Region-specific DNA hydroxymethylation along the malignant progression of IDH-mutant gliomas.

The majority of low-grade isocitrate dehydrogenase-mutant (IDHmt) gliomas undergo malignant progression (MP), but their underlying mechanism remains unclear. IDHmt gliomas exhibit global DNA methylation, and our previous report suggested that MP could be partly attributed to passive demethylation caused by accelerated cell cycles. However, during MP, there is also active demethylation mediated by ten-eleven translocation, such as DNA hydroxymethylation. Hydroxymethylation is reported to potentially contribute to gene expression regulation, but its role in MP remains under investigation. Therefore, we conducted a comprehensive analysis of hydroxymethylation during MP of IDHmt astrocytoma. Five primary/malignantly progressed IDHmt astrocytoma pairs were analyzed with oxidative bisulfite and the Infinium EPIC methylation array, detecting 5-hydroxymethyl cytosine at over 850,000 locations for region-specific hydroxymethylation assessment. Notably, we observed significant sharing of hydroxymethylated genomic regions during MP across the samples. Hydroxymethylated CpGs were enriched in open sea and intergenic regions (p < 0.001), and genes undergoing hydroxymethylation were significantly associated with cancer-related signaling pathways. RNA sequencing data integration identified 91 genes with significant positive/negative hydroxymethylation-expression correlations. Functional analysis suggested that positively correlated genes are involved in cell-cycle promotion, while negatively correlated ones are associated with antineoplastic functions. Analyses of The Cancer Genome Atlas clinical data on glioma were in line with these findings. Motif-enrichment analysis suggested the potential involvement of the transcription factor KLF4 in hydroxymethylation-based gene regulation. Our findings shed light on the significance of region-specific DNA hydroxymethylation in glioma MP and suggest its potential role in cancer-related gene expression and IDHmt glioma malignancy.

Transcriptomic and epigenetic dissection of spinal ependymoma (SP-EPN) identifies clinically relevant subtypes enriched for tumors with and without NF2 mutation.

Ependymomas encompass multiple clinically relevant tumor types based on localization and molecular profiles. Tumors of the methylation class "spinal ependymoma" (SP-EPN) represent the most common intramedullary neoplasms in children and adults. However, their developmental origin is ill-defined, molecular data are scarce, and the potential heterogeneity within SP-EPN remains unexplored. The only known recurrent genetic events in SP-EPN are loss of chromosome 22q and NF2 mutations, but neither types and frequency of these alterations nor their clinical relevance have been described in a large, epigenetically defined series. Transcriptomic (n = 72), epigenetic (n = 225), genetic (n = 134), and clinical data (n = 112) were integrated for a detailed molecular overview on SP-EPN. Additionally, we mapped SP-EPN transcriptomes to developmental atlases of the developing and adult spinal cord to uncover potential developmental origins of these tumors. The integration of transcriptomic ependymoma data with single-cell atlases of the spinal cord revealed that SP-EPN display the highest similarities to mature adult ependymal cells. Unsupervised hierarchical clustering of transcriptomic data together with integrated analysis of methylation profiles identified two molecular SP-EPN subtypes. Subtype A tumors primarily carried previously known germline or sporadic NF2 mutations together with 22q loss (bi-allelic NF2 loss), resulting in decreased NF2 expression. Furthermore, they more often presented as multilocular disease and demonstrated a significantly reduced progression-free survival as compared to SP-EP subtype B. In contrast, subtype B predominantly contained samples without NF2 mutation detected in sequencing together with 22q loss (monoallelic NF2 loss). These tumors showed regular NF2 expression but more extensive global copy number alterations. Based on integrated molecular profiling of a large multi-center cohort, we identified two distinct SP-EPN subtypes with important implications for genetic counseling, patient surveillance, and drug development priorities.

Genetic and epigenetic basis of hepatoblastoma diversity.

