Genome-wide DNA methylation profiles in noncancerous gastric mucosae with regard to Helicobacter pylori infection and the presence of gastric cancer

Background and Aims: To determine genome‐wide DNA methylation profiles induced by Helicobacter pylori (H. pylori) infection and to identify methylation markers in H. pylori‐induced gastric carcinogenesis. Methods: Gastric mucosae obtained from controls (n = 20) and patients with gastric cancer (n = 28) were included. A wide panel of CpG sites in cancer‐related genes (1505 CpG sites in 807 genes) was analyzed using Illumina bead array technology. Validation of the results of Illumina bead array technique was performed using methylation‐specific PCR method for four genes (MOS, DCC, CRK, and PTPN6). Results: The Illumina bead array showed that a total of 359 CpG sites (269 genes) were identified as differentially methylated by H. pylori infection (p < .0001). The correlation between methylation‐specific PCR and bead array analysis was significant (p < .0001, Spearman coefficient = 0.5054). Methylation profiles in noncancerous gastric mucosae of the patients with gastric cancer showed quite distinct patterns according to the presence or absence of the current H. pylori infection; however, 10 CpG sites were identified to be hypermethylated and three hypomethylated in association with the presence of gastric cancer regardless of H. pylori infection (p < .01). Conclusions: Genome‐wide methylation profiles showed a number of genes differentially methylated by H. pylori infection. Methylation profiles in noncancerous gastric mucosae from the patients with gastric cancer can be affected by H. pylori‐induced gastritis. Differentially methylated CpG sites in this study needs to be validated in a larger population using quantitative methylation‐specific PCR method.     

Osterix is a key target for mechanical signals in human thoracic ligament flavum cells

Mechanical stress is considered to be an important factor in the progression of thoracic ossification of the ligament flavum (TOLF). To elucidate the mechanism underlying mechanical stress-induced TOLF, we investigated the effect of stretching on cultured flavum ligament cells derived from TOLF and non-TOLF patients. We found that the mRNA expression of alkaline phosphatase (ALP), osteocalcin, Runx2, and osterix, but not that of Dlx5 and Msx2, was significantly increased by stretching in TOLF cells. In addition, the effect seems to be finely tuned by stretching-triggered activation of distinct mitogen-activated protein kinase cascades. Specifically, a p38 specific inhibitor, SB203580, significantly inhibited stretching-induced osterix expression as well as ALP activity, whereas a specific inhibitor of ERK1/2, U0126, prevented stretching-induced Runx2 expression. We showed that overexpression of osterix resulted in a significant increase of ALP activity in TOLF cells, and osterix-specific RNAi completely abrogated the stretching-induced ALP activity, indicating that osterix plays a key role in stretching-stimulated osteogenic effect in TOLF cells. These results suggest that mechanical stress plays important roles in the progression of TOLF through induction of osteogenic differentiation of TOLF cells, and our findings support that osterix functions as a molecular link between mechanostressing and osteogenic differentiation.     

Genome‐wide promoter methylation analysis identifies epigenetic silencing of MAPK13 in primary cutaneous melanoma

The involvement of epigenetic alterations in the pathogenesis of melanoma is increasingly recognized. Here, we performed genome‐wide DNA methylation analysis of primary cutaneous melanoma and benign melanocytic nevus interrogating 14 495 genes using BeadChip technology. This genome‐wide view of promoter methylation in primary cutaneous melanoma revealed an array of recurrent DNA methylation alterations with potential diagnostic applications. Among 106 frequently hypermethylated genes, there were many novel methylation targets and tumor suppressor genes. Highly recurrent methylation of the HOXA9, MAPK13, CDH11, PLEKHG6, PPP1R3C, and CLDN11 genes was established. Promoter methylation of MAPK13, encoding p38δ, was present in 67% of primary and 85% of metastatic melanomas. Restoration of MAPK13 expression in melanoma cells exhibiting epigenetic silencing of this gene reduced proliferation, indicative of tumor suppressive functions. This study demonstrates that DNA methylation alterations are widespread in melanoma and suggests that epigenetic silencing of MAPK13 contributes to melanoma progression.     

