Fixed genomic variation explains only a small proportion of the risk of adiposity. In animal models, maternal diet alters offspring body composition, accompanied by epigenetic changes in metabolic control genes. Little is known about whether such processes operate in humans.
RESEARCH DESIGN AND METHODS
Using Sequenom MassARRAY we measured the methylation status of 68 CpGs 5′ from five candidate genes in umbilical cord tissue DNA from healthy neonates. Methylation varied greatly at particular CpGs: for 31 CpGs with median methylation ≥5% and a 5–95% range ≥10%, we related methylation status to maternal pregnancy diet and to child’s adiposity at age 9 years. Replication was sought in a second independent cohort.
In cohort 1, retinoid X receptor-α (RXRA) chr9:136355885+ and endothelial nitric oxide synthase (eNOS) chr7:150315553+ methylation had independent associations with sex-adjusted childhood fat mass (exponentiated regression coefficient [β] 17% per SD change in methylation [95% CI 4–31], P = 0.009, n = 64, and β = 20% [9–32], P < 0.001, n = 66, respectively) and %fat mass (β = 10% [1–19], P = 0.023, n = 64 and β =12% [4–20], P = 0.002, n = 66, respectively). Regression analyses including sex and neonatal epigenetic marks explained >25% of the variance in childhood adiposity. Higher methylation of RXRA chr9:136355885+, but not of eNOS chr7:150315553+, was associated with lower maternal carbohydrate intake in early pregnancy, previously linked with higher neonatal adiposity in this population. In cohort 2, cord eNOS chr7:150315553+ methylation showed no association with adiposity, but RXRA chr9:136355885+ methylation showed similar associations with fat mass and %fat mass (β = 6% [2–10] and β = 4% [1–7], respectively, both P = 0.002, n = 239).
Our findings suggest a substantial component of metabolic disease risk has a prenatal developmental basis. Perinatal epigenetic analysis may have utility in identifying individual vulnerability to later obesity and metabolic disease.
Perturbation of DNA methylation is frequent in cancers and has emerged as an important mechanism involved in tumorigenesis. To determine how DNA methylation is modified in the genome of primary glioma, we used Methyl-DNA immunoprecipitation (MeDIP) and Nimblegen CpG promoter microarrays to identify differentially DNA methylation sequences between primary glioma and normal brain tissue samples.
MeDIP-chip technology was used to investigate the whole-genome differential methylation patterns in glioma and normal brain tissues. Subsequently, the promoter methylation status of eight candidate genes was validated in 40 glioma samples and 4 cell lines by Sequenom's MassARRAY system. Then, the epigenetically regulated expression of these genes and the potential mechanisms were examined by chromatin immunoprecipitation and quantitative real-time PCR.
A total of 524 hypermethylated and 104 hypomethylated regions were identified in glioma. Among them, 216 hypermethylated and 60 hypomethylated regions were mapped to the promoters of known genes related to a variety of important cellular processes. Eight promoter-hypermethylated genes (ANKDD1A, GAD1, HIST1H3E, PCDHA8, PCDHA13, PHOX2B, SIX3, and SST) were confirmed in primary glioma and cell lines. Aberrant promoter methylation and changed histone modifications were associated with their reduced expression in glioma. In addition, we found loss of heterozygosity (LOH) at the miR-185 locus located in the 22q11.2 in glioma and induction of miR-185 over-expression reduced global DNA methylation and induced the expression of the promoter-hypermethylated genes in glioma cells by directly targeting the DNA methyltransferases 1.
These comprehensive data may provide new insights into the epigenetic pathogenesis of human gliomas.
DNA methylation; MiR-185; Glioma; DNMT1
The insulin-like growth factor 2 (IGF2) and H19 imprinted genes control growth and body composition. Adverse in-utero environments have been associated with obesity-related diseases and linked with altered DNA methylation at the IGF2/H19 locus. Postnatally, methylation at the IGF2/H19 imprinting control region (ICR) has been linked with cerebellum weight. We aimed to investigate whether decreased IGF2/H19 ICR methylation is associated with decreased birth and childhood anthropometry and increased contemporaneous adiposity.
DNA methylation in peripheral blood (n = 315) at 17 years old was measured at 12 cytosine-phosphate-guanine sites (CpGs), analysed as Sequenom MassARRAY EpiTYPER units within the IGF2/H19 ICR. Birth size, childhood head circumference (HC) at six time-points and anthropometry at age 17 years were measured. DNA methylation was investigated for its association with anthropometry using linear regression.
