Identification of novel epigenetically modified genes in human melanoma via promoter methylation gene profiling

The inactivation of tumor-related genes through the aberrant methylation of promoter CpG islands is thought to contribute to tumor initiation and progression. We therefore investigated promoter methylation events involved in cutaneous melanoma by screening 30 genes of interest for evidence of promoter hypermethylation, examining 20 melanoma cell lines and 40 freshly procured melanoma samples. Utilizing quantitative methylation-specific PCR, we identified five genes (SOCS1, SOCS2, RAR-beta 2, TNFSF10C, and TNFSF10D) with hypermethylation frequencies ranging from 50% to 80% in melanoma cell lines as well as freshly procured tissue samples. Eighteen genes (LOX, RASSF1A, WFDC1, TM, APC, TFPI2, TNFSF10A, CDKN2A, MGMT, TIMP3, ASC, TPM1, IRF8, CIITA-PIV, CDH1, SYK, HOXB13, and DAPK1) were methylated at lower frequencies (2-30%). Two genes (CDKN1B and PTEN), previously reported as methylated in melanoma, and five other genes (RECK, IRF7, PAWR, TNFSF10B, and Rb) were not methylated in the samples screened here. Daughter melanoma cell lines showed identical methylation patterns when compared with original samples from which they were derived, as did synchronous metastatic lesions from the same patient. We identified four genes (TNFSF10C, TNFSF10D, LOX, and TPM1) that have never before been identified as hypermethylated in melanoma, with an overall methylation frequency of 60, 80, 50, and 10%, respectively, hypothesizing that these genes may play an important role in melanoma progression.     

Genome-wide DNA methylation profiling in rheumatoid arthritis identifies disease-associated methylation changes that are distinct to individual T- and B-lymphocyte populations

Changes to the DNA methylome have been described in patients with rheumatoid arthritis (RA). In previous work, we reported genome-wide methylation differences in T-lymphocyte and B-lymphocyte populations from healthy individuals. Now, using HumanMethylation450 BeadChips to interrogate genome-wide DNA methylation, we have determined disease-associated methylation changes in blood-derived T- and B-lymphocyte populations from 12 female patients with seropositive established RA, relative to 12 matched healthy individuals. Array data were analyzed using NIMBL software and bisulfite pyrosequencing was used to validate array candidates. Genome-wide DNA methylation, determined by analysis of LINE-1 sequences, revealed higher methylation in B-lymphocytes compared with T-lymphocytes (P ≤ 0.01), which is consistent with our findings in healthy individuals. Moreover, loci-specific methylation differences that distinguished T-lymphocytes from B-lymphocytes in healthy individuals were also apparent in RA patients. However, disease-associated methylation differences were also identified in RA. In these cases, we identified 509 and 252 CpGs in RA-derived T- and B-lymphocytes, respectively, that showed significant changes in methylation compared with their cognate healthy counterparts. Moreover, this included a restricted set of 32 CpGs in T-lymphocytes and 20 CpGs in B-lymphocytes (representing 15 and 10 genes, respectively, and including two, MGMT and CCS, that were common to both cell types) that displayed more substantial changes in methylation. These changes, apparent as hyper- or hypo-methylation, were independently confirmed by pyrosequencing analysis. Validation by pyrosequencing also revealed additional sites in some candidate genes that also displayed altered methylation in RA. In this first study of genome-wide DNA methylation in individual T- and B-lymphocyte populations in RA patients, we report disease-associated methylation changes that are distinct to each cell type and which support a role for discrete epigenetic regulation in this disease.     

