Showing papers in "Clinical Epigenetics in 2014"
TL;DR: The smoking score is a valuable tool for identification of true current smoking behaviour and Explanations for ethnic differences in DNA methylation in association with smoking may provide valuable clues to disease pathways.
Abstract: DNA methylation is strongly associated with smoking status at multiple sites across the genome. Studies have largely been restricted to European origin individuals yet the greatest increase in smoking is occurring in low income countries, such as the Indian subcontinent. We determined whether there are differences between South Asians and Europeans in smoking related loci, and if a smoking score, combining all smoking related DNA methylation scores, could differentiate smokers from non-smokers. Illumina HM450k BeadChip arrays were performed on 192 samples from the Southall And Brent REvisited (SABRE) cohort. Differential methylation in smokers was identified in 29 individual CpG sites at 18 unique loci. Interaction between smoking status and ethnic group was identified at the AHRR locus. Ethnic differences in DNA methylation were identified in non-smokers at two further loci, 6p21.33 and GNG12. With the exception of GFI1 and MYO1G these differences were largely unaffected by adjustment for cell composition. A smoking score based on methylation profile was constructed. Current smokers were identified with 100% sensitivity and 97% specificity in Europeans and with 80% sensitivity and 95% specificity in South Asians. Differences in ethnic groups were identified in both single CpG sites and combined smoking score. The smoking score is a valuable tool for identification of true current smoking behaviour. Explanations for ethnic differences in DNA methylation in association with smoking may provide valuable clues to disease pathways.
228 citations
TL;DR: It is suggested that this novel tool will facilitate selection of patients with suspected BWS for routine diagnostic testing and so improve the diagnosis of the disorder.
Abstract: Beckwith-Wiedemann syndrome (BWS), a congenital overgrowth disorder with variable expressivity and a predisposition to tumorigenesis, results from disordered expression and/or function of imprinted genes at chromosome 11p15.5. There are no generally agreed clinical diagnostic criteria, with molecular studies commonly performed to confirm diagnosis. In particular, methylation status analysis at two 11p15.5 imprinting control centres (IC1 and IC2) detects up to 80% of BWS cases (though low-level mosaicism may not be detected). In order to evaluate the relationship between the clinical presentation of suspected BWS and IC1/2 methylation abnormalities we reviewed the results of >1,000 referrals for molecular diagnostic testing. Out of 1,091 referrals, 507 (46.5%) had a positive diagnostic test for BWS. The frequency of tumours was 3.4% in those with a molecular diagnosis of BWS. Previously reported genotype-phenotype associations with paternal uniparental disomy, IC1, and IC2 epimutation groups were confirmed and potential novel associations detected. Predictive values of previously described clinical diagnostic criteria were compared and, although there were differences in their sensitivity and specificity, receiver operating characteristic (ROC) analysis demonstrated that these were not optimal in predicting 11p15.5 methylation abnormalities. Using logistic regression, we identified clinical features with the best predictive value for a positive methylation abnormality. Furthermore, we developed a weighted scoring system (sensitivity 75.9%, and specificity 81.8%) to prioritise patients presenting with the most common features of BWS, and ROC analysis demonstrated superior performance (area under the curve 0.85, 95% CI 0.83 to 0.87) compared to previous criteria. We suggest that this novel tool will facilitate selection of patients with suspected BWS for routine diagnostic testing and so improve the diagnosis of the disorder.
90 citations
TL;DR: The known functions of affected genes suggest that the observed epigenetic changes may underlie the amelioration of symptoms as well as accounting for certain adverse effects including the metabolic syndrome.
Abstract: The mechanism of action of olanzapine in treating schizophrenia is not clear. This research reports the effects of a therapeutic equivalent treatment of olanzapine on DNA methylation in a rat model in vivo. Genome-wide DNA methylation was assessed using a MeDIP-chip analysis. All methylated DNA immunoprecipitation (MeDIP), sample labelling, hybridization and processing were performed by Arraystar Inc (Rockville, MD, USA). The identified gene promoters showing significant alterations to DNA methylation were then subjected to Ingenuity Pathway Analysis (Ingenuity System Inc, CA, USA). The results show that olanzapine causes an increase in methylation in 1,140, 1,294 and 1,313 genes and a decrease in methylation in 633, 565 and 532 genes in the hippocampus, cerebellum and liver, respectively. Most genes affected are tissue specific. Only 41 affected genes (approximately 3%) showed an increase and no gene showed a decrease in methylation in all three tissues. Further, the two brain regions shared 123 affected genes (approximately 10%). The affected genes are enriched in pathways affecting dopamine signalling, molecular transport, nervous system development and functions in the hippocampus; ephrin receptor signalling and synaptic long-term potentiation in the cerebellum; and tissue morphology, cellular assembly and organization in the liver. Also, the affected genes included those previously implicated in psychosis. The known functions of affected genes suggest that the observed epigenetic changes may underlie the amelioration of symptoms as well as accounting for certain adverse effects including the metabolic syndrome. The results give insights into the mechanism of action of olanzapine, therapeutic effects and the side effects of antipsychotics.
73 citations
TL;DR: Demethylation of CD11a regulatory elements and subsequentCD11a overexpression in CD4+ T cells may mediate immunological abnormalities and fibrotic processes in SSc.
Abstract: Background
The pathogenesis and etiology of systemic sclerosis (SSc) are complex and poorly understood. To date, several studies have demonstrated that the activation of the immune system undoubtedly plays a pivotal role in SSc pathogenesis. Activated immune effector T cells contribute to the release of various pro-inflammatory cytokines and drive the SSc-specific autoantibody responses. This, and a profibrotic environment, are all-important components of abnormal active immune responses that can lead to pathological disorders of SSc. CD11a is essential to inflammatory and immune responses, regulating adhesive and co-stimulatory interactions between CD4+ T cells and other cells. Although CD11a is overexpressed in SSc patients, the mechanisms leading to this overexpression and its consequences remain unclear. DNA methylation, a main epigenetic modification, plays an important role in the regulation of gene expression and is involved in the pathogenesis of autoimmune diseases. This work aims to investigate the effect of DNA demethylation on CD11a expression in SSc CD4+ T cells and to determine its functional significance. CD11a expression was measured using RT-PCR and flow cytometry. Bisulfite sequencing was used to determine the methylation status of the CD11a regulatory region. CD4+ T cells were co-cultured with antigen-presenting cells, B cells, or fibroblasts with and without anti-CD11a, and proliferation of CD4+ T cells, IgG production by B cells, and expression levels of COL1A2 mRNA by fibroblasts were evaluated.