The principal component of IGF2/H19 ICR DNA methylation (representing mean methylation across all CpG units) positively correlated with skin fold thickness (at four CpG units) (P-values between 0.04 to 0.001) and subcutaneous adiposity (P = 0.023) at age 17, but not with weight, height, BMI, waist circumference or visceral adiposity. IGF2/H19 methylation did not associate with birth weight, length or HC, but CpG unit 13 to 14 methylation was negatively associated with HC between 1 and 10 years. β-coefficients of four out of five remaining CpG units also estimated lower methylation with increasing childhood HC.
As greater IGF2/H19 methylation was associated with greater subcutaneous fat measures, but not overall, visceral or central adiposity, we hypothesize that obesogenic pressures in youth result in excess fat being preferentially stored in peripheral fat depots via the IGF2/H19 domain. Secondly, as IGF2/H19 methylation was not associated with birth size but negatively with early childhood HC, we hypothesize that the HC may be a more sensitive marker of early life programming of the IGF axis and of fetal physiology than birth size. To verify this, investigations of the dynamics of IGF2/H19 methylation and expression from birth to adolescence are required.
Childhood; Fetal programming; DNA methylation; Insulin-like growth factor; Raine Study; Head circumference
Epigenetic changes such as aberrant DNA methylation and histone modification have been shown to play an important role in the tumorigenesis of malignant melanoma.
To identify novel tumor-specific differentially methylated regions (DMRs) in human malignant melanoma.
The aberrant methylation at 14 candidate human genomic regions identified through a mouse model study with quantitative DNA methylation analysis using the Sequenom MassARRAY system was performed.
The CpG island Exon 1 region of the Zygote arrest 1 (ZAR1) gene, which is responsible for oocyte-to-embryo transition, showed frequent aberrant methylation of 28 out of 30 (93%) melanoma surgical specimens, 16 of 17 (94%) melanoma cell lines, 0% of 4 normal human epidermal melanocyte (NHEM) cell lines, 0% of 10 melanocytic nevi and 100% of 51 various cancer cell lines. According to the real-time RT-PCR, the ZAR1 gene was overexpressed in part of the hypermethylated cell lines, while its low expression with bivalent histone methylation status was seen in unmethylated cell lines.
Our findings suggest that the ZAR1 intra-genic differentially methylated region would be a useful tumor marker for malignant melanoma and may be other type of cancers. The involvement of ZAR1 in the carcinogenesis of melanoma, still remains unclear, although we have examined tumorigenic capacities by exogenous full-length ZAR1 over-expression and siRNA knock-down experiments.
Background: Epigenetic modifications, such as DNA methylation, due to in utero exposures may play a critical role in early programming for childhood and adult illness. Maternal smoking is a major risk factor for multiple adverse health outcomes in children, but the underlying mechanisms are unclear.
Objective: We investigated epigenome-wide methylation in cord blood of newborns in relation to maternal smoking during pregnancy.
Methods: We examined maternal plasma cotinine (an objective biomarker of smoking) measured during pregnancy in relation to DNA methylation at 473,844 CpG sites (CpGs) in 1,062 newborn cord blood samples from the Norwegian Mother and Child Cohort Study (MoBa) using the Infinium HumanMethylation450 BeadChip (450K).
Results: We found differential DNA methylation at epigenome-wide statistical significance (p-value < 1.06 × 10–7) for 26 CpGs mapped to 10 genes. We replicated findings for CpGs in AHRR, CYP1A1, and GFI1 at strict Bonferroni-corrected statistical significance in a U.S. birth cohort. AHRR and CYP1A1 play a key role in the aryl hydrocarbon receptor signaling pathway, which mediates the detoxification of the components of tobacco smoke. GFI1 is involved in diverse developmental processes but has not previously been implicated in responses to tobacco smoke.
