HLA-DRB1 and HLA-DQB1 methylation changes promote the occurrence and progression of Kazakh ESCC

Human leukocyte antigen II (HLA-II) plays an important role in host immune responses to cancer cells. Changes in gene methylation may result in aberrant expression of HLA-II, serving a key role in the pathogenesis of Kazakh esophageal squamous cell carcinoma (ESCC). We analyzed the expression level of HLA-II (HLA-DP, -DQ, and -DR) by immunohistochemistry, as well as the methylation status of HLA-DRB1 and HLA-DQB1 by MassARRAY spectrometry in Xinjiang Kazakh ESCC. Expression of HLA-II in ESCC was significantly higher than that in cancer adjacent normal (ACN) samples (P < 0.05). Decreased HLA-II expression was closely associated with later clinical stages of ESCC (P < 0.05). Hypomethylation of HLA-DRB1 and hypermethylation of HLA-DQB1 was significantly correlated with occurrence of Kazakh ESCC (P < 0.01), and mainly manifested as hypomethylation of CpG9, CpG10-11, and CpG16 in HLA-DRB1 and hypermethylation of CpG6-7 and CpG16-17 in HLA-DQB1 (P < 0.01). Moreover, hypomethylation of HLA-DQB1 CpG6-7 correlated with poor differentiation in ESCCs, whereas hypermethylation of HLA-DRB1 CpG16 and hypomethylation of HLA-DQB1 CpG16-17 were significantly associated with later stages of ESCC (P < 0.05). A significant inverse association between HLA-DRB1 CpG9 methylation and HLA-II expression was found in ESCC (P < 0.05). These findings suggest aberrant HLA-DRB1 and HLA-DQB1 methylation contributes to the aberrant expression of HLA-II. These molecular changes may influence the immune response to specific tumor epitopes, promoting the occurrence and progression of Kazakh ESCC.     

HLA-DRB1*1501 and HLA-DQB1*0301 alleles are positively associated with HPV16 infection–related Kazakh esophageal squamous cell carcinoma in Xinjiang China

Multiple determinant factors are involved in the occurrence and progression of esophageal squamous cell carcinoma (ESCC). Human papillomavirus (HPV) and human leukocyte antigen (HLA) polymorphism were identified as important factors. This study examined the associations between the development of Kazakh ESCC and the determinant factors including HLA-DRB1*0901, 1501; DQB1*0301, 0602; high-risk HPV infection in the area of Xinjiang, China. 200 Kazakh patients with ESCC and 150 controls were recruited, and polymerase chain reaction (PCR) was performed to detect HLA-DRB1*0901, 1501 and DQB1*0301,0602 using sequence-specific primers (SSPs). HPV16 was detected in esophageal specimens using PCR. HPV16 infection rate in Kazakh ESCC case group was 41 %, significantly higher than that of control group 14 % (OR = 3.62; 95 % CI, 2.15–6.09; P < 0.001). A positive association between ESCC and HLA-DRB1*1501 (OR = 2.46, P < 0.0125) or HLA-DQB1*0301 (OR = 3.34, P < 0.0125) alleles was observed. Similar tendencies were observed for HLA-DRB1*1501 (OR = 3.095, P < 0.0125) and HLA-DQB1*0301 (OR = 2.410, P < 0.0125) alleles with HPV16-positive ESCC. HLA-DRB1*1501, HLA-DQB1*0301 and DQB1*0602 were significantly associated with ESCC when the age was ≥55 years (P < 0.0125 for all), whereas only HLA-DQB1*0301 was significantly associated with ESCC when the age was <55 years (P < 0.0125). HLA-DRB1*1501 and HLA-DQB1*0301 were significantly associated with an increase in ESCC occurrence in females (P < 0.0125), whereas only HLA-DQB1*0301 was significantly associated with ESCC in males. Moreover, the occurrence of HLA-DQB1*0602 gene in poorly differentiated ESCC group (68.8 %) was slightly higher than that of well-differentiated squamous cell carcinoma group (31.2 %). The difference was not statistically significant (P > 0.0125). The study suggests that HLA-DRB1*1501 and HLA-DQB1*0301 may influence the immune response to specific tumor and HPV-encoded epitopes and affect the risk of Kazakh ESCC in XinJiang, China.     

