Methylome Profiling Research Articles

Molecular diagnostic tools for the World Health Organization (WHO) 2021 classification of gliomas, glioneuronal and neuronal tumors; an EANO guideline.

In the 5th edition of the WHO CNS tumor classification (CNS5, 2021), multiple molecular characteristics became essential diagnostic criteria for many additional CNS tumor types. For those tumors, an integrated, 'histomolecular' diagnosis is required. A variety of approaches exists for determining the status of the underyling molecular markers. The present guideline focuses on the methods that can be used for assessment of the currently most informative diagnostic and prognostic molecular markers for the diagnosis of gliomas, glioneuronal and neuronal tumors. The main characteristics of the molecular methods are systematically discussed, followed by recommendations and information on available evidence levels for diagnostic measures. The recommendations cover DNA and RNA next-generation-sequencing, methylome profiling, and select assays for single/limited target analysis, including immunohistochemistry. Additionally, because of its importance as a predictive marker in IDH-wildtype glioblastomas, tools for the analysis of MGMT promoter status are covered. A structured overview of the different assays with their characteristics, especially their advantages and limitations, is provided, and requirements for input material and reporting of results are clarified. General aspects of molecular diagnostic testing regarding clinical relevance, accessibility, cost, implementation, regulatory and ethical aspects are discussed as well. Finally, we provide an outlook on new developments in the landscape of molecular testing technologies in neuro-oncology.

F.1 DNA methylome profiling identifies stability of IDH mutations throughout glioma evolution

Background: Isocitrate dehydrogenase (IDH) mutation status is a key diagnostic and prognostic feature of gliomas. There are conflicting reports regarding the stability of IDH mutations throughout glioma evolution and treatment. Here, we provide an institutional experience of patients with conflicting IDH mutation status longitudinally in order to determine if IDH mutation status changes over time. Methods: We retrospectively identified patients from 2009-2018 with immunohistochemistry (IHC)-recorded IDH mutation status discrepancies longitudinally. Archived frozen tissue samples were analyzed using methylation profiling, Sanger sequencing, and droplet digital PCR (ddPCR). Results were compared to the IHC-reported IDH mutation status. Results: We reviewed 1491 archived glioma samples including 91 patients with multiple tumour samples collected longitudinally. In all instances of IDH mutation discrepancy, we found reasonable explanations through multi-platform profiling that resolved the discrepancies. This included the presence of non-canonical IDH2 mutations identified through Sanger sequencing and perilesional tumour samples or reactive brain tissue identified through methylation profiling. Conclusions: Our findings support the hypothesis that IDH mutations occur early in gliomagenesis and are stable throughout glioma treatment and evolution. Our study highlights the importance of accurate surgical sampling and the role of DNA methylome profiling in diagnostically uncertain cases for integrated pathological and molecular diagnosis.

Integrated Multi-Omics Reveals Significant Roles of Non-Additively Expressed Small RNAs in Heterosis for Maize Plant Height.

Heterosis is a complex biological phenomenon regulated by genetic variations and epigenetic changes. However, the roles of small RNAs (sRNAs), an important epigenetic regulatory element, on plant heterosis are still poorly understood. Here, an integrative analysis was performed with sequencing data from multi-omics layers of maize hybrids and their two homologous parental lines to explore the potential underlying mechanisms of sRNAs in plant height (PH) heterosis. sRNAome analysis revealed that 59 (18.61%) microRNAs (miRNAs) and 64,534 (54.00%) 24-nt small interfering RNAs (siRNAs) clusters were non-additively expressed in hybrids. Transcriptome profiles showed that these non-additively expressed miRNAs regulated PH heterosis through activating genes involved in vegetative growth-related pathways while suppressing those related to reproductive and stress response pathways. DNA methylome profiles showed that non-additive methylation events were more likely to be induced by non-additively expressed siRNA clusters. Genes associated with low-parental expression (LPE) siRNAs and trans-chromosomal demethylation (TCdM) events were enriched in developmental processes as well as nutrients and energy metabolism, whereas genes associated with high-parental expression (HPE) siRNAs and trans-chromosomal methylation (TCM) events were gathered in stress response and organelle organization pathways. Our results provide insights into the expression and regulation patterns of sRNAs in hybrids and help to elucidate their potential targeting pathways contributing to PH heterosis.

