution for any solid tissue. EPISCORE applies a probabilistic epigenetic model of gene regulation to a single-cell RNA-seq tissue atlas to generate a tissue-specific DNA methylation reference matrix, allowing quantification of cell-type proportions and cell-type-specific differential methylation signals in bulk tissue data. We validate EPISCORE in multiple epigenome studies and tissue types.

Background: Alopecia areata (AA) is considered a highly heritable, T-cell-mediated autoimmune disease of the hair follicle. However, no convincing susceptibility gene has yet been pinpointed in the major histocompatibility complex (MHC), a genome region known to be associated with AA as compared to other regions., Methods: We engineered mice carrying AA risk allele identified by haplotype sequencing for the MHC region using allele-specific genome editing with the CRISPR/Cas9 system. Finally, we performed functional evaluations in the mice and AA patients with and without the risk allele., Findings: We identified a variant (rs142986308, p.Arg587Trp) in the coiled-coil alpha-helical rod protein 1 (CCHCR1) gene as the only non-synonymous variant in the AA risk haplotype. Furthermore, mice engineered to carry the risk allele displayed a hair loss phenotype. Transcriptomics further identified CCHCR1 as a novel component interacting with hair cortex keratin in hair shafts. Both, these alopecic mice and AA patients with the risk allele displayed morphologically impaired hair and comparable differential expression of hair-related genes, including hair keratin and keratin-associated proteins (KRTAPs)., Interpretation: Our results implicate CCHCR1 with the risk allele in a previously unidentified subtype of AA based on aberrant keratinization in addition to autoimmune events., Funding: This work was supported by JSPS KAKENHI (JP16K10177) and the NIHR UCLH Biomedical Research center (BRC84/CN/SB/5984)., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Epigenetic changes may contribute substantially to risks of diseases of aging. Previous studies reported seven methylation variable positions (MVPs) robustly associated with incident type 2 diabetes mellitus (T2DM). However, their causal roles in T2DM are unclear. In an incident T2DM case-cohort study nested within the population-based European Prospective Investigation into Cancer and Nutrition (EPIC)-Norfolk cohort, we used whole blood DNA collected at baseline, up to 11 years before T2DM onset, to investigate the role of methylation in the etiology of T2DM. We identified 15 novel MVPs with robust associations with incident T2DM and robustly confirmed three MVPs identified previously (near to TXNIP , ABCG1 , and SREBF1 ). All 18 MVPs showed directionally consistent associations with incident and prevalent T2DM in independent studies. Further conditional analyses suggested that the identified epigenetic signals appear related to T2DM via glucose and obesity-related pathways acting before the collection of baseline samples. We integrated genome-wide genetic data to identify methylation-associated quantitative trait loci robustly associated with 16 of the 18 MVPs and found one MVP, cg00574958 at CPT1A , with a possible direct causal role in T2DM. None of the implicated genes were previously highlighted by genetic association studies, suggesting that DNA methylation studies may reveal novel biological mechanisms involved in tissue responses to glycemia., (© 2019 by the American Diabetes Association.)

Epigenetic clocks comprise a set of CpG sites whose DNA methylation levels measure subject age. These clocks are acknowledged as a highly accurate molecular correlate of chronological age in humans and other vertebrates. Also, extensive research is aimed at their potential to quantify biological aging rates and test longevity or rejuvenating interventions. Here, we discuss key challenges to understand clock mechanisms and biomarker utility. This requires dissecting the drivers and regulators of age-related changes in single-cell, tissue- and disease-specific models, as well as exploring other epigenomic marks, longitudinal and diverse population studies, and non-human models. We also highlight important ethical issues in forensic age determination and predicting the trajectory of biological aging in an individual.

Summary: It is well recognized that cell-type heterogeneity hampers the interpretation of Epigenome-Wide Association Studies (EWAS). Many tools have emerged to address this issue, including several R/Bioconductor packages that infer cell-type composition. Here we present a web application for cell-type deconvolution, which offers the functionality of our EpiDISH Bioconductor/R package in a user-friendly GUI environment. Users can upload their data to infer cell-type composition and differentially methylated cytosines in individual cell-types (DMCTs) for a range of different tissues., Availability and Implementation: EpiDISH web server is implemented with Shiny in R, and is freely available at https://www.biosino.org/EpiDISH/., (© The Author(s) 2019. Published by Oxford University Press.)

Summary: The Illumina Infinium EPIC BeadChip is a new high-throughput array for DNA methylation analysis, extending the earlier 450k array by over 400 000 new sites. Previously, a method named eFORGE was developed to provide insights into cell type-specific and cell-composition effects for 450k data. Here, we present a significantly updated and improved version of eFORGE that can analyze both EPIC and 450k array data. New features include analysis of chromatin states, transcription factor motifs and DNase I footprints, providing tools for epigenome-wide association study interpretation and epigenome editing., Availability and Implementation: eFORGE v2.0 is implemented as a web tool available from https://eforge.altiusinstitute.org and https://eforge-tf.altiusinstitute.org/., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2019. Published by Oxford University Press.)