Epigenetic silencing of miR-203 in Kazakh patients with esophageal squamous cell carcinoma by MassARRAY spectrometry

Dysregulation of miR-203 by promoter methylation is associated with the development of various cancers. We aimed to explore the underlying link between promoter methylation and miR-203 expression in Kazakh esophageal squamous cell carcinoma (ESCC). MassARRAY® System spectrometry was used to quantitatively analyze the DNA methylation of 32 CpG sites within miR-203 in 99 Kazakh ESCC and 46 normal esophageal tissues (NETs) with similar population characteristics. We conducted real-time PCR to detect miR-203 expression levels and evaluated their association with methylation. Eleven CpG units within miR-203 promoter were frequently hypermethylated in ESCC compared with NETs (P < 0.05). The hypermethylation of several CpG units positively correlated with age, lower esophagus, constrictive type of ESCC, and moderately differentiated ESCC. Given the involvement of human papillomavirus (HPV) in etiology of ESCC was confirmed from our previous reports, herein we found that CpG units within miR-203 in HPV16-positive ESCC are more heavily methylated. Furthermore, miR-203 expression showed a nearly 4.5-fold decrease in ESCC than NETs (0.206 ± 0.336 vs. 0.908 ± 1.424, P < 0.001) and was significantly associated with lymph node metastasis (P = 0.012). The expression of miR-203 with 11 completely hypermethylated CpG units was approximately 6.5-fold lower than that with at least 1 unmethylated CpG unit (P < 0.001) and especially the CpG_15.16 and CpG_31.32 with higher methylation levels in ESCC tissues exhibited lower expression levels of miR-203, which indicated a reverse association between miR-203 methylation and expression. Hypermethylated miR-203 is a potential biomarker and targeted delivery of miR-203 could therefore serve as a preventive or therapeutic strategy for Kazakh ESCC.     

DNA Methylation in Cosmc Promoter Region and Aberrantly Glycosylated IgA1 Associated with Pediatric IgA Nephropathy

IgA nephropathy (IgAN) is one of the most common glomerular diseases leading to end-stage renal failure. Elevation of aberrantly glycosylated IgA1 is a key feature of it. The expression of the specific molecular chaperone of core1ß1, 3galactosyl transferase (Cosmc) is known to be reduced in IgAN. We aimed to investigate whether the methylation of CpG islands of Cosmc gene promoter region could act as a possible mechanism responsible for down-regulation of Cosmc and related higher secretion of aberrantly glycosylated IgA1in lymphocytes from children with IgA nephropathy. Three groups were included: IgAN children (n = 26), other renal diseases (n = 11) and healthy children (n = 13). B-lymphocytes were isolated and cultured, treated or not with IL-4 or 5-Aza-2’-deoxycytidine (AZA). The levels of DNA methylation of Cosmc promotor region were not significantly different between the lymphocytes of the three children populations (P = 0.113), but there were significant differences between IgAN lymphocytes and lymphocytes of the other two children populations after IL-4 (P<0.0001) or AZA (P<0.0001). Cosmc mRNA expression was low in IgAN lymphocytes compared to the other two groups (P<0.0001). The level of aberrantly glycosylated IgA1 was markedly higher in IgAN group compared to the other groups (P<0.0001). After treatment with IL-4, the levels of Cosmc DNA methylation and aberrantly glycosylated IgA1 in IgAN lymphocytes were remarkably higher than the other two groups (P<0.0001) with more markedly decreased Cosmc mRNA content (P<0.0001). After treatment with AZA, the levels in IgAN lymphocytes were decreased, but was still remarkably higher than the other two groups (P<0.0001), while Cosmc mRNA content in IgAN lymphocytes were more markedly increased than the other two groups (P<0.0001). The alteration of DNA methylation by IL-4 or AZA specifically correlates in IgAN lymphocytes with alterations in Cosmc mRNA expression and with the level of aberrantly glycosylated IgA1 (r = −0.948, r = 0. 707). Our results suggested that hypermethylation of Cosmc promoter region could be a key mechanism for the reduction of Cosmc mRNA expression in IgAN lymphocytes with associated increase in aberrantly glycosylated IgA1.     