Methylome and transcriptome data integration reveals aberrantly regulated genes in equine sarcoids

DNA methylation is a key mechanism in transcription regulation, and aberrant methylation is a common and important mechanism in tumor initiation, maintenance, and progression. To find genes that are aberrantly regulated by altered methylation in horse sarcoids, we used reduced representation bisulfite sequencing (RRBS) accompanied by RNA sequencing (RNA-Seq) for methylome (whole genome DNA methylation sequencing) and transcriptome profiling, respectively. We found that the DNA methylation level was generally lower in lesion samples than in controls. In the analyzed samples, a total of 14,692 differentially methylated sites (DMSs) in the context of CpG (where cytosine and guanine are separated by a phosphate), and 11,712 differentially expressed genes (DEGs) were identified. The integration of the methylome and transcriptome data suggests that aberrant DNA methylation may be involved in the deregulation of expression of the 493 genes in equine sarcoid. Furthermore, enrichment analysis of the genes demonstrated the activation of multiple molecular pathways related to extracellular matrix (ECM), oxidative phosphorylation (OXPHOS), immune response, and disease processes that can be related to tumor progression. The results provide further insight into the epigenetic alterations in equine sarcoids and provide a valuable resource for follow-up studies to identify biomarkers for predicting susceptibility to this common condition in horses.

DNA methylome profiling of blood to identify individuals in a pair of monozygotic twins

BackgroundShort tandem repeat (STR) markers cannot be used to distinguish between genetically identical monozygotic (MZ) twins, causing problems in a case with an MZ twin as a suspect. Many studies have shown that in older MZ twins, there are significant differences in overall content and genomic distribution of methylation.ObjectiveIn this study, we analyzed the DNA methylome profile of blood to identify recurrent differentially methylated CpG sites (DMCs) to discriminate between MZ twins.MethodsBlood samples were collected from 47 paired MZ twins. We performed the DNA methylation profiling using the HumanMethylation EPIC BeadChip platform and identified recurrent DMCs between MZ twins. Then, Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and motif enrichment analyses were performed to reveal the biological functions of recurrent DMCs. We collected DNA methylome data from the Gene Expression Omnibus (GEO) public database to verify the recurrent DMCs between MZ twins.ResultsWe identified recurrent DMCs between MZ twin samples and observed that they were enriched in immune-related genes. In addition, we verified our DMCs in a public dataset.ConclusionOur results suggest that the methylation level at recurrent DMCs between MZ twins may serve as a valuable biomarker for identification of individuals in a pair of MZ twins.

Evaluation of circulating tumor DNA by electropherogram analysis and methylome profiling in high-risk neuroblastomas.