Integrative analysis of multi-omics data is a powerful approach for gaining functional insights into biological and medical processes. Conducting these multifaceted analyses on human samples is often complicated by the fact that the raw sequencing output is rarely available under open access. The Personal Genome Project UK (PGP-UK) is one of few resources that recruits its participants under open consent and makes the resulting multi-omics data freely and openly available. As part of this resource, we describe the PGP-UK multi-omics reference panel consisting of ten genomic, methylomic and transcriptomic data. Specifically, we outline the data processing, quality control and validation procedures which were implemented to ensure data integrity and exclude sample mix-ups. In addition, we provide a REST API to facilitate the download of the entire PGP-UK dataset. The data are also available from two cloud-based environments, providing platforms for free integrated analysis. In conclusion, the genotype-validated PGP-UK multi-omics human reference panel described here provides a valuable new open access resource for integrated analyses in support of personal and medical genomics.

Whole genome bisulfite sequencing (WGBS), with its ability to interrogate methylation status at single CpG site resolution epigenome-wide, is a powerful technique for use in molecular experiments. Here, we aim to advance strategies for accurate and efficient WGBS for application in future large-scale epidemiological studies. We systematically compared the performance of three WGBS library preparation methods with low DNA input requirement (Swift Biosciences Accel-NGS, Illumina TruSeq and QIAGEN QIAseq) on two state-of-the-art sequencing platforms (Illumina NovaSeq and HiSeq X), and also assessed concordance between data generated by WGBS and methylation arrays. Swift achieved the highest proportion of CpG sites assayed and effective coverage at 26x (P < 0.001). TruSeq suffered from the highest proportion of PCR duplicates, while QIAseq failed to deliver across all quality metrics. There was little difference in performance between NovaSeq and HiSeq X, with the exception of higher read duplication rate on the NovaSeq (P < 0.05), likely attributable to the higher cluster densities on its flow cells. Systematic biases exist between WGBS and methylation arrays, with lower precision observed for WGBS across the range of depths investigated. To achieve a level of precision broadly comparable to the methylation array, a minimum coverage of 100x is recommended.

Background: Due to limited access to brain tissue, the precise mechanisms underlying neuro-axonal dysfunction in neurological disorders such as multiple sclerosis (MS) are largely unknown. In that context, profiling DNA methylation, which is a stable and cell type-specific regulatory epigenetic mark of genome activity, offers a unique opportunity to characterize the molecular mechanisms underpinning brain pathology in situ. We examined DNA methylation patterns of neuronal nuclei isolated from post-mortem brain tissue to infer processes that occur in neurons of MS patients., Results: We isolated subcortical neuronal nuclei from post-mortem white matter tissue of MS patients and non-neurological controls using flow cytometry. We examined bulk DNA methylation changes (total n = 29) and further disentangled true DNA methylation (5mC) from neuron-specific DNA hydroxymethylation (5hmC) (n = 17), using Illumina Infinium 450K arrays. We performed neuronal sub-type deconvolution using glutamate and GABA methylation profiles to further reduce neuronal sample heterogeneity. In total, we identified 2811 and 1534 significant (genome-wide adjusted P value < 0.05) differentially methylated and hydroxymethylated positions between MS patients and controls. We found striking hypo-5mC and hyper-5hmC changes occurring mainly within gene bodies, which correlated with reduced transcriptional activity, assessed using published RNAseq data from bulk brain tissue of MS patients and controls. Pathway analyses of the two cohorts implicated dysregulation of genes involved in axonal guidance and synaptic plasticity, with meta-analysis confirming CREB signalling as the most highly enriched pathway underlying these processes. We functionally investigated DNA methylation changes of CREB signalling-related genes by immunohistofluoresence of phosphorylated CREB in neurons from brain sections of a subcohort of MS patients and controls (n = 15). Notably, DNA methylation changes associated with a reduction of CREB activity in white matter neurons of MS patients compared to controls., Conclusions: Our data demonstrate that investigating 5mC and 5hmC modifications separately allows the discovery of a substantial fraction of changes occurring in neurons, which can escape traditional bisulfite-based DNA methylation analysis. Collectively, our findings indicate that neurons of MS patients acquire sustained hypo-5mC and hyper-5hmC, which may impair CREB-mediated neuro-axonal integrity, in turn relating to clinical symptoms.

As epigenetic studies become more common and lead to new insights into health and disease, the return of individual epigenetic results to research participants, in particular in large-scale epigenomic studies, will be of growing importance. Members of the International Human Epigenome Consortium (IHEC) Bioethics Workgroup considered the potential ethical, legal, and social issues (ELSI) involved in returning epigenetic research results and incidental findings in order to produce a set of 'Points-to-consider' (P-t-C) for the epigenetics research community. These P-t-C draw on existing guidance on the return of genetic research results, while also integrating the IHEC Bioethics Workgroup's ELSI research on and discussion of the issues associated with epigenetic data as well as the experience of a return of results pilot study by the Personal Genome Project UK (PGP-UK). Major challenges include how to determine the clinical validity and actionability of epigenetic results, and considerations related to environmental exposures and epigenetic marks, including circumstances warranting the sharing of results with family members and third parties. Interdisciplinary collaboration and good public communication regarding epigenetic risk will be important to advance the return of results framework for epigenetic science.