LMP gene promoter hypermethylation is a mechanism for its down regulation in Kazak’s esophageal squamous cell carcinomas

Abnormal hypermethylation of CpG islands not only associated with tumor suppressor genes can lead to repression of gene expression, but also contribute to escape of the tumor from immune surveillance and contribute significantly to tumorigenesis. In the present study, we studied the hypermethylation of low molecular-weight protein (LMP) gene and its regulation on protein expression in biopsies from resected tissues from Kazak’s esophageal squamous cell carcinoma (ESCC) patients and their neighboring normal tissues. LMP2 and LMP7 genes promoter region methylation sequences were maped in esophageal cancer cell line Eca109 by bisulfite-sequencing PCR and quantitative detection of methylated DNA from 30 pairs of Kazak’s ESCC and adjacent normal tissues by MassARRAY (Sequenom, San Diego, CA, USA) and LMP2 and LMP7 protein expression were analyzed with immunohistochemistry. In Eca109, we identified 6 CG sites methylated from all of 22 CpG sites of LMP7 gene. However, no methylation was found for LMP2. The analysis of the data resulted from the quantitative analysis of single CpG site methylation by Sequenom MassARRAY platform, has shown that the methylation level between two groups CpG sites (CpG_5, CpG_9, CpG_20, CpG_21 and CpG_20) from CpG_1, CpG_2, CpG_3, CpG_4, CpG_5, CpG_6, CpG_7, CpG_8, CpG_9, CpG_10.11, CpG_12.13.14, CpG_15.16.17.18, CpG_19, CpG_20, CpG_21 and CpG_22 significant differences between ESCC and neighboring normal tissues. The analysis of methylation level of whole target CpG fragment indicated that the methylation level of LMP7 was significant higher in ESCC (0.0517 ± 0.0357) than in neighboring normal tissues (0.0380 ± 0.0214, P < 0.05). there was a tendency of decreasing the LMP7 proteins expression as the increasing the methylation level of LMP7 gene promoter regions (F = 7.69, P = 0.041). The LMP7 gene promoter methylation and protein downregulation were correlated at high extent in Kazakh’s ESCC patients, and may explain the epigenetic regulation on gene expression.     

Identification of New Differentially Methylated Genes That Have Potential Functional Consequences in Prostate Cancer

Many differentially methylated genes have been identified in prostate cancer (PCa), primarily using candidate gene-based assays. Recently, several global DNA methylation profiles have been reported in PCa, however, each of these has weaknesses in terms of ability to observe global DNA methylation alterations in PCa. We hypothesize that there remains unidentified aberrant DNA methylation in PCa, which may be identified using higher resolution assay methods. We used the newly developed Illumina HumanMethylation450 BeadChip in PCa (n = 19) and adjacent normal tissues (n = 4) and combined these with gene expression data for identifying new DNA methylation that may have functional consequences in PCa development and progression. We also confirmed our methylation results in an independent data set. Two aberrant DNA methylation genes were validated among an additional 56 PCa samples and 55 adjacent normal tissues. A total 28,735 CpG sites showed significant differences in DNA methylation (FDR adjusted P<0.05), defined as a mean methylation difference of at least 20% between PCa and normal samples. Furthermore, a total of 122 genes had more than one differentially methylated CpG site in their promoter region and a gene expression pattern that was inverse to the direction of change in DNA methylation (e.g. decreased expression with increased methylation, and vice-versa). Aberrant DNA methylation of two genes, AOX1 and SPON2, were confirmed via bisulfate sequencing, with most of the respective CpG sites showing significant differences between tumor samples and normal tissues. The AOX1 promoter region showed hypermethylation in 92.6% of 54 tested PCa samples in contrast to only three out of 53 tested normal tissues. This study used a new BeadChip combined with gene expression data in PCa to identify novel differentially methylated CpG sites located within genes. The newly identified differentially methylated genes may be used as biomarkers for PCa diagnosis.     