Liquid biopsy has emerged as a promising, non-invasive diagnostic approach in oncology because the analysis of circulating tumor DNA (ctDNA) reflects the precise status of the disease at diagnosis, progression, and response to treatment. DNA methylation profiling is also a potential solution for sensitive and specific detection of many cancers. The combination of both approaches, DNA methylation analysis from ctDNA, provides an extremely useful and minimally invasive tool with high relevance in patients with childhood cancer. Neuroblastoma is an extracranial solid tumor most common in children and responsible for up to 15% of cancer-related deaths. This high death rate has prompted the scientific community to search for new therapeutic targets. DNA methylation also offers a new source for identifying these molecules. However, the limited blood sample size which can be obtained from children with cancer and the fact that ctDNA content may occasionally be diluted by non-tumor cell-free DNA (cfDNA) complicate optimal quantities of material for high-throughput sequencing studies. In this article, we present an improved method for ctDNA methylome studies of blood-derived plasma from high-risk neuroblastoma patients. We assessed the electropherogram profiles of ctDNA-containing samples suitable for methylome studies, using 10 ng of plasma-derived ctDNA from 126 samples of 86 high-risk neuroblastoma patients, and evaluated several bioinformatic approaches to analyze DNA methylation sequencing data. We demonstrated that enzymatic methyl-sequencing (EM-seq) outperformed bisulfite conversion-based method, based on the lower proportion of PCR duplicates and the higher percentage of unique mapping reads, mean coverage, and genome coverage. The analysis of the electropherogram profiles revealed the presence of nucleosomal multimers, and occasionally high molecular weight DNA. We established that 10% content of the mono-nucleosomal peak is sufficient ctDNA for successful detection of copy number variations and methylation profiles. Quantification of mono-nucleosomal peak also showed that samples at diagnosis contained a higher amount of ctDNA than relapse samples. Our results refine the use of electropherogram profiles to optimize sample selection for subsequent high-throughput analysis and support the use of liquid biopsy followed by enzymatic conversion of unmethylated cysteines to assess the methylomes of neuroblastoma patients.

Giant Cell Tumors With HMGA2::NCOR2 Fusion : Clinicopathologic, Molecular, and Epigenetic Study of a Distinct Entity.

Giant cell tumors (GCTs) with high mobility group AT-Hook 2 ( HMGA2 )::nuclear receptor corepressor 2 ( NCOR2 ) fusion are rare mesenchymal tumors of controversial nosology, which have been anecdotally reported to respond to CSFR1 inhibitors. Here, we performed a comprehensive study of 6 GCTs with HMGA2::NCOR2 fusion and explored their relationship with other giant cell-rich neoplasms. Tumors occurred in 4 females and 2 males ranging in age from 17 to 32 years old (median 24). Three lesions originated in subcutaneous soft tissue and 3 in bone. Tumor size ranged from 20 to 33mm (median 27mm). The lesions had a nodular/multinodular architecture and were composed of sheets of mononuclear "histiocytoid" cells with uniform nuclei intermingled with multinucleated giant cells. Mitotic activity was low and nuclear atypia and metaplastic bone were absent. Variable findings included necrosis, cystic degeneration, lymphocytic infiltrate (sometimes forming nodules), and xanthogranulomatous inflammation. On immunohistochemistry, all cases focally expressed pan-keratin and were negative with SATB2 and H3.3G34W. Whole RNA-sequencing was performed in all cases of GCT with HMGA2::NCOR2 fusion and a subset of giant cell-rich tumors (tenosynovial-GCT, n = 19 and "wild-type" GCT of soft tissue, n = 9). Hierarchical clustering of RNA-sequencing data showed that GCT with HMGA2::NCOR2 fusion formed a single cluster, independent of the other 2 entities. Methylome profiling showed similar results, but the distinction from "wild-type" GCT of soft tissue was less flagrant. Gene expression analysis showed similar levels of expression of the CSF1/CSFR1 axis between GCT with HMGA2::NCOR2 fusion and tenosynovial-GCT, supporting their potential sensitivity to CSFR1 inhibitors. Clinical follow-up was available for 5 patients (range: 10 to 64mo; median 32mo). Three patients (60%) experienced local recurrences, whereas none had distant metastases or died of disease. Overall, our study confirms and expands previous knowledge on GCT with HMGA2::NCOR2 fusion and supports its inclusion as an independent entity.

Multiplatform molecular profiling uncovers two subgroups of malignant peripheral nerve sheath tumors with distinct therapeutic vulnerabilities

