Distinguishing Lung Adenocarcinoma from Lung Squamous Cell Carcinoma by Two Hypomethylated and Three Hypermethylated Genes: A Meta-Analysis

Significant differences in the aberrant methylation of genes exist among various histological types of non-small cell lung cancer (NSCLC), which includes adenocarcinoma (AC) and squamous cell carcinoma (SCC). Different chemotherapeutic regimens should be administered to the two NSCLC subtypes due to their unique genetic and epigenetic profiles. The purpose of this meta-analysis was to generate a list of differentially methylated genes between AC and SCC. Our meta-analysis encompassed 151 studies on 108 genes among 12946 AC and 10243 SCC patients. Our results showed two hypomethylated genes (CDKN2A and MGMT) and three hypermethylated genes (CDH13, RUNX3 and APC) in ACs compared with SCCs. In addition, our results showed that the pooled specificity and sensitivity values of CDH13 and APC were higher than those of CDKN2A, MGMT and RUNX3. Our findings might provide an alternative method to distinguish between the two NSCLC subtypes.     

Genome-wide DNA methylation variability in adolescent monozygotic twins followed since birth

DNA methylation patterns are characterized by highly conserved developmental programs, but allow for divergent gene expression resulting from stochastic epigenetic drift or divergent environments. Genome-wide methylation studies in monozygotic (MZ) twins are providing insight into the extent of epigenetic variation that occurs, irrespective of genotype. However, little is known about the variability of DNA methylation patterns in adolescence, a period involving significant and rapid physical, emotional, social, and neurodevelopmental change. Here, we assessed genome-wide DNA methylation using the 450 K Illumina BeadChip in a sample of 37 MZ twin pairs followed longitudinally since birth to investigate: 1) the extent of variation in DNA methylation in identical genetic backgrounds in adolescence and; 2) whether these variations are randomly distributed or enriched in particular functional pathways. We also assessed stability of DNA methylation over 3–6 months to distinguish stable trait-like and variable state-like genes. A pathway analysis found high within-pair variability in genes associated with development, cellular mechanisms, tissue and cell morphology, and various disorders. Test-retest analyses performed in a sub-sample of 8 twin pairs demonstrated enrichment in gene pathways involved in organismal development, cellular growth and proliferation, cell signaling, and particular disorders. The variability found in functional gene pathways may plausibly underlie phenotypic differences in this adolescent MZ twin sample. Furthermore, we assessed stability of methylation over 3–6 months and found that some genes were stable while others were unstable, suggesting that the methylome remains dynamic in adolescence and that dynamic sites tend to be organized in certain gene pathways.     

Genome-wide DNA methylation variability in adolescent monozygotic twins followed since birth

DNA methylation patterns are characterized by highly conserved developmental programs, but allow for divergent gene expression resulting from stochastic epigenetic drift or divergent environments. Genome-wide methylation studies in monozygotic (MZ) twins are providing insight into the extent of epigenetic variation that occurs, irrespective of genotype. However, little is known about the variability of DNA methylation patterns in adolescence, a period involving significant and rapid physical, emotional, social, and neurodevelopmental change. Here, we assessed genome-wide DNA methylation using the 450?K Illumina BeadChip in a sample of 37 MZ twin pairs followed longitudinally since birth to investigate: 1) the extent of variation in DNA methylation in identical genetic backgrounds in adolescence and; 2) whether these variations are randomly distributed or enriched in particular functional pathways. We also assessed stability of DNA methylation over 3–6 months to distinguish stable trait-like and variable state-like genes. A pathway analysis found high within-pair variability in genes associated with development, cellular mechanisms, tissue and cell morphology, and various disorders. Test-retest analyses performed in a sub-sample of 8 twin pairs demonstrated enrichment in gene pathways involved in organismal development, cellular growth and proliferation, cell signaling, and particular disorders. The variability found in functional gene pathways may plausibly underlie phenotypic differences in this adolescent MZ twin sample. Furthermore, we assessed stability of methylation over 3–6 months and found that some genes were stable while others were unstable, suggesting that the methylome remains dynamic in adolescence and that dynamic sites tend to be organized in certain gene pathways.     

