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Objective: To evaluate the effect of infertility and intracytoplasmic sperm injection (ICSI) on DNA methylation of offspring., Design: Microarray analysis of DNA methylation in archived neonatal bloodspots of in vitro fertilization (IVF)/ICSI-conceived children compared with controls born to fertile and infertile parents., Setting: Academic research laboratory., Patient(s): Neonatal blood spots of 137 newborns conceived spontaneously, through intrauterine insemination (IUI), or through ICSI using fresh or cryopreserved (frozen) embryo transfer., Intervention(s): None., Main Outcome Measure(s): The Illumina Infinium HumanMethylation450k BeadChip assay determined genome-wide DNA methylation. Methylation differences between conception groups were detected using a Bioconductor package, ChAMP, in conjunction with Adjacent Site Clustering (A-clustering)., Result(s): The methylation profiles of assisted reproductive technology and IUI newborns were dramatically different from those of naturally (in vivo) conceived newborns. Interestingly, the profiles of ICSI-frozen (FET) and IUI infants were strikingly similar, suggesting that cryopreservation may temper some of the epigenetic aberrations induced by IVF or ICSI. The DNA methylation changes associated with IVF/ICSI culture conditions and/or parental infertility were detected at metastable epialleles, suggesting a lasting impact on a child's epigenome., Conclusion(s): Both infertility and ICSI alter DNA methylation at specific genomic loci, an effect that is mitigated to some extent by FET. The impact of assisted reproductive technology and/or fertility status on metastable epialleles in humans was uncovered. This study provides an expanded set of loci for future investigations on IVF populations., (Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Cancer has long been viewed as a genetic disease; however, epigenetic silencing as the result of aberrant promoter DNA methylation is frequently associated with cancer development, suggesting an epigenetic component to the disease. Nonetheless, it has remained unclear whether an epimutation (an aberrant change in epigenetic regulation) can induce tumorigenesis. Here, we exploited a functionally validated cis-acting regulatory element and devised a strategy to induce developmentally regulated genomic targeting of DNA methylation. We used this system to target DNA methylation within the p16(Ink4a) promoter in mice in vivo. Engineered p16(Ink4a) promoter hypermethylation led to transcriptional suppression in somatic tissues during aging and increased the incidence of spontaneous cancers in these mice. Further, mice carrying a germline p16(Ink4a) mutation in one allele and a somatic epimutation in the other had accelerated tumor onset and substantially shortened tumor-free survival. Taken together, these results provide direct functional evidence that p16(Ink4a) epimutation drives tumor formation and malignant progression and validate a targeted methylation approach to epigenetic engineering.

Analysis of DNA methylation patterns relies increasingly on sequencing-based profiling methods. The four most frequently used sequencing-based technologies are the bisulfite-based methods MethylC-seq and reduced representation bisulfite sequencing (RRBS), and the enrichment-based techniques methylated DNA immunoprecipitation sequencing (MeDIP-seq) and methylated DNA binding domain sequencing (MBD-seq). We applied all four methods to biological replicates of human embryonic stem cells to assess their genome-wide CpG coverage, resolution, cost, concordance and the influence of CpG density and genomic context. The methylation levels assessed by the two bisulfite methods were concordant (their difference did not exceed a given threshold) for 82% for CpGs and 99% of the non-CpG cytosines. Using binary methylation calls, the two enrichment methods were 99% concordant and regions assessed by all four methods were 97% concordant. We combined MeDIP-seq with methylation-sensitive restriction enzyme (MRE-seq) sequencing for comprehensive methylome coverage at lower cost. This, along with RNA-seq and ChIP-seq of the ES cells enabled us to detect regions with allele-specific epigenetic states, identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression.

Background: Animal models show that periconceptional supplementation with folic acid, vitamin B-12, choline, and betaine can induce differences in offspring phenotype mediated by epigenetic changes in DNA. In humans, altered DNA methylation patterns have been observed in offspring whose mothers were exposed to famine or who conceived in the Gambian rainy season. Objective: The objective was to understand the seasonality of DNA methylation patterns in rural Gambian women. We studied natural variations in dietary intake of nutrients involved in methyl-donor pathways and their effect on the respective metabolic biomarkers. Design: In 30 women of reproductive age (18-45 y), we monitored diets monthly for 1 y by using 48-h weighed records to measure intakes of choline, betaine, folate, methionine, riboflavin, and vitamins B-6 and B-12. Blood biomarkers of these nutrients, S-adenosylhomocysteine (SAH), S-adenosylmethionine (SAM), homocysteine, cysteine, and dimethylglycine were also assessed monthly. Results: Dietary intakes of riboflavin, folate, choline, and betaine varied significantly by season; the most dramatic variation was seen for betaine. All metabolic biomarkers showed significant seasonality, and vitamin B-6 and folate had the highest fluctuations. Correlations between dietary intakes and blood biomarkers were found for riboflavin, vitamin B-6, active vitamin B-12 (holotranscobalamin), and betaine. We observed a seasonal switch between the betaine and folate pathways and a probable limiting role of riboflavin in these processes and a higher SAM/SAH ratio during the rainy season. Conclusions: Naturally occurring seasonal variations in food-consumption patterns have a profound effect on methyl-donor biomarker status. The direction of these changes was consistent with previously reported differences in methylation of metastable epialleles. This trial was registered at www.clinicaltrials.gov as NCT01811641. [ABSTRACT FROM AUTHOR]

