CpG Island Methylation Profiles Research Articles

Epigenetic Findings in Twins with Esophageal Atresia.

Esophageal atresia (EA) is the most common malformation of the upper gastrointestinal tract. The estimated incidence of EA is 1 in 3500 births. EA is more frequently observed in boys and in twins. The exact cause of isolated EA remains unknown; a multifactorial etiology, including epigenetic gene expression modifications, is considered. The study included six pairs of twins (three pairs of monozygotic twins and three pairs of dizygotic twins) in which one child was born with EA as an isolated defect, while the other twin was healthy. DNA samples were obtained from the blood and esophageal tissue of the child with EA as well as from the blood of the healthy twin. The reduced representation bisulfite sequencing (RRBS) technique was employed for a whole-genome methylation analysis. The analyses focused on comparing the CpG island methylation profiles between patients with EA and their healthy siblings. Hypermethylation in the promoters of 219 genes and hypomethylation in the promoters of 78 genes were observed. A pathway enrichment analysis revealed the statistically significant differences in methylation profile of 10 hypermethylated genes in the Rho GTPase pathway, previously undescribed in the field of EA (ARHGAP36, ARHGAP4, ARHGAP6, ARHGEF6, ARHGEF9, FGD1, GDI1, MCF2, OCRL, and STARD8).

Identification of key methylation differentially expressed genes in posterior fossa ependymoma based on epigenomic and transcriptome analysis

BackgroundPosterior fossa ependymoma (EPN-PF) can be classified into Group A posterior fossa ependymoma (EPN-PFA) and Group B posterior fossa ependymoma (EPN-PFB) according to DNA CpG island methylation profile status and gene expression. EPN-PFA usually occurs in children younger than 5 years and has a poor prognosis.MethodsUsing epigenome and transcriptome microarray data, a multi-component weighted gene co-expression network analysis (WGCNA) was used to systematically identify the hub genes of EPN-PF. We downloaded two microarray datasets (GSE66354 and GSE114523) from the Gene Expression Omnibus (GEO) database. The Limma R package was used to identify differentially expressed genes (DEGs), and ChAMP R was used to analyze the differential methylation genes (DMGs) between EPN-PFA and EPN-PFB. GO and KEGG enrichment analyses were performed using the Metascape database.ResultsGO analysis showed that enriched genes were significantly enriched in the extracellular matrix organization, adaptive immune response, membrane raft, focal adhesion, NF-kappa B pathway, and axon guidance, as suggested by KEGG analysis. Through WGCNA, we found that MEblue had a significant correlation with EPN-PF (R = 0.69, P = 1 × 10–08) and selected the 180 hub genes in the blue module. By comparing the DEGs, DMGs, and hub genes in the co-expression network, we identified five hypermethylated, lower expressed genes in EPN-PFA (ATP4B, CCDC151, DMKN, SCN4B, and TUBA4B), and three of them were confirmed by IHC.ConclusionssGSEA and GSVA analysis indicated that these five hub genes could lead to poor prognosis by inducing hypoxia, PI3K-Akt-mTOR, and TNFα-NFKB pathways. Further study of these dysmethylated hub genes in EPN-PF and the pathways they participate in may provides new ideas for EPN-PF treatment.

Metformin modulates oncogenic expression of HOTAIR gene via promoter methylation and reverses epithelial-mesenchymal transition in MDA-MB-231 cells.

Epithelial-mesenchymal transition (EMT) is a biological event, which critically regulates migration and invasion of cancer cells. EMT is regulated by several protein and nonprotein factors (such as noncoding RNAs). HOTAIR is an oncogenic long noncoding RNAthat stimulates EMT in cancers. In the current study, we investigated the effect of metformin on EMT behavior and HOTAIR expression in MDA-MB-231 breast cancer cells. The minimal effective concentrations of metformin (10 and 20 mM) were obtained by the MTT test. Cell migration and invasion in the metformin-containing medium were assayed in the scratch assay and transwell test. Meaningful decreases in both cell migration and invasion were observed in the presence of metformin. Vimentin, snail, β-catenin, and HOTAIR transcripts were quantified by real-time polymerase chain reaction (PCR). Reduction in the expression of vimentin, β-catenin, and HOTAIR was detected as the result of metformin treatment, but the snail showed a constant expression. Western blottingrevealed the downregulation of vimentin and β-catenin proteins. HOTAIR promoter methylation pattern was also investigated in metformin-exposed cells using bisulfite sequencing PCR which the result showed differences in the methylation profile of CpG islands between the treated and untreated cells. In conclusion, metformin modulated oncogenic expression of the HOTAIR gene in the MDA-MB-231 cells. This downregulation was associated with the modification of promoter methylation patterns. Since HOTAIR induces EMT in breast cancer, HOTAIR decline might be one of the mechanisms by which metformin reverses EMT.