Genome-wide methylation profiling reveals Zinc finger protein 516 (ZNF516) and FK-506-binding protein 6 (FKBP6) promoters frequently methylated in cervical neoplasia,associated with HPV status and ethnicity in a Chilean population

Cervical cancer is a major health concern among women in Latin America due to its high incidence and mortality. Therefore, the discovery of molecular markers for cervical cancer screening and triage is imperative. The aim of this study was to use a genome wide DNA methylation approach to identify novel methylation biomarkers in cervical cancer. DNA from normal cervical mucosa and cervical cancer tissue samples from Chile was enriched with Methylated DNA Immunoprecipitation (MeDIP), hybridized to oligonucleotide methylation microarrays and analyzed with a stringent bioinformatics pipeline to identify differentially methylated regions (DMRs) as candidate biomarkers. Quantitative Methylation Specific PCR (qMSP) was used to study promoter methylation of candidate DMRs in clinical samples from two independent cohorts. HPV detection and genotyping were performed by Reverse Line Blot analysis. Bioinformatics analysis revealed GGTLA4, FKBP6, ZNF516, SAP130, and INTS1 to be differentially methylated in cancer and normal tissues in the Discovery cohort. In the Validation cohort FKBP6 promoter methylation had 73% sensitivity and 80% specificity (AUC = 0.80). ZNF516 promoter methylation was the best biomarker, with both sensitivity and specificity of 90% (AUC = 0.92), results subsequently corroborated in a Prevalence cohort. Together, ZNF516 and FKBP6 exhibited a sensitivity of 84% and specificity of 81%, when considering both cohorts. Our genome wide DNA methylation assessment approach (MeDIP-chip) successfully identified novel biomarkers that differentiate between cervical cancer and normal samples, after adjusting for age and HPV status. These biomarkers need to be further explored in case-control and prospective cohorts to validate them as cervical cancer biomarkers.     

Differentially methylated regions in maternal and paternal uniparental disomy for chromosome 7

DNA methylation is a hallmark of genomic imprinting and differentially methylated regions (DMRs) are found near and in imprinted genes. Imprinted genes are expressed only from the maternal or paternal allele and their normal balance can be disrupted by uniparental disomy (UPD), the inheritance of both chromosomes of a chromosome pair exclusively from only either the mother or the father. Maternal UPD for chromosome 7 (matUPD7) results in Silver-Russell syndrome (SRS) with typical features and growth retardation, but no gene has been conclusively implicated in SRS. In order to identify novel DMRs and putative imprinted genes on chromosome 7, we analyzed eight matUPD7 patients, a segmental matUPD7q31-qter, a rare patUPD7 case and ten controls on the Infinium HumanMethylation450K BeadChip with 30?017 CpG methylation probes for chromosome 7. Genome-scale analysis showed highly significant clustering of DMRs only on chromosome 7, including the known imprinted loci GRB10, SGCE/PEG10, and PEG/MEST. We found ten novel DMRs on chromosome 7, two DMRs for the predicted imprinted genes HOXA4 and GLI3 and one for the disputed imprinted gene PON1. Quantitative RT-PCR on blood RNA samples comparing matUPD7, patUPD7, and controls showed differential expression for three genes with novel DMRs, HOXA4, GLI3, and SVOPL. Allele specific expression analysis confirmed maternal only expression of SVOPL and imprinting of HOXA4 was supported by monoallelic expression. These results present the first comprehensive map of parent-of-origin specific DMRs on human chromosome 7, suggesting many new imprinted sites.     

Genome‐wide DNA methylation signatures of infection status in Trinidadian guppies (Poecilia reticulata)

Epigenetic modification, especially DNA methylation, can play an important role in mediating gene regulatory response to environmental stressors and may be a key process affecting phenotypic plasticity and adaptation. Parasites are potent stressors with profound physiological and ecological effects on their hosts, yet it remains unclear how parasites influence host methylation patterns. Here, we used a well‐studied host–parasite system, the guppy Poecilia reticulata and its ectoparasitic monogenean Gyrodactylus turnbulli to gain mechanistic insight into the dynamics of DNA methylation in host–parasite interactions. To explore this, we quantitatively measured genome‐wide DNA methylation in guppy skin tissue using reduced representation bisulphite sequencing and characterized differential methylation patterns in guppies during distinct phases of infection. We identified 365, 313, and 741 differentially methylated regions (DMRs) between infected and control fish in early infection, peak infection and recovery phases, respectively. The magnitude of the methylation difference was moderate in DMRs, with an average of 29% (early infection), 27% (peak infection) and 30% (recovery) differential methylation per DMR. Approximately 50% of DMRs overlapped with CpG islands, and over half of the DMRs overlapped with gene bodies, several of which encode proteins relevant to immune response. These findings provide the first evidence of an epigenetic signature of infection by ectoparasites and demonstrate the changing relationship between epigenetic variation and immune response in distinct phases of infection.     

