TY - JOUR A1 - Ghosh, Sujal A1 - Hönscheid, Andrea A1 - Dückers, Gregor A1 - Ginzel, Sebastian A1 - Gohlke, Holger A1 - Gombert, Michael A1 - Kempkes, Bettina A1 - Klapper, Wolfram A1 - Kuhlen, Michaela A1 - Laws, Hans-Jürgen A1 - Linka, René Martin A1 - Meisel, Roland A1 - Mielke, Christian A1 - Niehues, Tim A1 - Schindler, Detlev A1 - Schneider, Dominik A1 - Schuster, Friedhelm R. A1 - Speckmann, Carsten A1 - Borkhardt, Arndt T1 - Human RAD52 - a novel player in DNA repair in cancer and immunodeficiency JF - Haematologica N2 - No abstract available. KW - human medicine KW - DNA-Repair KW - cancer KW - immunodeficiency KW - RAD52 Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-180862 VL - 102 IS - 2 ER - TY - JOUR A1 - Hauer, Nadine N. A1 - Popp, Bernt A1 - Taher, Leila A1 - Vogl, Carina A1 - Dhandapany, Perundurai S. A1 - Büttner, Christian A1 - Uebe, Steffen A1 - Sticht, Heinrich A1 - Ferrazzi, Fulvia A1 - Ekici, Arif B. A1 - De Luca, Alessandro A1 - Klinger, Patrizia A1 - Kraus, Cornelia A1 - Zweier, Christiane A1 - Wiesener, Antje A1 - Abou Jamra, Rami A1 - Kunstmann, Erdmute A1 - Rauch, Anita A1 - Wieczorek, Dagmar A1 - Jung, Anna-Marie A1 - Rohrer, Tilman R. A1 - Zenker, Martin A1 - Doerr, Helmuth-Guenther A1 - Reis, André A1 - Thiel, Christian T. T1 - Evolutionary conserved networks of human height identify multiple Mendelian causes of short stature JF - European Journal of Human Genetics N2 - Height is a heritable and highly heterogeneous trait. Short stature affects 3% of the population and in most cases is genetic in origin. After excluding known causes, 67% of affected individuals remain without diagnosis. To identify novel candidate genes for short stature, we performed exome sequencing in 254 unrelated families with short stature of unknown cause and identified variants in 63 candidate genes in 92 (36%) independent families. Based on systematic characterization of variants and functional analysis including expression in chondrocytes, we classified 13 genes as strong candidates. Whereas variants in at least two families were detected for all 13 candidates, two genes had variants in 6 (UBR4) and 8 (LAMA5) families, respectively. To facilitate their characterization, we established a clustered network of 1025 known growth and short stature genes, which yielded 29 significantly enriched clusters, including skeletal system development, appendage development, metabolic processes, and ciliopathy. Eleven of the candidate genes mapped to 21 of these clusters, including CPZ, EDEM3, FBRS, IFT81, KCND1, PLXNA3, RASA3, SLC7A8, UBR4, USP45, and ZFHX3. Fifty additional growth-related candidates we identified await confirmation in other affected families. Our study identifies Mendelian forms of growth retardation as an important component of idiopathic short stature. KW - disease genetics KW - DNA sequencing KW - genetic counselling Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-227899 VL - 27 ER - TY - JOUR A1 - Ferreira, Manuel A. A1 - Gamazon, Eric R. A1 - Al-Ejeh, Fares A1 - Aittomäki, Kristiina A1 - Andrulis, Irene L. A1 - Anton-Culver, Hoda A1 - Arason, Adalgeir A1 - Arndt, Volker A1 - Aronson, Kristan J. A1 - Arun, Banu K. A1 - Asseryanis, Ella A1 - Azzollini, Jacopo A1 - Balmaña, Judith A1 - Barnes, Daniel R. A1 - Barrowdale, Daniel A1 - Beckmann, Matthias W. A1 - Behrens, Sabine A1 - Benitez, Javier A1 - Bermisheva, Marina A1 - Bialkowska, Katarzyna A1 - Blomqvist, Carl A1 - Bogdanova, Natalia V. A1 - Bojesen, Stig E. A1 - Bolla, Manjeet K. A1 - Borg, Ake A1 - Brauch, Hiltrud A1 - Brenner, Hermann A1 - Broeks, Annegien A1 - Burwinkel, Barbara A1 - Caldés, Trinidad A1 - Caligo, Maria A. A1 - Campa, Daniele A1 - Campbell, Ian A1 - Canzian, Federico A1 - Carter, Jonathan A1 - Carter, Brian D. A1 - Castelao, Jose E. A1 - Chang-Claude, Jenny A1 - Chanock, Stephen J. A1 - Christiansen, Hans A1 - Chung, Wendy K. A1 - Claes, Kathleen B. M. A1 - Clarke, Christine L. A1 - Couch, Fergus J. A1 - Cox, Angela A1 - Cross, Simon S. A1 - Czene, Kamila A1 - Daly, Mary B. A1 - de la Hoya, Miguel A1 - Dennis, Joe A1 - Devilee, Peter A1 - Diez, Orland A1 - Dörk, Thilo A1 - Dunning, Alison M. A1 - Dwek, Miriam A1 - Eccles, Diana M. A1 - Ejlertsen, Bent A1 - Ellberg, Carolina A1 - Engel, Christoph A1 - Eriksson, Mikael A1 - Fasching, Peter A. A1 - Fletcher, Olivia A1 - Flyger, Henrik A1 - Friedman, Eitan A1 - Frost, Debra A1 - Gabrielson, Marike A1 - Gago-Dominguez, Manuela A1 - Ganz, Patricia A. A1 - Gapstur, Susan M. A1 - Garber, Judy A1 - García-Closas, Montserrat A1 - García-Sáenz, José A. A1 - Gaudet, Mia M. A1 - Giles, Graham G. A1 - Glendon, Gord A1 - Godwin, Andrew K. A1 - Goldberg, Mark S. A1 - Goldgar, David E. A1 - González-Neira, Anna A1 - Greene, Mark H. A1 - Gronwald, Jacek A1 - Guenél, Pascal A1 - Haimann, Christopher A. A1 - Hall, Per A1 - Hamann, Ute A1 - He, Wei A1 - Heyworth, Jane A1 - Hogervorst, Frans B. L. A1 - Hollestelle, Antoinette A1 - Hoover, Robert N. A1 - Hopper, John L. A1 - Hulick, Peter J. A1 - Humphreys, Keith A1 - Imyanitov, Evgeny N. A1 - Isaacs, Claudine A1 - Jakimovska, Milena A1 - Jakubowska, Anna A1 - James, Paul A. A1 - Janavicius, Ramunas A1 - Jankowitz, Rachel C. A1 - John, Esther M. A1 - Johnson, Nichola A1 - Joseph, Vijai A1 - Karlan, Beth Y. A1 - Khusnutdinova, Elza A1 - Kiiski, Johanna I. A1 - Ko, Yon-Dschun A1 - Jones, Michael E. A1 - Konstantopoulou, Irene A1 - Kristensen, Vessela N. A1 - Laitman, Yael A1 - Lambrechts, Diether A1 - Lazaro, Conxi A1 - Leslie, Goska A1 - Lester, Jenny A1 - Lesueur, Fabienne A1 - Lindström, Sara A1 - Long, Jirong A1 - Loud, Jennifer T. A1 - Lubiński, Jan A1 - Makalic, Enes A1 - Mannermaa, Arto A1 - Manoochehri, Mehdi A1 - Margolin, Sara A1 - Maurer, Tabea A1 - Mavroudis, Dimitrios A1 - McGuffog, Lesley A1 - Meindl, Alfons A1 - Menon, Usha A1 - Michailidou, Kyriaki A1 - Miller, Austin A1 - Montagna, Marco A1 - Moreno, Fernando A1 - Moserle, Lidia A1 - Mulligan, Anna Marie A1 - Nathanson, Katherine L. A1 - Neuhausen, Susan L. A1 - Nevanlinna, Heli A1 - Nevelsteen, Ines A1 - Nielsen, Finn C. A1 - Nikitina-Zake, Liene A1 - Nussbaum, Robert L. A1 - Offit, Kenneth A1 - Olah, Edith A1 - Olopade, Olufunmilayo I. A1 - Olsson, Håkan A1 - Osorio, Ana A1 - Papp, Janos A1 - Park-Simon, Tjoung-Won A1 - Parsons, Michael T. A1 - Pedersen, Inge Sokilde A1 - Peixoto, Ana A1 - Peterlongo, Paolo A1 - Pharaoh, Paul D. P. A1 - Plaseska-Karanfilska, Dijana A1 - Poppe, Bruce A1 - Presneau, Nadege