Genome-Wide CpG Island Methylation Profiles of Cutaneous Skin with and without HPV Infection.

HPV infection is one of the most commonly transmitted diseases among the global population. While it can be asymptomatic, non-genital HPV infection often gives rise to cutaneous warts, which are benign growths arising from the epidermal layer of the skin. This study aimed to produce a global analysis of the ways in which cutaneous wart formation affected the CpG island methylome. The Infinium MethylationEPIC BeadChip microarray was utilized in order to quantitatively interrogate CpG island methylation in genomic DNA extracted from 24 paired wart and normal skin samples. Differential methylation analysis was carried out by means of assigning a combined rank score using RnBeads. The 1000 top-ranking CpG islands were then subject to Locus Overlap Analysis (LOLA) for enrichment of genomic ranges, while signaling pathway analysis was carried out on the top 100 differentially methylated CpG islands. Differential methylation analysis illustrated that the most differentially methylated CpG islands in warts lay within the ITGB5, DTNB, RBFOX3, SLC6A9, and C2orf27A genes. In addition, the most enriched genomic region sets in warts were Sheffield’s tissue-clustered DNase hypersensitive sites, ENCODE’s segmentation and transcription factor binding sites, codex sites, and the epigenome sites from cistrome. Lastly, signaling pathway analysis showed that the GRB2, GNB1, NTRK1, AXIN1, and SKI genes were the most common regulators of the genes associated with the top 100 most differentially methylated CpG islands in warts. Our study shows that HPV-induced cutaneous warts have a clear CpG island methylation profile that sets them apart from normal skin. Such a finding could account for the temporary nature of warts and the capacity for individuals to undergo clinical remission.

Abstract PR08: Overexpression and mutations of CXorf67 in “infant-type” posterior fossa type-A ependymomas

Abstract Our study aims to establish whether CXorf67 has a role in the pathogenesis of posterior fossa (PF) ependymoma. Among molecular groups of ependymoma defined by DNA methylation profiling, PF type-A (PFA) is the commonest. PFA ependymomas present mainly in infants and are difficult to treat, having a 10-year overall survival of approximately 40%. Published genomic studies have not identified a recurrent driver mutation in PFA ependymomas, but they do show widespread epigenetic alterations, including global loss of histone H3 K27-trimethlyation (H3K27-me3). Another childhood PF tumor, the diffuse pontine glioma (DPG), also shows loss of H3K27-me3. In DPGs, loss of H3K27-me3 is associated with H3 K27M mutation, but the mechanism in PFA ependymomas is not yet established. We reexamined published PF ependymoma sequencing data and discovered recurrent mutations in a novel gene, CXorf67. Targeted sequencing in a series of PFA ependymomas revealed CXorf67 mutations in 22/234 (9.4%). CXorf67 is a single-exon gene of unknown function. Its protein product is predicted to be “disordered,” apart from one region towards the N terminus. Mutations in PFA ependymomas are missense, and there is a mutation hotspot in the “ordered” region. CXorf67 mutations are not present in other molecular groups of ependymoma and are rare in other cancers. Analyzing the DNA methylation profiles of 675 PFA ependymomas, we have discovered two subgroups, PFA-1 and PFA-2, and nine subtypes among these two subgroups. All PFA subtypes harbor CXorf67 mutations with the exception of PFA-1f and PFA-2c. Targeted sequencing in our tumor series also revealed H3 K27M mutations in PFA ependymomas at a frequency of 3.9%. Two thirds of mutations in HIST1H3B, HIST1H3C, and H3F3A were found in PFA-1f ependymomas, among which H3 mutations were present at a frequency of 35%. Mutations in H3 genes and CXorf67 were mutually exclusive across our series of PFA ependymomas. We used Affymetrix u133v2 arrays to establish that CXorf67 is expressed at high levels in PFA ependymomas, in contrast to low levels in other ependymomas from the PF and supratentorial compartments. A mechanism for CXorf67 overexpression was revealed in a similar comparative analysis of CpG island methylation profiles, which showed that the promoter region of CXorf67 is hypomethylated in PFA tumors, but not in other ependymomas. Using immunohistochemical preparations, we detected expression of CXorf67 at the protein level in the nuclei of PFA ependymomas; PFB and supratentorial tumors were immunonegative. CXorf67 overexpression is found in all PFA molecular subtypes, except PFA-1f, which shows similar levels to those in supratentorial and PFB ependymomas. CXorf67 expression is unrelated to mutation status. Elevated CXorf67 expression is found in the Daoy and U2-OS cancer cell lines. We used immunoprecipitation (IP)/mass spectrometry (MS) to study proteins bound to CXorf67 in Daoy and U2-OS. Analysis of enriched peptides following immunoprecipitation of CXorf67 indicated that it binds EZH2, SUZ12, and EED, three core components of the PRC2 complex. Complementary immunoprecipitation of SUZ12 detected CXorf67. Detecting CXorf67 mutations in almost 10% of PFA ependymomas led to the discovery of overexpression related to promoter region hypomethylation in these tumors. Overexpression of CXorf67 was detected in all PFA ependymoma subtypes except PFA-1f, 35% of which harbor an H3 K27M mutation. The protein product of CXorf67 is found in tumor cell nuclei, where our IP/MS data suggest it is bound to PRC2. Our findings suggest that global loss of H3K27-me3 in PFA ependymomas could be related to overexpression of CXorf67 and its interaction with PRC2, except in tumors where alterations in histone H3 genes are responsible. However, the selective advantage of CXorf67 mutation is yet to be explained. Citation Format: Ji Wen, Jens-Martin Hübner, Wilda Orisme, Gang Wu, Bo Tang, Sujuan Jia, John Easton, Kelly Haupfear, Brian D. Freibaum, Hong Joo Kim, Anthony High, Junmin Peng, Ruth G. Tatevossian, J. Paul Taylor, Stefan M. Pfister, Jinghui Zhang, Kristian W. Pajtler, Marcel Kool, David W. Ellison. Overexpression and mutations of CXorf67 in “infant-type” posterior fossa type-A ependymomas [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr PR08.