ABCC9, NKAPL,and TMEM132C are potential diagnostic and prognostic markers in triple‐negative breast cancer

Triple‐negative breast cancer (TNBC) is a highly heterogeneous disease. The aim of this study is to identify the diagnostic and poor prognostic signatures in TNBC by exploring the aberrant DNA methylation and gene expression. Differential expression and methylation analysis of the TNBC and paracancer samples from The Cancer Genome Atlas were performed. Gene set enrichment and protein–protein interaction (PPI) network analysis was used to explore the mechanisms of TNBC. Methylation‐gene expression correlation analysis was performed, and multivariate Cox analysis and receiver operating characteristics analysis were used to further screen the hub genes for TNBC. We identified 1,525 differentially expressed genes and 150 differentially methylated genes between TNBC and paracancer samples. About 96.64% of the methylation sites were located on the CpG island. A total of 17 Gene Ontology biological process terms and 18 signal pathways were significantly enriched. GNG4, GNG11, PENK, MAOA, and AOX1 were identified as the core genes of the PPI network. Methylation‐expression correlations revealed that ABCC9 (cg06951626), NKAPL (cg18675097, cg01031101, and cg17384889), and TMEM132C (cg03530754) showed promise as diagnostic and prognostic markers in TNBC. ABCC9 (cg06951626), NKAPL (cg18675097, cg01031101, and cg17384889), and TMEM132C (cg03530754) were potential diagnostic and prognostic markers in TNBC.     

Genome‐wide mapping of cytosine methylation revealed dynamic DNA methylation patterns associated with genes and centromeres in rice

We conducted genome‐wide mapping of cytosine methylation using methylcytosine immunoprecipitation combined with Illumina sequencing. The chromosomal distribution pattern of methylated DNA is similar to the heterochromatin distribution pattern on rice chromosomes. The DNA methylation patterns of rice genes are similar to those in Arabidopsis thaliana, including distinct methylation patterns asssociated with gene bodies and promoters. The DNA sequences in the core domains of rice Cen4, Cen5 and Cen8 showed elevated methylation levels compared with sequences in the pericentromeric regions. In addition, elevated methylation levels were associated with the DNA sequences in the CENH3‐binding subdomains, compared with the sequences in the flanking H3 subdomains. In contrast, the centromeric domain of Cen11, which is composed exclusively of centromeric satellite DNA, is hypomethylated compared with the pericentromeric domains. Thus, the DNA sequences associated with functional centromeres can be either hypomethylated or hypermethylated. The methylation patterns of centromeric DNA appear to be correlated with the composition of the associated DNA sequences. We propose that both hypomethylation and hypermethylation of CENH3‐associated DNA sequences can serve as epigenetic marks to distinguish where CENH3 deposition will occur within the surrounding H3 chromatin.     

Genome-wide methylation profiling identifies novel methylated genes in neuroblastoma tumors

Neuroblastoma is a very heterogeneous tumor of childhood. The clinical spectra range from very aggressive metastatic disease to spontaneous regression, even without therapy. Aberrant DNA methylation pattern is a common feature of most cancers. For neuroblastoma, it has been demonstrated both for single genes as well as genome-wide, where a so-called methylator phenotype has been described. Here, we present a study using Illumina 450K methylation arrays on 60 neuroblastoma tumors. We show that aggressive tumors, characterized by International Neuroblastoma Risk Group (INRG) as stage M, are hypermethylated compared to low-grade tumors. On the contrary, INRG stage L tumors display more non-CpG methylation. The genes with the highest number of hypermethylated CpG sites in INRG M tumors are TERT, PCDHGA4, DLX5, and DLX6-AS1. Gene ontology analysis showed a representation of neuronal tumor relevant gene functions among the differentially methylated genes. For validation, we used a set of independent tumors previously analyzed with the Illumina 27K methylation arrays, which confirmed the differentially methylated sites. Top candidate genes with aberrant methylation were analyzed for altered gene expression through the R2 platform (http://r2.amc.nl), and for correlations between methylation and gene expression in a public dataset. Altered expression in nonsurvivors was found for the genes B3GALT4 and KIAA1949, CLIC5, DLX6-AS, TERT, and PIRT, and strongest correlations were found for TRIM36, KIAA0513, and PIRT. Our data indicate that methylation profiling can be used for patient stratification and informs on epigenetically deregulated genes with the potential of increasing our knowledge about the underlying mechanisms of tumor development.     

