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Pancreatic tumors are usually diagnosed at an advanced stage in the progression of the disease, thus reducing the survival chances of the patients. Non-invasive early detection would greatly enhance therapy and survival rates. Toward this aim, we investigated in a pilot study the power of methylation changes in whole blood as predictive markers for the detection of pancreatic tumors. We investigated methylation levels at selected CpG sites in the CpG rich regions at the promoter regions of p16, RARbeta, TNFRSF10C, APC, ACIN1, DAPK1, 3OST2, BCL2 and CD44 in the blood of 30 pancreatic tumor patients and in the blood of 49 matching controls. In addition, we studied LINE-1 and Alu repeats using degenerate amplification approach as a surrogate marker for genome-wide methylation. The site-specific methylation measurements at selected CpG sites were done by the SIRPH method. Our results show that in the patient’s blood, tumor suppressor genes were slightly but significantly higher methylated at several CpG sites, while repeats were slightly less methylated compared to control blood. This was found to be significantly associated with higher risk for pancreatic ductal adenocarcinoma. Additionally, high methylation levels at TNFRSCF10C were associated with positive perineural spread of tumor cells, while higher methylation levels of TNFRSF10C and ACIN1 were significantly associated with shorter survival. This pilot study shows that methylation changes in blood could provide a promising method for early detection of pancreatic tumors. However, larger studies must be carried out to explore the clinical usefulness of a whole blood methylation based test for non-invasive early detection of pancreatic tumors.

Background Abnormal sex chromosome numbers in humans are observed in Turner (45,X) and Klinefelter (47,XXY) syndromes. Both syndromes are associated with several clinical phenotypes, whose molecular mechanisms are obscure, and show a range of inter-individual penetrance. In order to understand the effect of abnormal numbers of X chromosome on the methylome and its correlation to the variable clinical phenotype, we performed a genome-wide methylation analysis using MeDIP and Illumina’s Infinium assay on individuals with four karyotypes: 45,X, 46,XY, 46,XX, and 47,XXY. Results DNA methylation changes were widespread on all autosomal chromosomes in 45,X and in 47,XXY individuals, with Turner individuals presenting five times more affected loci. Differentially methylated CpGs, in most cases, have intermediate methylation levels and tend to occur outside CpG islands, especially in individuals with Turner syndrome. The X inactivation process appears to be less effective in Klinefelter syndrome as methylation on the X was decreased compared to normal female samples. In a large number of individuals, we verified several loci by pyrosequencing and observed only weak inter-loci correlations between the verified regions. This suggests a certain stochastic/random contribution to the methylation changes at each locus. Interestingly, methylation patterns on some PAR2 loci differ between male and Turner syndrome individuals and between female and Klinefelter syndrome individuals, which possibly contributed to this distinguished and unique autosomal methylation patterns in Turner and Klinefelter syndrome individuals. Conclusions The presented data clearly show that gain or loss of an X chromosome results in different epigenetic effects, which are not necessary opposite.

Background Prenatal growth restriction and low birth weight have been linked to long-term alterations of health, presumably via adaptive modifications of the epigenome. Recent studies indicate a plasticity of the 11p15 epigenotype in response to environmental changes during early stages of human development. Study design We analyzed methylation levels at different 11p15 loci in 20 growth-discordant monozygotic twin pairs. Intrauterine development was discordant due to severe twin-to-twin transfusion syndrome (TTTS), which was treated by fetoscopic laser coagulation of communicating vessels before 25 weeks of gestation. Methylation levels at age 4 were determined in blood and buccal cell-derived DNA by the single nucleotide primer extension reaction ion pair reverse-phase high performance liquid chromatography (SNuPE IP RP HPLC) assay. Methylation at LINE-1 repeats was analyzed as an estimate of global methylation. Results In general, variance of locus-specific methylation levels appeared to be higher in buccal cell- as compared to blood cell-derived DNA samples. Paired analyses within the twin pairs revealed significant differences at only one CpG site (IGF2 dmr0 SN3 (blood), +1.9% in donors; P = 0.013). When plotting the twin pair-discordance in birth weight against the degree of discordance in site-specific methylation at age 4, only a few CpGs were found to interact (one CpG site each at IGF2dmr0 in blood/saliva DNA, one CpG at LINE-1 repeats in saliva DNA), with 26 to 36% of the intra-twin pair divergence at these sites explained by prenatal growth discordance. However, across the entire cohort of 40 children, site-specific methylation did not correlate with SD-scores for weight or length at birth. Insulin-like growth factor-II serum concentrations showed significant within-twin pair correlations at birth (R = 0.57) and at age 4 (R = 0.79), but did not differ between donors and recipients. They also did not correlate with the analyzed 11p15 methylation parameter

