Differential patterns of whole-genome DNA methylation in institutionalized children and children raised by their biological parents

Previous studies with nonhuman species have shown that animals exposed to early adversity show differential DNA methylation relative to comparison animals. The current study examined differential methylation among 14 children raised since birth in institutional care and 14 comparison children raised by their biological parents. Blood samples were taken from children in middle childhood. Analysis of whole-genome methylation patterns was performed using the Infinium HumanMethylation27 BeadChip assay (Illumina), which contains 27,578 CpG sites, covering approximately 14,000 gene promoters. Group differences were registered, which were characterized primarily by greater methylation in the institutionalized group relative to the comparison group, with most of these differences in genes involved in the control of immune response and cellular signaling systems, including a number of crucial players important for neural communication and brain development and functioning. The findings suggest that patterns of differential methylation seen in nonhuman species with altered maternal care are also characteristic of children who experience early maternal separation.     

Low frequency of imprinting defects in ICSI children born small for gestational age

Although there is an increased frequency of low birth weight after assisted reproduction, the mechanisms underlying this association are unclear. We have proposed that some of the children conceived by intracytoplasmic sperm injection (ICSI) with low birth weight might have an epimutation (faulty methylation pattern) in one of the imprinted genes involved in fetal growth control, eg, KCNQ1OT1, PEG1, PEG3, GTL2, IGF2/H19 and PLAGL1. Using bisulfite DNA sequencing and sequence-based quantitative methylation analysis (SeQMA), we determined the methylation pattern of these genes in buccal smears from 19 ICSI children born small for gestational age (SGA, birth weight <3rd percentile) and from 29 term-born normal weight children after spontaneous conception. We detected clear hypermethylation of KCNQ1OT1 and borderline hypermethylation of PEG1 in one and the same ICSI child. The other children and the parents of the affected child have normal methylation patterns. Imprinting defects appear to be a rare finding in ICSI children born SGA. Methylation of the paternal KCNQ1OT1 and PEG1 alleles may be a previously unrecognized cause of SGA. The epimutations found in the SGA child, whose father had oligozoospermia, probably result from an imprint erasure defect in the paternal germ line and therefore appear to be linked to the fertility problem of the father and not to in vitro fertilization/ICSI.     

Histamine metabolism in asthma

Histamine and 1-methyl-imidazole-4-acetic acid, the end product of histamine metabolism via the ring-N methylation pathway, were measured in urine from asthmatic and nonasthmatic children on a histamine-restricted diet. No significant differences were found between the relative output values that could distinguish the asthmatic from the nonasthmatic children. The intermediary metabolite, methylhistamine, was also measured in some of these urine specimens, and no obvious differences were found between the proportions of the three substances that would suggest impairment of the methylation pathway of histamine metabolism in asthma.     

FMR1基因启动子区CpG岛甲基化与智力低下的相关性研究

目的探讨智力低下基因1启动子区CpG岛甲基化与智力低下的相关性。方法应用甲基化PCR方法对79例智力低下儿童及79例正常儿童的外周血中FMRI基因启动子区CpG岛甲基化状态及（CGG）n进行检测。结果2例智力低下儿童的FMRI启动子区CpG岛发生甲基化,正常儿童基因CpG岛无甲基化;两组（CGG）n重复数分别为33—48、27—43,两者之间比较（P〉0．05）无统计学意义。结论FMRl基因启动子区CpG岛甲基化可能引起智力低下。     

脆性X综合征FMR1基因CGG重复序列与甲基化的检测

目的建立一种快速、可靠的脆性X综合征的群体筛查方法。方法应用热启动PCR和甲基化特异性PCR（MS-PCR）方法对62例智力低下儿童、12例父母外周血液以及5例高危胎儿的脐带血中FMR1基因CGG重复序列与甲基化状态进行检测。结果采用热启动PCR方法检测79例标本,77例标本的CGG重复数在21～40之间,与正常对照组无明显差异;2例标本未扩增出明显条带。采用MS-PCR方法检测出2例FMR1基因甲基化但CGG重复数在正常范围的患者。结论应用热启动PCR结合MS-PCR方法检测FMR1基因CGG重复数和甲基化,能提高诊断效率,可作为筛查脆性X综合征的首选方法。     