72 citations
TL;DR: An assay to identify changes in DNA methylation on the pathogenic distal 4q D4Z4 repeat can distinguish FSHD from healthy controls, differentiate F SHD1 from FSHd2, does not require HMW genomic DNA or PFGE, and can be performed on either cultured cells, tissue, blood, or saliva samples.
Abstract: Facioscapulohumeral muscular dystrophy (FSHD) is linked to chromatin relaxation due to epigenetic changes at the 4q35 D4Z4 macrosatellite array. Molecular diagnostic criteria for FSHD are complex and involve analysis of high molecular weight (HMW) genomic DNA isolated from lymphocytes, followed by multiple restriction digestions, pulse-field gel electrophoresis (PFGE), and Southern blotting. A subject is genetically diagnosed as FSHD1 if one of the 4q alleles shows a contraction in the D4Z4 array to below 11 repeats, while maintaining at least 1 repeat, and the contraction is in cis with a disease-permissive A-type subtelomere. FSHD2 is contraction-independent and cannot be diagnosed or excluded by this common genetic diagnostic procedure. However, FSHD1 and FSHD2 are linked by epigenetic deregulation, assayed as DNA hypomethylation, of the D4Z4 array on FSHD-permissive alleles. We have developed a PCR-based assay that identifies the epigenetic signature for both types of FSHD, distinguishing FSHD1 from FSHD2, and can be performed on genomic DNA isolated from blood, saliva, or cultured cells. Samples were obtained from healthy controls or patients clinically diagnosed with FSHD, and include both FSHD1 and FSHD2. The genomic DNAs were subjected to bisulfite sequencing analysis for the distal 4q D4Z4 repeat with an A-type subtelomere and the DUX4 5’ promoter region. We compared genomic DNA isolated from saliva and blood from the same individuals and found similar epigenetic signatures. DNA hypomethylation was restricted to the contracted 4qA chromosome in FSHD1 patients while healthy control subjects were hypermethylated. Candidates for FSHD2 showed extreme DNA hypomethylation on the 4qA DUX4 gene body as well as all analyzed DUX4 5’ sequences. Importantly, our assay does not amplify the D4Z4 arrays with non-permissive B-type subtelomeres and accurately excludes the arrays with non-permissive A-type subtelomeres. We have developed an assay to identify changes in DNA methylation on the pathogenic distal 4q D4Z4 repeat. We show that the DNA methylation profile of saliva reflects FSHD status. This assay can distinguish FSHD from healthy controls, differentiate FSHD1 from FSHD2, does not require HMW genomic DNA or PFGE, and can be performed on either cultured cells, tissue, blood, or saliva samples.
62 citations
TL;DR: This study provides new evidence that the ABCA1 epigenetic profile is associated with CAD and aging, and highlights that epigenetic modifications might be a significant molecular mechanism involved in the pathophysiological processesassociated with CAD.
Abstract: Background
Previous studies have suggested that DNA methylation contributes to coronary artery disease (CAD) risk variability. DNA hypermethylation at the ATP-binding cassette transporter A1 (ABCA1) gene, an important modulator of high-density lipoprotein cholesterol and reverse cholesterol transport, has been previously associated with plasma lipid levels, aging and CAD, but the association with CAD has yet to be replicated.
60 citations
TL;DR: The data suggest that it may be possible to design a very effective therapy for AML using agents that target the reversal of the following three epigenetic “lock” mechanisms that silence gene expression: DNA methylation, histonemethylation, and histone deacetylation.
Abstract: The silencing of tumor suppressor genes (TSGs) by aberrant DNA methylation occurs frequently in acute myeloid leukemia (AML). This epigenetic alteration can be reversed by 5-aza-2’-deoxcytidine (decitabine, 5-AZA-CdR). Although 5-AZA-CdR can induce complete remissions in patients with AML, most patients relapse. The effectiveness of this therapy may be limited by the inability of 5-AZA-CdR to reactivate all TSGs due to their silencing by other epigenetic mechanisms such as histone methylation or chromatin compaction. EZH2, a subunit of the polycomb repressive complex 2, catalyzes the methylation of histone H3 lysine 27 (H3K27) to H3K27me3. 3-Deazaneplanocin-A (DZNep), an inhibitor of methionine metabolism, can reactivate genes silenced by H3K27me3 by its inhibition of EZH2. In a previous report, we observed that 5-AZA-CdR, in combination with DZNep, shows synergistic antineoplastic action against AML cells. Gene silencing due to chromatin compaction is attributable to the action of histone deacetylases (HDAC). This mechanism of epigenetic gene silencing can be reversed by HDAC inhibitors such as trichostatin-A (TSA). Silent TSGs that cannot be reactivated by 5-AZA-CdR or DZNep have the potential to be reactivated by TSA. This provides a rationale for the use of HDAC inhibitors in combination with 5-AZA-CdR and DZNep to treat AML. The triple combination of 5-AZA-CdR, DZNep, and TSA induced a remarkable synergistic antineoplastic effect against human AML cells as demonstrated by an in vitro colony assay. This triple combination also showed a potent synergistic activation of several key TSGs as determined by real-time PCR. The triple combination was more effective than the combination of two agents or a single agent. Microarray analysis showed that the triple combination generated remarkable changes in global gene expression. Our data suggest that it may be possible to design a very effective therapy for AML using agents that target the reversal of the following three epigenetic “lock” mechanisms that silence gene expression: DNA methylation, histone methylation, and histone deacetylation. This approach merits serious consideration for clinical investigation in patients with advanced AML.
59 citations
TL;DR: The findings indicate that DNA methylation profiling contributes to the clarification of the heterogeneity in cytogenetically undefined ALL patient groups and could be implemented as a complementary method for diagnosis of ALL.