Conclusions: We identified a set of genes with methylation changes present at birth in children whose mothers smoked during pregnancy. This is the first study of differential methylation across the genome in relation to maternal smoking during pregnancy using the 450K platform. Our findings implicate epigenetic mechanisms in the pathogenesis of the adverse health outcomes associated with this important in utero exposure.
epigenetics; epigenome-wide; in utero; maternal smoking; methylation
The inheritance of DNA methylation patterns is a popular theory to explain the influence of parental genetic and environmental factors on the phenotype of their offspring but few studies have examined this relationship in humans. Using 120 paired maternal-umbilical cord blood samples randomly selected from a prospective birth cohort in Bangladesh, we quantified DNA methylation by pyrosequencing seven CpG positions in the promoter region of p16, four CpG positions in the promoter region of p53, LINE-1 and Alu. Positive correlations were observed between maternal and umbilical cord blood at p16, LINE-1, and Alu but not p53. Multiple linear regression models observed a significant association between maternal and umbilical cord blood at LINE-1 and Alu (LINE-1: β = 0.63, p<0.0001; Alu: β = 0.28, p = 0.009). After adjusting for multiple comparisons, maternal methylation of p16 at position 4 significantly predicted methylation at the same position in umbilical cord blood (β = 0.43, p = <0.0001). These models explained 48%, 5% and 16% of the observed variability in umbilical cord %5mC for LINE-1, Alu and p16 at position 4, respectively. These results suggest that DNA methylation in maternal blood was correlated with her offspring at LINE-1, Alu, and p16 but not p53. Additional studies are needed to confirm whether these observed associations were due to the inheritance of epigenetic events or the shared environment between mother and fetus. Future studies should also use a multi-generational family-based design that would quantify both maternal and paternal contributions to DNA methylation in offspring across more than one generation.
Major depression, because of its recurring and life-threatening nature, is one of the top 10 diseases for global disease burden. Major depression is still diagnosed on the basis of clinical symptoms in patients. The search for specific biological markers is of great importance to advance the method of diagnosis for depression. We examined the methylation profile of 2 CpG islands (I and IV) at the promoters of the brain-derived neurotrophic factor (BDNF) gene, which is well known to be involved in the pathophysiology of depression. We analyzed genomic DNA from peripheral blood of 20 Japanese patients with major depression and 18 healthy controls to identify an appropriate epigenetic biomarker to aid in the establishment of an objective system for the diagnosis of depression. Methylation rates at each CpG unit was measured using a MassArray® system (SEQUENOM), and 2-dimensional hierarchical clustering analyses were undertaken to determine the validity of these methylation profiles as a diagnostic biomarker. Analyses of the dendrogram from methylation profiles of CpG I, but not IV, demonstrated that classification of healthy controls and patients at the first branch completely matched the clinical diagnosis. Despite the small number of subjects, our results indicate that classification based on the DNA methylation profiles of CpG I of the BDNF gene may be a valuable diagnostic biomarker for major depression.
Risk for adverse neonatal outcome increases with declining gestational age (GA), and changes in DNA methylation may contribute to the relationship between GA and adverse health outcomes in offspring. To test this hypothesis, we evaluated the association between GA and more than 27,000 CpG sites in neonatal DNA extracted from umbilical cord blood from two prospectively-characterized cohorts: (1) a discovery cohort consisting of 259 neonates from women with a history of neuropsychiatric disorders and (2) a replication cohort consisting of 194 neonates of uncomplicated mothers. GA was determined by obstetrician report and maternal last menstrual period. The associations between proportion of DNA methylated and GA were evaluated by fitting a separate linear mixed effects model for each CpG site, adjusting for relevant covariates including neonatal sex, race, parity, birth weight percentile and chip effects. CpG sites in 39 genes were associated with GA (false discovery rate <0.05) in the discovery cohort. The same CpG sites in 25 of these genes replicated in the replication cohort, with each association replicating in the same direction. Notably, these CpG sites were located in genes previously implicated in labor and delivery (e.g., AVP, OXT, CRHBP and ESR1) or that may influence the risk for adverse health outcomes later in life (e.g., DUOX2, TMEM176A and CASP8). All associations were independent of method of delivery or induction of labor. These results suggest neonatal DNA methylation varies with GA even in term deliveries. The potential contribution of these changes to clinically significant postnatal outcomes warrants further investigation.