In molar tissues from patients with recurrent biparental hydatidiform moles, we could previously demonstrate that differentially methylated regions (DMRs) of four imprinted genes are abnormally methylated on the maternal alleles. It remained unclear if this abnormal methylation originated de novo in the molar tissues or if it is even recognizable in the patient somatic tissues. To address this question, we investigated the DNA methylation of four imprinted genes in total blood from the two sister-patients. Here, we show that both patients retain normal methylation levels at the DMRs of the four genes in blood tissues. For two maternally expressed genes, we could use informative SNPs to follow the inheritance of the abnormally methylated maternal alleles in the molar tissues. We find that the transmitted abnormally methylated maternal alleles to the moles originated from the maternal grandmother and that the same alleles are not methylated in the patients. Our data suggest that the abnormal methylation in familial biparental molar tissues was acquired de novo in the patients’germline as a result of a false reprogramming or during the postzygotic development of the conceptuses that led to moles.

Hemophilia A (HA) is caused by partial or total deficiency of F8 protein activity. In a small group, about 1.8% of patients with HA, no mutation is found in the F8 gene. Among this group, we report here on one patient with severe HA in whom no mRNA of the F8 gene was detected. Using 2 common polymorphisms in F8 exon 14, we were able to show that the same allele shared by the patient, his mother, and his sister was not detected by reverse transcription–polymerase chain reaction (RT-PCR) from total blood mRNA. Skewed X-chromosome inactivation in both the mother and the sister was excluded by studying the methylation profile of the androgen receptor gene (HUMARA locus). These findings strongly suggest that the cause of HA in this patient is either absence or rapid degradation of the F8 mRNA, which points to a novel mechanism leading to HA.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are neurogenetic disorders that are caused by the loss of function of imprinted genes in 15q11-q13. In a small group of patients, the disease is due to aberrant imprinting and gene silencing. Here, we describe the molecular analysis of 51 patients with PWS and 85 patients with AS who have such a defect. Seven patients with PWS (14%) and eight patients with AS (9%) were found to have an imprinting center (IC) deletion. Sequence analysis of 32 patients with PWS and no IC deletion and 66 patients with AS and no IC deletion did not reveal any point mutation in the critical IC elements. The presence of a faint methylated band in 27% of patients with AS and no IC deletion suggests that these patients are mosaic for an imprinting defect that occurred after fertilization. In patients with AS, the imprinting defect occurred on the chromosome that was inherited from either the maternal grandfather or grandmother; however, in all informative patients with PWS and no IC deletion, the imprinting defect occurred on the chromosome inherited from the paternal grandmother. These data suggest that this imprinting defect results from a failure to erase the maternal imprint during spermatogenesis.