Malignant peripheral nerve sheath tumor (MPNST) is a highly aggressive sarcoma, and a lethal neurofibromatosis type 1-related malignancy, with little progress made on treatment strategies. Here, we apply a multiplatform integrated molecular analysis on 108 tumors spanning the spectrum of peripheral nerve sheath tumors to identify candidate drivers of MPNST that can serve as therapeutic targets. Unsupervised analyses of methylome and transcriptome profiles identify two distinct subgroups of MPNSTs with unique targetable oncogenic programs. We establish two subgroups of MPNSTs: SHH pathway activation in MPNST-G1 and WNT/ß-catenin/CCND1 pathway activation in MPNST-G2. Single nuclei RNA sequencing characterizes the complex cellular architecture and demonstrate that malignant cells from MPNST-G1 and MPNST-G2 have neural crest-like and Schwann cell precursor-like cell characteristics, respectively. Further, in pre-clinical models of MPNST we confirm that inhibiting SHH pathway in MPNST-G1 prevent growth and malignant progression, providing the rational for investigating these treatments in clinical trials.

NKX3.1 immunohistochemistry and methylome profiling in mesenchymal chondrosarcoma: additional diagnostic value for a well-defined disease?

Mesenchymal chondrosarcoma (MCS) is a rare and highly aggressive tumour of soft tissue and bone that is defined by an underlying and highly specific fusion transcript involving HEY1 and NCOA2. Histologically, the tumours show a biphasic appearance consisting of an undifferentiated blue and round cell component as well as islands of highly differentiated cartilage. Particularly in core needle biopsies, the chondromatous component can be missed and the non-specific morphology and immunophenotype of the round cell component can cause diagnostic challenges. We applied NKX3.1 immunohistochemistry which was recently reported as a highly specific marker as well as methylome and copy number profiling to a set of 45 well characterised MCS cases to evaluate their potential diagnostic value. Methylome profiling revealed a highly distinct cluster for MCS. Notably, the findings were reproducible also when analysing the round cell and cartilaginous component separately. Furthermore, four outliers were identified by methylome profiling for which the diagnosis had to be revised. NKX3.1 immunohistochemistry showed positivity in 36% of tumours, the majority of which was rather focal and weak. Taken together, NKX3.1 expression showed a low sensitivity but a high specificity in our analysis. Methylome profiling on the other hand represents a sensitive, specific and reliable tool to support the diagnosis of MCS, particularly if only the round cell component is obtained in a biopsy and the diagnosis is not suspected. Furthermore, it can aid in confirming the diagnosis in case RNA sequencing for the HEY1::NCOA2 fusion transcript is not available.

Exercise is associated with younger methylome and transcriptome profiles in human skeletal muscle.

Exercise training prevents age-related decline in muscle function. Targeting epigenetic aging is a promising actionable mechanism and late-life exercise mitigates epigenetic aging in rodent muscle. Whether exercise training can decelerate, or reverse epigenetic aging in humans is unknown. Here, we performed a powerful meta-analysis of the methylome and transcriptome of an unprecedented number of human skeletal muscle samples (n = 3176). We show that: (1) individuals with higher baseline aerobic fitness have younger epigenetic and transcriptomic profiles, (2) exercise training leads to significant shifts of epigenetic and transcriptomic patterns toward a younger profile, and (3) muscle disuse "ages" the transcriptome. Higher fitness levels were associated with attenuated differential methylation and transcription during aging. Furthermore, both epigenetic and transcriptomic profiles shifted toward a younger state after exercise training interventions, while the transcriptome shifted toward an older state after forced muscle disuse. We demonstrate that exercise training targets many of the age-related transcripts and DNA methylation loci to maintain younger methylome and transcriptome profiles, specifically in genes related to muscle structure, metabolism, and mitochondrial function. Our comprehensive analysis will inform future studies aiming to identify the best combination of therapeutics and exercise regimes to optimize longevity.

Transcriptome and DNA methylome divergence of inflorescence development between 2 ecotypes in Panicum hallii.