Childhood outcomes of assisted reproductive technology

There is a large population of children conceived via assisted reproductive technology (ART), which continues to increase worldwide, without a clear understanding of associated long-term outcomes. ART children are more likely to be the result of multiple pregnancies, and thus to be born prematurely or low birthweight. There is growing evidence that ART children are phenotypically and biochemically different from naturally conceived children, but the mechanism(s) leading to these changes have not been elucidated. There is a possible increased risk of rare imprinted gene disorders in these children. However, it remains unclear whether more subtle changes in DNA methylation occur commonly, leading to differences in gene expression and phenotype in ART children. Although an increased risk of cancer among ART children has been reported, the role of ART in the development of cancer has not been demonstrated. Further research and ongoing surveillance of ART children is essential to better understand the possible effects of ART on the long-term health of this population.     

Assessing DNA methylation in the developing human intestinal epithelium: potential link to inflammatory bowel disease

DNA methylation is one of the major epigenetic mechanisms implicated in regulating cellular development and cell-type-specific gene expression. Here we performed simultaneous genome-wide DNA methylation and gene expression analysis on purified intestinal epithelial cells derived from human fetal gut, healthy pediatric biopsies, and children newly diagnosed with inflammatory bowel disease (IBD). Results were validated using pyrosequencing, real-time PCR, and immunostaining. The functional impact of DNA methylation changes on gene expression was assessed by employing in-vitro assays in intestinal cell lines. DNA methylation analyses allowed identification of 214 genes for which expression is regulated via DNA methylation, i.e. regulatory differentially methylated regions (rDMRs). Pathway and functional analysis of rDMRs suggested a critical role for DNA methylation in regulating gene expression and functional development of the human intestinal epithelium. Moreover, analysis performed on intestinal epithelium of children newly diagnosed with IBD revealed alterations in DNA methylation within genomic loci, which were found to overlap significantly with those undergoing methylation changes during intestinal development. Our study provides novel insights into the physiological role of DNA methylation in regulating functional maturation of the human intestinal epithelium. Moreover, we provide data linking developmentally acquired alterations in the DNA methylation profile to changes seen in pediatric IBD.     

Homozygous deletion of CDKN2A (p16, p14) and CDKN2B (p15) genes is a poor prognostic factor in adult but not in childhood B-lineage acute lymphoblastic leukemia: a comparative deletion and hypermethylation study

The biological behavior of childhood B-lineage acute lymphoblastic leukemia (B-ALL) is different from that of adults. We performed a comprehensive analysis of the deletion and the methylation profile of CDKN2A (hereafter identified separately as p16 and p14, for the different proteins encoded) and CDKN2B (hereafter p15) in 91 newly diagnosed B-ALL patients (61 children, 30 adults). The prognostic significance of the profiles of these genes and the association between alterations in these genes and known cytogenetic prognostic factors (BCR/ABL; ETV6/RUNX1, formerly TEL/AML1; MLL rearrangement; and ploidy changes of chromosomes) were also assessed. The prevalence of homozygous deletion, hemizygous deletion, and no deletion of the 9p21 region was 11.5%, 16.4%, and 72.1%, respectively, in children and 30.0%, 20.0%, and 50.0%, respectively, in adults; the higher incidence of homozygous deletion in adults was significant (P=0.029). Homozygous deletion was associated with poor overall survival in adults (P=0.019), but not in children. The incidence of promoter methylation of p16, p14, and p15 was 34.4%, 14.8%, and 34.4%, respectively, in children and 26.7%, 10.0%, and 40.0%, respectively, in adults, with no significant difference between the two groups. No significant association was observed between deletion and methylation or with known cytogenetic prognostic factors. The difference in incidence, distribution, and prognostic effect of homozygous deletion in children and adults may explain the prognostic disparity.     