A1 - Radice, Paolo A1 - Rantala, Johanna A1 - Rennert, Gad A1 - Risch, Harvey A. A1 - Saloustros, Emmanouil A1 - Sanden, Kristin A1 - Sawyer, Elinor J. A1 - Schmidt, Marjanka K. A1 - Schmutzler, Rita K. A1 - Sharma, Priyanka A1 - Shu, Xiao-Ou A1 - Simard, Jaques A1 - Singer, Christian F. A1 - Soucy, Penny A1 - Southey, Melissa C. A1 - Spinelli, John J. A1 - Spurdle, Amanda B. A1 - Stone, Jennifer A1 - Swerdlow, Anthony J. A1 - Tapper, William J. A1 - Taylor, Jack A. A1 - Teixeira, Manuel R. A1 - Terry, Mary Beth A1 - Teulé, Alex A1 - Thomassen, Mads A1 - Thöne, Kathrin A1 - Thull, Darcy L. A1 - Tischkowitz, Marc A1 - Toland, Amanda E. A1 - Torres, Diana A1 - Truong, Thérèse A1 - Tung, Nadine A1 - Vachon, Celine M. A1 - van Asperen, Christi J. A1 - van den Ouweland, Ans M. W. A1 - van Rensburg, Elizabeth J. A1 - Vega, Ana A1 - Viel, Alexandra A1 - Wang, Qin A1 - Wappenschmidt, Barbara A1 - Weitzel, Jeffrey N. A1 - Wendt, Camilla A1 - Winqvist, Robert A1 - Yang, Xiaohong R. A1 - Yannoukakos, Drakoulis A1 - Ziogas, Argyrios A1 - Kraft, Peter A1 - Antoniou, Antonis C. A1 - Zheng, Wei A1 - Easton, Douglas F. A1 - Milne, Roger L. A1 - Beesley, Jonathan A1 - Chenevix-Trench, Georgia T1 - Genome-wide association and transcriptome studies identify target genes and risk loci for breast cancer JF - Nature Communications N2 - Genome-wide association studies (GWAS) have identified more than 170 breast cancer susceptibility loci. Here we hypothesize that some risk-associated variants might act in non-breast tissues, specifically adipose tissue and immune cells from blood and spleen. Using expression quantitative trait loci (eQTL) reported in these tissues, we identify 26 previously unreported, likely target genes of overall breast cancer risk variants, and 17 for estrogen receptor (ER)-negative breast cancer, several with a known immune function. We determine the directional effect of gene expression on disease risk measured based on single and multiple eQTL. In addition, using a gene-based test of association that considers eQTL from multiple tissues, we identify seven (and four) regions with variants associated with overall (and ER-negative) breast cancer risk, which were not reported in previous GWAS. Further investigation of the function of the implicated genes in breast and immune cells may provide insights into the etiology of breast cancer. KW - cancer KW - genetics Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-228024 VL - 10 ER - TY - JOUR A1 - Dubail, Johanne A1 - Huber, Céline A1 - Chantepie, Sandrine A1 - Sonntag, Stephan A1 - Tüysüz, Beyhan A1 - Mihci, Ercan A1 - Gordon, Christopher T. A1 - Steichen-Gersdorf, Elisabeth A1 - Amiel, Jeanne A1 - Nur, Banu A1 - Stolte-Dijkstra, Irene A1 - van Eerde, Albertien M. A1 - van Gassen, Koen L. A1 - Breugem, Corstiaan C. A1 - Stegmann, Alexander A1 - Lekszas, Caroline A1 - Maroofian, Reza A1 - Karimiani, Ehsan Ghayoor A1 - Bruneel, Arnaud A1 - Seta, Nathalie A1 - Munnich, Arnold A1 - Papy-Garcia, Dulce A1 - De La Dure-Molla, Muriel A1 - Cormier-Daire, Valérie T1 - SLC10A7 mutations cause a skeletal dysplasia with amelogenesis imperfecta mediated by GAG biosynthesis defects JF - Nature Communications N2 - Skeletal dysplasia with multiple dislocations are severe disorders characterized by dislocations of large joints and short stature. The majority of them have been linked to pathogenic variants in genes encoding glycosyltransferases, sulfotransferases or epimerases required for glycosaminoglycan synthesis. Using exome sequencing, we identify homozygous mutations in SLC10A7 in six individuals with skeletal dysplasia with multiple dislocations and amelogenesis imperfecta. SLC10A7 encodes a 10-transmembrane-domain transporter located at the plasma membrane. Functional studies in vitro demonstrate that SLC10A7 mutations reduce SLC10A7 protein expression. We generate a Slc10a7−/− mouse model, which displays shortened long bones, growth plate disorganization and tooth enamel anomalies, recapitulating the human phenotype. Furthermore, we identify decreased heparan sulfate levels in Slc10a7−/− mouse cartilage and patient fibroblasts. Finally, we find an abnormal N-glycoprotein electrophoretic profile in patient blood samples. Together, our findings support the involvement of SLC10A7 in glycosaminoglycan synthesis and specifically in skeletal development. KW - bone development KW - disease genetics KW - medical genetics Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-226377 VL - 9 ER - TY - JOUR A1 - Luther, Christian H. A1 - Brandt, Philipp A1 - Vylkova, Slavena A1 - Dandekar, Thomas A1 - Müller, Tobias A1 - Dittrich, Marcus T1 - Integrated analysis of SR-like protein kinases Sky1 and Sky2 links signaling networks with transcriptional regulation in Candida albicans JF - Frontiers in Cellular and Infection Microbiology N2 - Fungal infections are a major global health burden where Candida albicans is among the most common fungal pathogen in humans and is a common cause of invasive candidiasis. Fungal phenotypes, such as those related to morphology, proliferation and virulence are mainly driven by gene expression, which is primarily regulated by kinase signaling cascades. Serine-arginine (SR) protein kinases are highly conserved among eukaryotes and are involved in major transcriptional processes in human and S. cerevisiae. Candida albicans harbors two SR protein kinases, while Sky2 is important for metabolic adaptation, Sky1 has similar functions as in S. cerevisiae. To investigate the role of these SR kinases for the regulation of transcriptional responses in C. albicans, we performed RNA sequencing of sky1Δ and sky2Δ and integrated a comprehensive phosphoproteome dataset of these mutants. Using a Systems Biology approach, we study transcriptional regulation in the context of kinase signaling networks. Transcriptomic enrichment analysis indicates that pathways involved in the regulation of gene expression are downregulated and mitochondrial processes are upregulated in sky1Δ. In sky2Δ, primarily metabolic processes are affected, especially for arginine, and we observed that arginine-induced hyphae formation is impaired in sky2Δ. In addition, our analysis identifies several transcription factors as potential drivers of the transcriptional response. Among these, a core set is shared between both kinase knockouts, but it appears to regulate different subsets of target genes. To elucidate these