The morphological diversity of the inflorescence determines flower and seed production, which is critical for plant adaptation. Hall's panicgrass (Panicum hallii, P. hallii) is a wild perennial grass that has been developed as a model to study perennial grass biology and adaptive evolution. Highly divergent inflorescences have evolved between the 2 major ecotypes in P. hallii, the upland ecotype (P. hallii var hallii, HAL2 genotype) with compact inflorescence and large seed and the lowland ecotype (P. hallii var filipes, FIL2 genotype) with an open inflorescence and small seed. Here we conducted a comparative analysis of the transcriptome and DNA methylome, an epigenetic mark that influences gene expression regulation, across different stages of inflorescence development using genomic references for each ecotype. Global transcriptome analysis of differentially expressed genes (DEGs) and co-expression modules underlying the inflorescence divergence revealed the potential role of cytokinin signaling in heterochronic changes. Comparing DNA methylome profiles revealed a remarkable level of differential DNA methylation associated with the evolution of P. hallii inflorescence. We found that a large proportion of differentially methylated regions (DMRs) were located in the flanking regulatory regions of genes. Intriguingly, we observed a substantial bias of CHH hypermethylation in the promoters of FIL2 genes. The integration of DEGs, DMRs, and Ka/Ks ratio results characterized the evolutionary features of DMR-associated DEGs that contribute to the divergence of the P. hallii inflorescence. This study provides insights into the transcriptome and epigenetic landscape of inflorescence divergence in P. hallii and a genomic resource for perennial grass biology.

Abstract 6218: NKX3.1 immunohistochemistry and methylome profiling in mesenchymal chondrosarcoma: additional diagnostic value for a well defined disease

Abstract Background: Mesenchymal chondrosarcoma (MCS) is a high-grade sarcoma characterized by a biphasic histological appearance. However, more often than not, only the poorly differentiated component is present on a biopsy, thus more entities enter the differential diagnosis. Since the small cell component is morphologically non-specific, the differential diagnoses are broad, such as Ewing sarcoma, rhabdomyosarcoma, poorly differentiated synovial sarcoma small cell carcinoma, lymphoma and also melanoma. Morevoer, although the HEY1::NCOA2 gene fusion is present in almost all MCS, some studies have shown alternative alterations in the absence of the HEY1::NCOA2 fusion, suggesting genomic heterogeneity in at least a small subset of MCS. Thus, new diagnostic aids would be helpful to confirm the diagnosis. While most of the immunohistochemical profile overlaps between these tumours, NKX3.1 was recently proposed as a useful diagnostic marker for MCS. Furthermore, studies using DNA-methylation profiling show that MCS forms a distinct methylation cluster. The aim of this study is to evaluate the utility of these new diagnostic tools. Methods: Slides from 45 samples with MCS were stained with NKX3.1 antibody (EP356, Cell Marque, Roche) and were also investigated by methylome analysis (Mehylation Epic BeadChips, Illumina). Depending on tissue availability, the cases were subjected to HEY1::NCOA2 gene fusion testing. Results: The methylation profile showed a distinct cluster for mesenchymal chondrosarcoma, in line with the previous studies. Moreover, these findings were reproduced even when submitting tissue solely from one component, either the cellular or the cartilaginous one. In addition, the copy number profile derived from the methylome analysis proves its utility in distinguishing MCS from its mimics. On the other hand, only 32.6% of cases were positive for NKX3.1. Four samples initially diagnosed as MCS were histologically re-evaluated due to an atypical methylome profile, a high amount of CNV and/or lack of validation of the gene fusion. Methylation profiling led to the re-classification of three cases, whereas the fourth sample could not be further classified. Conclusions: We conclude that the methylation analysis, along with the copy number profile, are reliable tools in terms of diagnosing MCS. On the contrary, the immunomarker NKX3.1 shows contradicting results, proving itself as an unreliable marker in this setting. Citation Format: Salome Glauser, Baptiste Ameline, Vanghelita Andrei, Dorothee Harder, Chantal Pauli, Marcel Trautmann, Wolfgang Hartmann, Daniel Baumhoer. NKX3.1 immunohistochemistry and methylome profiling in mesenchymal chondrosarcoma: additional diagnostic value for a well defined disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6218.