Allele-specific gene expression patterns in primary leukemic cells reveal regulation of gene expression by CpG site methylation

To identify genes that are regulated by cis-acting functional elements in acute lymphoblastic leukemia (ALL) we determined the allele-specific expression (ASE) levels of 2, 529 genes by genotyping a genome-wide panel of single nucleotide polymorphisms in RNA and DNA from bone marrow and blood samples of 197 children with ALL. Using a reproducible, quantitative genotyping method and stringent criteria for scoring ASE, we found that 16% of the analyzed genes display ASE in multiple ALL cell samples. For most of the genes, the level of ASE varied largely between the samples, from 1.4-fold overexpression of one allele to apparent monoallelic expression. For genes exhibiting ASE, 55% displayed bidirectional ASE in which overexpression of either of the two SNP alleles occurred. For bidirectional ASE we also observed overall higher levels of ASE and correlation with the methylation level of these sites. Our results demonstrate that CpG site methylation is one of the factors that regulates gene expression in ALL cells.     

IVF results in de novo DNA methylation and histone methylation at an Igf2-H19 imprinting epigenetic switch

Recent studies suggest that IVF and assisted reproduction technologies (ART) may result in abnormal genomic imprinting, leading to an increased frequency of Angelman syndrome (AS) and Beckwith-Weidemann syndrome (BWS) in IVF children. To learn how ART might alter the epigenome, we examined morulas and blastocysts derived from C57BL/6J X M. spretus F1 mice conceived in vivo and in vitro and determined the allelic expression of four imprinted genes: Igf2, H19, Cdkn1c and Slc221L. IVF-derived mouse embryos that were cultured in human tubal fluid (HTF) (Quinn's advantage) media displayed a high frequency of aberrant H19 imprinting, whereas in vivo and IVF embryos showed normal maternal expression of Cdkn1c and normal biallelic expression of Igf2 and Slc221L. Embryonic stem (ES) cells derived from IVF blastocysts also showed abnormal Igf2/H19 imprinting. Allele-specific bisulphite PCR reveals abnormal DNA methylation at a CCCTC-binding factor (CTCF) site in the imprinting control region (ICR), as the normally unmethylated maternal allele acquired a paternal methylation pattern. Chromatin immunoprecipitation (ChIP) assays indicate an increase of lysine 4 methylation (dimethyl Lys4-H3) on the paternal chromatin and a gain in lysine 9 methylation (trimethyl Lys9-H3) on the maternal chromatin at the same CTCF-binding site. Our results indicate that de novo DNA methylation on the maternal allele and allele-specific acquisition of histone methylation lead to aberrant Igf2/H19 imprinting in IVF-derived ES cells. We suggest that ART, which includes IVF and various culture media, might cause imprinting errors that involve both aberrant DNA methylation and histone methylation at an epigenetic switch of the Igf2-H19 gene region.     

Parental ages and levels of DNA methylation in the newborn are correlated

Background  

Changes in DNA methylation patterns with age frequently have been observed and implicated in the normal aging process and its associated increasing risk of disease, particularly cancer. Additionally, the offspring of older parents are at significantly increased risk of cancer, diabetes, and neurodevelopmental disorders. Only a proportion of these increased risks among the children of older parents can be attributed to nondisjunction and chromosomal rearrangements.     

SNRPN methylation patterns in germ cell tumors as a reflection of primordial germ cell development.

