Association of COMT genotypes with S-COMT promoter methylation in growth-discordant monozygotic twins and healthy adults

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

The role of microRNAs in defining LSECs cellular identity and in regulating F8 gene expression

Introduction: Coagulation Factor VIII (FVIII) plays a pivotal role in the coagulation cascade, and deficiencies in its levels, as seen in Hemophilia A, can lead to significant health implications. Liver sinusoidal endothelial cells (LSECs) are the main producers and contributors of FVIII in blood, a fact we have previously elucidated through mRNA expression profiling when comparing these cells to other endothelial cell types.Methods: Our current investigation focuses on small microRNAs, analyzing their distinct expression patterns across various endothelial cells and hepatocytes.Results: The outcome of this exploration underscores the discernible microRNAs expression differences that set LSECs apart from both hepatocytes (193 microRNAs at p &lt; 0.05) and other endothelial cells (72 microRNAs at p &lt; 0.05). Notably, the 134 and 35 overexpressed microRNAs in LSECs compared to hepatocytes and other endothelial cells, respectively, shed light on the unique functions of LSECs in the liver.Discussion: Our investigation identified a panel of 10 microRNAs (miR-429, miR-200b-3p, miR-200a-3p, miR-216b-5p, miR-1185-5p, miR-19b-3p, miR-192-5p, miR-122-5p, miR-30c-2-3p, and miR-30a-5p) that distinctly define LSEC identity. Furthermore, our scrutiny extended to microRNAs implicated in F8 regulation, revealing a subset (miR-122-5p, miR-214-3p, miR-204-3p, and miR-2682-5p) whose expression intricately correlates with F8 expression within LSECs. This microRNA cohort emerges as a crucial modulator of F8, both directly through suppression and indirect effects on established F8-related transcription factors. The above microRNAs emerged as potential targets for innovative therapies in Hemophilia A patients

A global disorder of imprinting in the human female germ line

Imprinted genes are expressed differently depending on whether they are carried by a chromosome of maternal or paternal origin. Correct imprinting is established by germline-specific modifications; failure of this process underlies several inherited human syndromes. All these imprinting control defects are cis-acting, disrupting establishment or maintenance of allele-specific epigenetic modifications across one contiguous segment of the genome. In contrast, we report here an inherited global imprinting defect. This recessive maternal-effect mutation disrupts the specification of imprints at multiple, non-contiguous loci, with the result that genes normally carrying a maternal methylation imprint assume a paternal epigenetic pattern on the maternal allele. The resulting conception is phenotypically indistinguishable from an androgenetic complete hydatidiform mole, in which abnormal extra-embryonic tissue proliferates while development of the embryo is absent or nearly so. This disorder offers a genetic route to the identification of trans-acting oocyte factors that mediate maternal imprint establishment

Methylation at Global LINE-1 Repeats in Human Blood Are Affected by Gender but Not by Age or Natural Hormone Cycles

Previously, we reported on inter-individual and gender specific variations of LINE-1 methylation in healthy individuals. In this study, we investigated whether this variability could be influenced by age or sex hormones in humans. To this end, we studied LINE-1 methylation in vivo in blood-derived DNA from individuals aged 18 to 64 years and from young healthy females at various hormone levels during the menstrual cycle. Our results show that no significant association with age was observed. However, the previously reported increase of LINE-1 methylation in males was reconfirmed. In females, although no correlation between LINE-1 or Alu methylation and hormone levels was observed, a significant stable individual specific level of methylation was noted. In vitro results largely confirmed these findings, as neither estrogen nor dihydrotestosterone affected LINE-1 or Alu methylation in Hek293T, HUVEC, or MDA-kb2 cell lines. In contrast, a decrease in methylation was observed in estrogen-treated T47-Kbluc cell lines strongly expressing estrogen receptor. The very low expression of estrogen receptor in blood cells could explain the observed insensitivity of methylation at LINE-1 to natural hormonal variations in females. In conclusion, neither natural cycle of hormones nor age has a detectable effect on the LINE-1 methylation in peripheral blood cells, while gender remains an important factor

F8 Inversions at Xq28 Causing Hemophilia A Are Associated With Specific Methylation Changes: Implication for Molecular Epigenetic Diagnosis