Abstract 3341: Preliminary clinical validation of COLO eDX, a novel plasma circulating tumor DNA methylation assay for detecting colorectal cancer

Abstract Introduction: Liquid biopsies with cell-free DNA (cfDNA) from plasma, are rapidly emerging as an important and minimally invasive approach for the early diagnosis of cancer. Here we present COLO eDX, as a novel methylation-specific droplet digital PCR (ddPCR)-based colorectal cancer (CRC) diagnostic tool. Methods: During the discovery phase, we analyzed DNA methylome profiles of more than 10,000 samples from CRC, normal colorectal mucosa, and other cancer types (the TCGA and GEO public databases). Through the multi-step biomarker discovery studies, we selected 14 CRC-specific hypermethylated CpG sites. Among them, 2 candidate sites were uniquely methylated in a CRC cell line among tested cancer cell lines from various organs including CRC (HCT-15), 2 hepatocellular carcinomas (Huh7 and SK-HEP-1), 2 lung cancers (A549 and HCC827), bladder cancer (HT-1376), prostate cancer (LNCap clone FGC), and breast cancer (T-47D). Through the methylation-specific ddPCR assay of paired tissue samples from the patients with CRC, we identified that single candidate was specifically hypermethylated in the most tumor tissues regardless of cancer stage. Copy numbers of the target region (TC) were divided by the copy numbers of the internal control (IC) to normalize. Results: We prospectively collected post-operative plasma samples from patients with CRC (N=23) who underwent surgery at Seoul National University Boramae Medical Center. According to the AJCC 8th staging system and risk groups allocation, 3 patients had standard risk and 8 patients had high risk for recurrence, among stage II patients. Among stage III patients, 3 patients had low risk and 6 patients had high risk for recurrence. 3 patients were stage IV at initial presentation. IC value in the stage IV patients (R2 resected) were significantly higher than that other groups (R0 resected), while there was no significant difference among stage II and stage III patients. Recurrence free survival for patients TC or IC value equal or higher than the median and those lower than the median were not statistically different. Since 3 patients in high risk stage III and 4 patients in high risk stage II patients did not receive the recommended adjuvant chemotherapy, comparison of RFS should be interpreted with caution. Conclusion: We have developed and validated the qualitative detection of methylation status of the bisulfite-converted target region in cfDNA. Further studies are warranted to evaluate the impact of COLO eDx on recurrence in a larger cohort of CRC patients. Citation Format: Jin-Soo Kim, Yun Young Lee, Seung Yeon Jung, Mi Young Kim, Rumi Shin, Jin Hyun Park, Joon An, Jinil Han, Seung Chul Heo, Youngho Moon. Preliminary clinical validation of COLO eDX, a novel plasma circulating tumor DNA methylation assay for detecting colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3341.

DNA methylome and transcriptome profiling reveal key electrophysiology and immune dysregulation in hypertrophic cardiomyopathy

ABSTRACT Hypertrophic cardiomyopathy (HCM) is the most common inherited heart disease. However, a detailed DNA methylation (DNAme) landscape has not yet been elucidated. Our study combined DNAme and transcriptome profiles for HCM myocardium and identify aberrant DNAme associated with altered myocardial function in HCM. The transcription of methylation-related genes did not significantly differ between HCM and normal myocardium. Nevertheless, the former had an altered DNAme profile compared with the latter. The hypermethylated and hypomethylated sites in HCM tissues had chromosomal distributions and functional enrichment of correlated genes differing from those of their normal tissue counterparts. The GO analysis of network underlying the genes correlated with DNAme alteration and differentially expressed genes (DEGs) shows functional clusters centred on immune cell function and muscle system processes. In KEGG analysis, only the calcium signalling pathway was enriched either by the genes correlated with changes in DNAme or DEGs. The protein-protein interactions (PPI) underlying the genes altered at both the DNAme and transcriptional highlighted two important functional clusters. One of these was related to the immune response and had the estrogen receptor-encoding ESR1 gene as its node. The other cluster comprised cardiac electrophysiology-related genes. Intelliectin-1 (ITLN1), a component of the innate immune system, was transcriptionally downregulated in HCM and had a hypermethylated site within 1500 bp upstream of the ITLN1 transcription start site. Estimates of immune infiltration demonstrated a relative decline in immune cell population diversity in HCM. A combination of DNAme and transcriptome profiles may help identify and develop new therapeutic targets for HCM.