genome-wide DNA methylation; gestational age; arginine vasopressin and oxytocin
Low birthweight, premature birth, intrauterine growth retardation, and maternal malnutrition have been related to an increased risk of cardiovascular disease, type 2 diabetes mellitus, obesity, and neuropsychiatric disorders later in life. Conversely, high birthweight has been linked to future risk of cancer. Global DNA methylation estimated by the methylation of repetitive sequences in the genome is an indicator of susceptibility to chronic diseases. We used data and biospecimens from an epigenetic birth cohort to explore the association between trajectories of fetal and maternal weight and LINE-1 methylation in 319 mother-child dyads. Newborns with low or high birthweight had significantly lower LINE-1 methylation levels in their cord blood compared to normal weight infants after adjusting for gestational age, sex of the child, maternal age at delivery, and maternal smoking during pregnancy (p = 0.007 and p = 0.036, respectively), but the magnitude of the difference was small. Infants born prematurely also had lower LINE-1 methylation levels in cord blood compared to term infants, and this difference, though small, was statistically significant (p = 0.004). We did not find important associations between maternal prepregnancy BMI or gestational weight gain and global methylation of the cord blood or fetal placental tissue. In conclusion, we found significant differences in cord blood LINE-1 methylation among newborns with low and high birthweight as well as among prematurely born infants. Future studies may elucidate whether chromosomal instabilities or other functional consequences of these changes contribute to the increased risk of chronic diseases among individuals with these characteristics.
Motivation: DNA cytosine methylation is an important epigenetic regulator, critical for mammalian development and the control of gene expression. Numerous techniques using either restriction enzyme or affinity-based approaches have been developed to interrogate cytosine methylation status genome-wide, however these assays must be validated by a more quantitative approach, such as MALDI-TOF mass spectrometry of bisulphite-converted DNA (commercialized as Sequenom's EpiTYPER assay using the MassArray system). Here, we present an R package (‘MassArray’) that assists in assay design and uses the standard Sequenom output file as the input to a pipeline of analyses not available as part of the commercial software. The tools in this package include bisulphite conversion efficiency calculation, sequence polymorphism flagging and visualization tools that combine multiple experimental replicates and create tracks for genome browser viewing.
Supplementary information: Supplementary data are available at Bioinformatics online.
The highly improved cognitive function is the most significant change in human evolutionary history. Recently, several large-scale studies reported the evolutionary roles of DNA methylation; however, the role of DNA methylation on brain evolution is largely unknown.
To test if DNA methylation has contributed to the evolution of human brain, with the use of MeDIP-Chip and SEQUENOM MassARRAY, we conducted a genome-wide analysis to identify differentially methylated regions (DMRs) in the brain between humans and rhesus macaques. We first identified a total of 150 candidate DMRs by the MeDIP-Chip method, among which 4 DMRs were confirmed by the MassARRAY analysis. All 4 DMRs are within or close to the CpG islands, and a MIR3 repeat element was identified in one DMR, but no repeat sequence was observed in the other 3 DMRs. For the 4 DMR genes, their proteins tend to be conserved and two genes have neural related functions. Bisulfite sequencing and phylogenetic comparison among human, chimpanzee, rhesus macaque and rat suggested several regions of lineage specific DNA methylation, including a human specific hypomethylated region in the promoter of K6IRS2 gene.
Our study provides a new angle of studying human brain evolution and understanding the evolutionary role of DNA methylation in the central nervous system. The results suggest that the patterns of DNA methylation in the brain are in general similar between humans and non-human primates, and only a few DMRs were identified.
DNA methylation; Brain evolution; Primates
Induction of an altered phenotype by prenatal under-nutrition involves changes in the epigenetic regulation of specific genes. We investigated the effect of feeding pregnant rats a protein-restricted (PR) diet with different amounts of folic acid on the methylation of individual CpG dinucleotides in the hepatic PPARα promoter in juvenile offspring, and the effect of the maternal PR diet on CpG methylation in adult offspring. Pregnant rats (n 5 / group) were fed 180g / kg casein (Control) or 90g / kg casein (PR) with 1mg / kg folic acid, or 90g / kg casein and 5 mg / kg folic acid (PRF). Offspring were killed on postnatal d34 (n 5 males and females / group) and d80 (n 5 males / group). Methylation of 16 CpG dinucleotides in the PPARα promoter was measured by pyrosequencing. Mean PPARα promoter methylation in the PR offspring (4.5%) was 26% lower than Controls (6.1%) due to specific reduction at CpG dinucleotides 2 (40%), 3 (43%), 4 (33%) and 16 (48 %) (P < 0.05). There was no significant difference in methylation at these CpGs between Control and PRF offspring. Methylation of CpGs 5 and 8 was higher (47% and 63%, respectively, P < 0.05) in the PRF offspring than Control or PR offspring. The methylation pattern in d80 PR offspring was comparable to d34 PR offspring. These data show for the first time that prenatal nutrition induces differential changes to the methylation of individual CpG dinucleotides in juvenile rats which persist in adults.