diverse regulatory patterns, we created network modules by integrating the data of site-specific protein phosphorylation and gene expression with kinase-substrate predictions and protein-protein interactions. These integrated signaling modules reveal shared parts but also highlight specific patterns characteristic for each kinase. Interestingly, the modules contain many proteins involved in fungal morphogenesis and stress response. Accordingly, experimental phenotyping shows a higher resistance to Hygromycin B for sky1Δ. Thus, our study demonstrates that a combination of computational approaches with integration of experimental data can offer a new systems biological perspective on the complex network of signaling and transcription. With that, the investigation of the interface between signaling and transcriptional regulation in C. albicans provides a deeper insight into how cellular mechanisms can shape the phenotype. KW - sky kinases KW - kinase signaling KW - network analysis KW - transcriptome KW - transcriptional regulation KW - phosphoproteome KW - Candida albicans Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-311771 SN - 2235-2988 VL - 13 ER - TY - THES A1 - Doll, Julia T1 - Identifizierung und Charakterisierung neuer, mit Hörstörungen assoziierter Gene und Varianten mittels Exom-Sequenzierung T1 - Identification and characterization of novel hearing loss associated genes and variants by Exome Sequencing N2 - Laut des aktuellen Reports der Weltgesundheitsorganisation sind ca. 466 Millionen Menschen weltweit von einer Hörstörung (HS) betroffen. Durch die enorme Heterogenität und die klinische Variabilität, die diese Erkrankung ausmacht, und viele bisher nicht mit HS assoziierte Gene, bleibt ein großer Teil der erblich bedingten HS in vielen Familien unaufgeklärt. Die Entwicklung moderner Techniken, wie die Next-Generation Sequenzierung (NGS) und der Fortschritt bei der Untersuchung von Modellorganismen trugen jedoch in den letzten Jahren immens dazu bei, neue Gene zu identifizieren, die innerhalb des auditorischen Signalwegs oder damit assoziierten Strukturen beteiligt sind. Die vorliegende Arbeit umfasst Ergebnisse dreier Veröffentlichungen, in denen iranische und pakistanische Familien und eine deutsche Familie mit erblich bedingter HS untersucht und neue, krankheitsverursachende Varianten identifiziert und funktionell charakterisiert wurden. Im ersten Abschnitt konnten zwei neue rezessive Varianten im CDC14A-Gen als krankheitsverursachend identifiziert werden, die zu einem potentiellen Funktionsverlust des kodierten Proteins in einer iranischen und einer pakistanischen Familie führen. Mit Hilfe einer funktionellen Charakterisierung auf RNA-Ebene (Spleiß-Assay und RT-qPCR) konnte der Funktionsverlust beider Varianten bestätigt werden. Der zweite Abschnitt umfasst eine deutsche Familie mit sieben von einer HS betroffenen Familienmitgliedern, in der eine heterozygote missense Variante in MYO3A identifiziert wurde. In der vorliegenden Arbeit konnte somit die erste autosomal dominante Variante in einer europäischen Familie mit einer bilingualen, sensorineuralen Hochtonschwerhörigkeit beschrieben werden und der dominante Charakter von MYO3A bestätigt werden. Im dritten Abschnitt konnten die krankheitsverursachenden Varianten in 13 Familien aus einer Kohorte mit 21 pakistanischen Familien mit einer syndromalen und nicht-syndromalen HS ausfindig gemacht werden. Hierbei wurden sowohl bekannte, als auch bisher nicht beschriebene Varianten detektiert. Die Aufklärungsrate innerhalb dieser Kohorte betrug 61,9% und es konnte somit das Spektrum syndromaler und nicht-syndromaler HS erweitert werden. Der letzte Abschnitt dieser Arbeit beschreibt eine iranische Familie mit einer milden HS und milden Intelligenzminderung, in der eine homozygote missense Variante im Kandidatengen DBN1 ausfindig gemacht wurde. Um die Funktion und die Auswirkungen eines potentiellen Verlusts des codierten Proteins Drebrin zu untersuchen, wurden immunhistochemische Färbungen und auditorische Messungen an Dbn1 Knockout (KO)-Mäusen durchgeführt. Hierbei konnte eine Expression innerhalb der Nervenfasern, die innere Haarzellen innervieren, nachgewiesen werden. Eine leicht verlängerte Latenz für die ABR-Welle IV in KO-Mäusen im Vergleich zum Wildtyp ergab den Hinweis auf einen Defekt innerhalb des zentralen auditorischen Signalwegs, der möglicherweise mit einer Sprachverarbeitungsstörung im Menschen korreliert. N2 - According to the latest World Health Organization report, approximately 466 million people are affected by hearing loss (HL) worldwide. Due to the enormous heterogeneity and clinical variability that comes with this disorder and the previously unassociated HL genes, a large proportion of hereditary HL remains unexplained in many families. However, the development of modern techniques such as next-generation sequencing (NGS) and constant progress in the study of model organisms contributed immensely to the identification of new genes involved within the auditory pathway or associated structures. This thesis includes results of three publications in which Iranian and Pakistani families and a German family with HL were studied and novel disease-causing variants were identified and characterized. In the first section, two novel recessive variants in the CDC14A gene were identified as disease-causing, leading to a loss-of-function of the encoded protein in an Iranian and a Pakistani family. Functional characterization on RNA level (splice assay and RT-qPCR) confirmed the loss of function of both variants. The second section includes a German family with seven family members affected by HL, in which a heterozygous missense variant in MYO3A was identified. Thus, the first autosomal dominant variant in a European family with a bilingual sensorineural high-frequency hearing loss could be described in the current work. In the third section, the disease-causing variants were established in 13 families from a cohort of 21 Pakistani families with syndromic and non-syndromic HL. Both known and previously undescribed variants were detected. The detection rate within this cohort was 61.9% and thus the spectrum of syndromic and non-syndromic HL could be expanded. The final section of this work describes an Iranian family with mild HL and mild intellectual disability in which a homozygous missense variant in the candidate gene DBN1 was detected. To investigate the function