DYNC1H1 gene methylation correlates with severity of spinal muscular atrophy.

Methylation profiles of CpG islands within the SLC23A2, CDK2AP1, and DYNC1H1 genes and their association with spinal muscular atrophy (SMA) severity were studied. High clinical heterogeneity of SMA suggests the existence of different factors modifying SMA phenotype with gene methylation as a plausible one. The genes picked up in our earlier genome-wide methylation studies of SMA patients demonstrated obvious differences in their methylation patterns, thus suggesting the likely involvement of their protein products in SMA development. Significantly decreased methylation of CpG islands within exon 37 of the DYNC1H1 gene was observed in patients with a severe SMA manifestation (type I) compared to mildly affected SMA patients (types III-IV). This finding provides new information on peculiarities of methylation in clinically different types of SMA patients and gives a clue for identification of new SMA modifiers.

MN1 overexpression is driven by loss of DNMT3B methylation activity in inv(16) pediatric AML.

In acute myeloid leukemia (AML), specific genomic aberrations induce aberrant methylation, thus directly influencing the transcriptional programing of leukemic cells. Therefore, therapies targeting epigenetic processes are advocated as a promising therapeutic tool for AML treatment. However, to develop new therapies, a comprehensive understanding of the mechanism(s) driving the epigenetic changes as a result of acquired genetic abnormalities is necessary. This understanding is still lacking. In this study, we performed genome-wide CpG-island methylation profiling on pediatric AML samples. Six differentially methylated genomic regions within two genes, discriminating inv(16)(p13;q22) from non-inv(16) pediatric AML samples, were identified. All six regions had a hypomethylated phenotype in inv(16) AML samples, and this was most prominent at the regions encompassing the meningioma (disrupted in balanced translocation) 1 (MN1) oncogene. MN1 expression primarily correlated with the methylation level of the 3' end of the MN1 exon-1 locus. Decitabine treatment of different cell lines showed that induced loss of methylation at the MN1 locus can result in an increase of MN1 expression, indicating that MN1 expression is coregulated by DNA methylation. To investigate this methylation-associated mechanism, we determined the expression of DNA methyltransferases in inv(16) AML. We found that DNMT3B expression was significantly lower in inv(16) samples. Furthermore, DNMT3B expression correlated negatively with MN1 expression in pediatric AML samples. Importantly, depletion of DNMT3B impaired remethylation efficiency of the MN1 exon-1 locus in AML cells after decitabine exposure. These findings identify DNMT3B as an important coregulator of MN1 methylation. Taken together, this study shows that the methylation level of the MN1 exon-1 locus regulates MN1 expression levels in inv(16) pediatric AML. This methylation level is dependent on DNMT3B, thus suggesting a role for DNMT3B in leukemogenesis in inv(16) AML, through MN1 methylation regulation.