Identification of a DNA Methylome Profile of Esophageal Squamous Cell Carcinoma and Potential Plasma Epigenetic Biomarkers for Early Diagnosis

DNA methylation is a critical epigenetic mechanism involved in key cellular processes. Its deregulation has been linked to many human cancers including esophageal squamous cell carcinoma (ESCC). This study was designed to explore the whole methylation status of ESCC and to identify potential plasma biomarkers for early diagnosis. We used Infinium Methylation 450k array to analyze ESCC tissues (n = 4), paired normal surrounding tissues (n = 4) and normal mucosa from healthy individuals (n = 4), and combined these with gene expression data from the GEO database. One hundred and sixty eight genes had differentially methylated CpG sites in their promoter region and a gene expression pattern inverse to the direction of change in DNA methylation. These genes were involved in several cancer-related pathways. Three genes were validated in additional 42 ESCC tissues and paired normal surrounding tissues. The methylation frequency of EPB41L3, GPX3, and COL14A1 were higher in tumor tissues than in normal surrounding tissues (P<0.017). The higher methylation frequency of EPB41l3 was correlated with large tumor size (P = 0.044) and advanced pT tumor stage (P = 0.001). The higher methylation frequency of GPX3 and COL14A1 were correlated with advanced pN tumor stage (P = 0.001 and P<0.001). The methylation of EPB41L3, GPX3, and COL14A1 genes were only found in ESCC patients'' plasma, but not in normal individuals upon testing 42 ESCC patients and 50 healthy individuals. Diagnostic sensitivity was increased when methylation of any of the 3 genes were counted (64.3% sensitivity and 100% specificity). These differentially methylated genes in plasma may be used as biomarkers for early diagnosis of ESCC.     

Modification of Epigenetic Patterns in Low Birth Weight Children: Importance of Hypomethylation of the ACE Gene Promoter

There is a growing body of evidence that epigenetic alterations are involved in the pathological mechanisms of many chronic disorders linked to fetal programming. Angiotensin-converting enzyme (ACE) appears as one candidate gene that brings new insights into the epigenetic control and later development of diseases. In this view, we have postulated that epigenetic modifications in the ACE gene might show different interactions between birth weight (BW), blood pressure levels, plasma ACE activity and ACE I/D polymorphism. To explore this hypothesis, we performed a cross-sectional study to evaluate the DNA methylation of 3 CpG sites using pyrosequencing within the ACE gene promoter of peripheral blood leukocytes from 45 LBW children compared with 70 NBW children. Our results have revealed that LBW children have lower methylation levels (P<0.001) in parallel with a higher ACE activity (P = 0.001). Adjusting for prematurity, gender, age, body mass index, and family history of cardiovascular disease did not alter these findings. We have also performed analyses of individual CpG sites. The frequency of DNA methylation was significantly different at two CpG sites (site 1: nucleotide position +555; and site 3: nucleotide position +563). In addition, we have found a significant inverse correlation between degree of DNA methylation and both ACE activity (P<0.001) and systolic blood pressure levels (P<0.001). We also observed that the methylation level was significantly lower in LBW children who are carriers of the DD genotype compared to NBW children with DD genotype (P<0.024). In conclusion, we are able to demonstrate that the hypomethylation in the 3 CpG sites of ACE gene promoter is associated with LBW in 6 to 12 year-old children. The magnitude of these epigenetic changes appears to be clinically important, which is supported by the observation that discrete changes in DNA methylation can affect systolic blood pressure and ACE protein activity levels.     