DNA methylation profiling of the vascular tissues in the setting of atherosclerosis

Currently, the question of epigenetic mechanisms of gene regulation in the context of cardiovascular diseases is of considerable interest. Here, DNA methylation profiles of vascular tissues of atherosclerotic patients have been analyzed for the first time using the Infinium Human Methylation27 BeadChip microarray (Illumina, United States). As the result, within 286 genes, 314 CpG sites that varied significantly in the level of DNA methylation between the tissue samples of carotid (in the area of atherosclerotic plaques and nearby macroscopically intact tissues of the vascular wall) and mammary arteries, as well as saphenous veins have been identified. The most pronounced differences in the methylation level was registered for CpG sites of homeobox genes HOXA2 and HOXD4, as well as the imprinted MEST gene. In particular, these genes were found to be hypomethylated in the carotid atherosclerotic plaques compared to their methylation patterns in intact tissues of internal mammary arteries and saphenous veins.     

Implication of DNA Demethylation and Bivalent Histone Modification for Selective Gene Regulation in Mouse Primordial Germ Cells

Primordial germ cells (PGCs) sequentially induce specific genes required for their development. We focused on epigenetic changes that regulate PGC-specific gene expression. mil-1, Blimp1, and Stella are preferentially expressed in PGCs, and their expression is upregulated during PGC differentiation. Here, we first determined DNA methylation status of mil-1, Blimp1, and Stella regulatory regions in epiblast and in PGCs, and found that they were hypomethylated in differentiating PGCs after E9.0, in which those genes were highly expressed. We used siRNA to inhibit a maintenance DNA methyltransferase, Dnmt1, in embryonic stem (ES) cells and found that the flanking regions of all three genes became hypomethylated and that expression of each gene increased 1.5- to 3-fold. In addition, we also found 1.5- to 5-fold increase of the PGC genes in the PGCLCs (PGC-like cells) induced form ES cells by knockdown of Dnmt1. We also obtained evidence showing that methylation of the regulatory region of mil-1 resulted in 2.5-fold decrease in expression in a reporter assay. Together, these results suggested that DNA demethylation does not play a major role on initial activation of the PGC genes in the nascent PGCs but contributed to enhancement of their expression in PGCs after E9.0. However, we also found that repression of representative somatic genes, Hoxa1 and Hoxb1, and a tissue-specific gene, Gfap, in PGCs was not dependent on DNA methylation; their flanking regions were hypomethylated, but their expression was not observed in PGCs at E13.5. Their promoter regions showed the bivalent histone modification in PGCs, that may be involved in repression of their expression. Our results indicated that epigenetic status of PGC genes and of somatic genes in PGCs were distinct, and suggested contribution of epigenetic mechanisms in regulation of the expression of a specific gene set in PGCs.     

Variable histone modifications at the Avy metastable epiallele

The ability of environmental factors to shape health and disease involves epigenetic mechanisms that mediate gene-environment interactions. Metastable epiallele genes are variably expressed in genetically identical individuals due to epigenetic modifications established during early development. DNA methylation within metastable epialleles is stochastic due to probabilistic reprogramming of epigenetic marks during embryogenesis. Maternal nutrition and environment have been shown to affect metastable epiallele methylation patterns and subsequent adult phenotype. Little is known, however, about the role of histone modifications in influencing metastable epiallele expression and phenotypic variation. Utilizing chromatin immunoprecipitation followed by qPCR, we observe variable histone patterns in the 5′ long terminal repeat (LTR) of the murine viable yellow agouti (A^vy) metastable epiallele. This region contains 6 CpG sites, which are variably methylated in isogenic A^vy/a offspring. Yellow mice, which are hypomethylated at the A^vy LTR and exhibit constitutive ectopic expression of Agouti (a), also display enrichment of H3 and H4 di-acetylation (p = 0.08 and 0.09, respectively). Pseudoagouti mice, in which A^vy hypermethylation is thought to silence ectopic expression, exhibit enrichment of H4K20 tri-methylation (p = 0.01). No differences are observed for H3K4 tri-methylation (p = 0.7), a modification often enriched in the promoter of active genes. These results show for the first time the presence of variable histone modifications at a metastable epiallele, indicating that DNA methylation acts in concert with histone modifications to affect inter-individual variation of metastable epiallele expression. Therefore, the potential for environmental factors to influence histone modifications, in addition to DNA methylation, should be addressed in environmental epigenomic studies.Key words: epigenetics, metastable epiallele, viable yellow agouti, histone, environmental epigenomics     