Hepatoblastoma (HB) is the most common pediatric liver malignancy; however, hereditary predisposition and acquired molecular aberrations related to HB clinicopathological diversity are not well understood. Here, we perform an integrative genomic profiling of 163 pediatric liver tumors (154 HBs and nine hepatocellular carcinomas) based on the data acquired from a cohort study (JPLT-2). The total number of somatic mutations is precious low (0.52/Mb on exonic regions) but correlated with age at diagnosis. Telomerase reverse transcriptase (TERT) promoter mutations are prevalent in the tween HBs, selective in the transitional liver cell tumor (TLCT, > 8 years old). DNA methylation profiling reveals that classical HBs are characterized by the specific hypomethylated enhancers, which are enriched with binding sites for ASCL2, a regulatory transcription factor for definitive endoderm in Wnt-pathway. Prolonged upregulation of ASCL2, as well as fetal-liver-like methylation patterns of IGF2 promoters, suggests their "cell of origin" derived from the premature hepatoblast, similar to intestinal epithelial cells, which are highly proliferative. Systematic molecular profiling of HB is a promising approach for understanding the epigenetic drivers of hepatoblast carcinogenesis and deriving clues for risk stratification.

A novel sensitive detection method for DNA methylation in circulating free DNA of pancreatic cancer.

Despite recent advances in clinical treatment, pancreatic cancer remains a highly lethal malignancy. In order to improve the survival rate of patients with pancreatic cancer, the development of non-invasive diagnostic methods using effective biomarkers is urgently needed. Here, we developed a highly sensitive method to detect DNA methylation in cell-free (cf)DNA samples based on the enrichment of methyl-CpG binding (MBD) protein coupled with a digital PCR method (MBD-ddPCR). Five DNA methylation markers for the diagnosis of pancreatic cancer were identified through DNA methylation microarray analysis in 37 pancreatic cancers. The sensitivity and specificity of the five markers were validated in another independent cohort of pancreatic cancers (100% and 100%, respectively; n = 46) as well as in The Cancer Genome Atlas data set (96% and 90%, respectively; n = 137). MBD-ddPCR analysis revealed that DNA methylation in at least one of the five markers was detected in 23 (49%) samples of cfDNA from 47 patients with pancreatic cancer. Further, a combination of DNA methylation markers and the KRAS mutation status improved the diagnostic capability of this method (sensitivity and specificity, 68% and 86%, respectively). Genome-wide MBD-sequencing analysis in cancer tissues and corresponding cfDNA revealed that more than 80% of methylated regions were overlapping; DNA methylation profiles of cancerous tissues and cfDNA significantly correlated with each other (R = 0.97). Our data indicate that newly developed MBD-ddPCR is a sensitive method to detect cfDNA methylation and that using five marker genes plus KRAS mutations may be useful for the detection of pancreatic cancers.

Defined lifestyle and germline factors predispose Asian populations to gastric cancer.

Germline and environmental effects on the development of gastric cancers (GC) and their ethnic differences have been poorly understood. Here, we performed genomic-scale trans-ethnic analysis of 531 GCs (319 Asian and 212 non-Asians). There was one distinct GC subclass with clear alcohol-associated mutation signature and strong Asian specificity, almost all of which were attributable to alcohol intake behavior, smoking habit, and Asian-specific defective ALDH2 allele. Alcohol-related GCs have low mutation burden and characteristic immunological profiles. In addition, we found frequent (7.4%) germline CDH1 variants among Japanese GCs, most of which were attributed to a few recurrent single-nucleotide variants shared by Japanese and Koreans, suggesting the existence of common ancestral events among East Asians. Specifically, approximately one-fifth of diffuse-type GCs were attributable to the combination of alcohol intake and defective ALDH2 allele or to CDH1 variants. These results revealed uncharacterized impacts of germline variants and lifestyles in the high incidence areas.

Structures and Inhibitory Activities for Interleukin-2 Production of Seasonally Variable Constituents in Flower Parts of Magnolia kobus at Different Growth Stages.