A New Approach to Evaluating Aberrant DNA Methylation Profiles in Hepatocellular Carcinoma as Potential Biomarkers

Hypermethylation of CpG islands in the promoter region of tumor suppressor genes (TSGs) and their subsequent silencing is thought to be one of the main mechanisms of carcinogenesis. MBD2b enrichment coupled with a NimbleGen array was applied to examine the genome-wide CpG island methylation profile of hepatocellular carcinoma (HCC). Hypermethylated DNA of 58 pairs of HCC and adjacent tissue samples was enriched and hybridized in the same array. Aberrant hypermethylated peaks of HCC and adjacent tissues were screened and annotated after data processing using NimbleScan2.5 and our newly developed Weighting and Scoring (WAS) method, respectively. Validation using bisulfite sequencing of randomly selected ANKRD45, APC, CDX1, HOXD3, PTGER and TUBB6 genes demonstrated significant hypermethylation modification in HCC samples, consistent with the array data.

Abstract 4433: Genome-wide CpG island methylation profiling analysis identifies an epigenetically dysregulated necroptosis mediator in osteosarcoma

Abstract BACKGROUND Aberrant CpG island (CGI) methylation with dysregulation of gene expression is a hallmark of cancer. To identify biologically-relevant targets of aberrant epigenetic silencing in osteosarcomas, we analyzed CGI genes exhibiting disproportionate high-frequency gain of methylation. METHODS Infinium HumanMethylation 450 Beadchip profiling quantitated methylation at 485,578 CpG sites in bisulfite-modified DNA of 16 primary human osteosarcomas (OS) samples. Diff scores (D), reflecting difference in prevalence of site-specific methylation for OS samples compared to pooled non-neoplastic controls, were analyzed for gene-associated CGI sites. Genes exhibiting disproportionate high frequency of gain of methylation (+D), were identified utilizing a program to rank order gene CGI loci along an observed/expected (Obs/Exp) hit distribution where “hits” were defined as DAverage > DThreshold. RIPK3 CGI hypermethylation was quantitated by Combined Bisulfite Restriction Analysis (COBRA) and by bisulfite sequencing in 5 OS cell lines (HOS, MNNGHOS, MG-63, G-292, U2OS) and in human mesenchymal cells (hMSC). RIPK3 expression was analyzed by q-RT PCR. To evaluate the effect of RIPK3 expression on cisplatin-associated cytotoxicity, G-292 cells were stably transfected with RIPK3 or the corresponding empty vector (EV). Cisplatin cytotoxicity was assayed in RIPK3 transfectants versus vector-only controls. RESULTS At DTh = 50, >2500/15,000 CGI genes in the OS samples exhibited gain of CGI methylation in excess of what would be expected based on a uniform distribution of methylation (i.e., Obs/Exp > 1). 169 genes exhibited hypermethylation “hits” at all CGI sites assayed (Obs/Exp = 11.95) including the receptor interacting serine-threonine kinase 3 gene (RIPK3). RIPK3 mediates necroptotic cell death and has been associated with cisplatin sensitivity. COBRA analysis of RIPK3 CGI showed extensive methylation in MNNGHOS, MG63 and G292 cells and partial methylation in HOS and U2OS. hMSCs were minimally methylated. COBRA data were congruent with the prevalence of methylation at 18 CpG sites within the CGI determined by bisulfite sequencing. Quantitation of RIPK3 mRNA expression by q-RT PCR confirmed transcriptional silencing in MNNGHOS, MG63, U2OS and G292 cells. G292 stable transfectants expressing RIPK3 were more sensitive (lower LD50) when cultured in cisplatin compared to vector-only controls. CONCLUSIONS Analysis of genome-wide methylation profiling data to identify CGI genes exhibiting a disproportionate gain of methylation in OS identified RIPK3 hypermethylation. Extensive RIPK3 CGI hypermethylation is associated with transcriptional silencing in OS cells. We further show that expression of RIPK3 is associated with enhanced cisplatin cytotoxicity in G-292 cells. These results suggest that methylation of RIPK3 may contribute to cisplatin resistance in OS. Citation Format: Aditya Sharma, Elaine Notis, Christine Mella, Steven J. Kuerbitz. Genome-wide CpG island methylation profiling analysis identifies an epigenetically dysregulated necroptosis mediator in osteosarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4433.

CpG island methylation profile in non-invasive oral rinse samples is predictive of oral and pharyngeal carcinoma

BackgroundThere are currently no screening tests in routine use for oral and pharyngeal cancer beyond visual inspection and palpation, which are provided on an opportunistic basis, indicating a need for development of novel methods for early detection, particularly in high-risk populations. We sought to address this need through comprehensive interrogation of CpG island methylation in oral rinse samples.MethodsWe used the Infinium HumanMethylation450 BeadArray to interrogate DNA methylation in oral rinse samples collected from 154 patients with incident oral or pharyngeal carcinoma prior to treatment and 72 cancer-free control subjects. Subjects were randomly allocated to either a training or a testing set. For each subject, average methylation was calculated for each CpG island represented on the array. We applied a semi-supervised recursively partitioned mixture model to the CpG island methylation data to identify a classifier for prediction of case status in the training set. We then applied the resultant classifier to the testing set for validation and to assess the predictive accuracy.ResultsWe identified a methylation classifier comprised of 22 CpG islands, which predicted oral and pharyngeal carcinoma with a high degree of accuracy (AUC = 0.92, 95 % CI 0.86, 0.98).ConclusionsThis novel methylation panel is a strong predictor of oral and pharyngeal carcinoma case status in oral rinse samples and may have utility in early detection and post-treatment follow-up.