We previously mapped a maternal locus responsible for biparental complete hydatidiform moles (BiCHMs) to 19q13.4. The two index patients had a total of 14 molar pregnancies, eight abortions at various developmental stages, and one 16-year-old healthy offspring. We suggested that the defective gene deregulates the expression of imprinted genes. Here, we report the methylation status of four imprinted genes in two BiCHMs from the two sisters, the 16-year-old normal offspring, and two sporadic BiCHMs from unrelated patients. Using two bisulfite-based methods, we demonstrate a general trend of abnormal hypomethylation at the paternally expressed genes, PEG3 and SNRPN, and hypermethylation at the maternally expressed genes, NESP55 and H19, in two to four BiCHMs. Using single nucleotide polymorphisms, we provide the first evidence that SNRPN, NESP55 and H19 are abnormally methylated on the maternal alleles in BiCHMs. We show, in the BiCHMs from the two sisters, that the abnormally methylated H19 allele is inherited from either the maternal grandmother or the maternal grandfather. These data suggest that the abnormal methylation in BiCHMs is not due to an error in erasing the parental imprinting marks but rather in the re-establishment of the new maternal marks during oogenesis or their postzygotic maintenance. The defective 19q13.4 locus may have led to the development of variable degrees of ‘faulty’ paternal marks on the maternal chromosomes.

Mutations in NLRP7 (NOD-like-receptor family, pyrin domain containing 7) are responsible for a type of recurrent pregnancy loss known as recurrent hydatidiform mole (HYDM1). This condition is characterized by abnormal growth of the placenta, a lack of proper embryonic development and abnormal methylation patterns at multiple imprinted loci in diploid biparental molar tissues. The role of NLRP7 protein in the disease manifestation is currently not clear. In order to better understand how the effects of HYDM1 are associated with mutations on the structure of NLRP7, we performed an inter-domain interaction screen using a yeast two-hybrid system. Additionally, we generated in silico structural models of NLRP7 in its non-activated and activated forms. Our observations from the yeast two-hybrid screen and modeling suggest that the NACHT-associated domain (NAD) of the NLRP7 protein is central to its oligomeric assembly. Upon activation, the NAD and a small part of the leucine rich repeat (LRR) of one molecule emerged out of the protective LRR domain and interact with the NACHT domain of the second molecule to form an oligomer. Furthermore, we investigated the molecular basis for the pathophysiological effect of four missense mutations, three HYDM1-causing and one rare non-synonymous variant, on the protein using confocal microscopy of transiently transfected NLRP7 in HEK293T cells and in silico structural analysis. We found that with the two clinically severe missense mutations, L398R and R693W, the normal molecule to molecule interaction was apparently affected thus decreasing their oligomerization potential while aggresome formation was increased; these changes could disturb the normal downstream functions of NLRP7 and therefore be a possible molecular effect underlying their pathophysiological impact.

In humans, considerable variation in methylation at single loci and repetitive elements in various cells and tissues is observed. Recently, several inter- and intra-tissue correlations for DNA methylation have been reported. To investigate the extent and reproducibility of such correlations, we investigated inter- and intra-tissue methylation correlations among seven different loci in 9 different tissues in a population of 100 healthy seven-week-old CD1 outbred mice. We used a highly quantitative approach to measure methylation levels to high accuracy at two single loci in the alpha-actin and myosine light chain promoters, at three differentially methylated regions of the Peg3, Snrpn and Lit1 genes associated with imprinted loci, and at two repetitive elements in the Line-1 and IAP-LTR genes in the various tissues. In this population of mice, methylation at several loci was sex-associated and intra-tissue correlations among the studied loci were observed for brain and spleen. Inter-tissue correlations were rarely observed. To investigate method-dependent experimental variability, we re-analyzed the same spleen and tongue samples using SIRPH and pyrosequencing methods and reconfirmed intra-tissue correlations for spleen and sex-associated correlations for DNA methylation for tongue. When we repeated DNA methylation measurements for a second mouse population raised under similar conditions three months later, we did not detect sex-associated or intra-tissues correlations. Additional studies that examine large numbers of loci may be required to further understand the factors that influence stability of DNA methylation.

