TY - JOUR A1 - Pfeiffer, Susanne A1 - Krüger, Jacqueline A1 - Maierhofer, Anna A1 - Böttcher, Yvonne A1 - Klöting, Nora A1 - El Hajj, Nady A1 - Schleinitz, Dorit A1 - Schön, Michael R. A1 - Dietrich, Arne A1 - Fasshauer, Mathias A1 - Lohmann, Tobias A1 - Dreßler, Miriam A1 - Stumvoll, Michael A1 - Haaf, Thomas A1 - Blüher, Matthias A1 - Kovacs, Peter T1 - Hypoxia-inducible factor 3A gene expression and methylation in adipose tissue is related to adipose tissue dysfunction JF - Scientific Reports N2 - Recently, a genome-wide analysis identified DNA methylation of the HIF3A (hypoxia-inducible factor 3A) as strongest correlate of BMI. Here we tested the hypothesis that HIF3A mRNA expression and CpG-sites methylation in adipose tissue (AT) and genetic variants in HIF3A are related to parameters of AT distribution and function. In paired samples of subcutaneous AT (SAT) and visceral AT (VAT) from 603 individuals, we measured HIF3A mRNA expression and analyzed its correlation with obesity and related traits. In subgroups of individuals, we investigated the effects on HIF3A genetic variants on its AT expression (N = 603) and methylation of CpG-sites (N = 87). HIF3A expression was significantly higher in SAT compared to VAT and correlated with obesity and parameters of AT dysfunction (including CRP and leucocytes count). HIF3A methylation at cg22891070 was significantly higher in VAT compared to SAT and correlated with BMI, abdominal SAT and VAT area. Rs8102595 showed a nominal significant association with AT HIF3A methylation levels as well as with obesity and fat distribution. HIF3A expression and methylation in AT are fat depot specific, related to obesity and AT dysfunction. Our data support the hypothesis that HIF pathways may play an important role in the development of AT dysfunction in obesity. KW - gene expression KW - adipose KW - hypoxia-inducible factor 3A KW - adipose tissue dysfunction KW - obesity Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-167662 VL - 6 IS - 27969 ER - TY - THES A1 - El Hajj, Nady T1 - Epimutations in Germ-Cell and Embryo Development: Possible Consequences for Assisted Reproduction T1 - Epimutationen in der Keimzell- und Embryonalentwicklung : Mögliche Konsequenzen für die assistierte Reproduktion N2 - Assisted reproductive technologies (ART) emerged in the late 1970’s as a therapy for human infertility. Up till now more than 3 million babies have been conceived through ART, demonstrating the safety and efficiency of the technique. Published reports showed an increase in the rate of imprinting disorders (Beckwith Wiedemann Syndrome, Angelman Syndrome, etc.) in babies born after ART. What are the effects imposed through ART and should researchers reassess its safety and implications on the future offspring? Throughout this thesis, I analyzed the methylation patterns of germ cells and embryos to determine whether in vitro maturation and in vitro fertilization have a negative impact on the epigenetic patterns. Furthermore, DNA methylation was compared between sperm of infertile and presumably fertile controls in order to understand whether epigenetic disturbances lead to infertility at the first place. The occurrence of methylation aberrations in germ cells of infertile patients could be transmitted to new-borns and then cause epigenetic disorders. In order to elucidate the imprinting status within single cells, I developed a new technique based on limiting dilution where bisulfite treated DNA is distributed across several wells before amplification. This allowed methylation measurement at the single allele level as well parent of origin detection. In a total of 141 sperm samples from couples undergoing in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) including 106 with male factor or combined infertility and 28 with female infertility, I detected a significant correlation between lower quality of semen parameters (sperm count, percentage