A seven-gene CpG-island methylation panel predicts breast cancer progression.

BackgroundDNA methylation regulates gene expression, through the inhibition/activation of gene transcription of methylated/unmethylated genes. Hence, DNA methylation profiling can capture pivotal features of gene expression in cancer tissues from patients at the time of diagnosis. In this work, we analyzed a breast cancer case series, to identify DNA methylation determinants of metastatic versus non-metastatic tumors.MethodsCpG-island methylation was evaluated on a 56-gene cancer-specific biomarker microarray in metastatic versus non-metastatic breast cancers in a multi-institutional case series of 123 breast cancer patients. Global statistical modeling and unsupervised hierarchical clustering were applied to identify a multi-gene binary classifier with high sensitivity and specificity. Network analysis was utilized to quantify the connectivity of the identified genes.ResultsSeven genes (BRCA1, DAPK1, MSH2, CDKN2A, PGR, PRKCDBP, RANKL) were found informative for prognosis of metastatic diffusion and were used to calculate classifier accuracy versus the entire data-set. Individual-gene performances showed sensitivities of 63–79 %, 53–84 % specificities, positive predictive values of 59–83 % and negative predictive values of 63–80 %. When modelled together, these seven genes reached a sensitivity of 93 %, 100 % specificity, a positive predictive value of 100 % and a negative predictive value of 93 %, with high statistical power. Unsupervised hierarchical clustering independently confirmed these findings, in close agreement with the accuracy measurements. Network analyses indicated tight interrelationship between the identified genes, suggesting this to be a functionally-coordinated module, linked to breast cancer progression.ConclusionsOur findings identify CpG-island methylation profiles with deep impact on clinical outcome, paving the way for use as novel prognostic assays in clinical settings.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1412-9) contains supplementary material, which is available to authorized users.

Identification of transgenic cloned dairy goats harboring human lactoferrin and methylation status of the imprinted gene IGF2R in their lungs.

Dairy goat is a good model for production of transgenic proteins in milk using somatic cell nuclear transfer (SCNT). However, animals produced from SCNT are often associated with lung deficiencies. We recently produced six transgenic cloned dairy goats harboring the human lactoferrin gene, including three live transgenic clones and three deceased transgenic clones that died from respiratory failure during the perinatal period. Imprinted genes are important regulators of lung growth, and may be subjected to faulty reprogramming. In the present study, first, microsatellite analysis, PCR, and DNA sequence identification were conducted to confirm that these three dead kids were genetically identical to the transgenic donor cells. Second, the CpG island methylation profile of the imprinted insulin-like growth factor receptor (IGF2R) gene was assessed in the lungs of the three dead transgenic kids and the normally produced kids using bisulfite sequencing PCR. In addition, the relative mRNA level of IGF2R was also determined by real-time PCR. Results showed that the IGF2R gene in the lungs of the dead cloned kids showed abnormal hypermethylation and higher mRNA expression levels than the control, indicating that aberrant DNA methylation reprogramming is one of the important factors in the death of transgenic cloned animals.

DNA Methylation Profiling of Pediatric AML Reveals That Hypomethylation of MN1 Is Characteristic of Inv(16) AML and a Driver of MN1 Overexpression