Genome-wide DNA methylation profiling in rheumatoid arthritis identifies disease-associated methylation changes that are distinct to individual T- and B-lymphocyte populations

Changes to the DNA methylome have been described in patients with rheumatoid arthritis (RA). In previous work, we reported genome-wide methylation differences in T-lymphocyte and B-lymphocyte populations from healthy individuals. Now, using HumanMethylation450 BeadChips to interrogate genome-wide DNA methylation, we have determined disease-associated methylation changes in blood-derived T- and B-lymphocyte populations from 12 female patients with seropositive established RA, relative to 12 matched healthy individuals. Array data were analyzed using NIMBL software and bisulfite pyrosequencing was used to validate array candidates. Genome-wide DNA methylation, determined by analysis of LINE-1 sequences, revealed higher methylation in B-lymphocytes compared with T-lymphocytes (P ≤ 0.01), which is consistent with our findings in healthy individuals. Moreover, loci-specific methylation differences that distinguished T-lymphocytes from B-lymphocytes in healthy individuals were also apparent in RA patients. However, disease-associated methylation differences were also identified in RA. In these cases, we identified 509 and 252 CpGs in RA-derived T- and B-lymphocytes, respectively, that showed significant changes in methylation compared with their cognate healthy counterparts. Moreover, this included a restricted set of 32 CpGs in T-lymphocytes and 20 CpGs in B-lymphocytes (representing 15 and 10 genes, respectively, and including two, MGMT and CCS, that were common to both cell types) that displayed more substantial changes in methylation. These changes, apparent as hyper- or hypo-methylation, were independently confirmed by pyrosequencing analysis. Validation by pyrosequencing also revealed additional sites in some candidate genes that also displayed altered methylation in RA. In this first study of genome-wide DNA methylation in individual T- and B-lymphocyte populations in RA patients, we report disease-associated methylation changes that are distinct to each cell type and which support a role for discrete epigenetic regulation in this disease.     

Expression and promoter methylation status of hMLH1, MGMT,APC, and CDH1 genes in patients with colon adenocarcinoma

Colorectal cancer (CRC) is the third most common cancer in men and the second in women worldwide. CRC development is the result of genetic and epigenetic alterations accumulation in the epithelial cells of colon mucosa. In the present study, DNA methylation, an epigenetic event, was evaluated in tumoral and matching normal epithelium in a cohort of 61 CRC patients. The results confirmed and expanded knowledge for the tumor suppressor genes hMLH1, MGMT, APC, and CDH1. Promoter methylation was observed for all the examined genes in different percentage. A total of 71% and 10% of the examined cases were found to be methylated in two or more and in all genes, respectively. mRNA and protein levels were also evaluated. Promoter methylation of hMLH1, MGMT, APC, and CDH1 genes was present at the early stages of tumor’s formation and it could also be detected in the normal mucosa. Correlations of the methylated genes with patient’s age and tumor’s clinicopathological characteristics were also observed. Our findings suggest that DNA methylation is a useful marker for tumor progression monitoring and that promoter methylation in certain genes is associated with more advanced tumor stage, poor differentiation, and metastasis.     

肝细胞癌中FHIT基因启动子甲基化状态分析及其与临床病理特征之间的关系研究

目的:分析肝细胞癌组织中FHIT基因启动子甲基化状态及其与FHIT基因表达和肝细胞癌临床病理特征的关系。方法:运用甲基化特异性PCR(MSP)方法分析肝细胞癌组织和癌旁正常肝脏组织中FHIT基因启动子甲基化状况;应用RT-PCT和Western免疫印迹方法检测FHIT基因mRNA和蛋白的表达情况;统计学分析FHIT基因启动子甲基化与肝细胞癌临床病理特征的关系。结果:MSP分析结果表明肝细胞癌组织中FHIT基因甲基化率(60.8%)显著高于癌旁正常组织中FHIT基因甲基化(16.2%;x2=31.071,P=0.000)。同时我们还发现:发生完全或者部分甲基化的肝细胞癌组织或者癌旁正常肝组织中FHIT基因mRNA和蛋白表达水平显著降低。FHIT基因启动子甲基化和肝细胞癌患者的临床分期和肝外转移情况密切相关(P=0.006和0.049),而与其他临床病理特征无相关性(P>0.05)。结论:FHIT基因甲基化是导致FHIT基因在肝细胞癌中失活的一个重要因素,与肝细胞癌的发生密切相关,有望成为肝细胞癌早期诊断的分子检测标志物和分子治疗新靶点。     

Methylation of progesterone receptor isoform A promoter in normal breast tissue: An epigenetic link between early age at menarche and risk of breast cancer?

