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Background ADAMs (a disintegrin and metalloproteinase) have long been associated with tumor progression. Recent findings indicate that members of the closely related ADAMTS (ADAMs with thrombospondin motifs) family are also critically involved in carcinogenesis. Gene silencing through DNA methylation at CpG loci around e.g. transcription start or enhancer sites is a major mechanism in cancer development. Here, we aimed at identifying genes of the ADAM and ADAMTS family showing altered DNA methylation in the development or colorectal cancer (CRC) and other epithelial tumors. Methods We investigated potential changes of DNA methylation affecting ADAM and ADAMTS genes in 117 CRC, 40 lung cancer (LC) and 15 oral squamous-cell carcinoma (SCC) samples. Tumor tissue was analyzed in comparison to adjacent non-malignant tissue of the same patients. The methylation status of 1145 CpGs in 51 ADAM and ADAMTS genes was measured with the HumanMethylation450 BeadChip Array. ADAMTS16 protein expression was analyzed in CRC samples by immunohistochemistry. Results In CRC, we identified 72 CpGs in 18 genes which were significantly affected by hyper- or hypomethylation in the tumor tissue compared to the adjacent non-malignant tissue. While notable/frequent alterations in methylation patterns within ADAM genes were not observed, conspicuous changes were found in ADAMTS16 and ADAMTS2 . To figure out whether these differences would be CRC specific, additional LC and SCC tissue samples were analyzed. Overall, 78 differentially methylated CpGs were found in LC and 29 in SCC. Strikingly, 8 CpGs located in the ADAMTS16 gene were commonly differentially methylated in all three cancer entities. Six CpGs in the promoter region were hypermethylated, whereas 2 CpGs in the gene body were hypomethylated indicative of gene silencing. In line with these findings, ADAMTS16 protein was strongly expressed in globlet cells and colonocytes in control tissue but not in CRC samples. Functional in vitro studies using the colorectal carcinoma cell line HT29 revealed that ADAMTS16 expression restrained tumor cell proliferation. Conclusions We identified ADAMTS16 as novel gene with cancer-specific promoter hypermethylation in CRC, LC and SCC patients implicating ADAMTS16 as potential biomarker for these tumors. Moreover, our results provide evidence that ADAMTS16 may have tumor suppressor properties.

Beckwith-Wiedemann syndrome (BWS; OMIM #130650) is an overgrowth syndrome caused by different genetic or epigenetic alterations affecting imprinted regions on chromosome 11p15.5. Here we report a family with multiple offspring affected with BWS including giant omphalocoeles in which maternal transmission of a chromosomal rearrangement including an inversion and two deletions leads to hypomethylation of the imprint control region 2 (ICR2). As the deletion includes the promoter and 5' part of the KCNQ1 gene, we suggest that transcription of this gene may be involved in establishing the maternal methylation imprint of the ICR2, which is located in intron 10 of KCNQ1.

Epigenetic mechanisms including DNA methylation are supposed to play a key role in fetal development. Here we have investigated fetal DNA-methylation levels of 27,578 CpG loci in 47 chorionic villi (CVS) and 16 amniotic cell (AC) samples. Methylation levels differed significantly between karyotypically normal AC and CVS for 2,014 genes. AC showed more extreme DNA-methylation levels of these genes than CVS and the differentially methylated genes are significantly enriched for processes characteristic for the different cell types sampled. Furthermore, we identified 404 genes differentially methylated in CVS with trisomy 21. These genes were significantly enriched for high CG dinucleotid (CpG) content and developmental processes associated with Down syndrome. Our study points to major tissue-specific differences of fetal DNA-methylation and gives rise to the hypothesis that part of the Down syndrome phenotype is epigenetically programmed in the first trimester of pregnancy.