of abnormal sperm, and percentage of motile sperm) and the rate of imprinting errors. ALU repeats displayed a higher methylation in sperm DNA of patients leading to a pregnancy and live birth, compared to patients in which pregnancy was not achieved or a spontaneous abortion occurred. A discriminant analysis based on ALU methylation allowed correct classification of >70% of cases. Preliminary data from illumina methylation arrays where more than 27,000 CpGs were analyzed determined that only a single CpG site from the open reading frame C14orf93 was significantly different between the infertile and presumably fertile control group. However, further improvements on data normalization might permit detection of other differentially methylated regions. Comparison of embryos after natural conception, in vitro fertilized embryos from superovulated oocytes, and embryos achieved through fertilization of in vitro cultured oocytes revealed no dramatic effect on the imprinting patterns of Igf2r, H19, and Snrpn. Oocyte cryotop vitrification did not result in a dramatic increase of imprinting mutations in oocytes even though the rate of sporadic methylation errors in single Snrpn CpGs were higher within the in-vitrified group. Collectively, the results I will present within this thesis suggest an increase in the rate of imprinting errors within the germ cells of infertile patients, in addition to a decrease in genome wide methylation of ALU repetitive elements. I did not observe a detrimental effect on the methylation patterns of oocytes and the resulting embryos using in vitro maturation of oocytes and/or standard IVF with in vivo grown superovulated oocytes. N2 - Assistierte Reproduktionstechniken (ART) wurden in den späten 1970er Jahren als Therapie für unfruchtbare Paare mit Kinderwunsch etabliert. Bis zum heutigen Tage wurden dank ART weltweit mehr als 3 Millionen Kinder geboren, ein eindrucksvoller Beweis für die Sicherheit und Effizienz dieser Methode. Dennoch zeigen veröffentlichte Studien einen Anstieg in der Rate von Imprinting-Erkrankungen (Beckwith Wiedemann-Syndrom, Angelman-Syndrom, etc.) bei Kindern, die nach assistierter Reproduktion geboren wurden. Es stellt sich die Frage, welche Effekte durch ART ausgelöst werden können und ob eine neue Einschätzung dieser Methode bezüglich ihrer gesundheitlichen Implikationen für künftige Generationen notwendig ist. In dieser Arbeit habe ich mögliche negative Effekte von in vitro-Maturation und -Fertili-sierung auf Methylierungsmuster humaner und muriner Keimzellen, sowie Maus-Embryonen untersucht. Aberrante DNA-Methylierungsmuster in Keimzellen von infertilen Patienten könnten auf die Neugeborenen übertragen werden und epigenetische Erkrankungen zur Folge haben. Ob epigenetische Störungen im Zusammenhang mit Infertilität stehen, wurde außerdem durch den Vergleich der DNA-Methylierung von Spermien infertiler und fertiler Männer untersucht. Um den Imprintigstatus auf Einzelzellebene zu bestimmen, habe ich basierend auf „Limiting Dilution“ eine neue Methode entwickelt. Bei diesem Verfahren wird Bisulfit-behandelte DNA vor der PCR-Amplifikation in mehrere Reaktionsgefässe verdünnt. Dies erlaubt die Methylierungsanalyse einzelner Allele und die Detektion elternspezifischer Methylierungsmuster. Mit insgesamt 141 Sperma-Proben von Paaren, die sich einer in vitro- Fertilisierung (IVF) oder einer Intrazytoplasmischen Spermieninjektion (ICSI) unterzogen hatten, davon 28 mit weiblicher und 106 mit männlicher oder kombinierter Unfruchtbarkeit, konnte ich einen positiven Zusammenhang zwischen der Rate an Imprinting-Fehlern und geringer Sperma-Qualität (gemessen an Standardparametern) ableiten. ALU-Sequenzen zeigten in Spermien-DNA von Patienten mit erfolgreicher Schwangerschaft und Geburt eine höhere Methylierung als von Patienten mit fehlgeschlagener Schwangerschaft oder Spontanabort. Eine auf der ALU-Methylierung basierende Diskriminanzanalyse