Extensive Natural Epigenetic Variation at a De Novo Originated Gene

Epigenetic variation, such as heritable changes of DNA methylation, can affect gene expression and thus phenotypes, but examples of natural epimutations are few and little is known about their stability and frequency in nature. Here, we report that the gene Qua-Quine Starch (QQS) of Arabidopsis thaliana, which is involved in starch metabolism and that originated de novo recently, is subject to frequent epigenetic variation in nature. Specifically, we show that expression of this gene varies considerably among natural accessions as well as within populations directly sampled from the wild, and we demonstrate that this variation correlates negatively with the DNA methylation level of repeated sequences located within the 5′end of the gene. Furthermore, we provide extensive evidence that DNA methylation and expression variants can be inherited for several generations and are not linked to DNA sequence changes. Taken together, these observations provide a first indication that de novo originated genes might be particularly prone to epigenetic variation in their initial stages of formation.     

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

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

Comparison of Genomic and Epigenomic Expression in Monozygotic Twins Discordant for Rett Syndrome

Monozygotic (identical) twins have been widely used in genetic studies to determine the relative contributions of heredity and the environment in human diseases. Discordance in disease manifestation between affected monozygotic twins has been attributed to either environmental factors or different patterns of X chromosome inactivation (XCI). However, recent studies have identified genetic and epigenetic differences between monozygotic twins, thereby challenging the accepted experimental model for distinguishing the effects of nature and nurture. Here, we report the genomic and epigenomic sequences in skin fibroblasts of a discordant monozygotic twin pair with Rett syndrome, an X-linked neurodevelopmental disorder characterized by autistic features, epileptic seizures, gait ataxia and stereotypical hand movements. The twins shared the same de novo mutation in exon 4 of the MECP2 gene (G269AfsX288), which was paternal in origin and occurred during spermatogenesis. The XCI patterns in the twins did not differ in lymphocytes, skin fibroblasts, and hair cells (which originate from ectoderm as does neuronal tissue). No reproducible differences were detected between the twins in single nucleotide polymorphisms (SNPs), insertion-deletion polymorphisms (indels), or copy number variations. Differences in DNA methylation between the twins were detected in fibroblasts in the upstream regions of genes involved in brain function and skeletal tissues such as Mohawk Homeobox (MKX), Brain-type Creatine Kinase (CKB), and FYN Tyrosine Kinase Protooncogene (FYN). The level of methylation in these upstream regions was inversely correlated with the level of gene expression. Thus, differences in DNA methylation patterns likely underlie the discordance in Rett phenotypes between the twins.     

Methylation of the CpG sites only on the sense strand of the APC gene is specific for hepatocellular carcinoma

Hypermethylation of the promoter of the tumor suppressor gene, adenomatous polyposis coli (APC), occurs in various malignancies, including hepatocellular carcinoma (HCC). However, reports on the specificity of the methylation of the APC gene for HCC have varied. To gain insight into how these variations occur, bisulfite PCR sequencing was performed to analyze the methylation status of both sense and antisense strands of the APC gene in samples of HCC tissue, matched adjacent non-HCC liver tissue, hepatitis, cirrhosis, and normal liver tissues. DNA derived from fetal liver and 12 nonhepatic normal tissue was also examined. These experiments revealed liver-specific, antisense strand-biased CpG methylation of the APC gene and suggested that, although methylation of the antisense strand of the APC gene exists in normal liver and other non-HCC disease liver tissue, methylation of the sense strand of the APC gene occurs predominantly in HCC. To determine the effect of the DNA strand on the specificity of the methylated APC gene as a biomarker for HCC detection, quantitative methylation-specific PCR assays for sense and antisense strand DNA were developed and performed on DNA isolated from HCC (n = 58), matched adjacent non-HCC (n = 58), cirrhosis (n = 41), and hepatitis (n = 39). Receiver operating characteristic curves were constructed. With the cutoff value set at the limit of detection, the specificity of sense and antisense strand methylation was 84% and 43%, respectively, and sensitivity was 67.2% and 72.4%, respectively. This result demonstrated that the identity of the methylated DNA strand impacted the specificity of APC for HCC detection. Interestingly, methylation of the sense strand of APC occurred in 40% of HCCs from patients with serum AFP levels less than 20 ng/mL, suggesting a potential role for APC as a biomarker to complement AFP in HCC screening.     