Industrial pollutans and epigenetic factors associated with cardiomyopathies

The article presents a review of current scientific articles related to a few particular questions of molecular-genetic and epigenetic pathogenesis of some cardiomyopathy types that can be the object of interest assuming potential associations with pathophysiological mechanisms of pollutants and industrial toxins impaction. The possible benefits may arise from that kind of approach due to the fact that toxic agents in question are generally recognized at the moment as one of the cardiovascular disease onset and progression factors. The need for multidirectional theoretical developments targeting potential pharmacology methods aimed to at least partially eliminate such a challenge becomes more and more apparent. We focused on mTOR function chains, translation factor eIF4E and splicing regulator RBM20 interconnected with RAAS and T3 regulatory cascades. A part of our attention was also concentrated on cardiomyocytes methylome profiling in connection with pollutant toxicodynamics and cardiomyopathies pathogenesis.

Low-dose arecoline regulates distinct core signaling pathways in oral submucous fibrosis and oral squamous cell carcinoma

BackgroundBetel nut chewing plays a role in the pathogenesis of oral submucous fibrosis (OSF) and oral squamous cell carcinoma (OSCC). As the major active ingredient of the betel nut, the effect of arecoline and its underlying mechanism to OSF and OSCC pathogenesis remain unclear.MethodsNext-generation sequencing-based transcriptome and dRRBS analysis were performed on OSF and OSCC cells under low-dose arecoline exposure. Functional analyses were performed to compare the different roles of arecoline during OSF and OSCC pathogenesis, and key genes were identified.ResultsIn this study, we identified that low-dose arecoline promoted cell proliferation of both NFs and OSCC cells via the acceleration of cell cycle progression, while high-dose arecoline was cytotoxic to both NFs and OSCC cells. We performed for the first time the transcriptome and methylome landscapes of NFs and OSCC cells under low-dose arecoline exposure. We found distinct transcriptome and methylome profiles mediated by low-dose arecoline in OSF and OSCC cells, as well as specific genes and signaling pathways associated with metabolic disorders induced by low-dose arecoline exposure. Additionally, low-dose arecoline displayed different functions at different stages, participating in the modulation of the extracellular matrix via Wnt signaling in NFs and epigenetic regulation in OSCC cells. After exposure to low-dose arecoline, the node roles of FMOD in NFs and histone gene clusters in OSCC cells were found. Meanwhile, some key methylated genes induced by arecoline were also identified, like PTPRM and FOXD3 in NFs, SALL3 and IRF8 in OSCC cells, indicating early molecular events mediated by arecoline during OSF and OSCC pathogenesis.ConclusionsThis study elucidated the contribution of low-dose arecoline to OSF and OSCC pathogenesis and identified key molecular events that could be targeted for further functional studies and their potential as biomarkers.

Long-read sequencing of diagnosis and post-therapy medulloblastoma reveals complex rearrangement patterns and epigenetic signatures

Cancer genomes harbor a broad spectrum of structural variants (SVs) driving tumorigenesis, a relevant subset of which escape discovery usingshort-read sequencing. We employed Oxford Nanopore Technologies (ONT) long-read sequencing in a paired diagnostic and post-therapy medulloblastoma to unravel the haplotype-resolved somatic genetic and epigenetic landscape. We assembled complex rearrangements, including a 1.55-Mbp chromothripsis event, and we uncover a complex SV pattern termed templated insertion (TI) thread, characterized by short (mostly <1 kb) insertions showing prevalent self-concatenation into highly amplified structures of up to 50 kbp in size. TI threads occur in 3% of cancers, with a prevalence up to 74% in liposarcoma, and frequent colocalization with chromothripsis. We also perform long-read-based methylome profiling and discover allele-specific methylation (ASM) effects, complex rearrangements exhibiting differential methylation, and differential promoter methylation in cancer-driver genes. Our study shows the advantage of long-read sequencing in the discovery and characterization of complex somatic rearrangements.