and effects of a potential loss of the encoded protein Drebrin, immunohistochemical staining and auditory measurements were performed in Dbn1 knockout mice. Here, expression within nerve fibers innervating inner hair cells was detected. A slightly prolonged latency for ABR wave IV in the auditory pathway in KO mice compared to wild type mice provided evidence for a defect within the central auditory pathway that might correlate with a speech processing disorder in humans. KW - Hörstörung KW - Exomsequenzierung KW - Konsanguinität KW - Hörstörungen Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-261097 ER - TY - THES A1 - Prell, Andreas T1 - The effects of paternal age on DNA methylation of developmentally important genes in human and bovine sperm T1 - Der väterliche Alterseffekt auf DNA-Methylierung in entwicklungsrelevanten Genen im menschlichen und bovinen Spermienepigenom N2 - Western societies are steadily becoming older undergoing a clear trend of delayed parenthood. Children of older fathers have an undeniably higher risk for certain neurodevelopmental disorders and other medical conditions. Changes in the epigenetic landscape and especially in DNA methylation patterns are likely to account for a portion of this inherited disease susceptibility. DNA methylation changes during the ageing process are a well-known epigenetic feature. These so-called age-DMRs exist in developmentally important genes in the methylome of several mammalian species. However, there is only a minor overlap between the age-DMR datasets of different studies. We therefore replicated age-DMRs (which were obtained from a genome wide technique) by applying a different technical approach in a larger sample number. Here, this study confirmed 10 age-DMRs in the human and 4 in the bovine sperm epigenome from a preliminary candidate list based on RRBS. For this purpose, we used bisulphite Pyrosequencing in 94 human and 36 bovine sperm samples. These Pyrosequencing results confirm RRBS as an effective and reliable method to screen for age-DMRs in the vertebrate genome. To decipher whether paternal age effects are an evolutionary conserved feature of mammalian development, we compared methylation patterns between human and bovine sperm in orthologous regulatory regions. We discovered that the level of methylation and the age effect are both species-specific and speculate that these methylation marks reflect the lineage-specific development of each species to hit evolutionary requirements and adaptation processes. Different methylation levels between species in developmentally important genes also imply a differing mutational burden, representing a potential driver for point mutations and consequently deviations in the underlying DNA sequence of different species. Using the example of different haplotypes, this study showed the great effect of single base variations on the methylation of adjacent CpGs. Nonetheless, this study could not provide further evidence or a mechanism for the transfer of epigenetic marks to future generations. Therefore, further research in tissues from the progeny of old and young fathers is required to determine if the observed methylation changes are transmitted to the next generation and if they are associated with altered transcriptional activity of the respective genes. This could provide a direct link between the methylome of sperm from elderly fathers and the development potential of the next generation. N2 - Unsere westliche Gesellschaft wird immer älter und unterliegt einem eindeutigen Trend zur verzögerten Elternschaft. Kinder von älteren Vätern haben ein unbestreitbar höheres Risiko für bestimmte neurologische Entwicklungsstörungen und andere Erkrankungen. Epigenetische Veränderungen insbesondere im DNA-Methylierungsmuster sind wahrscheinlich für einen Teil dieser vererbten Krankheitsanfälligkeit verantwortlich. Altersbedingte Veränderungen im DNA-Methylierungsmuster sind ein bekanntes und gut erforschtes Phänomen in der Epigenetik. Diese so genannten Alters-DMRs konnten im Methylom mehrerer Säugetierarten nachgewiesen werden, insbesondere in entwicklungsrelevanten Genen. Allerdings gibt es nur geringe Überschneidungen zwischen den Alters-DMR-Datensätzen verschiedener Studien. Unser Ziel war es daher, die Vertrauenswürdigkeit eines laborinternen Datensatzes, der auf Reduced Representation Bisulphite Sequencing (RRBS) basierte, zu erhöhen, indem wir einen anderen technischen Ansatz in einer unabhängigen Kohorte anwenden. Mit der Methode des Bisulphite Pyrosequencing konnten wir in dieser Studie 10 Alters-DMRs im menschlichen und 4 im Rinder-Spermienepigenom aus einer vorläufigen Kandidatenliste basierend auf RRBS validieren. Diese Ergebnisse bestätigen, dass RRBS eine wirksame und zuverlässige Methode ist, um neue Alters-DMRs im Wirbeltiergenom zu finden. Um festzustellen, ob väterliche Alterseffekte in der Evolution verschiedener Säugetierarten konserviert wurden, verglichen wir die Methylierungsmuster orthologer Regionen zwischen dem menschlichem und dem Rinderspermienepigenom. Es zeigte sich, dass sowohl der Methylierungsgrad als auch der Alterseffekt artspezifisch sind. Daher vermuten wir, dass diese Methylierungsmuster die artspezifische Entwicklung als Antwort auf evolutionäre Anforderungen und durchlebte Anpassungsprozesse darstellen. Diese unterschiedlichen Methylierungslevel zwischen verschiedenen Arten in entwicklungswichtigen Genen implizieren auch eine unterschiedliche Mutationslast, die einen potenziellen Treiber für Punktmutationen und folglich Abweichungen in der zugrunde liegenden DNA-Sequenz der verschiedenen Arten darstellt. Am Beispiel verschiedener Haplotypen konnten wir exemplarisch den ausgeprägten Einfluss bereits einzelner DNA-Basenvariationen auf die Methylierung benachbarter CpGs demonstrieren. Allerdings konnte diese Studie keinen weiteren Beweis oder einen Mechanismus für die Übertragung von epigenetischen Markierungen auf künftige Generationen liefern. Daher sind weitere Untersuchungen an embryonalen, fötalen und adulten Geweben von Nachkommen alter bzw. junger Väter unabdingbar, um festzustellen, ob die beobachteten Methylierungsveränderungen auch auf die nächste Generation übertragen werden. Ferner ist festzustellen, ob sie die Transkriptionsaktivität der betroffenen Gene beeinflussen. Dies könnte einen direkten Einfluss des Spermienmethyloms älterer Väter auf das Entwicklungspotenzial der nächsten Generation