Abstract: We present a method that utilizes DNA methylation profiling for prediction of the cytogenetic subtypes of acute lymphoblastic leukemia (ALL) cells from pediatric ALL patients. The primary aim of our study was to improve risk stratification of ALL patients into treatment groups using DNA methylation as a complement to current diagnostic methods. A secondary aim was to gain insight into the functional role of DNA methylation in ALL. We used the methylation status of ~450,000 CpG sites in 546 well-characterized patients with T-ALL or seven recurrent B-cell precursor ALL subtypes to design and validate sensitive and accurate DNA methylation classifiers. After repeated cross-validation, a final classifier was derived that consisted of only 246 CpG sites. The mean sensitivity and specificity of the classifier across the known subtypes was 0.90 and 0.99, respectively. We then used DNA methylation classification to screen for subtype membership of 210 patients with undefined karyotype (normal or no result) or non-recurrent cytogenetic aberrations (‘other’ subtype). Nearly half (n = 106) of the patients lacking cytogenetic subgrouping displayed highly similar methylation profiles as the patients in the known recurrent groups. We verified the subtype of 20% of the newly classified patients by examination of diagnostic karyotypes, array-based copy number analysis, and detection of fusion genes by quantitative polymerase chain reaction (PCR) and RNA-sequencing (RNA-seq). Using RNA-seq data from ALL patients where cytogenetic subtype and DNA methylation classification did not agree, we discovered several novel fusion genes involving ETV6, RUNX1, and PAX5. Our findings indicate that DNA methylation profiling contributes to the clarification of the heterogeneity in cytogenetically undefined ALL patient groups and could be implemented as a complementary method for diagnosis of ALL. The results of our study provide clues to the origin and development of leukemic transformation. The methylation status of the CpG sites constituting the classifiers also highlight relevant biological characteristics in otherwise unclassified ALL patients.
56 citations
TL;DR: The interaction of DNA methylation and SNPs in Th2 pathway genes is likely to contribute to asthma risk, and this effect may vary with age.
Abstract: Genetic effects on asthma of genes in the T-helper 2 (Th2) pathway may interact with epigenetic factors including DNA methylation. We hypothesized that interactions between genetic variants and methylation in genes in this pathway (IL4, IL4R, IL13, GATA3, and STAT6) influence asthma risk, that such influences are age-dependent, and that methylation of some CpG sites changes over time in accordance with asthma transition. We tested these hypotheses in subsamples of girls from a population-based birth cohort established on the Isle of Wight, UK, in 1989. Logistic regression models were applied to test the interaction effect of DNA methylation and SNP on asthma within each of the five genes. Bootstrapping was used to assess the models identified. From 1,361 models fitted at each age of 10 and 18 years, 8 models, including 4 CpGs and 8 SNPs, showed potential associations with asthma risk. Of the 4 CpGs, methylation of cg26937798 (IL4R) and cg23943829 (IL4) changes between ages 10 and 18 (both higher at 10; P = 9.14 × 10−6 and 1.07 × 10−5, respectively). At age 10, the odds of asthma tended to decrease as cg12405139 (GATA3) methylation increased (log-OR = −12.15; P = 0.049); this effect disappeared by age 18. At age 18, methylation of cg09791102 (IL4R) was associated with higher risk of asthma among subjects with genotype GG compared to AG (P = 0.003), increased cg26937798 methylation among subjects with rs3024685 (IL4R) genotype AA (P = 0.003) or rs8832 (IL4R) genotype GG (P = 0.01) was associated with a lower asthma risk; these CpGs had no effect at age 10. Increasing cg26937798 methylation over time possibly reduced the risk of positive asthma transition (asthma-free at age 10 → asthma at age 18; log-OR = −3.11; P = 0.069) and increased the likelihood of negative transition (asthma at age 10 → asthma-free at age 18; log-OR = 3.97; P = 0.074). The interaction of DNA methylation and SNPs in Th2 pathway genes is likely to contribute to asthma risk. This effect may vary with age. Methylation of some CpGs changed over time, which may influence asthma transition.
56 citations
TL;DR: The authors' data revealed highly dynamic cfDNA methylation profiles in support of HBV-related HCC development and identified a panel of DMGs that are predictive for the early, middle and late stages of H CC development, which are potential markers for theEarly detection of HCC as well as the screening of high-risk populations.
Abstract: An important model of hepatocellular carcinoma (HCC) that has been described in southeast Asia includes the transition from chronic hepatitis B infection (CHB) to liver cirrhosis (LC) and, finally, to HCC. The genome-wide methylation profiling of plasma cell-free DNA (cfDNA) has not previously been used to assess HCC development. Using MethylCap-seq, we analyzed the genome-wide cfDNA methylation profiles by separately pooling healthy control (HC), CHB, LC and HCC samples and independently validating the library data for the tissue DNA and cfDNA by MSP, qMSP and Multiplex-BSP-seq. The dynamic features of cfDNA methylation coincided with the natural course of HCC development. Data mining revealed the presence of 240, 272 and 286 differentially methylated genes (DMGs) corresponding to the early, middle and late stages of HCC progression, respectively. The validation of the DNA and cfDNA results in independent tissues identified three DMGs, including ZNF300, SLC22A20 and SHISA7, with the potential for distinguishing between CHB and LC as well as between LC and HCC. The area under the curve (AUC) ranged from 0.65 to 0.80, and the odds ratio (OR) values ranged from 5.18 to 14.2. Our data revealed highly dynamic cfDNA methylation profiles in support of HBV-related HCC development. We have identified a panel of DMGs that are predictive for the early, middle and late stages of HCC development, and these are potential markers for the early detection of HCC as well as the screening of high-risk populations.
51 citations
TL;DR: The epigenetic modulators AZA, TSA, SFN, and SAM may provide opportunities for cancer prevention by regulating the components of epigenetic gene-silencing machinery especially DNMTs and MBDs.
Abstract: DNA methylation mediates gene silencing primarily by inducing repressive chromatin architecture via a common theme of interaction involving methyl-CpG binding (MBD) proteins, histone modifying enzymes and chromatin remodelling complexes. Hence, targeted inhibition of MBD protein function is now considered a potential therapeutic alternative for thwarting DNA hypermethylation prompted neoplastic progress. We have analyzed the gene and protein expression level of the principal factors responsible for gene silencing, that is, DNMT and MBD proteins in MCF-7 and MDA-MB-231 breast cancer cell lines after treatment with various epigenetic drugs. Our study reveals that the epigenetic modulators affect the expression levels at both transcript and protein levels as well as encourage growth arrest and apoptosis in MCF-7 and MDA-MB-231 cells. AZA, TSA, SFN, and SAM inhibit cell growth in MCF-7 and MDA-MB-231 cell lines in a dose-dependent manner, that is, with increasing concentrations of drugs the cell viability gradually decreases. All the epigenetic modulators promote apoptotic cell death, as is evident form increased chromatin condensation which is a distinct characteristic of apoptotic cells. From FACS analysis, it is also clear that these drugs induce G2-M arrest and apoptosis in breast cancer cells. Further, transcript and protein level expression of MBDs and DNMTs is also affected - after treatment with epigenetic drugs; the level of transcripts/mRNA of MBDs and DNMTs has consistently increased in general. The increase in level of gene expression is substantiated at the protein level also where treated cells show higher expression of DNMT1, DNMT3A, DNMT3B, and MBD proteins in comparison to untreated cells. In case of tissue samples, the expression of different DNMTs is tissue stage-specific. DNMT1 exhibits significantly higher expression in the metastatic stage, whereas, DNMT3A and DNMT3B have higher expression in the primary stage in comparison to the metastatic samples. The epigenetic modulators AZA, TSA, SFN, and SAM may provide opportunities for cancer prevention by regulating the components of epigenetic gene-silencing machinery especially DNMTs and MBDs.