Primary refractory and relapsed pediatric acute myeloid leukemia (AML) still lead to a significant number of childhood cancer deaths, despite the current chemotherapeutic regimens. AML leukemogenesis is driven by collaborative genetic abnormalities that induce hematopoietic maturation arrest and cell proliferation. Particular AML-associated maturation inhibiting aberrations are known to target chromatin regulators, thus directly influencing the transcriptional program of leukemic cells. Therapies targeting epigenetic processes, e.g. with hypomethylation-inducing agents, are therefore becoming an attractive therapeutic strategy in adult AML. AML biology in children is not equivalent to that of adults, thus methylation patterns seen in adult AML cannot be extrapolated to pediatric AML. Therefore there is a need to unravel the mechanism behind changes in epigenetic processes as the result of AML-causing genetic abnormalities in order to develop new drugs for pediatric AML.We hypothesized that pediatric AML samples have distinct DNA-methylation patterns which may provide a rationale for treatment with demethylating agents in specific pediatric AML subtypes. Furthermore, these differences in methylation could be characteristic for AML subgroups and that particular methylation patterns drive the expression of specific genes which may play a key role in the tumorigenesis of these AML leukemias.We performed genome-wide CpG-island methylation profiling on a representative and molecularly characterized cohort of pediatric patients with de novo AML. Empirical Bayes Wilcoxon rank-sum test showed that AML patients carrying inv(16)(p13;q22) (n=9) have distinct DNA methylation patterns when compared to non-inv(16) AML patients (n=143) (consisting mainly of MLL-rearranged, t(8;21), t(15;17), t(8;16) AML and AML cases with a normal karyotype). The MN1 gene ranked as most significantly differentially methylated in inv(16) AML compared to non-inv(16) AML, with inv(16) AML cases having significantly (p=2x10-6) lower methylation levels compared to non-inv(16) AML cases. Hypomethylation of specific regions of the MN1-associated CpG-island was confirmed by methylation specific PCR and bisulfite sequencing. Subsequent gene expression (GEP) data on 294 pediatric AML patients showed that MN1 was 8 fold higher expressed in patients carrying inv(16) compared to all other patients (9.9, n=35 vs 6.9, n=259, p<0.001). Furthermore, integrating GEP and methylation array data showed that MN1 expression negatively correlated (ρs= 0.82, p=0.011) with methylation levels, which is in agreement with the biological assumption of methylation and gene expression.Since genes known to regulate DNA methylation have frequently been shown to be mutated in adult AML we determined whether a decreased expression of DNA methyltransferases, DNMT1, DNMT2, DNMT3A, DNMT3B, could be the cause of a hypometylated MN1 locus in inv(16) AML. Our findings show that only DNMT3B expression was significantly (p=8x10-15) lower in inv(16) cases compared to non-inv(16) cases. To test whether hypomethylation of the MN1 CpG-island and the overexpression of MN1 is the result of decreased DNMT1 expression, HL60 cells which express negligible levels of MN1 were treated with the DNMT1 inhibitor Decitabine. This showed that treatment of HL60 cells with Decitabine led to increase of MN1 transcript levels, however, not as high as those observed in patient samples. This suggests that DNMT1 activity may not be the only DNA methyltransferase influencing expression of MN1 in inv(16) patients. Interestingly, we observed a high (ρs= 0.42) correlation between MN1 methylation and DNMT3B expression, which suggests DNMT3B could be an important DNA methyltransferase involved in regulating MN1expression.Overall we show that pediatric AML patients carrying and inv(16) have a characteristic DNA methylation pattern compared to other AML patients carrying specific cytogenetic aberrations. Furthermore, our data suggest that hypomethylation of the MN1 gene is an underlying mechanism for high MN1 expression in inv(16)(p13;q22) patients possibly regulated by multiple DNA methyltransferases. DisclosuresNo relevant conflicts of interest to declare.

Abstract IA04: EWS-FLI1, polycomb proteins and stem cells: Clues to the developmental origins of Ewing sarcoma

Abstract IA04: EWS-FLI1, polycomb proteins and stem cells: Clues to the developmental origins of Ewing sarcoma

Methylation Profile of the Promoter CpG Islands of Death Associated Protein-Kinase in Nephroblastoma

Background : Nephroblastoma (Wilms Tumor) is the most common malignant, solid, extracranial tumor of childhood which has a complex etiology, displaying genetic and epigenetic changes, including loss of imprinting and tumor suppressor gene silencing. Methylation of promoter regions of CpG-rich sites is an important mechanism for silencing of tumor suppressor genes. Death Associated Protein Kinase (DAPK) is a pro-apoptotic, calcium/calmodulin-regulated protein kinase that methylation profile of the promoter CpG islands is a target for cancer. DAPK is not studied in nephroblastoma yet. The aim of this study is to establish the methylation profile of the promoter CpG islands of DAPK that might play role in tumorigenesis and chemotherapy effect of nephroblastoma. Patients and Methods : Methylation spesific PCR was performed to detect the methylation profile of the promoter CpG islands of DAPK in pre and post chemotherapy biopsy and operation tissues of the same cases of nephroblastoma (n=8), in persistant subcapsular nephrogenic zone (PSNZ) of kidney obtained from postnatal term autopsy cases (n=6). PSNZ is a blastic focus generally multifoca. It resembles nephroblastoma in morphology. This zone exists in embriology but differentiates to renal cortex after term delivery. Chi- square test was used in statistical analysis. Results : As detected by methylation specific PCR, DAPK methylation rate was low in nephroblastoma biopsy (25%) and after chemotherapy tissues (37.5%). It was 50% in PSNZ cases. The methylation status changed in only one case that became methylated after chemotherapy. There is no statistical difference between PSNZ and nephroblastoma cases. Conclusions : DAPK methylation does not seem to play a role in chemotherapy effect on nephroblastoma. It does not seem to play role in persistance of subcapsular nephrogenic zone in normal kidney. Further studies with large case series including nephroblastic rest tissue, chemotherapy resistant cases, normal renal cortical tissue and with wide panel of methylation status of genes are recommended.

