Methylierungsanalysen und Charakterisierung epigenetischer Regulationselemente bei Imprintingerkrankungen

Genomisches Imprinting ist ein epigenetischer Prozess, der zu einer monoallelischen elternspezifischen Expression bestimmter Gene führt. Die Prägung dieser Gene findet bereits in der jeweiligen elterlichen Keimbahn statt. Fehler bei diesem Prozess können zu Veränderungen von Genexpressionsmustern und somit zu Imprintingerkrankungen wie Angelman- oder Prader-Willi-Syndrom, Beckwith-Wiedemann- oder upd(14)pat-Syndrom führen. Die Rolle von cis- und transagierenden Faktoren bei diesen Prozessen ist bisher noch nicht vollständig aufgeklärt. Im Rahmen der vorliegenden Arbeit wurden daher patientenbezogene Ansätze verfolgt, um z.B. epigenetische Regulationsmechanismen näher zu untersuchen. Dabei wurde unter anderem die hier etablierte Methode der hochquantitativen DNA-Methylierungsanalyse genutzt. Ein Kollektiv von AS- und PWS-Patienten mit Imprintingdefekt wurde mittels Affymetrix SNP Array 6.0 genomweit auf Kopienzahlveränderungen untersucht. Es konnten dabei jedoch keine relevanten Veränderungen in cis oder in trans beobachtet werden. Im Rahmen dieser Arbeit war es möglich, ein Kind mit multiplen Imprintingdefekten zu untersuchen. Erste Analysen zeigten, dass der Patient eine Häufung aberranter DNA-Methylierung an geimprinteten und nicht geimprinteten Loci aufweist. Elf Kandidatengene wurden mittels der hier etablierten hochquantitativen Hochdurchsatz-Bisulfitsequenzierung auf dem Roche/454 GS Junior näher untersucht. Nur das Gen FAM50B wies dabei eine allelspezifische Methylierung auf. Außerdem konnte bei zwei hier untersuchten Genen (TSPO und CHP2) durch Trennung der Allele beim Patienten gezeigt werden, dass beide parentalen Allele von den Methylierungsveränderungen betroffen sind. Dies weist darauf hin, dass es zu einem Fehler bei der Aufrechterhaltung der Methylierungsimprints gekommen ist. Weiterhin konnte in der vorliegenden Arbeit bei einer BWS-Patientin mit einer Hypermethylierung der ICR1 auf Chromosom 11p15.5 eine Deletion in cis identifiziert und molekulargenetisch charakterisiert werden. Die 2,2 kb große Deletion führt zum Verlust von drei der sechs CTCF-Bindestellen innerhalb der ICR1 und konnte auch bei drei gesunden Familienmitgliedern nachgewiesen werden. Bei dieser und fünf weiteren Familien mit unterschiedlichen ICR1-Mikrodeletionen wurden Methylierungsanalysen mittels Hochdurchsatz-Bisulfitsequenzierung durchgeführt. Die Ergebnisse zeigten, dass die 2,2 kb große Deletion mit einer leichten Hypermethylierung der verbliebenen CTCF-Bindestellen oder auch einer normalen Methylierung einhergeht. Bei Patienten mit 1,4 und 1,8 kb großen Deletionen, die zum Verlust von einer oder zwei CTCF-Bindestellen führten, zeigte sich hingegen eine starke Hypermethylierung. Die Ergebnisse dieser in vivo Methylierungsanalyse sowie weiterer in vitro Untersuchungen, stützen die zuvor postulierte These, dass die räumliche Anordnung der verbliebenen CTCF-Bindestellen die Bindung von CTCF und damit die Aufrechterhaltung des unmethylierten Zustands des maternalen Allels beeinflusst. Bei einer weiteren Familie mit BWS aufgrund einer Punktmutation einer der OCT4-Bindestellen innerhalb der ICR1 wurde die Methylierung mittels hochquantitativer Hochdurchsatz-Bisulfitsequenzierung untersucht. Dabei konnte gezeigt werden, dass der Grad der Hypermethylierung über die Generationen hinweg zunimmt und mit der phänotypischen Ausprägung korreliert. Im Rahmen dieser Arbeit konnten bei zwei Patienten mit upd(14)pat-Syndrom Deletionen in der chromosomalen Region 14q32 mit einer Größe von 165 bzw. 5,8 kb identifiziert werden. Beide Deletionen führen dabei zum Verlust der MEG3-DMR, während die IG-DMR intakt bleibt. Die 5,8 kb große Deletion konnte auch bei einer ebenfalls betroffenen Schwester und der phänotypisch unauffälligen Mutter beobachtet werden, die diese Deletion im Mosaik auf ihrem paternalen Allel trägt. Wie erwartet konnten mittels Hochdurchsatz-Bisulfitsequenzierung bei allen Patienten nur methylierte Einzelsequenzen der MEG3-DMR detektiert werden. Bei der Mutter konnten aufgrund des Mosaikstatus der Deletion im Verhältnis weniger Einzelsequenzen vom methylierten paternalen Allel detektiert werden als vom unmethylierten maternalen Allel. Die IG-DMR hingegen zeigte, mit Ausnahme einiger bestimmter