Background/Aim: Recently, a set of studies addressed the question of the prevalence of aberrant methylation in surrogate tissues, such as peripheral blood leukocytes. Toward this aim, we conducted a case-control pilot study to investigate aberrant methylation in leukocytes of gastric cancer patients. Materials and Methods: The SNuPE combined with ion pair reverse phase HPLC (SIRPH method) was used to examine site-specific methylation status at selected CpG sites of the promoter regions of APC, ACIN1, BCL2, CD44, DAPK1, CDKN2A, RARB, TNFRSF10C HS3ST2 and of LINE-1, Alu repeats. Results: We observed that in the patients, tumor suppressor genes were slightly but significantly higher methylated at several CpG sites, while DNA repetitive elements were slightly less methylated compared to controls. This was found to be significantly associated with higher prevalence for gastric cancer. Conclusion: These results suggest that larger studies must be carried-out to explore the biological significance and clinical usefulness of leukocyte DNA as non-invasive detection tool for gastric cancer.

LINE-1 repeats account for ∼17% of the human genome. Little is known about their individual methylation patterns, because their repetitive, almost identical sequences make them difficult to be individually targeted. Here, we used bisulfite conversion to study methylation at individual LINE-1 repeats. The loci studied included 39 X-linked loci and 5 autosomal loci. On the X chromosome in women, we found statistically significant less methylation at almost all L1Hs compared with men. Methylation at L1P and L1M did not correlate with the inactivation status of the host DNA, while the majority of L1Hs that were possible to be studied lie in inactivated regions. To investigate whether the male–female differences at L1Hs on the X are linked to the inactivation process itself rather than to a mere influence of gender, we analyzed six of the L1Hs loci on the X chromosome in Turners and Klinefelters which have female and male phenotype, respectively, but with reversed number of X chromosomes. We could confirm that all samples with two X chromosomes are hypomethylated at the L1Hs loci. Therefore, the inactive X is hypomethylated at L1Hs; the latter could play an exclusive role in the X chromosome inactivation process. At autosomal L1Hs, methylation levels showed a correlation tendency between methylation level and genome size, with higher methylation observed at most loci in individuals with one X chromosome and the lowest in XXY individuals. In summary, loci-specific LINE-1 methylation levels show considerable plasticity and depend on genomic position and constitution.

The intron 22 inversion represents the most prevalentfactor VIII gene defect in severe hemophilia A, accounting for about 40% of all mutations. It is hypothesized that the inversion mutations occur almost exclusively in germ cells during meiotic cell division by intrachromosomal recombination between 1 of 2 telomeric copies of the Int22h region and its intragenic homologue. The majority of inversion mutations originate in male germ cells, where the lack of bivalent formation may facilitate flipping of the telomeric end of the single X chromosome. This is the first intron 22 inversion that presents as a somatic mosaicism in a female, affecting only about 50% of lymphocyte and fibroblast cells of the proposita. Supposing a post-zygotic de novo mutation as the usual cause of somatic mosaicism, the finding would imply that the intron 22 inversion mutation is not restricted to meiotic cell divisions but can also occur during mitotic cell divisions, either in germ cell precursors or in somatic cells.

The levels of genomic DNA methylation in vertebrate species display a wide range of developmental dynamics. Here, we show that in contrast to mice, the paternal genome of the amphibian, Xenopus laevis, is not subjected to active demethylation of 5-methyl cytosine immediately after fertilization. High levels of methylation in the DNA of both oocyte and sperm are maintained in the early embryo but progressively decline during the cleavage stages. As a result, the Xenopus genome has its lowest methylation content at the midblastula transition (MBT) and during subsequent gastrulation. Between blastula and gastrula stages, we detect a loss of methylation at individual Xenopus gene promoters (TFIIIA, Xbra, and c-Myc II) that are activated at MBT. No changes are observed in the methylation patterns of repeated sequences, genes that are inactive at MBT, or in the coding regions of individual genes. In embryos that are depleted of the maintenance methyltransferase enzyme (xDnmt1), these developmentally programmed changes in promoter methylation are disrupted, which may account for the altered patterns of gene expression that occur in these embryos. Our results suggest that DNA methylation has a role in regulating the timing of gene activation at MBT in Xenopus laevis embryos.