Emerging evidence indicates that some altered patterns of methylation that occur in breast tumors may also be found in breast tissue of healthy women in relation to the breast cancer (BC) risk factors. Progesterone receptor (PR) isoform α is a crucial regulator of breast hormone responsiveness and its hypermethylation plays an important role in the initiation and development of breast tumors. However, such a methylation change in healthy women and its link with the different risk factors has not yet been investigated. In the present study, we aimed to examine the relationship of possible methylation changes within a critical region in the promoter CpG island of PGR-α (progesterone receptor α) gene in the healthy women with a set of reproductive and nonreproductive BC risk factors. The breast tissues were collected from 120 cancer-free women who had undergone cosmetic mammoplasty. The genomic DNA was extracted from the breast tissues and the methylation level of PGR-α promoter CpG island was determined by using MeDIP-qPCR assay. Using regression analysis, we found that increasing menarche age is inversely associated with the high methylation of PGR-α promoter ( β = −0.790, SE = 0.362; P = 0.031). Although lactating women had more methylation than nonlactating women (P = 0.026, the t test), this result was not confirmed by regression models. Such an observation may be helpful in better understanding of the underlying mechanisms by which early age at menarche increases the risk of BC. However, this perspective requires further validations in larger studies of more subjects as well as the inclusion of other related genes.     

Features and trend of loss of promoter-associated CpG islands in the human and mouse genomes

CpG islands (CGIs) are often considered as gene markers, but the number of CGIs varies among mammalian genomes that have similar numbers of genes. In this study, we investigated the distribution of CGIs in the promoter regions of 3,197 human-mouse orthologous gene pairs and found that the mouse genome has notably fewer CGIs in the promoter regions and less pronounced CGI characteristics than does the human genome. We further inferred CGI's ancestral state using the dog genome as a reference and examined the nucleotide substitution pattern and the mutational direction in the conserved regions of human and mouse CGIs. The results reveal many losses of CGIs in both genomes but the loss rate in the mouse lineage is two to four times the rate in the human lineage. We found an intriguing feature of CGI loss, namely that the loss of a CGI usually starts from erosion at the both edges and gradually moves towards the center. We found functional bias in the genes that have lost promoter-associated CGIs in the human or mouse lineage. Finally, our analysis indicates that the association of CGIs with housekeeping genes is not as strong as previously estimated. Our study provides a detailed view of the evolution of promoter-associated CGIs in the human and mouse genomes and our findings are helpful for understanding the evolution of mammalian genomes and the role of CGIs in gene function.     

Differential promoter methylation and histone modification contribute to the brain specific expression of the mouse Mbu-1 gene

Mbu-1 (Csrnp-3) is a mouse gene that was identified in our previous study as showing highly restricted expression to the central nervous system. In this study, to elucidate the regulatory mechanism for tissue specificity of the gene, epigenetic approaches that identify the profiles of CpG methylation, as well as histone modifications at the promoter region were conducted. Methylation-specific PCR revealed that the CpG sites in brain tissues from embryo to adult stages showed virtually no methylation (0.052–0.67%). Lung (9.0%) and pancreas (3.0%) also showed lower levels. Other tissues such as liver, kidney, and heart showed much higher methylation levels ranging from approximately 39-93%. Treatment of 5-aza-2′-deoxycytidine (5-Aza-dC) significantly decreased promoter methylation, reactivating Mbu-1 expression in NG108-15 and Neuro-2a neuronal cells. Chromatin immunoprecipitation assay revealed that 5-Aza-dC decreased levels of acetylated H3K9 and methylated H3K4, and increased methylated H3K9. This result indicates that CpG methylation converses with histone modifications in an opposing sense of regulating Mbu-1 expression.     