CpG-Dinukleotide, eine größtenteils normale Methylierung. Die hier gewonnenen Ergebnisse stützten die zuvor postulierte Hypothese der hierarchischen Interaktion der IG-DMR mit der ihr untergeordneten MEG3-DMR und liefern weitere Erkenntnisse über die heterogene Struktur der Methylierung an der IG-DMR.Genomic imprinting is an epigenetic process leading to a monoallelic parent-of-origin specific expression of certain genes. The imprint of these genes is already set in the parental germline. Mistakes during this process can lead to changes in gene expression, and thus, to imprinting diseases like Angelman, Prader-Willi, Beckwith-Wiedemann or upd(14)pat syndrome. The role of cis- and trans-acting factors in these processes is not completely elucidated. In the work at hand, patient-based approaches were pursued to investigate these mechanisms of epigenetic regulation using state-of-the-art methods and a here newly established highly quantitative DNA methylation analysis. A group of AS and PWS patients with an imprinting defect was screened for genome-wide copy number variations using the Affymetrix SNP Array 6.0. No relevant changes in cis or in trans could be identified by this approach. As another part of this thesis a patient with multiple imprinting defects could be investigated. First analyses showed that the patient harboured an accumulation of methylation changes at imprinted and non-imprinted loci. Eleven candidate genes were chosen for further investigation via the here established deep bisulfite sequencing on the Roche/454 GS Junior system. Only the FAM50B gene exhibited allele-specific methylation. Furthermore, allele separation was possible for two genes (TSPO and CHP2) in the patient. The result showed that both parental alleles were equally affected by the methylation changes in the patient, pointing to a defect in imprint maintenance. Additionally in a BWS patient with an imprinting control region 1 (ICR1) hypermethylation on chromosome 11p15.5, a deletion in cis could be identified. Further analyses revealed that the 2.2 kb deletion leads to the loss of three of the six CTCF-binding sites within the ICR1, and it is also present in three healthy family members. Methylation analyses in this and five further families with different ICR1 microdeletions were conducted via deep bisulfite sequencing. The results showed that the 2.2 kb deletion can lead to a mild hypermethylation or a normal methylation of the remaining CTCF-binding sites in ICR1. In contrast, smaller 1.4 and 1.8 kb deletions that abolished only one or two CTCF-binding sites, respectively, led to a severe hypermethylation of the remaining sites within the ICR1. The results of this in vivo methylation analysis as well as further in vitro analyses support the previously postulated hypothesis that the spatial arrangement of the remaining CTCF-binding sites plays an important role in the binding of CTCF, and thus, in the maintenance of the unmethylated state of the maternal allele. In another family with BWS caused by a point mutation within an OCT binding site in the ICR1 methylation analyses were also conducted using deep bisulfite sequencing. It could be shown that the degree of hypermethylation increases over generations and correlates to the phenotypical outcome. As a further part of this thesis, two patients with a upd(14)pat syndrome caused by deletions within the chromosomal region 14q32 could be identified. The deletions have sizes of 165 kb and 5.8 kb and lead to loss of the MEG3-DMR, but the IG-DMR remains intact. The 5.8 kb deletion was also detected in the affected sister and in the phenotypically unremarkable mother, who carries the deletion in a mosaic state on her paternal allele. As expected, only completely methylated deep bisulfite sequencing reads could be obtained for the MEG3-DMR for all upd(14)pat syndrome patients. For the mother with the mosaic deletion, a smaller proportion of reads was obtained from the methylated paternal allele than from the unmethylated maternal allele. In all upd(14)pat patients the methylation at the IG-DMR was mostly normal with the exception of a few certain CpG dinucleotides. These results support the previously postulated hypothesis of a hierarchical interaction of the two DMRs where the IG-DMR governs the MEG3-DMR and yield new insights into the heterogeneous methylation at the IG-DMR