Cover Image, Volume 62, Issue 4

The cover image is based on the Research Article Plasma cell-free DNA methylome profiling in pre- and postsurgery oral cavity squamous cell carcinoma by Krupal B. Patel et al., https://doi.org/10.1002/mc.23501.

A variability in response of osteoclasts to zoledronic acid is mediated by smoking-associated modification in the DNA methylome

BackgroundClinical trials have shown zoledronic acid as a potent bisphosphonate in preventing bone loss, but with varying potency between patients. Human osteoclasts ex vivo reportedly displayed a variable sensitivity to zoledronic acid > 200-fold, determined by the half-maximal inhibitory concentration (IC50), with cigarette smoking as one of the reported contributors to this variation. To reveal the molecular basis of the smoking-mediated variation on treatment sensitivity, we performed a DNA methylome profiling on whole blood cells from 34 healthy female blood donors. Multiple regression models were fitted to associate DNA methylation with ex vivo determined IC50 values, smoking, and their interaction adjusting for age and cell compositions.ResultsWe identified 59 CpGs displaying genome-wide significance (p < 1e−08) with a false discovery rate (FDR) < 0.05 for the smoking-dependent association with IC50. Among them, 3 CpGs have p < 1e−08 and FDR < 2e−03. By comparing with genome-wide association studies, 15 significant CpGs were locally enriched (within < 50,000 bp) by SNPs associated with bone and body size measures. Furthermore, through a replication analysis using data from a published multi-omics association study on bone mineral density (BMD), we could validate that 29 out of the 59 CpGs were in close vicinity of genomic sites significantly associated with BMD. Gene Ontology (GO) analysis on genes linked to the 59 CpGs displaying smoking-dependent association with IC50, detected 18 significant GO terms including cation:cation antiporter activity, extracellular matrix conferring tensile strength, ligand–gated ion channel activity, etc.ConclusionsOur results suggest that smoking mediates individual sensitivity to zoledronic acid treatment through epigenetic regulation. Our novel findings could have important clinical implications since DNA methylation analysis may enable personalized zoledronic acid treatment.

Histone H3-wild type diffuse midline gliomas with H3K27me3 loss are a distinct entity with exclusive EGFR or ACVR1 mutation and differential methylation of homeobox genes

Diffuse midline gliomas (DMG) harbouring H3K27M mutation are paediatric tumours with a dismal outcome. Recently, a new subtype of midline gliomas has been described with similar features to DMG, including loss of H3K27 trimethylation, but lacking the canonical H3K27M mutation (H3-WT). Here, we report a cohort of five H3-WT tumours profiled by whole-genome sequencing, RNA sequencing and DNA methylation profiling and combine their analysis with previously published cases. We show that these tumours have recurrent and mutually exclusive mutations in either ACVR1 or EGFR and are characterised by high expression of EZHIP associated to its promoter hypomethylation. Affected patients share a similar poor prognosis as patients with H3K27M DMG. Global molecular analysis of H3-WT and H3K27M DMG reveal distinct transcriptome and methylome profiles including differential methylation of homeobox genes involved in development and cellular differentiation. Patients have distinct clinical features, with a trend demonstrating ACVR1 mutations occurring in H3-WT tumours at an older age. This in-depth exploration of H3-WT tumours further characterises this novel DMG, H3K27-altered sub-group, characterised by a specific immunohistochemistry profile with H3K27me3 loss, wild-type H3K27M and positive EZHIP. It also gives new insights into the possible mechanism and pathway regulation in these tumours, potentially opening new therapeutic avenues for these tumours which have no known effective treatment.This study has been retrospectively registered on clinicaltrial.gov on 8 November 2017 under the registration number NCT03336931 (https://clinicaltrials.gov/ct2/show/NCT03336931).