Studies examining altered imprinted gene expression in cancer compare the observed expression pattern to the normal expression pattern for a given tissue of origin, usually the somatic expression pattern for the imprinted gene. Germ cell tumors (GCTs), however, require a developmental stage-dependent comparison. To explore using methylation as an indicator of germ cell development, we determined the pattern of methylation at the 5' untranslated region of SNRPN in 89 GCTs from both children and adults. Fifty-one of 84 tumors (60.7%) (12/30 (40%) of cultured pediatric GCTs, 23/36 (63.9%) of frozen adult GCTs, and 16/23 (69.5%) of frozen pediatric GCTs, with five samples having results from both cultured and uncultured material) demonstrated a nonsomatic methylation pattern after dual digestion with XbaI, NotI, and Southern blot analysis. In contrast, only 2 of 18 (11%) control samples (16 non-GCTs and 2 normal ovaries) exhibited a nonsomatic pattern. In both cases, the result was shown to be due to copy number differences between maternal and paternal homologs, unlike the GCTs in which there was no evidence of an uneven homolog number. A comparison of the data for only the gonadal GCTs and the control data showed a highly significant difference in the proportion of tumors with methylation alterations at this locus (P = 0.0000539). Since there is no published evidence of the involvement of SNRPN methylation changes in the development of malignancy, the data suggest that the methylation pattern of SNRPN in GCTs reflects that of the primordial germ cell giving rise to the tumor.     

Methylation profiling of CpG islands in human breast cancer cells

CpG island hypermethylation is known to be associated with gene silencing in cancer. This epigenetic event is generally accepted as a stochastic process in tumor cells resulting from aberrant DNA methyltransferase (DNA-MTase) activities. Specific patterns of CpG island methylation could result from clonal selection of cells having growth advantages due to silencing of associated tumor suppressor genes. Alternatively, methylation patterns may be determined by other, as yet unidentified factors. To explore further the underlying mechanisms, we developed a novel array-based method, called differential methylation hybridization (DMH), which allows a genome- wide screening of hypermethylated CpG islands in tumor cells. DMH was used to determine the methylation status of >276 CpG island loci in a group of breast cancer cell lines. Between 5 and 14% of these loci were hypermethylated extensively in these cells relative to a normal control. Pattern analysis of 30 positive loci by Southern hybridization indicated that CpG islands might differ in their susceptibility to hypermethylation. Loci exhibiting pre-existing methylation in normal controls were more susceptible to de novo methylation in these cancer cells than loci without this condition. In addition, these cell lines exhibited different intrinsic abilities to methylate CpG islands not directly associated with methyltransferase activities. Our study provides evidence that, aside from random DNA-MTase action, additional cellular factors exist that govern aberrant methylation in breast cancer cells.      

Differential DNA methylation profiles of peripheral blood mononuclear cells in allergic asthmatic children following dust mite immunotherapy

Background/PurposeAllergen-specific immunotherapy (SIT) is now considered curative to allergic diseases such as asthma. Mechanistically, our previous work showed DNA hypermethylation of cytokine genes, in T-helper cells, in allergic asthmatic children treated with allergen-SIT. In this study, we extended to work to assess possible changes in the DNA methylomes of peripheral blood mononuclear cells (PBMCs), isolated from mite allergen-SIT asthmatic children, to explore further the underlying methylation changes.MethodsThirteen allergic asthmatic children who received Der p-SIT, 12 non-SIT allergic asthmatic controls, and 12 healthy controls were enrolled. Bisulfite-converted DNA from Der p-stimulated PBMCs was analyzed using Human Methylation 450 k BeadChip. Pyrosequencing and quantitative real-time PCR were used to validate the DNA methylation levels and the gene expression of individual samples.ResultsWe identified 108 significantly differentially methylated regions (DMRs) unique to Der p-treated PBMCs, with 53 probes linked to demethylated DMRs, and 55 probes linked to methylated DMRs. Three associated genes (BCL6, HSPG2, and HSP90AA1), of selected DMRs, were subjected to bisulfite pyrosequencing. Of these, BCL6 showed significant hypomethylation, while HSPG2 and HSP90AA1 were hypermethylated in SIT group, compared to the AA group. Furthermore, SIT group had significantly higher gene expression of BCL6 and lower gene expression of HSPG2. KEGG pathway analysis further revealed DMR genes involved in ECM-receptor interactions, asthma, and antigen processing and presentation pathways.ConclusionsSeveral DNA regions showed DNA methylation altered by Der p specific immunotherapy, indicating desensitization-associated methylomes. Genes belonging to these SIT-altered pathways may represent therapeutic targets for better clinical management of asthma.     