DNA methylation and expression profiles of the brain-derived neurotrophic factor (BDNF) and dopamine transporter (DAT1) genes in patients with schizophrenia

Methylation and expression profile of CpG islands were examined in the promoters of the brain-derived neurotrophic factor (BDNF) and dopamine transporter (DAT1) genes. These are well known to be involved in the pathophysiology of psychiatric disorders such as schizophrenia. Genomic DNA was extracted from peripheral blood of 80 patients with schizophrenia and 71 healthy controls. Methylation pattern was studied by Methylation-Specific PCR. RNA expression analysis was done on extracted RNA from blood samples from patients suffering from schizophrenia (n = 17) and healthy controls (n = 17). Frequency of the BDNF gene methylation was highlighted as a statistically significant relationship between cases and controls regarding decreased risk of disease in comparison to unmethylated patterns (OR = 0.24; 95 % CI = 1.11-0.50; P = 0.00007). For the DAT1 gene, this relationship was insignificant in 61 cases (76.25 %) and 52 controls (73.23 %) (OR = 1.17; 95 % CI = 0.53-2.61). Estimates of relative gene expression revealed a statistically significant association of the BDNF gene between schizophrenic patients and healthy controls (Mean ± SD: 13.3920 ± 15.19 and 0.437 ± 0.328, P = 0.0001) respectively; however, it was not significant for the DAT1 gene. This first hand evidence, regarding BDNF and DAT1 gene methylation and their expression profile with risk of schizophrenia, indicated a significant function for the BDNF gene in the development of schizophrenia. However, further populations with large sample sizes need to be studied to verify the exact role of BDNF in mental disorders such as schizophrenia.

Abstract 4521: Differential whole genome methylation profile in a responder and a non-responder to chemotherapy in colorectal cancer patients

Abstract Background: Colorectal adenoma (CRA) to cancer (CRC) progression involves in epigenetic changes including DNA methylation. Here we performed whole genome Bisulfite Next-Generation sequencing (WGBSNGS) on a normal, a tubular adenoma and a tumor patients’ tissue DNA to elucidate epigenetic drivers in normal to cancer progression. Patients and Methods: Genomic DNA was isolated from fresh frozen tissues from a patient with normal colon, from a tubular adenoma lesion (&amp;lt;0.5 cm) and from a carcinoma. A WGBSNGS at &amp;gt;20X depth was performed on these DNA samples., Alignment, mapping and CpG methylation analyses were done. Pyrosequencing and immunohistochemistry (IHC) was used to confirm selected CpG methylation. Results: We identified 94 unique CpG sites hypermethylated in 4 novel genes in adenoma compared to normal and 180 unique CpG sites in 7 genes in cancer. Ingenuity pathway analysis (IPA) showed that the methylated genes including a novel gene ATXN7L1 were involved in WNT pathway, ubiquotlation process and cross-talk with B-catenin, in the adenoma. Three of the hypermethylated genes in cancer were, Loc100506436: a novel gene with 17 unique CpG sites (EGFR pathway, cRNA CpG site hypermethylated is part of MEK1/2), GPNMB with 7 unique CpG sites (a G protein) and TNFAIP2 with 8 unique CpG sites (its role was noted in Head and neck cancer). IHC confirmed the lack of expression in cancer. Conclusion: This work provides insight into differential CpG island methylation profiles in CRA, CRC vs. normal colon tissue and provides a window into the more complex factors, including alteration of known and novel genes as a multiple CpG methylation drivers in colorectal carcinogenesis. Investigations into the possible roles of the novel gene targets in the context of early and prognostic methylation biomarker are underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4521. doi:1538-7445.AM2012-4521

Abstract 994: Colorectal cancer, adenoma and normal whole genome methylation sequencing in Africa Americans