Enhanced expression of lipogenic genes may contribute to hyperglycemia and alterations in plasma lipids in response to dietary iron deficiency

Iron deficiency (ID) remains a public health concern affecting ~25% of the world’s population. Metabolic consequences of ID include elevated plasma glucose concentrations consistent with increased reliance on glucose as a metabolic substrate, though the mechanisms controlling these responses remain unclear. To further characterize the metabolic response to ID, weanling male Sprague–Dawley rats were fed either a control (C; 40 mg Fe/kg diet) or iron-deficient (ID; 3 mg Fe/kg diet) diet or were pair-fed (PF) the C diet to the level of intake of the ID group for 21 days. In addition to reductions in hemoglobin, hematocrit, and plasma iron, the ID group also exhibited higher percent body fat and plasma triglycerides compared to the PF group. Steady-state levels of both plasma glucose and insulin increased 40 and 45%, respectively, in the ID group compared to the PF group. Plasma cortisol levels were decreased 67% in the ID group compared to the PF diet group. The systematic evaluation of the expression of genes involved in insulin signaling, glucose metabolism, and fatty acid metabolism in the liver and skeletal muscle revealed significant alterations in the expression of 48 and 52 genes in these tissues, respectively. A significant concurrent increase in lipogenic gene expression and decrease in gene expression related to β-oxidation in both the liver and skeletal muscle, in combination with differential tissue expression of genes involved in glucose metabolism, provides novel insight into the adaptive metabolic response in rodent models of severe iron deficiency anemia.     

Clinical and public health research using methylated DNA Immunoprecipitation (MeDIP): A comparison of commercially available kits to examine differential DNA methylation across the genome

The methylated DNA immunoprecipitation method (MeDIP) is a genome-wide, high-resolution approach that detects DNA methylation with oligonucleotide tiling arrays or high throughput sequencing platforms. A simplified high-throughput MeDIP assay will enable translational research studies in clinics and populations, which will greatly enhance our understanding of the human methylome. We compared three commercial kits, MagMeDIP Kit TM (Diagenode), Methylated-DNA IP Kit (Zymo Research) and Methylamp? Methylated DNA Capture Kit (Epigentek), in order to identify which one has better reliability and sensitivity for genomic DNA enrichment. Each kit was used to enrich two samples, one from fresh tissue and one from a cell line, with two different DNA amounts. The enrichment efficiency of each kit was evaluated by agarose gel band intensity after Nco I digestion and by reaction yield of methylated DNA. A successful enrichment is expected to have a 1:4 to 10:1 conversion ratio and a yield of 80% or higher. We also evaluated the hybridization efficiency to genome-wide methylation arrays in a separate cohort of tissue samples. We observed that the MagMeDIP kit had the highest yield for the two DNA amounts and for both the tissue and cell line samples, as well as for the positive control. In addition, the DNA was successfully enriched from a 1:4 to 10:1 ratio. Therefore, the MagMeDIP kit is a useful research tool that will enable clinical and public health genome-wide DNA methylation studies.     

Clinical and public health research using methylated DNA immunoprecipitation (MeDIP): a comparison of commercially available kits to examine differential DNA methylation across the genome