Genome‐wide screen of promoter methylation analysis of ES cells and ES derived epidermal‐like cells

Embryonic stem cells (ESCs) are a population of pluripotent cells which can differentiate into different cell types. However, there are few reports with regard to differentiate ESCs into epidermal cells in vitro. In this study, we aimed to investigate differentially methylated promoters involved in process of differentiation from ESCs into epidermal‐like cells (ELCs) induced by human amnion. We successfully induced ESCs into ELCs, which expressed the surface markers of CK19, CK15 and β1‐integrin. With MeDIP‐chip arrays, we identified 3435 gene promoters to be differentially methylated, involving 894 HCP (high CpG‐containing promoter), 974 ICP (intermediate CpG‐containing promoter) and 1567 LCP (low CpG‐containing promoter) among all the 17 500 DNA methylation regions of gene promoters in both ESCs and ELCs. Gene oncology and pathway analysis demonstrated that these genes were involved in all the three categories of GO enrichment analysis, including biological process, molecular function and cellular component. All these data suggested that embryonic stem cells can differentiate into epidermal‐like cells and promoter methylation is of great importance in this process. Copyright © 2015 John Wiley & Sons, Ltd.     

Aberrant DNA methylation of WNT pathway genes in the development and progression of CIMP-negative colorectal cancer

The WNT signaling pathway has an essential role in colorectal carcinogenesis and progression, which involves a cascade of genetic and epigenetic changes. We aimed to analyze DNA methylation affecting the WNT pathway genes in colorectal carcinogenesis in promoter and gene body regions using whole methylome analysis in 9 colorectal cancer, 15 adenoma, and 6 normal tumor adjacent tissue (NAT) samples by methyl capture sequencing. Functional methylation was confirmed on 5-aza-2′-deoxycytidine-treated colorectal cancer cell line datasets. In parallel with the DNA methylation analysis, mutations of WNT pathway genes (APC, β-catenin/CTNNB1) were analyzed by 454 sequencing on GS Junior platform. Most differentially methylated CpG sites were localized in gene body regions (95% of WNT pathway genes). In the promoter regions, 33 of the 160 analyzed WNT pathway genes were differentially methylated in colorectal cancer vs. normal, including hypermethylated AXIN2, CHP1, PRICKLE1, SFRP1, SFRP2, SOX17, and hypomethylated CACYBP, CTNNB1, MYC; 44 genes in adenoma vs. NAT; and 41 genes in colorectal cancer vs. adenoma comparisons. Hypermethylation of AXIN2, DKK1, VANGL1, and WNT5A gene promoters was higher, while those of SOX17, PRICKLE1, DAAM2, and MYC was lower in colon carcinoma compared to adenoma. Inverse correlation between expression and methylation was confirmed in 23 genes, including APC, CHP1, PRICKLE1, PSEN1, and SFRP1. Differential methylation affected both canonical and noncanonical WNT pathway genes in colorectal normal-adenoma-carcinoma sequence. Aberrant DNA methylation appears already in adenomas as an early event of colorectal carcinogenesis.     

Genome-scale analysis of DNA methylation in colorectal cancer using Infinium HumanMethylation450 BeadChips

Illumina’s Infinium HumanMethylation450 BeadChip arrays were used to examine genome-wide DNA methylation profiles in 22 sample pairs from colorectal cancer (CRC) and adjacent tissues and 19 colon tissue samples from cancer-free donors. We show that the methylation profiles of tumors and healthy tissue samples can be clearly distinguished from one another and that the main source of methylation variability is associated with disease status. We used different statistical approaches to evaluate the methylation data. In general, at the CpG-site level, we found that common CRC-specific methylation patterns consist of at least 15,667 CpG sites that were significantly different from either adjacent healthy tissue or tissue from cancer-free subjects. Of these sites, 10,342 were hypermethylated in CRC, and 5,325 were hypomethylated. Hypermethylated sites were common in the maximum number of sample pairs and were mostly located in CpG islands, where they were significantly enriched for differentially methylated regions known to be cancer-specific. In contrast, hypomethylated sites were mostly located in CpG shores and were generally sample-specific. Despite the considerable variability in methylation data, we selected a panel of 14 highly robust candidates showing methylation marks in genes SND1, ADHFE1, OPLAH, TLX2, C1orf70, ZFP64, NR5A2, and COL4A. This set was successfully cross-validated using methylation data from 209 CRC samples and 38 healthy tissue samples from The Cancer Genome Atlas consortium (AUC = 0.981 [95% CI: 0.9677–0.9939], sensitivity = 100% and specificity = 82%). In summary, this study reports a large number of loci with novel differential methylation statuses, some of which may serve as candidate markers for diagnostic purposes.