Fetal programming; epigenetic; rat; PPARα
DNA methylation, an epigenetic alteration typically occurring early in cancer development, could aid in the molecular diagnosis of melanoma. We determined technical feasibility for high-throughput DNA-methylation array-based profiling using formalin-fixed paraffin-embedded tissues for selection of candidate DNA-methylation differences between melanomas and nevi. Promoter methylation was evaluated in 27 common benign nevi and 22 primary invasive melanomas using a 1505 CpG site microarray. Unsupervised hierarchical clustering distinguished melanomas from nevi; 26 CpG sites in 22 genes were identified with significantly different methylation levels between melanomas and nevi after adjustment for age, sex, and multiple comparisons and with β-value differences of ≥0.2. Prediction analysis for microarrays identified 12 CpG loci that were highly predictive of melanoma, with area under the receiver operating characteristic curves of >0.95. Of our panel of 22 genes, 14 were statistically significant in an independent sample set of 29 nevi (including dysplastic nevi) and 25 primary invasive melanomas after adjustment for age, sex, and multiple comparisons. This first report of a DNA-methylation signature discriminating melanomas from nevi indicates that DNA methylation appears promising as an additional tool for enhancing melanoma diagnosis.
melanoma; nevi; methylation profiling; diagnostic markers
DNA methylation, an epigenetic alteration typically occurring early in cancer development, could aid in the molecular diagnosis of melanoma. We determined technical feasibility for high-throughput DNA-methylation array-based profiling using formalin-fixed paraffin-embedded tissues for selection of candidate DNA-methylation differences between melanomas and nevi. Promoter methylation was evaluated in 27 common benign nevi and 22 primary invasive melanomas using a 1505 CpG-site microarray. Unsupervised hierarchical clustering distinguished melanomas from nevi; and 26 CpG sites in 22 genes were identified with significantly different methylation levels between melanomas and nevi after adjustment for age, sex, and multiple comparisons and with β-value differences of ≥ 0.2. Prediction Analysis for Microarrays identified 12 CpG loci that were highly predictive of melanoma, with area under the receiver operating characteristic curves of greater than 0.95. Of our panel of 22 genes, 14 were statistically significant in an independent sample set of 29 nevi (including dysplastic nevi) and 25 primary invasive melanomas after adjustment for age, sex, and multiple comparisons. This first report of a DNA-methylation signature discriminating melanomas from nevi indicates that DNA methylation appears promising as an additional tool for enhancing melanoma diagnosis.
melanoma; nevi; methylation profiling; diagnostic markers
Methylation levels of long interspersed nucleotide elements (LINE-1) are representative of genome-wide methylation status and play an important role in maintaining genomic stability and gene expression. To derive insight into the association between genome-wide methylation status and tetralogy of fallot (TOF), we compared the methylation status of LINE-1 element between TOF patients and controls. The methylation of the NKX 2–5, HAND 1, and TBX 20 promoter regions was also evaluated.
Genomic DNA from right ventricular tissue samples was obtained from 32 patients with TOF and 15 control subjects. Sequenom MassARRAY platform was performed to examine the methylation levels of LINE-1, NKX2-5, HAND1 and TBX20. Mann–Whitney U test was used to compare differences in methylation levels between two groups.
The methylation level of LINE-1 was significantly lower in patients with TOF, with a median of 57.95% (interquartile range [IQR]: 56.10%–60.04%), as opposed to 59.70% in controls (IQR: 59.00%–61.30%; P = 0.0021). The highest LINE-1 methylation level was 61.3%. The risk of TOF increased in subjects with the lowest methylation levels (less than or equal to 59.0%; OR = 14.7, 95% CI: 1.8–117.7, P = 0.014) and in those with medium methylation levels (59.0%–61.3%; OR = 2.0, 95% CI: 0.3–14.2, P = 0.65). An ROC curve analysis showed a relatively high accuracy of using the LINE-1 methylation level in predicting the presence of TOF (AUC = 0.78, 95% CI: 0.65–0.91; P = 0.002). The association of the LINE-1 methylation level with TOF was only observed in males (P = 0.006) and not in females (P = 0.25). Neither age nor gender was found to be associated with the LINE-1 methylation level in patients or controls. Higher methylation levels of NKX2-5 and HAND1 and lower methylation levels of TBX20 were also observed in patients with TOF than in controls. No association was found between the methylation levels of NKX2-5, HAND1 and TBX 20 with the LINE-1 methylation level.