No evidence for intervention-associated DNA methylation changes in monocytes of patients with posttraumatic stress disorder

DNA methylation patterns can be responsive to environmental influences. This observation has sparked interest in the potential for psychological interventions to influence epigenetic processes. Recent studies have observed correlations between DNA methylation changes and therapy outcome. However, most did not control for changes in cell composition. This study had two aims: first, we sought to replicate therapy-associated changes in DNA methylation of commonly assessed candidate genes in isolated monocytes from 60 female patients with post-traumatic stress disorder (PTSD). Our second, exploratory goal was to identify novel genomic regions with substantial pre-to-post intervention DNA methylation changes by performing whole-genome bisulfite sequencing (WGBS) in two patients with PTSD. Equivalence testing and Bayesian analyses provided evidence against physiologically meaningful intervention-associated DNA methylation changes in monocytes of PTSD patients in commonly investigated target genes (NR3C1, FKBP5, SLC6A4, OXTR). Furthermore, WGBS yielded only a limited set of candidate regions with suggestive evidence of differential DNA methylation pre- to post-therapy. These differential DNA methylation patterns did not prove replicable when investigated in the entire cohort. We conclude that there is no evidence for major, recurrent intervention-associated DNA methylation changes in the investigated genes in monocytes of patients with PTSD

Recommendations for a nomenclature system for reporting methylation aberrations in imprinted domains

The analysis of DNA methylation has become routine in the pipeline for diagnosis of imprinting disorders, with many publications reporting aberrant methylation associated with imprinted differentially methylated regions (DMRs). However, comparisons between these studies are routinely hampered by the lack of consistency in reporting sites of methylation evaluated. To avoid confusion surrounding nomenclature, special care is needed to communicate results accurately, especially between scientists and other health care professionals. Within the European Network for Human Congenital Imprinting Disorders we have discussed these issues and designed a nomenclature for naming imprinted DMRs as well as for reporting methylation values. We apply these recommendations for imprinted DMRs that are commonly assayed in clinical laboratories and show how they support standardized database submission. The recommendations are in line with existing recommendations, most importantly the Human Genome Variation Society nomenclature, and should facilitate accurate reporting and data exchange among laboratories and thereby help to avoid future confusion

No evidence for intervention-associated DNA methylation changes in monocytes of patients with posttraumatic stress disorder.

DNA methylation patterns can be responsive to environmental influences. This observation has sparked interest in the potential for psychological interventions to influence epigenetic processes. Recent studies have observed correlations between DNA methylation changes and therapy outcome. However, most did not control for changes in cell composition. This study had two aims: first, we sought to replicate therapy-associated changes in DNA methylation of commonly assessed candidate genes in isolated monocytes from 60 female patients with post-traumatic stress disorder (PTSD). Our second, exploratory goal was to identify novel genomic regions with substantial pre-to-post intervention DNA methylation changes by performing whole-genome bisulfite sequencing (WGBS) in two patients with PTSD. Equivalence testing and Bayesian analyses provided evidence against physiologically meaningful intervention-associated DNA methylation changes in monocytes of PTSD patients in commonly investigated target genes (NR3C1, FKBP5, SLC6A4, OXTR). Furthermore, WGBS yielded only a limited set of candidate regions with suggestive evidence of differential DNA methylation pre- to post-therapy. These differential DNA methylation patterns did not prove replicable when investigated in the entire cohort. We conclude that there is no evidence for major, recurrent intervention-associated DNA methylation changes in the investigated genes in monocytes of patients with PTSD

Mutations in NLRP5 are associated with reproductive wastage and multilocus imprinting disorders in humans