belegen. KW - Epigenetik KW - Spermium KW - Methylierung KW - DNA-Methylation KW - Paternal age effect Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-347866 ER - TY - THES A1 - Riemens, Renzo J. M. T1 - Neuroepigenomics in Alzheimer’s disease: The single cell ADds T1 - Neuroepigenomik bei der Alzheimer-Krankheit: Die Einzelzell ADds N2 - Die Forschung, die in dieser Arbeit zusammengestellt wird, kann in zwei Teile geteilt werden. Der erste Teil, bestehend aus vier Kapiteln, konzentriert sich auf die Rolle der epigenetischen Dysregulation in der Ätiopathophysiologie der sporadischen Alzheimer-Krankheit (sAD). Neben Einblicken in die neuesten Entwicklungen in neuroepigenomischen Studien zu dieser Krankheit geht der erste Teil der Arbeit auch auf verbleibende Herausforderungen ein und gibt einen Ausblick auf mögliche Entwicklungen auf diesem Gebiet. Der zweite Teil, der drei weitere Kapitel umfasst, konzentriert sich auf die Anwendung von auf induzierten pluripotenten Stammzellen (iPSC) basierenden Krankheitsmodellen für das Studium der AD, einschließlich, aber nicht beschränkt auf mechanistische Studien zur epigenetischen Dysregulation unter Verwendung dieser Plattform. Neben der Skizzierung der bisherigen Forschung mit iPSC-basierten Modellen für sAD gibt der zweite Teil der Arbeit auch Einblicke in die Gewinnung krankheitsrelevanter Nervenkulturen auf Basis der gezielten Differenzierung von iPSCs und beinhaltet darüber hinaus einen experimentellen Ansatz für den Aufbau eines solchen Modellsystems. N2 - The research that is compiled in this thesis can be divided in two parts. The first part, consisting of four chapters, is centered around the role of epigenetic dysregulation in the etiopathophysiology of sporadic alzheimer's disease (sAD). In addition to providing insights into the most recent developments in neuroepigenomic studies of this disease, the first part of the thesis also touches upon remaining challenges, and provides a future outlook on possible developments in the field. The second part, which includes three more chapters, is focused on the application of induced pluripotent stem cell (iPSC)-based disease models for the study of AD, including but not limited to mechanistic studies on epigenetic dysregulation using this platform. Aside from outlining the research that has been conducted using iPSC-based models for sAD to date, the second part of the thesis also provides insights into the acquisition of disease-relevant neural cultures based on directed differentiation of iPSCs, and furthermore includes an experimental approach for the establishment of such a model system. KW - Epigenetik KW - Alzheimerkrankheit KW - Neuroepigenomics KW - Alzheimer's disease Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-254574 SN - 978-94-6423-524-1 ER - TY - THES A1 - Klein, Andreas T1 - Der altersabhängige Verlust der Geschlechtschromosomen beim Menschen unter Einwirkung von 5-Azadeoxycytidin T1 - Age related loss of human sex chromosomes induced by 5-azadeoxycytidine N2 - Die vorliegende Arbeit untersucht, ob mit zunehmendem Alter während der Mitose häufiger Geschlechtschromsomen verlorengehen. Die Beobachtungen erfolgten an Lymphozytenkulturen gesunder weiblicher und männlicher Probanden aus drei verschiedenen Altersgruppen. Unter Zugabe von 5-Azadeoxycytidin, einem Nukleosidanalogon, ergab sich in den höheren Altersgruppen ein verstärktes Auftreten von Mikronuklei. Mikronuklei enthalten Chromosomen oder -bruchstücke, die während der Mitose nicht in die Tochterzellkerne integriert wurden. Mittels in situ Hybridisierung konnte in den Mikronuklei der Frauen zu 5,5 Prozent ein X-Chromosom, bei den Männern mit 10,7 Prozent überzufällig häufig ein Y-Chromosom nachgewiesen werden. Zwischen den einzelnen Altersstufen änderte sich dieser Anteil nicht wesentlich. 5-Azadeoxycytidin wird als Nukleosidanalogon während der Replikation in die DNA eingebaut und verhindert die Methylierung des Tochterstrangs, da ein Kohlenstoffatom im Pyrimidinrings durch ein Stickstoffatom substituiert ist. Wahrscheinlich resultiert aus der Hyomethylierung eine falsche "Verpackung" des Gonosoms während der Mitose, dadurch erfolgt eine fehlerhafte Aufteilung des Chromosoms mit Bildung eines Mikronukleus. N2 - In lymphocyte cultures, the number of aneuploid cell nuclei increases with age. A preferential loss of sex chromosomes is supected. Lymphocyte cultures of female and male probands were treated with 5-azadeoxycytidine and an increased frequency of micronuclei in relation to age was observed. In situ hybridizations with X-specific and Y-specific DNA probes were carried out, and 5.5 per cent of female and 10.7 per cent of male micronuclei contained a sex chromosom. 5-azadeoxycytidine induces hypomethylation of the DNA and undercondensation in the heterochromatic regions of chromosomes 1, 9, 15, 16, and Y. The results suggest that in 5-azadeoxycytidine-treated cultures the Y-chromosome is lagged during mitosis, with subsequent chromosome loss via micronucleus formation. This mechanism may explain hypoploidy of sex chromosomes with ageing. KW - Altern KW - Geschlechtschromosomen KW - Chromosomenverlust KW - 5-Azadeoxycytidin KW - Mikronukleus KW - ageing KW - sex chromosomes KW - chromosome loss KW - 5-azadeoxycytidine micronucleus Y1 - 2001 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-327714 N1 - Dieses Dokument wurde aus Datenschutzgründen - ohne inhaltliche Änderungen - erneut veröffentlicht. Die ursprüngliche Veröffentlichung war am 12.08.2002 ER - TY - JOUR A1 - Pluta, Natalie A1 - Hoffjan, Sabine A1 - Zimmer, Frederic A1 - Köhler, Cornelia A1 - Lücke, Thomas A1 - Mohr, Jennifer A1 - Vorgerd, Matthias A1 - Nguyen, Hoa Huu Phuc A1 - Atlan, David A1 - Wolf, Beat A1 - Zaum, Ann-Kathrin A1 - Rost, Simone T1 - Homozygous inversion on chromosome 13 involving SGCG detected by short read whole genome sequencing in a patient suffering from limb-girdle muscular dystrophy JF - Genes N2 - New techniques in molecular genetic diagnostics now allow for accurate diagnosis in a large proportion of patients with muscular diseases. Nevertheless, many patients remain unsolved, although the clinical history and/or the muscle biopsy give a clear indication of the involved genes. In many cases, there is a strong suspicion that the cause must lie in unexplored gene areas, such as deep-intronic or other non-coding regions. In order to find these changes, next-generation