Epigenetic,Genetic and Environmental Interactions in Esophageal Squamous Cell Carcinoma from Northeast India

Background

Esophageal squamous cell carcinoma (ESCC) develops as a result of complex epigenetic, genetic and environmental interactions. Epigenetic changes like, promoter hypermethylation of multiple tumour suppressor genes are frequent events in cancer, and certain habit-related carcinogens are thought to be capable of inducing aberrant methylation. However, the effects of environmental carcinogens depend upon the level of metabolism by carcinogen metabolizing enzymes. As such key interactions between habits related factors and carcinogen metabolizing gene polymorphisms towards modulating promoter methylation of genes are likely. However, this remains largely unexplored in ESCC. Here, we studied the interaction of various habits related factors and polymorphism of GSTM1/GSTT1 genes towards inducing promoter hypermethylation of multiple tumour suppressor genes.

Methodology/Principal Findings

The study included 112 ESCC cases and 130 age and gender matched controls. Conditional logistic regression was used to calculate odds ratios (OR) and multifactor dimensionality reduction (MDR) was used to explore high order interactions. Tobacco chewing and smoking were the major individual risk factors of ESCC after adjusting for all potential confounding factors. With regards to methylation status, significantly higher methylation frequencies were observed in tobacco chewers than non chewers for all the four genes under study (p<0.01). In logistic regression analysis, betel quid chewing, alcohol consumption and null GSTT1 genotypes imparted maximum risk for ESCC without promoter hypermethylation. Whereas, tobacco chewing, smoking and GSTT1 null variants were the most important risk factors for ESCC with promoter hypermethylation. MDR analysis revealed two predictor models for ESCC with promoter hypermethylation (Tobacco chewing/Smoking/Betel quid chewing/GSTT1 null) and ESCC without promoter hypermethylation (Betel quid chewing/Alcohol/GSTT1) with TBA of 0.69 and 0.75 respectively and CVC of 10/10 in both models.

Conclusion

Our study identified a possible interaction between tobacco consumption and carcinogen metabolizing gene polymorphisms towards modulating promoter methylation of tumour suppressor genes in ESCC.     

Hypermethylation of the CpG Island Near the G4C2 Repeat in ALS with a C9orf72 Expansion

The G₄C₂ repeat expansion in C9orf72 is the most common known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). We tested the hypothesis that the repeat expansion causes aberrant CpG methylation near the G₄C₂ repeat, which could be responsible for the downregulation of gene expression. We investigated the CpG methylation profile by two methods using genomic DNA from the blood of individuals with ALS (37 expansion carriers and 64 noncarriers), normal controls (n = 76), and family members of 7 ALS probands with the expansion. We report that hypermethylation of the CpG island 5′ of the G₄C₂ repeat is associated with the presence of the expansion (p < 0.0001). A higher degree of methylation was significantly correlated with a shorter disease duration (p < 0.01), associated with familial ALS (p = 0.009) and segregated with the expansion in 7 investigated families. Notably, we did not detect methylation for either normal or intermediate alleles (up to 43 repeats), bringing to question the current cutoff of 30 repeats for pathological alleles. Our study raises several important questions for the future investigation of large data sets, such as whether the degree of methylation corresponds to clinical presentation (ALS versus FTLD).     