Diverse DNA structural variations (SVs) in human cancers and several other diseases are well documented. For genomic inversions in particular, the disease causing mechanism may not be clear, especially if the inversion border does not cross a coding sequence. Understanding about the molecular processes of these inverted genomic sequences, in a mainly epigenetic context, may provide additional information regarding sequence-specific regulation of gene expression in human diseases. Herein, we study one such inversion hotspot at Xq28, which leads to the disruption of F8 gene and results in hemophilia A phenotype. To determine the epigenetic consequence of this rearrangement, we evaluated DNA methylation levels of 12 CpG rich regions with the coverage of 550 kb by using bisulfite-pyrosequencing and next-generation sequencing (NGS)-based bisulfite re-sequencing enrichment assay. Our results show that this inversion prone area harbors widespread methylation changes at the studied regions. However, only 5/12 regions showed significant methylation changes, specifically in case of intron 1 inversion (two regions), intron 22 inversion (two regions) and one common region in both inversions. Interestingly, these aberrant methylated regions were found to be overlapping with the inversion proximities. In addition, two CpG sites reached 100% sensitivity and specificity to discriminate wild type from intron 22 and intron 1 inversion samples. While we found age to be an influencing factor on methylation levels at some regions, covariate analysis still confirms the differential methylation induced by inversion, regardless of age. The hemophilia A methylation inversion “HAMI” assay provides an advantage over conventional PCR-based methods, which may not detect novel rare genomic rearrangements. Taken together, we showed that genomic inversions in the F8 (Xq28) region are associated with detectable changes in methylation levels and can be used as an epigenetic diagnostic marker

Whole Blood DNA Aberrant Methylation in Pancreatic Adenocarcinoma Shows Association with the Course of the Disease: A Pilot Study

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

An 11p15 Imprinting Centre Region 2 Deletion in a Family with Beckwith Wiedemann Syndrome Provides Insights into Imprinting Control at CDKN1C

We report a three generation family with Beckwith Wiedemann syndrome (BWS) in whom we have identified a 330 kb deletion within the KCNQ1 locus, encompassing the 11p15.5 Imprinting Centre II (IC2). The deletion arose on the paternal chromosome in the first generation and was only associated with BWS when transmitted maternally to subsequent generations. The deletion on the maternal chromosome was associated with a lower median level of CDKN1C expression in the peripheral blood of affected individuals when compared to a cohort of unaffected controls (p<0.05), however was not significantly different to the expression levels in BWS cases with loss of methylation (LOM) within IC2 (p<0.78). Moreover the individual with a deletion on the paternal chromosome did not show evidence of elevated CDKN1C expression or features of Russell Silver syndrome. These observations support a model invoking the deletion of enhancer elements required for CDKN1C expression lying within or close to the imprinting centre and importantly extend and validate a single observation from an earlier study. Analysis of 94 cases with IC2 loss of methylation revealed that KCNQ1 deletion is a rare cause of loss of maternal methylation, occurring in only 3% of cases, or in 1.5% of BWS overall

Characterization of cis- and trans-acting elements in the imprinted human SNURF-SNRPN locus

The imprinted SNRPN locus is a complex transcriptional unit that encodes the SNURF and SmN polypeptides as well as multiple non-coding RNAs. SNRPN is located within the Prader-Willi and Angelman syndrome (PWS/AS) region that contains multiple imprinted genes, which are coordinately regulated by a bipartite imprinting center (IC). The SNRPN 5′ region co-localizes with the PWS-IC and contains two DNase I hypersensitive sites, DHS1 at the SNRPN promoter, and DHS2 within intron 1, exclusively on the paternally inherited chromosome. We have examined DHS1 and DHS2 to identify cis- and trans-acting regulatory elements within the endogenous SNRPN 5′ region. Analysis of DHS1 by in vivo footprinting and chromatin immunoprecipitation identified allele-specific interaction with multiple regulatory proteins, including NRF-1, which regulates genes involved in mitochondrial and metabolic functions. DHS2 acted as an enhancer of the SNRPN promoter and contained a highly conserved region that showed allele-specific interaction with unphosphorylated RNA polymerase II, YY1, Sp1 and NRF-1, further suggesting a key role for NRF-1 in regulation of the SNRPN locus. We propose that one or more of the regulatory elements identified in this study may also contribute to PWS-IC function

Functional roles of the factor VIII B domain

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74800/1/j.1365-2516.2009.02026.x.pd

AKT activity orchestrates marginal zone B cell development in mice and humans.

The signals controlling marginal zone (MZ) and follicular (FO) B cell development remain incompletely understood. Here, we show that AKT orchestrates MZ B cell formation in mice and humans. Genetic models that increase AKT signaling in B cells or abolish its impact on FoxO transcription factors highlight the AKT-FoxO axis as an on-off switch for MZ B cell formation in mice. In humans, splenic immunoglobulin (Ig) D &lt;sup&gt;+&lt;/sup&gt; CD27 &lt;sup&gt;+&lt;/sup&gt; B cells, proposed as an MZ B cell equivalent, display higher AKT signaling than naive IgD &lt;sup&gt;+&lt;/sup&gt; CD27 &lt;sup&gt;-&lt;/sup&gt; and memory IgD &lt;sup&gt;-&lt;/sup&gt; CD27 &lt;sup&gt;+&lt;/sup&gt; B cells and develop in an AKT-dependent manner from their precursors in vitro, underlining the conservation of this developmental pathway. Consistently, CD148 is identified as a receptor indicative of the level of AKT signaling in B cells, expressed at a higher level in MZ B cells than FO B cells in mice as well as humans