TL;DR: This study shows for the first time that T1D patients had decreased DNA methylation levels in the IGFBP1 gene and further implies that increased circulating IGFBP-1 levels are associated with T 1D and DN.
Abstract: Background: Clinical observations have demonstrated that high levels of circulating insulin-like growth factor binding protein-1 (IGFBP-1) are associated with type 1 diabetes (T1D), whereas low serum IGFBP-1 levels are associated with the risk of type 2 diabetes (T2D). Recently, we reported that increased DNA methylation levels in the IGFBP1 gene were associated with T2D. In the present study, we evaluated the epigenetic changes of IGFBP1 in T1D and diabetic nephropathy (DN). Results: In total, 778 Swedish individuals, including T1D patients with or without DN and subjects with the normal glucose tolerance (NGT), were involved in the study. IGFBP1 methylation levels in genomic DNA extracted from peripheral blood were analyzed with bisulfite pyrosequencing. Serum IGFBP-1 levels were measured with radioimmunoassay. We found that DNA methylation levels in the IGFBP1 gene were decreased (15.6% versus 16.9%; P< 0.001), whereas serum IGFBP-1 levels were increased (31 versus 24 μg/L, P = 0.003) in T1D patients compared with NGT subjects. Furthermore, T1D patients with DN had increased circulating IGFBP-1 concentration compared with the patients without DN (52 versus 28 μg/L; P = 0.006). However, no difference of the IGFBP1 DNA methylation levels between T1D patients with and without DN was observed. Conclusions: This study shows for the first time that T1D patients had decreased DNA methylation levels in the IGFBP1 gene and further implies that increased circulating IGFBP-1 levels are associated with T1D and DN.
TL;DR: Analysis of methylation data from the Illumina HumanMethylation450 array shows that genotype at 1,069 highly informative loci, and both alcohol and smoking consumption information, can be derived from the array data.
Abstract: Background: Genome-wide methylation arrays are increasingly used tools in studies of complex medical disorders. Because of their expense and potential utility to the scientific community, current federal policy dictates that data from these arrays, like those from genome-wide genotyping arrays, be deposited in publicly available databases. Unlike the genotyping information, access to the expression data is not restricted. An underlying supposition in the current nonrestricted access to methylation data is the belief that protected health and personal identifying information cannot be simultaneously extracted from these arrays. Results: In this communication, we analyze methylation data from the Illumina HumanMethylation450 array and show that genotype at 1,069 highly informative loci, and both alcohol and smoking consumption information, can be derived from the array data. Conclusions: We conclude that both potentially personally identifying information and substance-use histories can be simultaneously derived from methylation array data. Because access to genetic information about a database subject or one of their relatives is critical to the de-identification process, this risk of de-identification is limited at the current time. We propose that access to genome-wide methylation data be restricted to institutionally approved investigators who accede to data use agreements prohibiting re-identification.
TL;DR: The data support the idea that the main cause of the prevalent downregulation of DLK1 and MEG3 in urothelial carcinoma is epigenetic silencing across the 14q32 imprinted gene cluster, resulting in the unusual concomitant inactivation of oppositely expressed and imprinted genes.
Abstract: Background
The two oppositely imprinted and expressed genes, DLK1 and MEG3, are located in the same gene cluster at 14q32. Previous studies in bladder cancer have suggested that tumor suppressor genes are located in this region, but these have not been identified.
TL;DR: The study characterized the genome-wide DNA methylation patterns in PC and identified DMRs that were distributed among various genomic contexts that might influence the expression of corresponding genes or transcripts to promote PC and serve as diagnostic biomarkers or therapeutic targets for PC.
Abstract: Extensive reprogramming and dysregulation of DNA methylation is an important characteristic of pancreatic cancer (PC). Our study aimed to characterize the genomic methylation patterns in various genomic contexts of PC. The methyl capture sequencing (methylCap-seq) method was used to map differently methylated regions (DMRs) in pooled samples from ten PC tissues and ten adjacent non-tumor (PN) tissues. A selection of DMRs was validated in an independent set of PC and PN samples using methylation-specific PCR (MSP), bisulfite sequencing PCR (BSP), and methylation sensitive restriction enzyme-based qPCR (MSRE-qPCR). The mRNA and expressed sequence tag (EST) expression of the corresponding genes was investigated using RT-qPCR. A total of 1,131 PC-specific and 727 PN-specific hypermethylated DMRs were identified in association with CpG islands (CGIs), including gene-associated CGIs and orphan CGIs; 2,955 PC-specific and 2,386 PN-specific hypermethylated DMRs were associated with gene promoters, including promoters containing or lacking CGIs. Moreover, 1,744 PC-specific and 1,488 PN-specific hypermethylated DMRs were found to be associated with CGIs or CGI shores. These results suggested that aberrant hypermethylation in PC typically occurs in regions surrounding the transcription start site (TSS). The BSP, MSP, MSRE-qPCR, and RT-qPCR data indicated that the aberrant DNA methylation in PC tissue and in PC cell lines was associated with gene (or corresponding EST) expression. Our study characterized the genome-wide DNA methylation patterns in PC and identified DMRs that were distributed among various genomic contexts that might influence the expression of corresponding genes or transcripts to promote PC. These DMRs might serve as diagnostic biomarkers or therapeutic targets for PC.
TL;DR: Integrated analysis indicated that CpG site location, heterogeneous or autogenous controls, and the proportion of adenocarcinoma in samples were the most significant heterogeneity sources.