Lower LINE-1 methylation levels are associated with increased risk of TOF and may provide important clues for the development of TOF.
LINE-1 methylation; Tetralogy of fallot; Infants
Impaired flexibility in the use of substrates for energy production in the heart is implicated in cardiomyopathy. We investigated the effect of maternal protein restriction during pregnancy in rats on the transcription of key genes in cardiac lipid and carbohydrate metabolism in the offspring. Rats were fed protein-sufficient or protein-restricted (PR) diets during pregnancy. Triacylglycerol concentration in adult (day 105) heart was altered by maternal protein intake contingent on post-weaning fat intake and sex. mRNA Expression of peroxisomal proliferator-activated receptor (PPAR)-α and carnitine palmitoyltransferase-1 was increased by the maternal PR diet in adult, but not neonatal, offspring. PPARα promoter methylation was lower in adult and neonatal heart from PR offspring. These findings suggest that prenatal nutrition alters the future transcriptional regulation of cardiac energy metabolism in the offspring through changes in epigenetic regulation of specific genes. However, changes in gene functional changes may not be apparent in early life.
Heart; protein-restriction; PPAR; epigenetic; cardiomyopathy
Epidemiological data indicate that children conceived in vitro have a greater relative risk of low birth-weight, major and minor birth defects, and rare disorders involving imprinted genes, suggesting that epigenetic changes may be associated with assisted reproduction. We examined DNA methylation at more than 700 genes (1536 CpG sites) in placenta and cord blood and measured gene expression levels of a subset of genes that differed in methylation levels between children conceived in vitro versus in vivo. Our results suggest that in vitro conception is associated with lower mean methylation at CpG sites in placenta and higher mean methylation at CpG sites in cord blood. We also find that in vitro conception-associated DNA methylation differences are associated with gene expression differences at both imprinted and non-imprinted genes. The range of inter-individual variation in gene expression of the in vitro and in vivo groups overlaps substantially but some individuals from the in vitro group differ from the in vivo group mean by more than two standard deviations. Several of the genes whose expression differs between the two groups have been implicated in chronic metabolic disorders, such as obesity and type II diabetes. These findings suggest that there may be epigenetic differences in the gametes or early embryos derived from couples undergoing treatment for infertility. Alternatively, assisted reproduction technology may have an effect on global patterns of DNA methylation and gene expression. In either case, these differences or changes may affect long-term patterns of gene expression.
Methylation of CpG islands associated with genes can affect the expression of the proximal gene, and methylation of non-associated CpG islands correlates to genomic instability. This epigenetic modification has been shown to be important in many pathologies, from development and disease to cancer. We report the development of a novel high-resolution microarray that detects the methylation status of over 25 000 CpG islands in the human genome. Experiments were performed to demonstrate low system noise in the methodology and that the array probes have a high signal to noise ratio. Methylation measurements between different cell lines were validated demonstrating the accuracy of measurement. We then identified alterations in CpG islands, both those associated with gene promoters, as well as non-promoter-associated islands in a set of breast and ovarian tumors. We demonstrate that this methodology accurately identifies methylation profiles in cancer and in principle it can differentiate any CpG methylation alterations and can be adapted to analyze other species.
Background: Arsenic is an epigenetic toxicant and could influence fetal developmental programming.
Objectives: We evaluated the association between arsenic exposure and DNA methylation in maternal and umbilical cord leukocytes.
Methods: Drinking-water and urine samples were collected when women were at ≤ 28 weeks gestation; the samples were analyzed for arsenic using inductively coupled plasma mass spectrometry. DNA methylation at CpG sites in p16 (n = 7) and p53 (n = 4), and in LINE-1 and Alu repetitive elements (3 CpG sites in each), was quantified using pyrosequencing in 113 pairs of maternal and umbilical blood samples. We used general linear models to evaluate the relationship between DNA methylation and tertiles of arsenic exposure.
