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The use of next generation sequencing has expanded our view on whole mammalian methylome patterns. In particular, it provides a genome-wide insight of local DNA methylation diversity at single nucleotide level and enables the examination of single chromosome sequence sections at a sufficient statistical power. We describe a bisulfite-based sequence profiling pipeline, Bi-PROF, which is based on the 454 GS-FLX Titanium technology that allows to obtain up to one million sequence stretches at single base pair resolution without laborious subcloning. To illustrate the performance of the experimental workflow connected to a bioinformatics program pipeline (BiQ Analyzer HT) we present a test analysis set of 68 different epigenetic marker regions (amplicons) in five individual patient-derived xenograft tissue samples of colorectal cancer and one healthy colon epithelium sample as a control. After the 454 GS-FLX Titanium run, sequence read processing and sample decoding, the obtained alignments are quality controlled and statistically evaluated. Comprehensive methylation pattern interpretation (profiling) assessed by analyzing 10 (2)-10 (4) sequence reads per amplicon allows an unprecedented deep view on pattern formation and methylation marker heterogeneity in tissues concerned by complex diseases like cancer.

BACKGROUND: Low birth weight is associated with an increased adult metabolic disease risk. It is widely discussed that poor intra-uterine conditions could induce long-lasting epigenetic modifications, leading to systemic changes in regulation of metabolic genes. To address this, we acquire genome-wide DNA methylation profiles from saliva DNA in a unique cohort of 17 monozygotic monochorionic female twins very discordant for birth weight. We examine if adverse prenatal growth conditions experienced by the smaller co-twins lead to long-lasting DNA methylation changes. RESULTS: Overall, co-twins show very similar genome-wide DNA methylation profiles. Since observed differences are almost exclusively caused by variable cellular composition, an original marker-based adjustment strategy was developed to eliminate such variation at affected CpGs. Among adjusted and unchanged CpGs 3153 are differentially methylated between the heavy and light co-twins at nominal significance, of which 45 show sensible absolute mean beta-value differences. Deep bisulfite sequencing of eight such loci reveals that differences remained in the range of technical variation, arguing against a reproducible biological effect. Analysis of methylation in repetitive elements using methylation-dependent primer extension assays also indicates no significant intra-pair differences. CONCLUSIONS: Severe intra-uterine growth differences observed within these monozygotic twins are not associated with long-lasting DNA methylation differences in cells composing saliva, detectable with up-to-date technologies. Additionally, our results indicate that uneven cell type composition can lead to spurious results and should be addressed in epigenomic studies. ispartof: Genome biology vol:14 issue:5 ispartof: location:England status: published

BACKGROUND Assisted reproductive technologies (ART) such as in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) are believed to destabilise genomic imprints. An increased frequency of Beckwith-Wiedemann syndrome in children born after ART has been reported. Other, mostly epidemiological, studies argue against this finding. OBJECTIVE To examine the effect of ART on the stability of DNA methylation imprints, DNA was extracted from maternal peripheral blood (MPB), umbilical cord blood (UCB) and amnion/chorion tissue (ACT) of 185 phenotypically normal children (77 ICSI, 35 IVF, and 73 spontaneous conceptions). Using bisulfite based technologies 10 differentially methylated regions (DMRs) were analysed, including KvDMR1, H19, SNRPN, MEST, GRB10, DLK1/MEG3 IG-DMR, GNAS NESP55, GNAS NESPas, GNAS XL-alpha-s and GNAS Exon1A. RESULTS Methylation indices (MI) do not reveal any significant differences at nine DMRs among the conception groups in neither MPB, UCB nor in ACT. The only slightly variable DMR was that of MEST. Here the mean MI was higher in UCB and MPB of IVF cases (mean MI+/-SD: 0.41+/-0.03 (UCB) and 0.40+/-0.03 (MPB)) compared to the ICSI (0.38+/-0.03, p=0.003 (UCB); 0.37+/-0.04, p=0.0007 (MPB)) or spontaneous cases (0.38+/-0.03, p=0.003 (UCB); 0.38+/-0.04, p=0.02 (MPB)). Weak but suggestive correlations between DMRs were, however, found between MPB, UCB and ACT. CONCLUSION This study supports the notion that children conceived by ART do not show a higher degree of imprint variability and hence do not have an a priori higher risk for imprinting disorders.