Methylation at CPT1A locus is associated with lipoprotein subfraction profiles

Lipoprotein subfractions help discriminate cardiometabolic disease risk. Genetic loci validated as associating with lipoprotein measures do not account for a large proportion of the individual variation in lipoprotein measures. We hypothesized that DNA methylation levels across the genome contribute to interindividual variation in lipoprotein measures. Using data from participants of the Genetics of Lipid Lowering Drugs and Diet Network (n = 663 for discovery and n = 331 for replication stages, respectively), we conducted the first systematic screen of the genome to determine associations between methylation status at ∼470,000 cytosine-guanine dinucleotide (CpG) sites in CD4⁺ T cells and 14 lipoprotein subfraction measures. We modeled associations between methylation at each CpG site and each lipoprotein measure separately using linear mixed models, adjusted for age, sex, study site, cell purity, and family structure. We identified two CpGs, both in the carnitine palmitoyltransferase-1A (CPT1A) gene, which reached significant levels of association with VLDL and LDL subfraction parameters in both discovery and replication phases (P < 1.1 × 10⁻⁷ in the discovery phase, P < .004 in the replication phase, and P < 1.1 × 10⁻¹² in the full sample). CPT1A is regulated by PPARα, a ligand for drugs used to reduce CVD. Our associations between methylation in CPT1A and lipoprotein measures highlight the epigenetic role of this gene in metabolic dysfunction.     

Prenatal antidepressant exposure associated with CYP2E1 DNA methylation change in neonates

Some but not all neonates are affected by prenatal exposure to serotonin reuptake inhibitor antidepressants (SRI) and maternal mood disturbances. Distinguishing the impact of these 2 exposures is challenging and raises critical questions about whether pharmacological, genetic, or epigenetic factors can explain the spectrum of reported outcomes. Using unbiased DNA methylation array measurements followed by a detailed candidate gene approach, we examined whether prenatal SRI exposure was associated with neonatal DNA methylation changes and whether such changes were associated with differences in birth outcomes. Prenatal SRI exposure was first associated with increased DNA methylation status primarily at CYP2E1(β_Non-exposed = 0.06, β_SRI-exposed = 0.30, FDR = 0); however, this finding could not be distinguished from the potential impact of prenatal maternal depressed mood. Then, using pyrosequencing of CYP2E1 regulatory regions in an expanded cohort, higher DNA methylation status—both the mean across 16 CpG sites (P < 0.01) and at each specific CpG site (P < 0.05)—was associated with exposure to lower 3rd trimester maternal depressed mood symptoms only in the SRI-exposed neonates, indicating a maternal mood x SRI exposure interaction. In addition, higher DNA methylation levels at CpG2 (P = 0.04), CpG9 (P = 0.04) and CpG10 (P = 0.02), in the interrogated CYP2E1 region, were associated with increased birth weight independently of prenatal maternal mood, SRI drug exposure, or gestational age at birth. Prenatal SRI antidepressant exposure and maternal depressed mood were associated with altered neonatal CYP2E1 DNA methylation status, which, in turn, appeared to be associated with birth weight.     

Genome-wide differentially methylated genes in prostate cancer tissues from African-American and Caucasian men

Increasing evidence suggests that aberrant DNA methylation changes may contribute to prostate cancer (PCa) ethnic disparity. To comprehensively identify DNA methylation alterations in PCa disparity, we used the Illumina 450K methylation platform to interrogate the methylation status of 485,577 CpG sites focusing on gene-associated regions of the human genome. Genomic DNA from African-American (AA; 7 normal and 3 cancers) and Caucasian (Cau; 8 normal and 3 cancers) was used in the analysis. Hierarchical clustering analysis identified probe-sets unique to AA and Cau samples, as well as common to both. We selected 25 promoter-associated novel CpG sites most differentially methylated by race (fold change > 1.5-fold; adjusted P < 0.05) and compared the β-value of these sites provided by the Illumina, Inc. array with quantitative methylation obtained by pyrosequencing in 7 prostate cell lines. We found very good concordance of the methylation levels between β-value and pyrosequencing. Gene expression analysis using qRT-PCR in a subset of 8 genes after treatment with 5-aza-2′-deoxycytidine and/or trichostatin showed up-regulation of gene expression in PCa cells. Quantitative analysis of 4 genes, SNRPN, SHANK2, MST1R, and ABCG5, in matched normal and PCa tissues derived from AA and Cau PCa patients demonstrated differential promoter methylation and concomitant differences in mRNA expression in prostate tissues from AA vs. Cau. Regression analysis in normal and PCa tissues as a function of race showed significantly higher methylation prevalence for SNRPN (P = 0.012), MST1R (P = 0.038), and ABCG5 (P < 0.0002) for AA vs. Cau samples. We selected the ABCG5 and SNRPN genes and verified their biological functions by Western blot analysis and siRNA gene knockout effects on cell proliferation and invasion in 4 PCa cell lines (2 AA and 2 Cau patients-derived lines). Knockdown of either ABCG5 or SNRPN resulted in a significant decrease in both invasion and proliferation in Cau PCa cell lines but we did not observe these remarkable loss-of-function effects in AA PCa cell lines. Our study demonstrates how differential genome-wide DNA methylation levels influence gene expression and biological functions in AA and Cau PCa.     