Results: Mean (± SD) drinking-water arsenic concentration was 14.8 ± 36.2 μg/L (range: < 1–230 μg/L). Methylation in LINE-1 increased by 1.36% [95% confidence interval (CI): 0.52, 2.21%] and 1.08% (95% CI: 0.07, 2.10%) in umbilical cord and maternal leukocytes, respectively, in association with the highest versus lowest tertile of total urinary arsenic per gram creatinine. Arsenic exposure was also associated with higher methylation of some of the tested CpG sites in the promoter region of p16 in umbilical cord and maternal leukocytes. No associations were observed for Alu or p53 methylation.
Conclusions: Exposure to higher levels of arsenic was positively associated with DNA methylation in LINE-1 repeated elements, and to a lesser degree at CpG sites within the promoter region of the tumor suppressor gene p16. Associations were observed in both maternal and fetal leukocytes. Future research is needed to confirm these results and determine if these small increases in methylation are associated with any health effects.
Alu; arsenic; developmental programming; DNA methylation; environmental exposures; epigenetics; in utero exposure; LINE-1; p16; p53
Epigenetic regulation of tumor suppressor genes (TSGs) has been shown to play a central role in melanomagenesis. By integrating gene expression and methylation array analysis we identified novel candidate genes frequently methylated in melanoma. We validated the methylation status of the most promising genes using highly sensitive Sequenom Epityper assays in a large panel of melanoma cell lines and resected melanomas, and compared the findings with those from cultured melanocytes. We found transcript levels of UCHL1, COL1A2, THBS1 and TNFRSF10D were inversely correlated with promoter methylation. For THBS1 and UCHL1 the effect of this methylation on expression was confirmed at the protein level. Identification of these candidate TSGs and future research designed to understand how their silencing is related to melanoma development will increase our understanding of the etiology of this cancer and may provide tools for its early diagnosis.
The TOB1 gene, mapped on 17q21, is a member of the BTG/Tob family. In breast cancer it has been identified as a candidate tumor suppressor gene. However, whether TOB1 is a bona fide tumor suppressor and downregulated in hepatocellular carcinoma (HCC) remains unclear. In addition, whether its expression is regulated through methylation requires investigation. In the present study, we therefore analyzed the expression of TOB1 in HCC and its methylation levels in human HCC and breast cancer. No significant difference in the expression levels of TOB1 was observed between tumor tissues and adjacent normal tissues in HCC. Quantitative methylation analysis by MassArray revealed no significant differences at single CpG sites or in the global promoter region, and all these CpG sites shared a similar methylation pattern in HCC and breast cancer. Moreover, 5-aza-2′-deoxycytidine treatment of three tumor cell lines did not cause elevation of TOB1 mRNA in HepG2 cell lines. Based on these data, we speculate that TOB1 may be a candidate non-tumor suppressor gene in HCC. Furthermore, the clinical outcome was not correlated with TOB1 expression or expression rate. In addition, TOB1 expression or expression rate was not correlated with the overall survival (OS) rates or cumulative recurrence rates. Taken together, we suggest that TOB1 does not act as a tumor suppressor in HCC.
TOB1; tumor suppressor gene; hepatocellular carcinoma
AIM: To investigated whether sall3 transcription was regulated by promoter CpG island hypermethylation in hepatocellular carcinoma (HCC).
METHODS: The cell lines Huh7, HepG2, SK-HEP1, SMMC7721, Bel7402, QGY7703 and a cohort of 38 HCC tissue specimens and corresponding nontumorous tissues were subjected to analysis for sall3 promoter CpG island methylation and mRNA transcription. sall3 promoter CpG island methylation levels were determined using the MassARRAY platform and mRNA transcription levels of the gene were detected by quantitative real-time polymerase chain reaction.
RESULTS: The levels of sall3 mRNA were decreased by more than twofold in 33 of 38 tumor tissues compared to adjacent noncancerous tissues. Among these 33 tumor tissues with lower levels of sall3 mRNA, 24 showed higher levels of methylation. Based on these results, we hypothesized that the decrease in sall3 mRNA transcription level was likely due to promoter CpG island hypermethylation. Changes in sall3 mRNA transcription and promoter CpG island methylation were determined in the above six cell lines after treatment with 0, 0.1, 0.5 and 2.5 μmol 5-aza-2-deoxycytidine, a demethylating agent. Promoter CpG island methylation levels decreased in a dose-dependent manner in all six cell lines, while the mRNA transcription level increased dose-dependently in Huh7, HepG2, SK-HEP1 and SMMC7721 cells and irregularly in Bel7402 and QGY7703 cells.