Abstract Background: Colorectal adenoma (CRA) to cancer (CRC) progression involves epigenetic changes including DNA methylation. Here we performed whole genome Bisulfite Next-Generation Sequencing (WGBNGS) on a normal, a tubular adenoma and a tumor patients’ tissue DNA to elucidate epigenetic drivers in normal to cancer progression. Patients and Methods: Genomic DNA was isolated from fresh frozen tissues from a patient with normal colon, from a tubular adenoma lesion (20X depth was performed on these DNA samples., Alignment, mapping and CpG methylation analyses were done. Pyrosequencing and immunohistochemistry (IHC) were used to confirm selected methylation target genes. Results: We identified 94 unique CpG sites hypermethylated in 4 novel genes in adenoma compared to normal and 180 unique CpG sites in 7 genes in cancer. Ingenuity pathway analysis (IPA) showed that the methylated genes including a novel gene ATXN7L1 were involved in WNT pathway, ubiquitination process and cross-talk with B-catenin, in the adenoma. Three of the hypermethylated genes in cancer were, Loc100506436: a novel gene with 17 unique CpG sites (EGFR pathway, cRNA CpG site hypermethylated is part of MEK1/2), GPNMB with 7 unique CpG sites (membrane protein likely in complexes with integrins) and TNFAIP2 with 8 unique CpG sites (its role was noted in Head and neck cancer). IHC confirmed the lack of GPNMB expression in cancer tissues. Conclusion: This work provides insight into differential CpG island methylation profiles in CRA, CRC vs. normal colon tissue and provides a window into the more complex factors, including alteration of known and novel genes as multiple CpG methylation drivers in colorectal carcinogenesis. Investigations into the possible roles of the novel genes targets in the context of early and prognostic methylation biomarker are underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 994. doi:1538-7445.AM2012-994

CpG Island Methylation Profile of Estrogen Receptor Alpha in Iranian Females with Triple Negative or Non-triple Negative Breast Cancer: New Marker of Poor Prognosis

One decade early onset of the breast cancer in Iranian females was reported but the basis of the observed difference has remained unclear and difference in gene silencing by epigenetic processes is suggested. Hence, this study was sought to map the methylation status of ER gene CpG islands and its impact on clinicopathological factors of triple negative and non-triple negative ductal cell carcinoma of the breast in Iranian females. Surgically resected formalin-fixed paraffin-embedded breast tissues from sixty Iranian women with confirmed invasive ductal carcinoma were assessed by methylation-specific PCR using primer sets encompassing some of the 29 CpGs across the ER gene CpG island. The estrogen and progesterone receptors, Her-2 overexpression, and nuclear accumulation of P53 were examined using immunohistochemistry (IHC). Methylated ER3, ER4, and ER5 were found in 41.7, 11.3, and 43.3% of the samples, respectively. Significantly higher methylation of ER4 was found in the tumors with nuclear accumulation of P53, and significantly higher methylation of ER5 was found in patients with lymph node involvement and tumor with bigger size or higher grades. Furthermore, significantly higher rate of ER5 methylation was found in patients with Her-2+ tumors and in postmenopausal patients with ER-, PgR-, or ER-/PgR- tumors. However, no significant difference in ERs methylation status was found between triple negative and non-triple negative tumors in pre- and postmenopausal patients. Findings revealed that aberrant hypermethylation of ER-a gene frequently occur in Iranian women with invasive ductal cell carcinoma of the breast. However, methylation of different CpG islands produced a diverse impact on the prognosis of breast cancer, and ER5 was found to be the most frequently methylated region in the Iranian women, and could serve as a marker of poor prognosis.

Genome-Wide CpG Island Profiling of Intraductal Papillary Mucinous Neoplasms of the Pancreas

Intraductal papillary mucinous neoplasms (IPMN) are precursors to infiltrating pancreatic ductal adenocarcinomas. Widespread epigenetic alterations are characteristic of many cancers, yet few studies have systematically analyzed epigenetic alterations of neoplastic precursors. Our goal was to conduct genome-wide CpG island methylation profiling to identify aberrantly methylated loci in IPMNs. We compared the CpG island methylation profiles of six IPMNs to normal primary pancreatic duct samples using methylation CpG island amplification (MCA) and Agilent CpG island microarray (MCAM) analysis. When selected 13 genes identified as differentially methylated by MCAM for methylation-specific PCR (MSP) analysis in an independent set of IPMNs and normal pancreas samples and conducted expression analysis of selected genes. We identified 2,259 loci as differentially methylated in at least one of six IPMNs including 245 genes hypermethylated in IPMNs with high-grade dysplasia compared with normal pancreatic duct samples. Eleven of 13 genes evaluated by MSP were more commonly methylated in 61 IPMNs than in 43 normal pancreas samples. Several genes (BNIP3, PTCHD2, SOX17, NXPH1, EBF3) were significantly more likely to be methylated in IPMNs with high-grade than with low-grade dysplasia. One gene, SOX17, showed loss of protein expression by immunohistochemistry in 22% (19 of 88) of IPMNs. The most specific marker, BNIP3, was not methylated in any IPMNs with low-grade dysplasia or in normal pancreas samples. IPMNs undergo extensive aberrant CpG island hypermethylation. The detection of genes selectively methylated in high-grade IPMNs such as BNIP3 may have use in the clinical evaluation of IPMNs.