Transitional mutations at CpG dinucleotides account for approximately a third of all point mutations. These mutations probably arise through spontaneous deamination of 5-methylcytosine. Studies of CpG mutation rates in disease-linked genes, such as factor VIII and FGFR3, have indicated that they more frequently originate in male than in female germ cells. It has been speculated that these sex-biased mutation rates might be a consequence of sex-specific methylation differences between the female and the male germ lines. Using the bisulfite-based genomic-sequencing method, we investigated the methylation status of the human factor VIII and FGFR3 genes in mature male and female germ cells. With the exception of a single CpG, both genes were found to be equally and highly methylated in oocytes and spermatocytes. Whereas these observations strongly support the notion that DNA methylation is the major determining factor for recurrent CpG germ-line mutations in patients with hemophilia and achondroplasia, the higher mutation rate in the male germ line is apparently not a simple reflection of sex-specific methylation differences.

X chromosome inactivation is controlled by the cis-acting X-inactivation centre (Xic). In addition to initiating inactivation, Xic, which includes the Xist gene, is involved in both a counting process that senses the number of X chromosomes and the choice of X chromosome to inactivate. Controlling elements lying 3′ to Xist include the DXPas34 locus. Deletion of DXPas34 in undifferentiated embryonic stem (ES) cells eliminates expression of both Xist and the antisense transcript Tsix, thought to initiatefrom a CpG island lying close to, but telomeric to, the DXPas34 locus itself. Deletion of DXPas34 leads to non-random inactivation on ES cell differentiation and disrupts imprinted X-inactivation in vivo. In order to investigate the role of methylation at DXPas34 in the initial steps of X-inactivation, we studied its methylation status during pre- and post-implantation embryonic development and ES cell differentiation, using the bisulphite sequencing technique. Analysis of the methylation status of both the DXPas34 locus and the associated downstream CpG island shows that extensive hypermethylation of the DXPas34 locus is a relatively late event in differentiation and embryogenesis. We conclude that methylation of DXPas34 cannot be the X chromosome imprint, nor can it be involved in the parent-of-origin effects associated with deletion of the DXPas34 locus and the neighbouring CpG island.

Background Catechol-O-Methyltransferase (COMT) plays a key role in dopamine and estrogen metabolism. Recently, COMT haplotypes rather than the single polymorphism Val158Met have been reported to underlie differences in protein expression by modulating mRNA secondary structure. So far, studies investigating the epigenetic variability of the S-COMT (soluble COMT) promoter region mainly focused on phenotypical aspects, and results have been controversial. Methods We assessed S-COMT promoter methylation in saliva and blood derived DNA with regard to early pre- and postnatal growth as well as to genotype for polymorphisms rs6269, rs4633, and rs4680 (Val158Met) in 20 monozygotic twin pairs (mean age 4 years), who were discordant for intrauterine development due to severe feto-fetal-transfusion syndrome. Methylation levels of two previously reported partially methylated cytosines were determined by the quantitative SIRPH (SNuPE- IP RP HPLC) assay. Results Overall, we observed a high variability of S-COMT promoter methylation, which did not correlate with individual differences in the pre- or postnatal growth pattern. Within the twin pairs however we noted a distinct similarity that could be linked to underlying COMT genotypes. This association was subsequently confirmed in a cohort of 93 unrelated adult controls. Interestingly, 158Val-alleles were found at both ends of the epigenotypical range, which is in accordance with a recently proposed model of COMT haplotypes corresponding to a continuum of phenotypical variability. Conclusion The strong heritable component of S-COMT promoter methylation found in our study needs to be considered in future approaches that focus on interactions between COMT epigenotype and phenotype.