Abstract: Adenomatous polyposis coli (APC) has been reported to be a candidate tumor suppressor in many cancers. However, the diagnostic role of APC promoter methylation in non-small cell lung cancer (NSCLC) remains unclear. We systematically integrated published articles and DNA methylation microarray data to investigate the diagnostic performance of the APC methylation test for NSCLC. Two thousand two hundred and fifty-nine NSCLC tumor samples and 1,039 controls were collected from 17 published studies and TCGA NSCLC data. The association between APC promoter methylation and NSCLC was evaluated in a meta-analysis. An independent DNA methylation microarray dataset from TCGA project, in which five CpG sites located in the promoter region of APC were involved, was used to validate the results of the meta-analysis. A significant association was observed between APC promoter hypermethylation and NSCLC, with an aggregated odds ratio (OR) of 3.79 (95% CI: 2.22 to 6.45) in a random effects model. Pooled sensitivity and specificity were 0.548 (95% CI: 0.42 to 0.67, P < 0.0001) and 0.776 (95% CI: 0.62 to 0.88, P < 0.0001), respectively. Each of the five CpG sites was much better in prediction (area under the curve, AUC: 0.71 to 0.73) in lung adenocarcinoma (Ad) than in lung squamous cell carcinoma (Sc) (AUC: 0.45 to 0.61). The AUCs of the logistic prediction model based on these five CpGs were 0.73 and 0.60 for Ad and Sc, respectively. Integrated analysis indicated that CpG site location, heterogeneous or autogenous controls, and the proportion of adenocarcinoma in samples were the most significant heterogeneity sources. The methylation status of APC promoter was strongly associated with NSCLC, especially adenocarcinoma. The APC methylation test could be applied in the clinical diagnosis of lung adenocarcinoma.
TL;DR: Comparison of probes between periodontitis and normal gingival tissues showed that the mean methylation scores and the frequency of methylated probes were significantly lower in genes related to the immune process, indicating that DNA methylation must be modulating chromatin regions and, consequently, modulating the mRNA transcription of immune-inflammatory genes related withperiodontitis, impacting the prognosis of disease.
Abstract: Background: Chronic periodontitis represents a complex disease that is hard to control and is not completely understood. Evidence from past studies suggests that there is a key role for DNA methylation in the pathogenesis of periodontitis. However, all reports have applied technologies that investigate genes in a low throughput. In order to advance in the knowledge of the disease, we analyzed DNA methylation variations associated with gene transcription using a high-throughput assay. Infinium® HumanMethylation450 (Illumina) was performed on gingival samples from 12 periodontitis cases and 11 age-matched healthy individuals. Methylation data of 1,284 immune-related genes and 1,038 cell cycle-related genes from Gene Ontology (GO) and 575 genes from a dataset of stably expressed genes (genes with consistent expression in different physiological states and tissues) were extracted from a microarray dataset and analyzed using bioinformatics tools. DNA methylation variations ranging from �2,000 to +2,000 bp from the transcription start site (TSS) were analyzed, and the results were tested against a differential expression microarray dataset between healthy and periodontitis gingival tissues. Differences were evaluated using tests from the R Statistical Project. Results: The comparison of probes between periodontitis and normal gingival tissues showed that the mean methylation scores and the frequency of methylated probes were significantly lower in genes related to the immune process. In the immune group, these parameters were negatively correlated with gene expression (Mann-Whitney test, p<2.2e�16). Conclusions: Our results show that variations in DNA methylation between healthy and periodontitis cases are higher in genes related to the immune-inflammatory process. Thus, DNA methylation must be modulating chromatin regions and, consequently, modulating the mRNA transcription of immune-inflammatory genes related with periodontitis, impacting the prognosis of disease.
TL;DR: These findings highlight significant age, sex, and tissue-related differences in IFNγ promoter methylation that further the understanding of methylation in the allergic asthma pathway and in the application of biomarkers in clinical research.
Abstract: Asthma is associated with allergic sensitization in about half of all cases, and asthma phenotypes can vary by age and sex. DNA methylation in the promoter of the allergy regulatory gene interferon gamma (IFNγ) has been linked to the maintenance of allergic immune function in human cell and mouse models. We hypothesized that IFNγ promoter methylation at two well-studied, key cytosine phosphate guanine (CpG) sites (-186 and -54), may differ by age, sex, and airway versus systemic tissue in a cohort of 74 allergic asthmatics. After sampling buccal cells, a surrogate for airway epithelial cells, and CD4+ lymphocytes, we found that CD4+ lymphocyte methylation was significantly higher in children compared to adults at both CpG sites (P <0.01). Buccal cell methylation was significantly higher in children at CpG -186 (P = 0.03) but not CpG -54 (P = 0.66). Methylation was higher in males compared to females at both CpG sites in CD4+ lymphocytes (-186: P <0.01, -54: P = 0.02) but not buccal cells (-186: P = 0.14, -54: P = 0.60). In addition, methylation was lower in CD4+ lymphocytes compared to buccal cells (P <0.01) and neighboring CpG sites were strongly correlated in CD4+ lymphocytes (r = 0.84, P <0.01) and weakly correlated in buccal cells (r = 0.24, P = 0.04). At CpG -186, there was significant correlation between CD4+ lymphocytes and buccal cells (r = 0.24, P = 0.04) but not at CpG -54 (r = -0.03, P = 0.78). These findings highlight significant age, sex, and tissue-related differences in IFNγ promoter methylation that further our understanding of methylation in the allergic asthma pathway and in the application of biomarkers in clinical research.
TL;DR: The differential inflammatory response in mice mammary tissue during intramammary infection and the altered epigenetic context induced by two closely related strains of S. aureus are addressed to understand how certain bacteria can evade the immune surveillance and cause sustained infection while others are rapidly cleared from the host body.