Treatment with recombinant human growth hormone (rhGH) has been consistently reported to induce transcriptional changes in various human tissues including peripheral blood. For other hormones it has been shown that the induction of such transcriptional effects is conferred or at least accompanied by DNA-methylation changes. To analyse effects of short term rhGH treatment on the DNA-methylome we investigated a total of 24 patients at baseline and after 4-day rhGH stimulation. We performed array-based DNA-methylation profiling of paired peripheral blood mononuclear cell samples followed by targeted validation using bisulfite pyrosequencing. Unsupervised analysis of DNA-methylation in this short-term treated cohort revealed clustering according to individuals rather than treatment. Supervised analysis identified 239 CpGs as significantly differentially methylated between baseline and rhGH-stimulated samples (p<0.0001, unadjusted paired t-test), which nevertheless did not retain significance after adjustment for multiple testing. An individualized evaluation strategy led to the identification of 2350 CpG and 3 CpH sites showing methylation differences of at least 10% in more than 2 of the 24 analyzed sample pairs. To investigate the long term effects of rhGH treatment on the DNA-methylome, we analyzed peripheral blood cells from an independent cohort of 36 rhGH treated children born small for gestational age (SGA) as compared to 18 untreated controls. Median treatment interval was 33 months. In line with the groupwise comparison in the short-term treated cohort no differentially methylated targets reached the level of significance in the long-term treated cohort. We identified marked intra-individual responses of DNA-methylation to short-term rhGH treatment. These responses seem to be predominately associated with immunologic functions and show considerable inter-individual heterogeneity. The latter is likely the cause for the lack of a rhGH induced homogeneous DNA-methylation signature after short- and long-term treatment, which nevertheless is well in line with generally assumed safety of rhGH treatment.

Background Uniparental disomy of certain chromosomes are associated with a group of well-known genetic syndromes referred to as imprinting disorders. However, the extreme form of uniparental disomy affecting the whole genome is usually not compatible with life, with the exception of very rare cases of patients with mosaic genome-wide uniparental disomy reported in the literature. Results We here report on a fetus with intrauterine growth retardation and malformations observed on prenatal ultrasound leading to invasive prenatal testing. By cytogenetic (conventional karyotyping), molecular cytogenetic (QF-PCR, FISH, array), and methylation (MS-MLPA) analyses of amniotic fluid, we detected mosaicism for one cell line with genome-wide maternal uniparental disomy and a second diploid cell line of biparental inheritance with trisomy X due to paternal isodisomy X. As expected for this constellation, we observed DNA methylation changes at all imprinted loci investigated. Conclusions This report adds new information on phenotypic outcome of mosaic genome-wide maternal uniparental disomy leading to an extreme form of multilocus imprinting disturbance. Moreover, the findings highlight the technical challenges of detecting these rare chromosome disorders prenatally.

Various genes located at imprinted loci and regulated by epigenetic mechanisms are involved in the control of growth and differentiation. The broad phenotypic variability of imprinting disorders suggests that individuals with inborn errors of imprinting might remain undetected among patients born small for gestational age (SGA). We evaluated quantitative DNA methylation analysis at differentially methylated regions (DMRs) of 10 imprinted loci (PLAGL1, IGF2R DMR2, GRB10, H19 DMR, IGF2, MEG3, NDN, SNRPN, NESP, NESPAS) by bisulphite pyrosequencing in 98 patients born SGA and 50 controls. For IGF2R DMR2, methylation patterns of additional 47 parent pairs and one mother (95 individuals) of patients included in the SGA cohort were analyzed. In six out of 98 patients born SGA, we detected DNA methylation changes at single loci. In one child, the diagnosis of upd(14)mat syndrome owing to an epimutation of the MEG3 locus in 14q32 could be established. The remaining five patients showed hypomethylation at GRB10 (n=2), hypomethylation at the H19 3CTCF-binding site (n=1), hypermethylation at NDN (n=1) and hypermethylation at IGF2 (n=1). IGF2R DMR2 hypermethylation was detected in five patients, six parents of patients in the SGA cohort and two controls. We conclude that aberrant methylation at imprinted loci in children born SGA exists but seems to be rare if known imprinting syndromes are excluded. Further investigations on the physiological variations and the functional consequences of the detected aberrant methylation are necessary before final conclusions on the clinical impact can be drawn.