sequencing (NGS) methods are constantly evolving, making it possible to sequence entire genomes to reveal these previously uninvestigated regions. Here, we present a young woman who was strongly suspected of having a so far genetically unsolved sarcoglycanopathy based on her clinical history and muscle biopsy. Using short read whole genome sequencing (WGS), a homozygous inversion on chromosome 13 involving SGCG and LINC00621 was detected. The breakpoint in intron 2 of SGCG led to the absence of γ-sarcoglycan, resulting in the manifestation of autosomal recessive limb-girdle muscular dystrophy 5 (LGMDR5) in the young woman. KW - inversion KW - sarcoglycanopathy KW - whole genome sequencing (WGS) KW - next generation sequencing (NGS) KW - LGMDR5 KW - muscle disease KW - genetic diagnostics Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-288122 SN - 2073-4425 VL - 13 IS - 10 ER - TY - JOUR A1 - Nanda, Indrajit A1 - Schröder, Sarah K. A1 - Steinlein, Claus A1 - Haaf, Thomas A1 - Buhl, Eva M. A1 - Grimm, Domink G. A1 - Weiskirchen, Ralf T1 - Rat hepatic stellate cell line CFSC-2G: genetic markers and short tandem repeat profile useful for cell line authentication JF - Cells N2 - Hepatic stellate cells (HSCs) are also known as lipocytes, fat-storing cells, perisinusoidal cells, or Ito cells. These liver-specific mesenchymal cells represent about 5% to 8% of all liver cells, playing a key role in maintaining the microenvironment of the hepatic sinusoid. Upon chronic liver injury or in primary culture, these cells become activated and transdifferentiate into a contractile phenotype, i.e., the myofibroblast, capable of producing and secreting large quantities of extracellular matrix compounds. Based on their central role in the initiation and progression of chronic liver diseases, cultured HSCs are valuable in vitro tools to study molecular and cellular aspects of liver diseases. However, the isolation of these cells requires special equipment, trained personnel, and in some cases needs approval from respective authorities. To overcome these limitations, several immortalized HSC lines were established. One of these cell lines is CFSC, which was originally established from cirrhotic rat livers induced by carbon tetrachloride. First introduced in 1991, this cell line and derivatives thereof (i.e., CFSC-2G, CFSC-3H, CFSC-5H, and CFSC-8B) are now used in many laboratories as an established in vitro HSC model. We here describe molecular features that are suitable for cell authentication. Importantly, chromosome banding and multicolor spectral karyotyping (SKY) analysis demonstrate that the CFSC-2G genome has accumulated extensive chromosome rearrangements and most chromosomes exist in multiple copies producing a pseudo-triploid karyotype. Furthermore, our study documents a defined short tandem repeat (STR) profile including 31 species-specific markers, and a list of genes expressed in CFSC-2G established by bulk mRNA next-generation sequencing (NGS). KW - liver KW - extracellular matrix KW - hepatic stellate cell KW - myofibroblast KW - fibrosis KW - stress fibers KW - spectral karyotyping KW - rhodamine–phalloidin stain KW - next-generation sequencing KW - STR profile Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-288067 SN - 2073-4409 VL - 11 IS - 18 ER - TY - JOUR A1 - Bauer, Benedikt A1 - Mally, Angela A1 - Liedtke, Daniel T1 - Zebrafish embryos and larvae as alternative animal models for toxicity testing JF - International Journal of Molecular Sciences N2 - Prerequisite to any biological laboratory assay employing living animals is consideration about its necessity, feasibility, ethics and the potential harm caused during an experiment. The imperative of these thoughts has led to the formulation of the 3R-principle, which today is a pivotal scientific standard of animal experimentation worldwide. The rising amount of laboratory investigations utilizing living animals throughout the last decades, either for regulatory concerns or for basic science, demands the development of alternative methods in accordance with 3R to help reduce experiments in mammals. This demand has resulted in investigation of additional vertebrate species displaying favourable biological properties. One prominent species among these is the zebrafish (Danio rerio), as these small laboratory ray-finned fish are well established in science today and feature outstanding biological characteristics. In this review, we highlight the advantages and general prerequisites of zebrafish embryos and larvae before free-feeding stages for toxicological testing, with a particular focus on cardio-, neuro, hepato- and nephrotoxicity. Furthermore, we discuss toxicokinetics, current advances in utilizing zebrafish for organ toxicity testing and highlight how advanced laboratory methods (such as automation, advanced imaging and genetic techniques) can refine future toxicological studies in this species. KW - danio rerio KW - alternative methods KW - organ toxicity KW - 3R KW - transgenic animals Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284225 SN - 1422-0067 VL - 22 IS - 24 ER - TY - JOUR A1 - Doll, Julia A1 - Kolb, Susanne A1 - Schnapp, Linda A1 - Rad, Aboulfazl A1 - Rüschendorf, Franz A1 - Khan, Imran A1 - Adli, Abolfazl A1 - Hasanzadeh, Atefeh A1 - Liedtke, Daniel A1 - Knaup, Sabine A1 - Hofrichter, Michaela AH A1 - Müller, Tobias A1 - Dittrich, Marcus A1 - Kong, Il-Keun A1 - Kim, Hyung-Goo A1 - Haaf, Thomas A1 - Vona, Barbara T1 - Novel loss-of-function variants in CDC14A are associated with recessive sensorineural hearing loss in Iranian and Pakistani patients JF - International Journal of Molecular Sciences N2 - CDC14A encodes the Cell Division Cycle 14A protein and has been associated with autosomal recessive non-syndromic hearing loss (DFNB32), as well as hearing impairment and infertile male syndrome (HIIMS) since 2016. To date, only nine variants have been associated in patients whose initial symptoms included moderate-to-profound hearing impairment. Exome analysis of Iranian and Pakistani probands who both showed bilateral, sensorineural hearing loss revealed a novel splice site variant (c.1421+2T>C, p.?) that disrupts the splice donor site and a novel frameshift variant (c.1041dup, p.Ser348Glnfs*2) in the gene CDC14A, respectively. To evaluate the pathogenicity of both loss-of-function variants, we analyzed the effects of both variants on the RNA-level. The