Magnolia Flower is a crude drug used for the treatment of headaches, toothaches, and nasal congestion. Here, we focused on Magnolia kobus, one of the botanical origins of Magnolia Flower, and collected the flower parts at different growth stages to compare chemical compositions and investigate potential inhibitory activities against interleukin-2 (IL-2) production in murine splenic T cells. After determining the structures, we examined the inhibitory effects of the constituents of the bud, the medicinal part of the crude drug, against IL-2 production. We first extracted the flower parts of M. kobus from the bud to fallen bloom stages and analysed the chemical compositions to identify the constituents characteristic to the buds. We found that the inhibitory activity of the buds against IL-2 production was more potent than that of the blooms. We isolated two known compounds, tiliroside (1) and syringin (2), characteristic to the buds from the methanol (MeOH) extract of Magnolia Flower. Moreover, we examined the inhibitory activities of both compounds against IL-2 production and found that tiliroside (1) but not syringin (2), showed strong inhibitory activity against IL-2 production and inhibited its mRNA expression. Thus, our strategy to examine the relationship between chemical compositions and biological activities during plant maturation could not only contribute to the scientific evaluation of medicinal parts of crude drugs but also assist in identifying biologically active constituents that have not yet been reported.

Publisher Correction: DNA demethylation is associated with malignant progression of lower-grade gliomas.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

DNA demethylation is associated with malignant progression of lower-grade gliomas.

To elucidate the mechanisms of malignant progression of lower-grade glioma, molecular profiling using methylation array, whole-exome sequencing, and RNA sequencing was performed for 122, 36 and 31 gliomas, respectively. This cohort included 24 matched pairs of initial lower-grade gliomas and recurrent tumors, most of which showed malignant progression. Nearly half of IDH-mutant glioblastomas that had progressed from lower-grade gliomas exhibited characteristic partial DNA demethylation in previously methylated genomic regions of their corresponding initial tumors, which had the glioma CpG island methylator phenotype (G-CIMP). In these glioblastomas, cell cycle-related genes, RB and PI3K-AKT pathway genes were frequently altered. Notably, late-replicating domain was significantly enriched in the demethylated regions that were mostly located in non-regulatory regions, suggesting that the loss of DNA methylation during malignant transformation may involve mainly passive demethylation due to a delay in maintenance of methylation during accelerated cell division. Nonetheless, a limited number of genes including IGF2BP3, which potentially drives cell proliferation, were presumed to be upregulated due to demethylation of their promoter. Our data indicated that demethylation of the G-CIMP profile found in a subset of recurrent gliomas reflects accelerated cell divisions accompanied by malignant transformation. Oncogenic genes activated by such epigenetic change represent potential therapeutic targets.

Distinct molecular profile of diffuse cerebellar gliomas.

Recent studies have demonstrated that tumor-driving alterations are often different among gliomas that originated from different brain regions and have underscored the importance of analyzing molecular characteristics of gliomas stratified by brain region. Therefore, to elucidate molecular characteristics of diffuse cerebellar gliomas (DCGs), 27 adult, mostly glioblastoma cases were analyzed. Comprehensive analysis using whole-exome sequencing, RNA sequencing, and Infinium methylation array (n = 17) demonstrated their distinct molecular profile compared to gliomas in other brain regions. Frequent mutations in chromatin-modifier genes were identified including, noticeably, a truncating mutation in SETD2 (n = 4), which resulted in loss of H3K36 trimethylation and was mutually exclusive with H3F3A K27M mutation (n = 3), suggesting that epigenetic dysregulation may lead to DCG tumorigenesis. Alterations that cause loss of p53 function including TP53 mutation (n = 9), PPM1D mutation (n = 2), and a novel type of PPM1D fusion (n = 1), were also frequent. On the other hand, mutations and copy number changes commonly observed in cerebral gliomas were infrequent. DNA methylation profile analysis demonstrated that all DCGs except for those with H3F3A mutations were categorized in the "RTK I (PDGFRA)" group, and those DCGs had a gene expression signature that was highly associated with PDGFRA. Furthermore, compared with the data of 315 gliomas derived from different brain regions, promoter methylation of transcription factors genes associated with glial development showed a characteristic pattern presumably reflecting their tumor origin. Notably, SOX10, a key transcription factor associated with oligodendroglial differentiation and PDGFRA regulation, was up-regulated in both DCG and H3 K27M-mutant diffuse midline glioma, suggesting their developmental and biological commonality. In contrast, SOX10 was silenced by promoter methylation in most cerebral gliomas. These findings may suggest potential tailored targeted therapy for gliomas according to their brain region, in addition to providing molecular clues to identify the region-related cellular origin of DCGs.