Although chronic myeloid leukemia (CML) can be effectively treated using BCR-ABL1 kinase inhibitors, resistance due to kinase alterations or to BCR-ABL1 independent mechanisms remain a therapeutic challenge. For the latter, the underlying mechanisms are widely discussed; for instance, gene expression changes, epigenetic factors and alternative signaling pathway activation. In the present study, in vitro-CML cell models of resistance against the tyrosine kinase inhibitors (TKIs) imatinib (0.5 and 2 µM) and nilotinib (0.1 µM) with biological replicates were generated to identify novel mechanisms of resistance. Subsequently, genome-wide mRNA expression and DNA methylation were analyzed. While mRNA expression patterns differed largely between biological replicates, there was an overlap of 71 genes differentially expressed between cells resistant against imatinib or nilotinib. Moreover, all TKI resistant cell lines demonstrated a slight hypermethylation compared with native cells. In a combined analysis of 151 genes differentially expressed in the biological replicates of imatinib resistance, cell adhesion signaling, in particular the cellular matrix protein fibronectin 1 (FN1), was significantly dysregulated. This gene was also downregulated in nilotinib resistance. Further analyses showed significant FN1-downregulation in imatinib resistance on mRNA (P<0.001) and protein level (P<0.001). SiRNA-mediated FN1-knockdown in native cells reduced cell adhesion (P=0.02), decreased imatinib susceptibility visible by higher Ki-67 expression (1.5-fold, P=0.04) and increased cell number (1.5-fold, P=0.03). Vice versa, recovery of FN1-expression in imatinib resistant cells was sufficient to partially restore the response to imatinib. Overall, these results suggested a role of cell adhesion signaling and fibronectin 1 in TKI resistant CML and a potential target for novel strategies in treatment of resistant CML.

BackgroundGenomic imprinting results from the resistance of germline epigenetic marks to reprogramming in the early embryo for a small number of mammalian genes. Genetic, epigenetic or environmental insults that prevent imprints from evading reprogramming may result in imprinting disorders, which impact growth, development, behaviour and metabolism. We aimed to identify genetic defects causing imprinting disorders by whole-exome sequencing in families with one or more members affected by multilocus imprinting disturbance.MethodsWhole-exome sequencing was performed in 38 pedigrees where probands had multilocus imprinting disturbance, in five of whom maternal variants in NLRP5 have previously been found.ResultsWe now report 15 further pedigrees in which offspring had disturbance of imprinting, while their mothers had rare, predicted-deleterious variants in maternal effect genes, including NLRP2, NLRP7 and PADI6. As well as clinical features of well-recognised imprinting disorders, some offspring had additional features including developmental delay, behavioural problems and discordant monozygotic twinning, while some mothers had reproductive problems including pregnancy loss.ConclusionThe identification of 20 putative maternal effect variants in 38 families affected by multilocus imprinting disorders adds to the evidence that maternal genetic factors affect oocyte fitness and thus offspring development. Testing for maternal-effect genetic variants should be considered in families affected by atypical imprinting disorders.

Bernhard Radlwimmer and colleagues report whole-genome bisulfite sequencing of 13 Burkitt lymphomas and nine follicular lymphomas. They find that both types of germinal center B cell lymphomas show global hypomethylation compared to normal germinal center B cell precursors and identify regions of differential methylation that correlate with somatic mutations and differential gene expression. Although Burkitt lymphomas and follicular lymphomas both have features of germinal center B cells, they are biologically and clinically quite distinct. Here we performed whole-genome bisulfite, genome and transcriptome sequencing in 13 IG - MYC translocation–positive Burkitt lymphoma, nine BCL2 translocation–positive follicular lymphoma and four normal germinal center B cell samples. Comparison of Burkitt and follicular lymphoma samples showed differential methylation of intragenic regions that strongly correlated with expression of associated genes, for example, genes active in germinal center dark-zone and light-zone B cells. Integrative pathway analyses of regions differentially methylated in Burkitt and follicular lymphomas implicated DNA methylation as cooperating with somatic mutation of sphingosine phosphate signaling, as well as the TCF3-ID3 and SWI/SNF complexes, in a large fraction of Burkitt lymphomas. Taken together, our results demonstrate a tight connection between somatic mutation, DNA methylation and transcriptional control in key B cell pathways deregulated differentially in Burkitt lymphoma and other germinal center B cell lymphomas.