Abstract: There is renewed interest towards understanding the host-pathogen interaction in the light of epigenetic modifications. Although epithelial tissue is the major site for host-pathogen interactions, there is handful of studies to show how epithelial cells respond to pathogens. Bacterial infection in the mammary gland parenchyma induces local and subsequently systemic inflammation that results in a complex disease called mastitis. Globally Staphylococcus aureus is the single largest mastitis pathogen and the infection can ultimately result in either subclinical or chronic and sometimes lifelong infection. In the present report we have addressed the differential inflammatory response in mice mammary tissue during intramammary infection and the altered epigenetic context induced by two closely related strains of S. aureus, isolated from field samples. Immunohistochemical and immunoblotting analysis showed strain specific hyperacetylation at histone H3K9 and H3K14 residues. Global gene expression analysis in S. aureus infected mice mammary tissue revealed a selective set of upregulated genes that significantly correlated with the promoter specific, histone H3K14 acetylation. Furthermore, we have identified several differentially expressed known miRNAs and 3 novel miRNAs in S. aureus infected mice mammary tissue by small RNA sequencing. By employing these gene expression data, an attempt has been made to delineate the gene regulatory networks in the strain specific inflammatory response. Apparently, one of the isolates of S. aureus activated the NF-κB signaling leading to drastic inflammatory response and induction of immune surveillance, which could possibly lead to rapid clearance of the pathogen. The other strain repressed most of the inflammatory response, which might help in its sustenance in the host tissue. Taken together, our studies shed substantial lights to understand the mechanisms of strain specific differential inflammatory response to S. aureus infection during mastitis. In a broader perspective this study also paves the way to understand how certain bacteria can evade the immune surveillance and cause sustained infection while others are rapidly cleared from the host body.
TL;DR: It is demonstrated that pathophysiological leucine appears to be antagonistic to insulin, promotes glucose uptake (and not glycogen synthesis), but results in hepatic cell triglyceride (TG) accumulation, and evidence that myostatin regulation of AMP-activated protein kinase (AMPK) is a key pathway in the metabolic effects elicited by excessLeucine.
Abstract: Elevated plasma levels of the branched-chain amino acid (BCAA) leucine are associated with obesity and insulin resistance (IR), and thus the propensity for type 2 diabetes mellitus development. However, other clinical studies suggest the contradictory view that leucine may in fact offer a degree of protection against metabolic syndrome. Aiming to resolve this apparent paradox, we assessed the effect of leucine supplementation on the metabolism of human hepatic HepG2 cells. We demonstrate that pathophysiological leucine appears to be antagonistic to insulin, promotes glucose uptake (and not glycogen synthesis), but results in hepatic cell triglyceride (TG) accumulation. Further, we provide evidence that myostatin (MSTN) regulation of AMP-activated protein kinase (AMPK) is a key pathway in the metabolic effects elicited by excess leucine. Finally, we report associated changes in miRNA expression (some species previously linked to metabolic disease etiology), suggesting that epigenetic processes may contribute to these effects. Collectively, our observations suggest leucine may be both ‘friend’ and ‘foe’ in the context of metabolic syndrome, promoting glucose sequestration and driving lipid accumulation in liver cells. These observations provide insight into the clinical consequences of excess plasma leucine, particularly for hyperglycemia, IR and nonalcoholic fatty liver disease (NAFLD).
TL;DR: A two-stage model consisting of genetic variants in the GATA3 gene, OCP use, age at menarche, and DNA-M may explain how sex hormones in women can increase the asthma prevalence after puberty.
Abstract: The prevalence of asthma in girls increases after puberty. Previous studies have detected associations between sex hormones and asthma, as well as between sex hormones and T helper 2 (Th2) asthma-typical immune responses. Therefore, we hypothesized that exogenous or endogenous sex hormone exposure (represented by oral contraceptive pill (OCP) use and early menarche, respectively) are associated with DNA methylation (DNA-M) of the Th2 transcription factor gene, GATA3, in turn affecting the risk of asthma in girls, possibly in interaction with genetic variants. Blood samples were collected from 245 female participants aged 18 years randomly selected for methylation analysis from the Isle of Wight birth cohort, UK. Information on use of OCPs, age at menarche, and concurrent asthma were assessed by questionnaire. Genome-wide DNA-M was determined using the Illumina Infinium HumanMethylation450 beadchip. In a first stage, we tested the interaction between sex hormone exposure and genetic variants on DNA-M of specific cytosine-phosphate-guanine (CpG) sites. In a second stage, we determined whether these CpG sites interact with genetic variants in GATA3 to explain the risk of asthma. Interactions between OCP use and seven single nucleotide polymorphisms (SNPs) of GATA3 were analyzed for 14 CpG sites (stage 1). The interaction between OCP use and SNP rs1269486 was found to be associated with the methylation level of cg17124583 (P = 0.002, false discovery rate (FDR) adjusted P = 0.04). DNA-M of this same CpG site was also influenced by the interaction between age at menarche and rs1269486 (P = 0.0017). In stage 2, we found that cg17124583 modified the association of SNP rs422628 with asthma risk at the age of 18 years (P = 0.006, FDR adjusted P = 0.04). Subjects with genotype AG showed an increase in average risk ratio (RR) from 0.31 (95% CI: 0.10 to 0.8) to 11.65 (95% CI: 1.71 to 79.5) when methylation level increased from 0.02 to 0.12, relative to genotype AA. A two-stage model consisting of genetic variants in the GATA3 gene, OCP use, age at menarche, and DNA-M may explain how sex hormones in women can increase the asthma prevalence after puberty.
TL;DR: In OTSCC, there is little evidence of significant or frequent hypermethylation of many loci reported to be commonly methylated, and the critical need for quantification of methylation levels and its impact on correlative analyses is demonstrated.
Abstract: Background: DNA hypermethylation is reported as a frequent event and prognostic marker in head and neck squamous cell carcinomas (HNSCC). Methylation has been commonly assessed with non-quantitative methodologies, such as methylation-specific PCR (MSP). We investigated previously reported hypermethylated genes with quantitative methodology in oral tongue squamous cell carcinomas (OTSCC). Results: The methylation status of 12 genes in 115 OTSCC samples was assessed by one or more of three quantitative analyses: methylation sensitive high resolution melting (MS-HRM), sensitive-melting analysis after real time-methylation specific PCR (SMART-MSP), and bisulfite pyrosequencing. In contrast to much of the literature, either no or infrequent locus-specific methylation was identified by MS-HRM for DAPK1, RASSF1A, MGMT, MLH1, APC, CDH1, CDH13, BRCA1, ERCC1 ,a ndATM. The most frequently methylated loci were RUNX3 (18/108 methylated) and ABO (22/107 methylated). Interrogation of the Cancer Genome Atlas (TCGA) HNSCC cohort confirmed the frequency of significant methylation for the loci investigated. Heterogeneous methylation of RUNX3 (18/108) and ABO (22/107) detected by MS-HRM, conferred significantly worse survival (P=0.01, and P=0.03). However, following quantification of methylation levels using pyrosequencing, only four tumors had significant quantities (>15%) of RUNX3 methylation which correlated with a worse patient outcome (P <0.001), while the prognostic significance of ABO hypermethylation was lost. RUNX3 methylation was not prognostic for the TCGA cohort (P=0.76). Conclusions: We demonstrated the critical need for quantification of methylation levels and its impact on correlative analyses. In OTSCC, we found little evidence of significant or frequent hypermethylation of many loci reported to be commonly methylated. It is likely that previous reports have overestimated the frequency of significant methylation events as a consequence of the use of non-quantitative methodology.