Hypermethylation of tumor suppressor genes involved in critical regulatory pathways for developing a blood-based test in breast cancer

Background

Aberrant DNA methylation patterns might be used as a biomarker for diagnosis and management of cancer patients.

Methods and Findings

To achieve a gene panel for developing a breast cancer blood-based test we quantitatively assessed the DNA methylation proportion of 248 CpG sites per sample (total of 31,248 sites in all analyzed samples) on 10 candidate genes (APC, BIN1, BMP6, BRCA1, CST6, ESR-b, GSTP1, P16, P21 and TIMP3). The number of 126 samples consisting of two different cohorts was used (first cohort: plasma samples from breast cancer patients and normal controls; second cohort: triple matched samples including cancerous tissue, matched normal tissue and serum samples). In the first cohort, circulating cell free methylated DNA of the 8 tumor suppressor genes (TSGs) was significantly higher in patients with breast cancer compared to normal controls (P<0.01). In the second cohort containing triple matched samples, seven genes showed concordant hypermethylated profile in tumor tissue and serum samples compared to normal tissue (P<0.05). Using eight genes as a panel to develop a blood-based test for breast cancer, a sensitivity and specificity of more than 90% could be achieved in distinguishing between tumor and normal samples.

Conclusions

Our study suggests that the selected TSG panel combined with the high-throughput technology might be a useful tool to develop epigenetic based predictive and prognostic biomarker for breast cancer relying on pathologic methylation changes in tumor tissue, as well as in circulation.     

Methylomic analysis of monozygotic twins discordant for childhood psychotic symptoms

Childhood psychotic symptoms are associated with increased rates of schizophrenia, other psychiatric disorders, and suicide attempts in adulthood; thus, elucidating early risk indicators is crucial to target prevention efforts. There is considerable discordance for psychotic symptoms between monozygotic twins, indicating that child-specific non-genetic factors must be involved. Epigenetic processes may constitute one of these factors and have not yet been investigated in relation to childhood psychotic symptoms. Therefore, this study explored whether differences in DNA methylation at age 10 were associated with monozygotic twin discordance for psychotic symptoms at age 12. The Environmental Risk (E-Risk) Longitudinal Twin Study cohort of 2,232 children (1,116 twin pairs) was assessed for age-12 psychotic symptoms and 24 monozygotic twin pairs discordant for symptoms were identified for methylomic comparison. Children provided buccal samples at ages 5 and 10. DNA was bisulfite modified and DNA methylation was quantified using the Infinium HumanMethylation450 array. Differentially methylated positions (DMPs) associated with psychotic symptoms were subsequently tested in post-mortem prefrontal cortex tissue from adult schizophrenia patients and age-matched controls. Site-specific DNA methylation differences were observed at age 10 between monozygotic twins discordant for age-12 psychotic symptoms. Similar DMPs were not found at age 5. The top-ranked psychosis-associated DMP (cg23933044), located in the promoter of the C5ORF42 gene, was also hypomethylated in post-mortem prefrontal cortex brain tissue from schizophrenia patients compared to unaffected controls. These data tentatively suggest that epigenetic variation in peripheral tissue is associated with childhood psychotic symptoms and may indicate susceptibility to schizophrenia and other mental health problems.     