konnte mehr als 70% aller Fälle korrekt klassifizieren. Vorläufige Daten aus Experimenten mit Illumina Methylierungs-Arrays mit einer Auflösung von mehr als 27.000 CpG-Positionen identifizierten einen signifikanten Gruppenunterschied zwischen Patienten- und Kontrollgruppe für eine CpG-Position innerhalb des offenen Leserahmens C14orf93. Verbesserungen der Datenauswertung (Normalisierung, Testung etc.) sollten die Entdeckung weiterer differenziell methylierter Regionen erlauben. Der Vergleich von Mausembryos aus natürlicher Konzeption, aus in vitro kultivierten und fertilisierten Oozyten und aus in vitro Fertilisation nach Superovulation zeigte keine dramatischen Effekte auf die Imprinting-Muster der geprägten Gene Igf2r, H19 und Snrpn. Das gilt auch für Cryo-Top vitrifizierte Oozyten, wenn auch die Rate sporadischer Methylierungsfehler einzelner CpG-Positionen in Snrpn etwas höher war als in den Kontrollgruppen. Zusammengenommen lassen die in dieser Arbeit präsentieren Resultate auf eine Zunahme an Imprinting-Fehlern und eine genomweite Abnahme der Methylierung repetitiver ALU-Sequenzen in den Keimzellen infertiler Patienten schließen. KW - Reproduktionsmedizin KW - Epigenotypus KW - Mutation KW - assistierte Reproduktion KW - Keimzell- und Embryonalentwicklung KW - Epimutation KW - Epigenetics KW - Asisted Reproduction KW - Imprinting Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-65995 ER - TY - JOUR A1 - Haertle, Larissa A1 - El Hajj, Nady A1 - Dittrich, Marcus A1 - Müller, Tobias A1 - Nanda, Indrajit A1 - Lehnen, Harald A1 - Haaf, Thomas T1 - Epigenetic signatures of gestational diabetes mellitus on cord blood methylation JF - Clinical Epigenetics N2 - Background: Intrauterine exposure to gestational diabetes mellitus (GDM) confers a lifelong increased risk for metabolic and other complex disorders to the offspring. GDM-induced epigenetic modifications modulating gene regulation and persisting into later life are generally assumed to mediate these elevated disease susceptibilities. To identify candidate genes for fetal programming, we compared genome-wide methylation patterns of fetal cord bloods (FCBs) from GDM and control pregnancies. Methods and results: Using Illumina’s 450K methylation arrays and following correction for multiple testing, 65 CpG sites (52 associated with genes) displayed significant methylation differences between GDM and control samples. Four candidate genes, ATP5A1, MFAP4, PRKCH, and SLC17A4, from our methylation screen and one, HIF3A, from the literature were validated by bisulfite pyrosequencing. The effects remained significant after adjustment for the confounding factors maternal BMI, gestational week, and fetal sex in a multivariate regression model. In general, GDM effects on FCB methylation were more pronounced in women with insulin-dependent GDM who had a more severe metabolic phenotype than women with dietetically treated GDM. Conclusions: Our study supports an association between maternal GDM and the epigenetic status of the exposed offspring. Consistent with a multifactorial disease model, the observed FCB methylation changes are of small effect size but affect multiple genes/loci. The identified genes are primary candidates for transmitting GDM effects to the next generation. They also may provide useful biomarkers for the diagnosis, prognosis, and treatment of adverse prenatal exposures. KW - fetal programming KW - insulin treatment KW - DNA methylation KW - fetal cord blood KW - gestational diabetes mellitus Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-159459 VL - 9 IS - 28 ER - TY - JOUR A1 - El Hajj, Nady A1 - Dittrich, Marcus A1 - Böck, Julia A1 - Kraus, Theo F. J. A1 - Nanda, Indrajit A1 - Müller, Tobias A1 - Seidmann, Larissa A1 - Tralau, Tim A1 - Galetzka, Danuta A1 - Schneider, Eberhard A1 - Haaf, Thomas T1 - Epigenetic dysregulation in the developing Down syndrome cortex JF - Epigenetics N2 - Using