TL;DR: The multiple myeloma subgroup with an aberrant PTEN status had a prevalence of the component IgG, Salmon Durie stage I, lower lactate dehydrogenase levels, intermediate-standard cytogenetic risk and longer overall survival with the respect to the unmethylated subgroup.
Abstract: Aberrant DNA methylation of promoter region CpG islands is an alternative mechanism that leads to genetic defects in the inactivation of tumor suppressor genes during myelomagenesis. The aim of this study was to examine the promoter methylation status of the phosphates and tensin homologue on chromosome 10 (PTEN) gene in a cohort of multiple myeloma patients. The PTEN gene was hypermethylated in 7 out of 58 (12%) primary myeloma samples. The correlation between functional inactivation and PTEN mRNA levels was not statistically significant. The multiple myeloma subgroup with an aberrant PTEN status had a prevalence of the component IgG, Salmon Durie stage I, lower lactate dehydrogenase levels, intermediate-standard cytogenetic risk and longer overall survival with the respect to the unmethylated subgroup. This is the first report demonstrating the presence of PTEN promoter hypermethylation in multiple myeloma.
TL;DR: The data suggest that methylation of H3K9 can occur either as a consequence of a specific repressive event such as T-antigen binding to Site I or as a result of a general repression of transcription in the presence of active replication.
Abstract: We have recently shown that T-antigen binding to Site I results in the replication-dependent introduction of H3K9me1 into SV40 chromatin late in infection. Since H3K9me2 and H3K9me3 are also present late in infection, we determined whether their presence was also related to the status of ongoing transcription and replication. Transcription was either inhibited with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB) or stimulated with sodium butyrate and the effects on histone modifications early and late in infection determined. The role of DNA replication was determined by concomitant inhibition of replication with aphidicolin. We observed that H3K9me2/me3 was specifically introduced when transcription was inhibited during active replication. The introduction of H3K9me2/me3 that occurred when transcription was inhibited was partially blocked when replication was also inhibited. The introduction of H3K9me2/me3 did not require the presence of H3K9me1 since similar results were obtained with the mutant cs1085 whose chromatin contains very little H3K9me1. Our data suggest that methylation of H3K9 can occur either as a consequence of a specific repressive event such as T-antigen binding to Site I or as a result of a general repression of transcription in the presence of active replication. The results suggest that the nonproductive generation of transcription complexes as occurs following DRB treatment may be recognized by a ‘proof reading’ mechanism, which leads to the specific introduction of H3K9me2 and H3K9me3.
TL;DR: In tumors with low tumor suppressionor gene expression, decitabine may be associated with increased expression of the tumor suppressor genes FHIT, FUS1, and WWOX, but not PTEN.
Abstract: Background: Since tumor suppressor gene function may be lost through hypermethylation, we assessed whether the demethylating agent decitabine could increase tumor suppressor gene expression clinically. For fragile histidine triad (FHIT), WW domain-containing oxidoreductase (WWOX), fused in sarcoma-1 (FUS1) and phosphatase and tensin homolog (PTEN), immunohistochemistry scores from pre- and post-decitabine tumor biopsies (25 patients) were correlated with methylation of the long interspersed nuclear element-1 (LINE-1) repetitive DNA element (as a surrogate for global DNA methylation) and with tumor regression. Results: With negative staining pre-decitabine (score = 0), the number of patients converting to positive staining post-decitabine was 1 of 1 for FHIT, 3 of 6 for WWOX, 2 of 3 for FUS1 and 1 of 10 for PTEN. In tumors with low pre-decitabine tumor suppressor gene scores (≤150), expression was higher post-treatment in 8 of 8 cases for FHIT (P= 0.014), 7 of 17 for WWOX (P = 0.0547), 7 of 12 for FUS1 (P = 0.0726), and 1 of 16 for PTEN (P= 0.2034). If FHIT, WWOX and FUS1 were considered together, median pre- versus post-decitabine scores were 60 versus 100 (P= 0.0002). Overall, tumor suppressor gene expression change did not correlate with LINE-1 demethylation, although tumors converting from negative to positive had a median decrease in LINE-1 methylation of 24%, compared to 6% in those not converting (P = 0.069). Five of 15 fully evaluable patients had reductions in tumor diameter (range 0.2% to 33.4%). Of these, three had simultaneous increases in three tumor suppressor genes (including the two patients with the greatest tumor regression) compared to 2 of 10 with tumor growth (P =0 .25). Conclusions: In tumors with low tumor suppressor gene expression, decitabine may be associated with increased expression of the tumor suppressor genes FHIT, FUS1, and WWOX, but not PTEN.
TL;DR: It is shown that the expression of several genes and the chromatin landscape become altered upon HBV infection, including global hypoacetylation of H2A.Z and H3K9, which could contribute to the HBV-induced pathomechanism in nontransformed hepatocytes.