Coordinated changes in AHRR methylation in lymphoblasts and pulmonary macrophages from smokers

Smoking is associated with a wide variety of adverse health outcomes including cancer, chronic obstructive pulmonary disease, diabetes, depression, and heart disease. Unfortunately, the molecular mechanisms through which these effects are conveyed are not clearly understood. To examine the potential role of epigenetic factors in these processes, we examined the relationship of smoking to genome wide methylation and gene expression using biomaterial from two independent samples, lymphoblast DNA and RNA (n = 119) and lung alveolar macrophage DNA (n = 19). We found that in both samples current smoking status was associated with significant changes in DNA methylation, in particular at the aryl hydrocarbon receptor repressor (AHRR), a known tumor suppressor. Both baseline DNA methylation and smoker associated DNA methylation signatures at AHRR were highly correlated (r = 0.94 and 0.45, respectively). DNA methylation at the most differentially methylated AHRR CpG residue in both samples, cg0557592, was significantly associated with AHRR gene expression. Pathway analysis of lymphoblast data (genes with most significant methylation changes) demonstrated enrichment in protein kinase C pathways and in TGF beta signaling pathways. For alveolar macrophages, pathway analysis demonstrated alterations in inflammation-related processes. We conclude that smoking is associated with functionally significant genome wide changes in DNA methylation in both lymphoblasts and pulmonary macrophages and that further integrated investigations of these epigenetic effects of smoking on carcinogenesis and other related co-morbidities are indicated.     

Dietary fat quality impacts genome-wide DNA methylation patterns in a cross-sectional study of Greek preadolescents

The type and the amount of dietary fat have a significant influence on the metabolic pathways involved in the development of obesity, metabolic syndrome, diabetes type 2 and cardiovascular diseases. However, it is unknown to what extent this modulation is achieved through DNA methylation. We assessed the effects of cholesterol intake, the proportion of energy intake derived from fat, the ratio of polyunsaturated fatty acids (PUFA) to saturated fatty acids (SFA), the ratio of monounsaturated fatty acids (MUFA) to SFA, and the ratio of MUFA+PUFA to SFA on genome-wide DNA methylation patterns in normal-weight and obese children. We determined the genome-wide methylation profile in the blood of 69 Greek preadolescents (∼10 years old) as well as their dietary intake for two consecutive weekdays and one weekend day. The methylation levels of one CpG island shore and four sites were significantly correlated with total fat intake. The methylation levels of 2 islands, 11 island shores and 16 sites were significantly correlated with PUFA/SFA; of 9 islands, 26 island shores and 158 sites with MUFA/SFA; and of 10 islands, 40 island shores and 130 sites with (MUFA+PUFA)/SFA. We found significant gene enrichment in 34 pathways for PUFA/SFA, including the leptin pathway, and a significant enrichment in 5 pathways for (MUFA+PUFA)/SFA. Our results suggest that specific changes in DNA methylation may have an important role in the mechanisms involved in the physiological responses to different types of dietary fat.     

Silver–Russell syndrome due to paternal H19/IGF2 hypomethylation in a twin girl born after in vitro fertilization

Silver–Russell syndrome (SRS) is a clinically and genetically heterogeneous syndrome characterized by severe intrauterine and postnatal growth retardation, facial dysmorphism and body asymmetry. One of the main molecular mechanisms leading to the syndrome involves methylation abnormalities of chromosome 11p15. In the last decades, an increase of imprinting disorders have been reported in children born from assisted reproductive technology (ART); however there is currently little evidence linking SRS and ART. Only few infants with SRS born using ART, supported by molecular analysis, have been described. We report on a twin‐girl conceived using intracytoplasmic sperm injection (ICSI) diagnosed with SRS. Molecular studies revealed a hypomethylation of the paternal H19/IGF2 Imprinting Control Region. Her twin sister had a normal prenatal and postnatal growth and a normal methylation pattern of the chromosome 11p15. This is the second reported case of a twin infant with SRS conceived using ART with hypomethylation of H19/IGF2; it provides additional evidence of a possible relationship between ART procedures and methylation defects observed in SRS. Given the clinical heterogeneity of SRS, and the increased risk of multiple and preterm births in the ART‐conceived children, it is possible that a number of cases of SRS remains undiagnosed in this population. Future studies should investigate the possible link between ART and SRS, in order to better understand the causes of epimutations in ART pregnancies, and to help clinicians to adequately counsel parents who approach to ART and to assess the opportunity of a long‐term follow‐up of children conceived using ART. © 2013 Wiley Periodicals, Inc.     