Variable histone modifications at the Avy metastable epiallele

The ability of environmental factors to shape health and disease involves epigenetic mechanisms that mediate gene-environment interactions. Metastable epiallele genes are variably expressed in genetically identical individuals due to epigenetic modifications established during early development. DNA methylation within metastable epialleles is stochastic due to probabilistic reprogramming of epigenetic marks during embryogenesis. Maternal nutrition and environment have been shown to affect metastable epiallele methylation patterns and subsequent adult phenotype. Little is known, however, about the role of histone modifications in influencing metastable epiallele expression and phenotypic variation. Utilizing chromatin immunoprecipitation followed by qPCR, we observe variable histone patterns in the 5’ long terminal repeat (LTR) of the murine viable yellow agouti (A^vy) metastable epiallele. This region contains 6 CpG sites, which are variably methylated in isogenic A^vy/a offspring. Yellow mice, which are hypomethylated at the Avy LTR and exhibit constitutive ectopic expression of agouti (a), also display enrichment of H3 and H4 di-acetylation (p=0.08 and 0.09, respectively). Pseudoagouti mice, in which A^vy hypermethylation is thought to silence ectopic expression, exhibit enrichment of H4K20 tri-methylation (p=0.01). No differences are observed for H3K4 tri-methylation (p=0.7), a modification often enriched in the promoter of active genes. These results show for the first time the presence of variable histone modifications at a metastable epiallele, indicating that DNA methylation acts in concert with histone modifications to affect inter-individual variation of metastable epiallele expression. Therefore, the potential for environmental factors to influence histone modifications, in addition to DNA methylation, should be addressed in environmental epigenomic studies.     

Frequent and distinct aberrations of DNA methylation patterns in fibrolamellar carcinoma of the liver

Background

Gene silencing due to aberrant DNA methylation is a frequent event in hepatocellular carcinoma (HCC) and also in hepatocellular adenoma (HCA). However, very little is known about epigenetic defects in fibrolamellar carcinoma (FLC), a rare variant of hepatocellular carcinoma that displays distinct clinical and morphological features.

Methodology/Principal Findings

We analyzed the methylation status of the APC, CDH1, cyclinD2, GSTπ1, hsa-mir-9-1, hsa-mir-9-2, and RASSF1A gene in a series of 15 FLC and paired normal liver tissue specimens by quantitative high-resolution pyrosequencing. Results were compared with common HCC arising in non-cirrhotic liver (n = 10). Frequent aberrant hypermethylation was found for the cyclinD2 (19%) and the RASSF1A (38%) gene as well as for the microRNA genes mir-9-1 (13%) and mir-9-2 (33%). In contrast to common HCC the APC and CDH1 (E-cadherin) genes were found devoid of any DNA methylation in FLC, whereas the GSTπ1 gene showed comparable DNA methylation in tumor and surrounding tissue at a moderate level. Changes in global DNA methylation level were measured by analyzing methylation status of the highly repetitive LINE-1 sequences. No evidence of global hypomethylation could be found in FLCs, whereas HCCs without cirrhosis showed a significant reduction in global methylation level as described previously.

Conclusions

FLCs display frequent and distinct gene-specific hypermethylation in the absence of significant global hypomethylation indicating that these two epigenetic aberrations are induced by different pathways and that full-blown malignancy can develop in the absence of global loss of DNA methylation. Only quantitative DNA methylation detection methodology was able to identify these differences.     

Divergent methylation pattern in adult stage between two forms of Tetranychus urticae (Acari: Tetranychidae)

The two‐spotted spider mite, Tetranychus urticae Koch has two forms: green form and red form. Understanding the molecular basis of how these two forms established without divergent genetic background is an intriguing area. As a well‐known epigenetic process, DNA methylation has particularly important roles in gene regulation and developmental variation across diverse organisms that do not alter genetic background. Here, to investigate whether DNA methylation could be associated with different phenotypic consequences in the two forms of T. urticae, we surveyed the genome‐wide cytosine methylation status and expression level of DNA methyltransferase 3 (Tudnmt3) throughout their entire life cycle. Methylation‐sensitive amplification polymorphism (MSAP) analyses of 585 loci revealed variable methylation patterns in the different developmental stages. In particular, principal coordinates analysis (PCoA) indicates a significant epigenetic differentiation between female adults of the two forms. The gene expression of Tudnmt3 was detected in all examined developmental stages, which was significantly different in the adult stage of the two forms. Together, our results reveal the epigenetic distance between the two forms of T. urticae, suggesting that DNA methylation might be implicated in different developmental demands, and contribute to different phenotypes in the adult stage of these two forms.