Abstract: Virus-host interactions result in altered gene expression profiles in host cell nuclei and enable virus particle production, thus obligatorily involving changes in their epigenomes. Neither such epigenome changes nor their response to antiviral treatment have been extensively studied to date, although viral infections are known to contribute to the long-term development of severe secondary diseases, for example, hepatocellular carcinoma. This may be causally linked to virus-induced persistent plastic chromatin deformations. We studied whether impaired hepatitis B virus (HBV) replication can lead to the restitution of epigenome signatures hypothesizing that hepatocytes alternatively could adopt a ‘memory’ state of the infection; that is, the chromatin could persist in a HBV-induced configuration potentially inheritable between dividing hepatocytes. We therefore determined epigenomic signatures and gene expression changes altered by HBV and the effects of suppressed HBV replication in nontransformed hepatocytes of newborn mice. Further we investigated differential histone acetyltransferase and histone deacetylase activities in HBV-negative and HBVpositive hepatocytes, as well as the effects of HBV suppression on gene expression and the chromatin landscape. We show that the expression of several genes and the chromatin landscape become altered upon HBV infection, including global hypoacetylation of H2A.Z and H3K9. Reporter assays monitoring the activities of histone acetyltransferases or histone deacetylases, respectively, suggest that hypoacetylation most probably depends on elevated sirtuin deacetylase activity, but not on class I/II histone deacetylases. Using Micrococcus nuclease to study the chromatin accessibility in met murine-D3 and hepatitis B virus met murine hepatocytes, we demonstrate that the observed differences in H2A.Z/H3K9 acetylation lead to global chromatin structure changes. At all selected sites examined by chromatin immunoprecipitation and quantitative real-time PCR, these effects can be partly restituted via the nucleoside analog reverse transcriptase inhibitor 3TC or using anti-HBV microRNA-like molecules. Increased sirtuin activity might lead to global histone hypoacetylation signatures, which could contribute to the HBV-induced pathomechanism in nontransformed hepatocytes. Using several techniques to suppress HBV replication, we observed restituted gene expression and chromatin signature patterns reminiscent of noninfected hepatocytes. Importantly, ectopic expression of antiviral short-hairpin RNA, but not microRNA-like molecules, provoked intolerable off-target effects on the gene expression level.
TL;DR: There was increased expression of some transporters that may play a role in chemotherapy uptake after decitabine administration, and there was a trend towards an inverse correlation between change in SLC19A1 methylation and change in RFC1 expression.
Abstract: Background
In 31 solid tumor patients treated with the demethylating agent decitabine, we performed tumor biopsies before and after the first cycle of decitabine and used immunohistochemistry (IHC) to assess whether decitabine increased expression of various membrane transporters. Resistance to chemotherapy may arise due to promoter methylation/downregulation of expression of transporters required for drug uptake, and decitabine can reverse resistance in vitro. The endocytosis regulator RhoA, the folate carriers FOLR1 and RFC1, and the glucose transporter GLUT4 were assessed.
TL;DR: In a cohort of 20 growth-discordant monozygotic twin pairs, severe alteration in placental blood supply due to TTTS appears to leave only weak, if any, epigenetic marks at the analyzed CpG sites at 11p15.
Abstract: Prenatal growth restriction and low birth weight have been linked to long-term alterations of health, presumably via adaptive modifications of the epigenome. Recent studies indicate a plasticity of the 11p15 epigenotype in response to environmental changes during early stages of human development. We analyzed methylation levels at different 11p15 loci in 20 growth-discordant monozygotic twin pairs. Intrauterine development was discordant due to severe twin-to-twin transfusion syndrome (TTTS), which was treated by fetoscopic laser coagulation of communicating vessels before 25 weeks of gestation. Methylation levels at age 4 were determined in blood and buccal cell-derived DNA by the single nucleotide primer extension reaction ion pair reverse-phase high performance liquid chromatography (SNuPE IP RP HPLC) assay. Methylation at LINE-1 repeats was analyzed as an estimate of global methylation. In general, variance of locus-specific methylation levels appeared to be higher in buccal cell- as compared to blood cell-derived DNA samples. Paired analyses within the twin pairs revealed significant differences at only one CpG site (IGF2 dmr0 SN3 (blood), +1.9% in donors; P = 0.013). When plotting the twin pair-discordance in birth weight against the degree of discordance in site-specific methylation at age 4, only a few CpGs were found to interact (one CpG site each at IGF2dmr0 in blood/saliva DNA, one CpG at LINE-1 repeats in saliva DNA), with 26 to 36% of the intra-twin pair divergence at these sites explained by prenatal growth discordance. However, across the entire cohort of 40 children, site-specific methylation did not correlate with SD-scores for weight or length at birth. Insulin-like growth factor-II serum concentrations showed significant within-twin pair correlations at birth (R = 0.57) and at age 4 (R = 0.79), but did not differ between donors and recipients. They also did not correlate with the analyzed 11p15 methylation parameters. In a cohort of 20 growth-discordant monozygotic twin pairs, severe alteration in placental blood supply due to TTTS appears to leave only weak, if any, epigenetic marks at the analyzed CpG sites at 11p15.
TL;DR: The results suggest that global hypomethylation is not present in rectal ACF and argues against the existence of LINE-1 methylation field defect in sporadic colon cancer.
Abstract: Background
Aberrant crypt foci (ACF) are considered the first identifiable preneoplastic lesion in colorectal cancer (CRC), and have been proposed as a potential biomarker for CRC risk. Global DNA hypomethylation is an early event in colorectal carcinogenesis, and long interspersed nuclear element-1 (LINE-1) methylation status is a well-known surrogate marker for genome-wide DNA methylation levels. Despite the gradual increase in DNA hypomethylation in the adenoma–carcinoma sequence, LINE-1 methylation in ACF has never been studied. Moreover, recent studies have reported a field defect for LINE-1 hypomethylation, suggesting that LINE-1 methylation status in normal mucosa could be used to stratify CRC risk and tailor preventive strategies. Thus, we assessed LINE-1 status by pyrosequencing in rectal ACF and paired normal colorectal mucosa from individuals with sporadic colon cancer (CC) (n = 35) or adenoma (n = 42), and from healthy controls (n = 70).
TL;DR: The mechanism of action of the allelic variation in the canine Cox-2 promoter most likely involves variations in the extent of epigenetic downregulation of this gene.
Abstract: Background: Novel allelic variants in the promoter of the canine cyclooxygenase-2 (Cox-2) gene are associated with renal dysplasia (RD). These variants consist of either deletions of putative SP1 transcription factor-binding sites or insertions of tandem repeats of SP1-binding sites located in the CpG island just upstream of the ATG translation initiation site. The canine Cox-2 gene was studied because Cox-2-deficient mice have renal abnormalities and a pathology that is strikingly similar to RD in dogs. Findings: The allelic variants were associated with hypermethylation of the Cox-2 promoter only in clinical cases of RD. The wild-type allele was never methylated, even in clinical cases that were heterozygous for a mutant allele. In cases that were biopsy-negative, the promoter remained unmethylated, regardless of the genotype. Methylated DNA was found in DNA from various adult tissues of dogs with clinical RD. Conclusions: The mechanism of action of the allelic variation in the canine Cox-2 promoter most likely involves variation in the extent of epigenetic downregulation of this gene. This epigenetic downregulation must have occurred early in development because methylated Cox-2 promoter DNA sequences are found in various adult tissues.