splice variant was characterized using a minigene assay. Altered expression levels due to the c.1041dup variant were assessed using RT-qPCR. In summary, cDNA analysis confirmed that the c.1421+2T>C variant activates a cryptic splice site, resulting in a truncated transcript (c.1414_1421del, p.Val472Leufs*20) and the c.1041dup variant results in a defective transcript that is likely degraded by nonsense-mediated mRNA decay. The present study functionally characterizes two variants and provides further confirmatory evidence that CDC14A is associated with a rare form of hereditary hearing loss. KW - CDC14A KW - DFNB32 KW - autosomal recessive hearing loss KW - exome sequencing KW - splicing KW - frameshift KW - non-sense mediated mRNA decay Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-285142 SN - 1422-0067 VL - 21 IS - 1 ER - TY - JOUR A1 - Dumont, Martine A1 - Weber-Lassalle, Nana A1 - Joly-Beauparlant, Charles A1 - Ernst, Corinna A1 - Droit, Arnaud A1 - Feng, Bing-Jian A1 - Dubois, Stéphane A1 - Collin-Deschesnes, Annie-Claude A1 - Soucy, Penny A1 - Vallée, Maxime A1 - Fournier, Frédéric A1 - Lemaçon, Audrey A1 - Adank, Muriel A. A1 - Allen, Jamie A1 - Altmüller, Janine A1 - Arnold, Norbert A1 - Ausems, Margreet G. E. M. A1 - Berutti, Riccardo A1 - Bolla, Manjeet K. A1 - Bull, Shelley A1 - Carvalho, Sara A1 - Cornelissen, Sten A1 - Dufault, Michael R. A1 - Dunning, Alison M. A1 - Engel, Christoph A1 - Gehrig, Andrea A1 - Geurts-Giele, Willemina R. R. A1 - Gieger, Christian A1 - Green, Jessica A1 - Hackmann, Karl A1 - Helmy, Mohamed A1 - Hentschel, Julia A1 - Hogervorst, Frans B. L. A1 - Hollestelle, Antoinette A1 - Hooning, Maartje J. A1 - Horváth, Judit A1 - Ikram, M. Arfan A1 - Kaulfuß, Silke A1 - Keeman, Renske A1 - Kuang, Da A1 - Luccarini, Craig A1 - Maier, Wolfgang A1 - Martens, John W. M. A1 - Niederacher, Dieter A1 - Nürnberg, Peter A1 - Ott, Claus-Eric A1 - Peters, Annette A1 - Pharoah, Paul D. P. A1 - Ramirez, Alfredo A1 - Ramser, Juliane A1 - Riedel-Heller, Steffi A1 - Schmidt, Gunnar A1 - Shah, Mitul A1 - Scherer, Martin A1 - Stäbler, Antje A1 - Strom, Tim M. A1 - Sutter, Christian A1 - Thiele, Holger A1 - van Asperen, Christi J. A1 - van der Kolk, Lizet A1 - van der Luijt, Rob B. A1 - Volk, Alexander E. A1 - Wagner, Michael A1 - Waisfisz, Quinten A1 - Wang, Qin A1 - Wang-Gohrke, Shan A1 - Weber, Bernhard H. F. A1 - Devilee, Peter A1 - Tavtigian, Sean A1 - Bader, Gary D. A1 - Meindl, Alfons A1 - Goldgar, David E. A1 - Andrulis, Irene L. A1 - Schmutzler, Rita K. A1 - Easton, Douglas F. A1 - Schmidt, Marjanka K. A1 - Hahnen, Eric A1 - Simard, Jacques T1 - Uncovering the contribution of moderate-penetrance susceptibility genes to breast cancer by whole-exome sequencing and targeted enrichment sequencing of candidate genes in women of European ancestry JF - Cancers N2 - Rare variants in at least 10 genes, including BRCA1, BRCA2, PALB2, ATM, and CHEK2, are associated with increased risk of breast cancer; however, these variants, in combination with common variants identified through genome-wide association studies, explain only a fraction of the familial aggregation of the disease. To identify further susceptibility genes, we performed a two-stage whole-exome sequencing study. In the discovery stage, samples from 1528 breast cancer cases enriched for breast cancer susceptibility and 3733 geographically matched unaffected controls were sequenced. Using five different filtering and gene prioritization strategies, 198 genes were selected for further validation. These genes, and a panel of 32 known or suspected breast cancer susceptibility genes, were assessed in a validation set of 6211 cases and 6019 controls for their association with risk of breast cancer overall, and by estrogen receptor (ER) disease subtypes, using gene burden tests applied to loss-of-function and rare missense variants. Twenty genes showed nominal evidence of association (p-value < 0.05) with either overall or subtype-specific breast cancer. Our study had the statistical power to detect susceptibility genes with effect sizes similar to ATM, CHEK2, and PALB2, however, it was underpowered to identify genes in which susceptibility variants are rarer or confer smaller effect sizes. Larger sample sizes would be required in order to identify such genes. KW - breast cancer KW - genetic susceptibility KW - whole-exome sequencing KW - moderate-penetrance genes Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-281768 SN - 2072-6694 VL - 14 IS - 14 ER - TY - JOUR A1 - Rolfes, Muriel A1 - Borde, Julika A1 - Möllenhoff, Kathrin A1 - Kayali, Mohamad A1 - Ernst, Corinna A1 - Gehrig, Andrea A1 - Sutter, Christian A1 - Ramser, Juliane A1 - Niederacher, Dieter A1 - Horváth, Judit A1 - Arnold, Norbert A1 - Meindl, Alfons A1 - Auber, Bernd A1 - Rump, Andreas A1 - Wang-Gohrke, Shan A1 - Ritter, Julia A1 - Hentschel, Julia A1 - Thiele, Holger A1 - Altmüller, Janine A1 - Nürnberg, Peter A1 - Rhiem, Kerstin A1 - Engel, Christoph A1 - Wappenschmidt, Barbara A1 - Schmutzler, Rita K. A1 - Hahnen, Eric A1 - Hauke, Jan T1 - Prevalence of cancer predisposition germline variants in male breast cancer patients: results of the German Consortium for Hereditary Breast and Ovarian Cancer JF - Cancers N2 - Male breast cancer (mBC) is associated with a high prevalence of pathogenic variants (PVs) in the BRCA2 gene; however, data regarding other BC predisposition genes are limited. In this retrospective multicenter study, we investigated the prevalence of PVs in BRCA1/2 and 23 non-BRCA1/2 genes using a sample of 614 patients with mBC, recruited through the centers of the German Consortium for Hereditary Breast and Ovarian Cancer. A high proportion of patients with mBC carried PVs in BRCA2 (23.0%, 142/614) and BRCA1 (4.6%, 28/614). The prevalence of BRCA1/2 PVs was 11.0% in patients with mBC without a family history of breast and/or ovarian cancer. Patients with BRCA1/2 PVs did not show an earlier disease onset than those without. The predominant clinical presentation of tumor phenotypes was estrogen receptor (ER)-positive, progesterone receptor (PR)-positive, and HER2-negative (77.7%); further, 10.2% of the tumors were triple-positive, and 1.2% were triple-negative. No association was found between ER/PR/HER2 status and BRCA1/2 PV occurrence. Comparing the prevalence of protein-truncating variants (PTVs) between patients with mBC and control data (ExAC, n = 27,173) revealed significant associations