Base-Resolution Analysis of 5-Hydroxymethylcytosine by One-Pot Bisulfite-Free Chemical Conversion with Peroxotungstate.

5-Hydroxymethylcytosine (hmC) is an essential intermediate in the active DNA demethylation pathway. Here we report a new base-resolution method for measuring hmC by combining peroxotungstate-mediated oxidation and sequencing analysis. We reveal that an oxidized product of hmC, trihydroxylated thymine (thT), tolerated the incorporation of dATP as a substrate in the process of DNA polymerase elongation. By comparing the results of Sanger sequencing before and after the oxidation, we observed that hmC sites on single-stranded DNAs could be discriminated from unmethylated cytosines. We found that a thermal cycle condition during peroxotungstate treatment enhanced the oxidation reaction of hmC in double-stranded DNA. Furthermore, Illumina sequencing analysis of hmC-containing synthetic genome fragments enabled us to identify simultaneously the positions of hmC in base resolution. This bisulfite-free simple hmC detection technique could facilitate the acquisition of epigenomic information.

Population-genetic nature of copy number variations in the human genome.

Copy number variations (CNVs) are universal genetic variations, and their association with disease has been increasingly recognized. We designed high-density microarrays for CNVs, and detected 3000-4000 CNVs (4-6% of the genomic sequence) per population that included CNVs previously missed because of smaller sizes and residing in segmental duplications. The patterns of CNVs across individuals were surprisingly simple at the kilo-base scale, suggesting the applicability of a simple genetic analysis for these genetic loci. We utilized the probabilistic theory to determine integer copy numbers of CNVs and employed a recently developed phasing tool to estimate the population frequencies of integer copy number alleles and CNV-SNP haplotypes. The results showed a tendency toward a lower frequency of CNV alleles and that most of our CNVs were explained only by zero-, one- and two-copy alleles. Using the estimated population frequencies, we found several CNV regions with exceptionally high population differentiation. Investigation of CNV-SNP linkage disequilibrium (LD) for 500-900 bi- and multi-allelic CNVs per population revealed that previous conflicting reports on bi-allelic LD were unexpectedly consistent and explained by an LD increase correlated with deletion-allele frequencies. Typically, the bi-allelic LD was lower than SNP-SNP LD, whereas the multi-allelic LD was somewhat stronger than the bi-allelic LD. After further investigation of tag SNPs for CNVs, we conclude that the customary tagging strategy for disease association studies can be applicable for common deletion CNVs, but direct interrogation is needed for other types of CNVs.

The coalescence time of sampled genes in the structured coalescent model.

The aim of this article is to investigate the distribution of the coalescence time (T) for sampled genes in the structured coalescent. We obtain some exact solutions for small samples and approximate distributions for n sampled genes in strong and weak migration. We also conduct computer simulation to evaluate efficiencies of these approximations and show the dependency of the distribution of the coalescence time on the geographical structure and the intensity of migration. In a panmictic population, we prove that the conditional distribution of the coalescence time given the number of segregating sites (S) among sampled genes is given by the weighted mean of the convolution of gamma distributions. We also study the joint distribution of T and S in the structured coalescent model and show some exact solutions.