Illumina 450K arrays, 1.85% of all analyzed CpG sites were significantly hypermethylated and 0.31% hypomethylated in fetal Down syndrome (DS) cortex throughout the genome. The methylation changes on chromosome 21 appeared to be balanced between hypo- and hyper-methylation, whereas, consistent with prior reports, all other chromosomes showed 3-11times more hyper- than hypo-methylated sites. Reduced NRSF/REST expression due to upregulation of DYRK1A (on chromosome 21q22.13) and methylation of REST binding sites during early developmental stages may contribute to this genome-wide excess of hypermethylated sites. Upregulation of DNMT3L (on chromosome 21q22.4) could lead to de novo methylation in neuroprogenitors, which then persists in the fetal DS brain where DNMT3A and DNMT3B become downregulated. The vast majority of differentially methylated promoters and genes was hypermethylated in DS and located outside chromosome 21, including the protocadherin gamma (PCDHG) cluster on chromosome 5q31, which is crucial for neural circuit formation in the developing brain. Bisulfite pyrosequencing and targeted RNA sequencing showed that several genes of PCDHG subfamilies A and B are hypermethylated and transcriptionally downregulated in fetal DS cortex. Decreased PCDHG expression is expected to reduce dendrite arborization and growth in cortical neurons. Since constitutive hypermethylation of PCDHG and other genes affects multiple tissues, including blood, it may provide useful biomarkers for DS brain development and pharmacologic targets for therapeutic interventions. KW - trisomy 21 KW - DNA methylation KW - Down syndrome KW - fetal brain development KW - frontal cortex KW - protocadherin gamma cluster Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-191239 VL - 11 IS - 8 ER - TY - JOUR A1 - Schneider, Eberhard A1 - Dittrich, Marcus A1 - Böck, Julia A1 - Nanda, Indrajit A1 - Müller, Tobias A1 - Seidmann, Larissa A1 - Tralau, Tim A1 - Galetzka, Danuta A1 - El Hajj, Nady A1 - Haaf, Thomas T1 - CpG sites with continuously increasing or decreasing methylation from early to late human fetal brain development JF - Gene N2 - Normal human brain development is dependent on highly dynamic epigenetic processes for spatial and temporal gene regulation. Recent work identified wide-spread changes in DNA methylation during fetal brain development. We profiled CpG methylation in frontal cortex of 27 fetuses from gestational weeks 12-42, using Illumina 450K methylation arrays. Sites showing genome-wide significant correlation with gestational age were compared to a publicly available data set from gestational weeks 3-26. Altogether, we identified 2016 matching developmentally regulated differentially methylated positions (m-dDMPs): 1767 m-dDMPs were hypermethylated and 1149 hypomethylated during fetal development. M-dDMPs are underrepresented in CpG islands and gene promoters, and enriched in gene bodies. They appear to cluster in certain chromosome regions. M-dDMPs are significantly enriched in autism-associated genes and CpGs. Our results promote the idea that reduced methylation dynamics during fetal brain development may predispose to autism. In addition, m-dDMPs are enriched in genes with human-specific brain expression patterns and/or histone modifications. Collectively, we defined a subset of dDMPs exhibiting constant methylation changes from early to late pregnancy. The same epigenetic mechanisms involving methylation changes in cis-regulatory regions may have been adopted for human brain evolution and ontogeny. KW - Autism spectrum disorders KW - DNA methylation KW - Genome KW - Autism KW - Frontal cortex KW - Human prefrontal cortex KW - Gene-expression KW - Schizophrenia KW - Patterns KW - Transcription KW - Epigenetics KW - Environment KW - Fetal brain development KW - DNA methylation dynamics KW - Methylome Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-186936 VL - 592 IS - 1 ER -