In Ovo Injection of Betaine Affects Hepatic Cholesterol Metabolism through Epigenetic Gene Regulation in Newly Hatched Chicks

Betaine is reported to regulate hepatic cholesterol metabolism in mammals. Chicken eggs contain considerable amount of betaine, yet it remains unknown whether and how betaine in the egg affects hepatic cholesterol metabolism in chicks. In this study, eggs were injected with betaine at 2.5 mg/egg and the hepatic cholesterol metabolism was investigated in newly hatched chicks. Betaine did not affect body weight or liver weight, but significantly increased the serum concentration (P < 0.05) and the hepatic content (P < 0.01) of cholesterol. Accordingly, the cholesterol biosynthetic enzyme HMGCR was up-regulated (P < 0.05 for both mRNA and protein), while CYP7A1 which converts cholesterol to bile acids was down-regulated (P < 0.05 for mRNA and P = 0.07 for protein). Moreover, hepatic protein content of the sterol-regulatory element binding protein 1 which regulates cholesterol and lipid biosynthesis, and the mRNA abundance of ATP binding cassette sub-family A member 1 (ABCA1) which mediates cholesterol counter transport were significantly (P < 0.05) increased in betaine-treated chicks. Meanwhile, hepatic protein contents of DNA methyltransferases 1 and adenosylhomocysteinase-like 1 were increased (P < 0.05), which was associated with global genomic DNA hypermethylation (P < 0.05) and diminished gene repression mark histone H3 lysine 27 trimethylation (P < 0.05). Furthermore, CpG methylation level on gene promoters was found to be increased (P < 0.05) for CYP7A1 yet decreased (P < 0.05) for ABCA1. These results indicate that in ovo betaine injection regulates hepatic cholesterol metabolism in chicks through epigenetic mechanisms including DNA and histone methylations.     

A Preliminary Study of the Relationship between Promoter Methylation of the ABCG1, GALNT2 and HMGCR Genes and Coronary Heart Disease

Aims

To investigate the association of ABCG1, GALNT2 and HMGCR genes promoter DNA methylation with coronary heart disease (CHD) and explore the interaction between their methylation status and the CHD patients'' clinical characteristics in Han Chinese population.