The role of CpG island methylation in normal development and cell differentiation is of keen interest, but remains poorly understood. We performed comprehensive DNA methylation profiling of promoter regions in normal peripheral blood by methylated CpG island amplification in combination with microarrays. This technique s allowed us to simultaneously determine the methylation status of 6,177 genes, 92% of which include dense CpG islands. Among these 5,549 autosomal genes with dense CpG island promoters, we have identified 4.0% genes that are nearly completely methylated in normal blood, providing another exception to the general rule that CpG island methylation in normal tissue is limited to X inactivation and imprinted genes. We examined seven genes in detail, including ANKRD30A, FLJ40201, INSL6, SOHLH2, FTMT, C12orf12, and DPPA5. Dense promoter CpG island methylation and gene silencing were found in normal tissues studied except testis and sperm. In both tissues, bisulfite cloning and sequencing identified cells carrying unmethylated alleles. Interestingly, hypomethylation of several genes was associated with gene activation in cancer. Furthermore, reactivation of silenced genes could be induced after treatment with a DNA demethylating agent or in a cell line lacking DNMT1 and/or DNMT3b. Sequence analysis identified five motifs significantly enriched in this class of genes, suggesting that cis-regulatory elements may facilitate preferential methylation at these promoter CpG islands. We have identified a group of non-X-linked bona fide promoter CpG islands that are densely methylated in normal somatic tissues, escape methylation in germline cells, and for which DNA methylation is a primary mechanism of tissue-specific gene silencing. [ABSTRACT FROM AUTHOR]

Methylation of DNA occurs at cytosines within CpG (cytosine-guanine) dinucleotides and is 1 of several epigenetic mechanisms that serve to establish and maintain tissue-specific patterns of gene expression. The methyl groups transferred in mammalian DNA methylation reactions are ultimately derived from methionine. High dietary methionine intake might therefore be expected to increase DNA methylation. Because of the circular nature of the methionine cycle, however, methionine excess may actually impair DNA methylation by inhibiting remethylation of homocysteine. Although little is known regarding the effect of dietary methionine supplementation on mammalian DNA methylation, the available data suggest that methionine supplementation can induce hypermethylation of DNA in specific genomic regions. Because locus-specific DNA hypomethylation is implicated in the etiology of various cancers and developmental syndromes, clinical trials of ‘promethylation’ dietary supplements are already under way. However, aberrant hypermethylation of DNA could be deleterious. It is therefore important to determine whether dietary supplementation with methionine can effectively support therapeutic maintenance of DNA methylation without causing excessive and potentially adverse locus-specific hypermethylation. In the viable yellow agouti (Avy mouse, maternal diet affects the coat color distribution of offspring by perturbing the establishment of methylation at the Avy metastable epiallele. Hence, the Avy mouse can be employed as a sensitive epigenetic biosensor to assess the effects of dietary methionine supplementation on locus-specific DNA methylation. Recent developments in epigenomic approaches that survey locus-specific DNA methylation on a genome-wide scale offer broader opportunities to assess the effects of high methionine intake on mammalian epigenomes. [ABSTRACT FROM AUTHOR]

Early nutrition affects adult metabolism in humans and other mammals, potentially via persistent alterations in DNA methylation. With viable yellow agouti (A[sup vy]) mice, which harbor a transposable element in the agouti gene, we tested the hypothesis that the metastable methylation status of specific transposable element insertion sites renders them epigenetically labile to early methyl donor nutrition. Our results show that dietary methyl supplementation of a/a dams with extra folic acid, vitamin B[sub 12], choline, and betaine alter the phenotype of their A[sup vy]/a offspring via increased CpG methylation at the A[sup vy] locus and that the epigenetic metastability which confers this lability is due to the A[sup vy] transposable element. These findings suggest that dietary supplementation, long presumed to be purely beneficial, may have unintended deleterious influences on the establishment of epigenetic gene regulation in humans. [ABSTRACT FROM AUTHOR]

Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) are persistent organic pollutants which may alter prenatal development, potentially through epigenetic modifications. Prior studies examining PFOS/PFOA and DNA methylation have relatively few subjects (n < 200) and inconsistent results. We examined relations of PFOA/PFOS with DNA methylation among 597 neonates in the Upstate KIDS cohort study. PFOA/PFOS were quantified in newborn dried blood spots (DBS) using high-performance liquid chromatography/tandem mass spectrometry. DNA methylation was measured using the Infinium MethylationEPIC BeadChip with DNA extracted from DBS. Robust linear regression was used to examine the associations of PFOA/PFOS with DNA methylation at individual CpG sites. Covariates included sample plate, estimated cell type, epigenetically derived ancestry, infant sex and plurality, indicators of maternal socioeconomic status, and prior pregnancy loss. In supplemental analysis, we restricted the analysis to 2242 CpG sites previously identified as Correlated Regions of Systemic Interindividual Variation (CoRSIVs) which include metastable epialleles. At FDR<0.05, PFOA concentration >90th percentile was related to DNA methylation at cg15557840, near SCRT2, SRXN1 ; PFOS>90th percentile was related to 2 CpG sites in a sex-specific manner (cg19039925 in GVIN1 in boys and cg05754408 in ZNF26 in girls). When analysis was restricted to CoRSIVs, log-scaled, continuous PFOS concentration was related to DNA methylation at cg03278866 within PTBP1. In conclusion, there was limited evidence of an association between high concentrations of PFOA/PFOS and DNA methylation in newborn DBS in the Upstate KIDS cohort. These findings merit replication in populations with a higher median concentration of PFOA/PFOS. • PFOS and PFOA were measured in newborn dried blood spots (DBS). • DNA was extracted from DBS to study methylation. • High PFOA/PFOS (>90th percentile) is related to DNA methylation at select CpG sites. • Findings are limited and need replication in cohorts with higher PFOA/PFOS exposure. [ABSTRACT FROM AUTHOR]