The methylated DNA immunoprecipitation method (MeDIP) is a genome-wide, high-resolution approach that detects DNA methylation with oligonucleotide tiling arrays or high throughput sequencing platforms. A simplified high-throughput MeDIP assay will enable translational research studies in clinics and populations, which will greatly enhance our understanding of the human methylome. We compared three commercial kits, MagMeDIP Kit TM (Diagenode), Methylated-DNA IP Kit (Zymo Research) and Methylamp™ Methylated DNA Capture Kit (Epigentek), in order to identify which one has better reliability and sensitivity for genomic DNA enrichment. Each kit was used to enrich two samples, one from fresh tissue and one from a cell line, with two different DNA amounts. The enrichment efficiency of each kit was evaluated by agarose gel band intensity after Nco I digestion and by reaction yield of methylated DNA. A successful enrichment is expected to have a 1:4 to 10:1 conversion ratio and a yield of 80% or higher. We also evaluated the hybridization efficiency to genome-wide methylation arrays in a separate cohort of tissue samples. We observed that the MagMeDIP kit had the highest yield for the two DNA amounts and for both the tissue and cell line samples, as well as for the positive control. In addition, the DNA was successfully enriched from a 1:4 to 10:1 ratio. Therefore, the MagMeDIP kit is a useful research tool that will enable clinical and public health genome-wide DNA methylation studies.     

Promoter methylation and expression of the VANGL2 gene in the myocardium of pediatric patients with Tetralogy of Fallot

Background: Tetralogy of Fallot (ToF) is the most common form of cyanotic congenital heart disease and is a major cause of significant morbidity and mortality. VANGL2 is a critical gene in the planar cell polarity pathway that plays an important role in the development of the heart. This study investigates the methylation status of the promoter region of VANGL2 and the expression pattern of VANGL2 in cardiac tissue. Methods: The promoter region of VANGL2 was sequenced in 200 ToF patients and 400 control subjects. Methylation levels were measured in four regions of the VANGL2 promoter (B1‐1: ?282 bp ～ ?117 bp, B1–2: ?117 bp ～ 41 bp, B2: 8 bp ～ 157 bp, B3: 132 bp ～ 401 bp) by bisulfite sequencing PCR in the right ventricular outflow tract of the myocardium. Quantitative real‐time PCR and immunohistochemistry were used to detect the mRNA and protein expression levels, respectively. Results: No mutations were found in the promoter region, but two SNPs (rs11582932 T>G, rs11265385 T>G) were found in ToF patients and controls with similar frequencies (p > 0.05). The overall methylation status of the VANGL2 promoter was significantly higher in ToF patients than in controls (p = 0.0234). Specifically, the methylation levels of regions B1‐1 and B3 were significantly higher in ToF patients (p = 0.0042, p = 0.0418). Both the VANGL2 mRNA and protein levels were significantly lower in ToF patients than in controls (p < 0.05). Conclusion: The aberrant VANGL2 promoter methylation and the decreased gene expression in ToF patients may provide important clues for the development of ToF. Birth Defects Research (Part A) 100:973–984, 2014. © 2014 Wiley Periodicals, Inc.     

Methylation of the <Emphasis Type="Italic">RASSF1A</Emphasis> promoter region and the allelic imbalance frequencies in chromosome 3 critical regions correlate with progression of clear cell renal carcinoma

The short arm of chromosome 3 (3p) contains several critical regions that have increased frequencies of allelic deletions and harbor a set of tumor suppressor genes. In particular, the range of functions performed by RASSF1A (LUCA region, 3p21.31) includes those potentially associated with carcinogenesis. Among 3p genes, RASSF1A has the highest methylation frequency in epithelial tumors of various locations. For the first time, two different methods (methylation-specific PCR and methylation-sensitive restriction analysis) independently showed that the methylation level of the CpG island in the RASSF1A promoter region significantly correlated with grade and clinical stage of clear cell renal cell carcinoma (RCC). An analysis of 23 3p polymorphic markers in a representative set of 80 RCC cases characterized clinically and histologically revealed that RCC progression significantly correlated with the frequency of allelic imbalances in some critical regions of 3p (LUCA and AP20), but not in 3p as a whole. These data suggest that RCC progression is associated with the methylation of the RASSF1A promoter and, possibly, with structural and functional alterations in other 3p genes. In addition, significant correlation between RASSF1A methylation and allelic losses at the nearby polymorphic marker locus suggests the “two hit” model for the inactivation of this tumor suppressor gene in RCC.