Methods and results

Methylation-specific polymerase chain reaction (MSP) technology was used to examine the role of the aberrant gene promoter methylation in CHD in Han Chinese population. A total of 85 CHD patients and 54 participants without CHD confirmed by angiography were recruited. 82.8% of the participants with ABCG1 gene promoter hypermethylation have CHD, while only 17.4% of the participants without hypermethylation have it. The average age of the participants with GALNT2 gene promoter hypermethylation is 62.10±8.21, while that of the participants without hypermethylation is 57.28±9.87; in the former group, 75.4% of the participants have CHD, compared to only 50% in the latter group. As for the HMGCR gene, the average age of the participants with promoter hypermethylation is 63.24±8.10 and that of the participants without hypermethylation is 57.79±9.55; its promoter hypermethylation is likely to be related to smoking. Our results indicated a significant statistical association of promoter methylation of the ABCG1 gene with increased risk of CHD (OR = 19.966; 95% CI, 7.319–54.468; P ^*<0.001; P ^*: adjusted for age, gender, smoking, lipid level, hypertension, and diabetes). Similar results were obtained for that of the GALNT2 gene (OR = 2.978; 95% CI, 1.335–6.646; P ^* = 0.008), but not of HMGCR gene (OR = 1.388; 95% CI, 0.572–3.371; P ^* = 0.469).

Conclusions

The present work provides evidence to support the association of promoter DNA methylation status with the risk profile of CHD. Our data indicates that promoter DNA hypermethylation of the ABCG1 and GALNT2 genes, but not the HMGCR gene, is associated with an increased risk of CHD. CHD, smoking and aging are likely to be the important factors influencing DNA hypermethylation.     

Breast Cancer DNA Methylation Profiles Are Associated with Tumor Size and Alcohol and Folate Intake

Although tumor size and lymph node involvement are the current cornerstones of breast cancer prognosis, they have not been extensively explored in relation to tumor methylation attributes in conjunction with other tumor and patient dietary and hormonal characteristics. Using primary breast tumors from 162 (AJCC stage I–IV) women from the Kaiser Division of Research Pathways Study and the Illumina GoldenGate methylation bead-array platform, we measured 1,413 autosomal CpG loci associated with 773 cancer-related genes and validated select CpG loci with Sequenom EpiTYPER. Tumor grade, size, estrogen and progesterone receptor status, and triple negative status were significantly (Q-values <0.05) associated with altered methylation of 209, 74, 183, 69, and 130 loci, respectively. Unsupervised clustering, using a recursively partitioned mixture model (RPMM), of all autosomal CpG loci revealed eight distinct methylation classes. Methylation class membership was significantly associated with patient race (P<0.02) and tumor size (P<0.001) in univariate tests. Using multinomial logistic regression to adjust for potential confounders, patient age and tumor size, as well as known disease risk factors of alcohol intake and total dietary folate, were all significantly (P<0.0001) associated with methylation class membership. Breast cancer prognostic characteristics and risk-related exposures appear to be associated with gene-specific tumor methylation, as well as overall methylation patterns.     

Epigenetics of a tandem DNA repeat: chromatin DNaseI sensitivity and opposite methylation changes in cancers

DNA methylation and chromatin DNaseI sensitivity were analyzed in and adjacent to D4Z4 repeat arrays, which consist of 1 to ~100 tandem 3.3-kb units at subtelomeric 4q and 10q. D4Z4 displayed hypomethylation in some cancers and hypermethylation in others relative to normal tissues. Surprisingly, in cancers with extensive D4Z4 methylation there was a barrier to hypermethylation spreading to the beginning of this disease-associated array (facioscapulohumeral muscular dystrophy, FSHD) despite sequence conservation in repeat units throughout the array. We infer a different chromatin structure at the proximal end of the array than at interior repeats, consistent with results from chromatin DNaseI sensitivity assays indicating a boundary element near the beginning of the array. The relative chromatin DNaseI sensitivity in FSHD and control myoblasts and lymphoblasts was as follows: a non-genic D4Z4-adjacent sequence (p13E-11, array-proximal)> untranscribed gene standards > D4Z4 arrays> constitutive heterochromatin (satellite 2; P < 10⁻⁴ for all comparisons). Cancers displaying D4Z4 hypermethylation also exhibited a hypermethylation-resistant subregion within the 3.3-kb D4Z4 repeat units. This subregion contains runs of G that form G-quadruplexes in vitro. Unusual DNA structures might contribute to topological constraints that link short 4q D4Z4 arrays to FSHD and make long ones phenotypically neutral.