CONCLUSION: These results indicated that promoter CpG island hypermethylation contributes to the downregulation of sall3 mRNA transcription in HCC.
Hepatocellular carcinoma; sall3; Aberrant methylation; Down regulation mRNA transcription
DNA promoter methylation is an epigenetic phenomenon for long-term gene silencing during tumorigenesis. The purpose of this study is to identify novel hypermethylated loci associated with clinicopathologic variables in endometrioid endometrial carcinomas.
To find hypermethylated promoter loci, we used differential methylation hybridization coupling with microarray and further validated by combined bisulfite restriction analysis and MassARRAY assay. Methylation levels of candidate loci were corrected with clinicopathologic factors of endometrial carcinomas.
Increased promoter methylation of CIDE, HAAO and RXFP3 was detected in endometrial carcinomas compared with adjacent normal tissues, and was associated with decreased gene expression of all three genes. In a clinical cohort, promoter hypermethylation on CIDEA, HAAO and RXFP3 was detected in 85, 63 and 71% of endometrial carcinomas, respectively (n=118, P<0.001) compared with uninvolved normal endometrium. Methylation status of CIDEA, HAAO and RXFP3 had significant association with microsatellite instability in tumors (P<0.001). Furthermore, methylation levels of HAAO were further found to relate to disease-free survivals (P=0.034).
Hypermethylation of CIDEA, HAAO and RXFP3 promoter regions appears to be a frequent event in endometrial carcinomas. Hypermethylation at these loci is strongly associated with microsatellite instability status. Moreover, HAAO methylation predicts disease-free survival in this cohort of patients with endometrioid endometrial cancer.
Endometrial carcinoma; Hypermethylation; CIDEA; HAAO; RXFP3
To verify whether the leptin gene epigenetic (DNA methylation) profile is altered in the offspring of mothers with gestational impaired glucose tolerance (IGT).
RESEARCH DESIGN AND METHODS
Placental tissues and maternal and cord blood samples were obtained from 48 women at term including 23 subjects with gestational IGT. Leptin DNA methylation, gene expression levels, and circulating concentration were measured using the Sequenom EpiTYPER system, quantitative real-time RT-PCR, and enzyme-linked immunosorbent assay, respectively. IGT was assessed after a 75-g oral glucose tolerance test (OGTT) at 24–28 weeks of gestation.
We have shown that placental leptin gene DNA methylation levels were correlated with glucose levels (2-h post-OGTT) in women with IGT (fetal side: ρ = −0.44, P ≤ 0.05; maternal side: ρ = 0.53, P ≤ 0.01) and with decreased leptin gene expression (n = 48; ρ ≥ −0.30, P ≤ 0.05) in the whole cohort. Placental leptin mRNA levels accounted for 16% of the variance in maternal circulating leptin concentration (P < 0.05).
IGT during pregnancy was associated with leptin gene DNA methylation adaptations with potential functional impacts. These epigenetic changes provide novel mechanisms that could contribute to explaining the detrimental health effects associated with fetal programming, such as long-term increased risk of developing obesity and type 2 diabetes.
Tissue specific differentially methylated regions (TDMRs) were identified and localized in the mouse genome using second generation virtual RLGS (vRLGS). Sequenom MassARRAY quantitative methylation analysis was used to confirm and determine the fine structure of tissue specific differences in DNA methylation. TDMRs have a broad distribution of locations to intragenic and intergenic regions including both CpG islands, and non-CpG islands regions. Somewhat surprising, there is a strong bias for TDMR location in non-promoter intragenic regions. Although some TDMRs are within or close to repeat sequences, overall they are less frequently associated with repetitive elements than expected from a random distribution. Many TDMRs are methylated at early developmental stages, but unmethylated later, suggesting active or passive demethylation, or expansions of populations of cells with unmethylated TDMRs. This is notable during postnatal testis differentiation where many testis-specific TDMRs become progressively “demethylated”. These results suggest that methylation changes during development are dynamic, involve demethylation and methylation, and may occur at late stages of embryonic development or even postnatally.
DNA; Methylation; Epigenesis; Genetic; Gene silencing; Embryonic stem cells; Developmental biology; Mouse