This is the final version. It first appeared at http://www.nature.com/ncomms/2015/150901/ncomms9086/full/ncomms9086.html.Human-imprinting disorders are congenital disorders of growth, development and metabolism, associated with disturbance of parent of origin-specific DNA methylation at imprinted loci across the genome. Some imprinting disorders have higher than expected prevalence of monozygotic twinning, of assisted reproductive technology among parents, and of disturbance of multiple imprinted loci, for which few causative trans-acting mutations have been found. Here we report mutations in NLRP5 in five mothers of individuals affected by multilocus imprinting disturbance. Maternal-effect mutations of other human NLRP genes, NLRP7 and NLRP2, cause familial biparental hydatidiform mole and multilocus imprinting disturbance, respectively. Offspring of mothers with NLRP5 mutations have heterogenous clinical and epigenetic features, but cases include a discordant monozygotic twin pair, individuals with idiopathic developmental delay and autism, and families affected by infertility and reproductive wastage. NLRP5 mutations suggest connections between maternal reproductive fitness, early zygotic development and genomic imprinting.L.E.D. and F.I.R. were supported by the Medical Research Council (MR/J000329/1). J.B., K.B., B.H., L.S. M.B. and T.E. were supported by Bundesministerium fu?r Bildung und Forschung (grant number 01GM1513A and 01GM1513C) and C.T. was supported by an Ipsen Fellowship Grant. The cohort ?Imprinting Disorders-Finding out Why? was accrued through the support of the Newlife Foundation for Disabled Children and through support from the Wessex NIHR clinical research network and NIHR Wellcome Southampton clinical research facility. Funding for DNA collection and methylation analysis of normal control samples was provided in part by the National Institutes of Health R01 AI091905-01, R01 AI061471 and R01 HL082925. ERM thanks Action Medical Research for support

De novo missense variants in FBXO11 alter its protein expression and subcellular localization.

Recently, we and others identified de novo FBXO11 variants as causative for a variable neurodevelopmental disorder (NDD). We now assembled clinical and mutational information on 23 additional individuals. The phenotypic spectrum remains highly variable, with developmental delay and/or intellectual disability as the core feature and behavioral anomalies, hypotonia and various facial dysmorphism as frequent aspects. The mutational spectrum includes intragenic deletions, likely gene disrupting and missense variants distributed across the protein. To further characterize the functional consequences of FBXO11 missense variants, we analyzed their effects on protein expression and localization by overexpression of 17 different mutant constructs in HEK293 and HeLa cells. We found that the majority of missense variants resulted in subcellular mislocalization and/or reduced FBXO11 protein expression levels. For instance, variants located in the nuclear localization signal and the N-terminal F-Box domain lead to altered subcellular localization with exclusion from the nucleus or the formation of cytoplasmic aggregates and to reduced protein levels in western blot. In contrast, variants localized in the C-terminal Zn-finger UBR domain lead to an accumulation in the cytoplasm without alteration of protein levels. Together with the mutational data our functional results suggest that most missense variants likely lead to a loss of the original FBXO11 function and thereby highlight haploinsufficiency as the most likely disease mechanism for FBXO11-associated NDDs

De novo missense variants in FBXO11 alter its protein expression and subcellular localization.

Recently, we and others identified de novo FBXO11 variants as causative for a variable neurodevelopmental disorder (NDD). We now assembled clinical and mutational information on 23 additional individuals. The phenotypic spectrum remains highly variable, with developmental delay and/or intellectual disability as the core feature and behavioral anomalies, hypotonia and various facial dysmorphism as frequent aspects. The mutational spectrum includes intragenic deletions, likely gene disrupting and missense variants distributed across the protein. To further characterize the functional consequences of FBXO11 missense variants, we analyzed their effects on protein expression and localization by overexpression of 17 different mutant constructs in HEK293 and HeLa cells. We found that the majority of missense variants resulted in subcellular mislocalization and/or reduced FBXO11 protein expression levels. For instance, variants located in the nuclear localization signal and the N-terminal F-Box domain lead to altered subcellular localization with exclusion from the nucleus or the formation of cytoplasmic aggregates and to reduced protein levels in western blot. In contrast, variants localized in the C-terminal Zn-finger UBR domain lead to an accumulation in the cytoplasm without alteration of protein levels. Together with the mutational data our functional results suggest that most missense variants likely lead to a loss of the original FBXO11 function and thereby highlight haploinsufficiency as the most likely disease mechanism for FBXO11-associated NDDs