DNA methylation profiling reveals novel biomarkers and important roles for DNA methyltransferases in prostate cancer

Candidate gene-based studies have identified a handful of aberrant CpG DNA methylation events in prostate cancer. However, DNA methylation profiles have not been compared on a large scale between prostate tumor and normal prostate, and the mechanisms behind these alterations are unknown. In this study, we quantitatively profiled 95 primary prostate tumors and 86 benign adjacent prostate tissue samples for their DNA methylation levels at 26,333 CpGs representing 14,104 gene promoters by using the Illumina HumanMethylation27 platform. A 2-class Significance Analysis of this data set revealed 5912 CpG sites with increased DNA methylation and 2151 CpG sites with decreased DNA methylation in tumors (FDR < 0.8%). Prediction Analysis of this data set identified 87 CpGs that are the most predictive diagnostic methylation biomarkers of prostate cancer. By integrating available clinical follow-up data, we also identified 69 prognostic DNA methylation alterations that correlate with biochemical recurrence of the tumor. To identify the mechanisms responsible for these genome-wide DNA methylation alterations, we measured the gene expression levels of several DNA methyltransferases (DNMTs) and their interacting proteins by TaqMan qPCR and observed increased expression of DNMT3A2, DNMT3B, and EZH2 in tumors. Subsequent transient transfection assays in cultured primary prostate cells revealed that DNMT3B1 and DNMT3B2 overexpression resulted in increased methylation of a substantial subset of CpG sites that showed tumor-specific increased methylation.     

Leukotriene B4 modulates phospholipid methylation and chemotaxis in human polymorphonuclear leukocytes

Formation of phosphatidylcholine from phosphatidylethanolamine via the S-adenosylmethionine (AdoMet) pathway has been shown to be required for signal transduction of receptor-ligand interactions in a variety of cells. These interactions result in the remodeling of phospholipid pools and phospholipase activation. To extend these observations and to explore the role of the phosphatidylcholine synthesis pathway in transduction of the leukotriene B4 (LTB4) receptor-ligand response, we examined phospholipid methylation in human polymorphonuclear leukocytes (PMN) following stimulation by LTB4, a potent chemotactic agent that is a metabolite of arachidonic acid. At early time points (approximately 3-10 min), formation of methylated phospholipids was enhanced following LTB4 stimulation. The LTB4 analogs 6-trans LTB4 as well as LTB4 epimers induced less methylation compared with LTB4, and the potencies of these analogs in inducing methylation correlated with their diminished ability to induce chemotaxis. Furthermore, the ability of these agonists to induce methylation also correlated with the binding affinity of these agents to the LTB4 receptors on these cells. Synthesis of phosphatidylcholine by the choline transferase pathway was not affected by LTB4. Inhibition of the AdoMet reaction with 3- deazaadenosine, L-homocysteine homolactone, or erythro-9-[2-hydroxy-3-nonyl] adenine (EHNA) abrogated LTB4-induced phospholipid methylation and the chemotactic response. The potencies of these inhibitors in blocking phospholipid methylation also correlated with their ability to abrogate the LTB4-induced chemotactic response. These data suggest that phospholipid methylation and phospholipase activation play an important role in transduction of the LTB4 receptor-ligand interaction in PMN, which results in chemotaxis.