of PTVs in both BRCA1 and BRCA2 with mBC (BRCA1: OR = 17.04, 95% CI = 10.54–26.82, p < 10\(^{−5}\); BRCA2: OR = 77.71, 95% CI = 58.71–102.33, p < 10\(^{−5}\)). A case-control investigation of 23 non-BRCA1/2 genes in 340 BRCA1/2-negative patients and ExAC controls revealed significant associations of PTVs in CHEK2, PALB2, and ATM with mBC (CHEK2: OR = 3.78, 95% CI = 1.59–7.71, p = 0.002; PALB2: OR = 14.77, 95% CI = 5.02–36.02, p < 10\(^{−5}\); ATM: OR = 3.36, 95% CI = 0.89–8.96, p = 0.04). Overall, our findings support the benefit of multi-gene panel testing in patients with mBC irrespective of their family history, age at disease onset, and tumor phenotype. KW - breast neoplasms KW - male breast cancer KW - breast cancer predisposition genes KW - genetic testing KW - familial breast cancer Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-281758 SN - 2072-6694 VL - 14 IS - 13 ER - TY - JOUR A1 - Nanda, Indrajit A1 - Steinlein, Claus A1 - Haaf, Thomas A1 - Buhl, Eva M. A1 - Grimm, Domink G. A1 - Friedman, Scott L. A1 - Meurer, Steffen K. A1 - Schröder, Sarah K. A1 - Weiskirchen, Ralf T1 - Genetic characterization of rat hepatic stellate cell line HSC-T6 for in vitro cell line authentication JF - Cells N2 - Immortalized hepatic stellate cells (HSCs) established from mouse, rat, and humans are valuable in vitro models for the biomedical investigation of liver biology. These cell lines are homogenous, thereby providing consistent and reproducible results. They grow more robustly than primary HSCs and provide an unlimited supply of proteins or nucleic acids for biochemical studies. Moreover, they can overcome ethical concerns associated with the use of animal and human tissue and allow for fostering of the 3R principle of replacement, reduction, and refinement proposed in 1959 by William M. S. Russell and Rex L. Burch. Nevertheless, working with continuous cell lines also has some disadvantages. In particular, there are ample examples in which genetic drift and cell misidentification has led to invalid data. Therefore, many journals and granting agencies now recommend proper cell line authentication. We herein describe the genetic characterization of the rat HSC line HSC-T6, which was introduced as a new in vitro model for the study of retinoid metabolism. The consensus chromosome markers, outlined primarily through multicolor spectral karyotyping (SKY), demonstrate that apart from the large derivative chromosome 1 (RNO1), at least two additional chromosomes (RNO4 and RNO7) are found to be in three copies in all metaphases. Additionally, we have defined a short tandem repeat (STR) profile for HSC-T6, including 31 species-specific markers. The typical features of these cells have been further determined by electron microscopy, Western blotting, and Rhodamine-Phalloidin staining. Finally, we have analyzed the transcriptome of HSC-T6 cells by mRNA sequencing (mRNA-Seq) using next generation sequencing (NGS). KW - liver KW - extracellular matrix KW - hepatic stellate cell KW - myofibroblast KW - fibrosis KW - in vitro model KW - SKY analysis KW - phalloidin stain KW - next generation sequencing KW - STR profile Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-275178 SN - 2073-4409 VL - 11 IS - 11 ER - TY - JOUR A1 - Prell, Andreas A1 - Sen, Mustafa Orkun A1 - Potabattula, Ramya A1 - Bernhardt, Laura A1 - Dittrich, Marcus A1 - Hahn, Thomas A1 - Schorsch, Martin A1 - Zacchini, Federica A1 - Ptak, Grazyna Ewa A1 - Niemann, Heiner A1 - Haaf, Thomas T1 - Species-specific paternal age effects and sperm methylation levels of developmentally important genes JF - Cells N2 - A growing number of sperm methylome analyses have identified genomic loci that are susceptible to paternal age effects in a variety of mammalian species, including human, bovine, and mouse. However, there is little overlap between different data sets. Here, we studied whether or not paternal age effects on the sperm epigenome have been conserved in mammalian evolution and compared methylation patterns of orthologous regulatory regions (mainly gene promoters) containing both conserved and non-conserved CpG sites in 94 human, 36 bovine, and 94 mouse sperm samples, using bisulfite pyrosequencing. We discovered three (NFKB2, RASGEF1C, and RPL6) age-related differentially methylated regions (ageDMRs) in humans, four (CHD7, HDAC11, PAK1, and PTK2B) in bovines, and three (Def6, Nrxn2, and Tbx19) in mice. Remarkably, the identified sperm ageDMRs were all species-specific. Most ageDMRs were in genomic regions with medium methylation levels and large methylation variation. Orthologous regions in species not showing this age effect were either hypermethylated (>80%) or hypomethylated (<20%). In humans and mice, ageDMRs lost methylation, whereas bovine ageDMRs gained methylation with age. Our results are in line with the hypothesis that sperm ageDMRs are in regions under epigenomic evolution and may be part of an epigenetic mechanism(s) for lineage-specific environmental adaptations and provide a solid basis for studies on downstream effects in the genes analyzed here. KW - age-related differentially methylated regions (ageDMRs) KW - bisulfite pyrosequencing KW - mammalian male germline KW - paternal age effect KW - species-specific epigenetic marks KW - sperm DNA methylation Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-262301 SN - 2073-4409 VL - 11 IS - 4 ER - TY - JOUR A1 - Wagenhäuser, Laura A1 - Rickert, Vanessa A1 - Sommer, Claudia A1 - Wanner, Christoph A1 - Nordbeck, Peter A1 - Rost, Simone A1 - Üçeyler, Nurcan T1 - X-chromosomal inactivation patterns in women with Fabry disease JF - Molecular Genetics & Genomic Medicine N2 - Background Although Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations in the α-galactosidase A gene (GLA), women may develop severe symptoms. We investigated X-chromosomal inactivation patterns (XCI) as a potential determinant of symptom severity in FD women. Patients and Methods We included 95 women with mutations in GLA (n = 18 with variants of unknown pathogenicity) and 50 related men, and collected mouth epithelial cells, venous blood, and skin fibroblasts for XCI analysis using the methylation status of the androgen receptor gene. The mutated X-chromosome was identified by comparison of samples from relatives. Patients underwent genotype categorization and deep clinical phenotyping of symptom severity. Results 43/95 (45%) women carried mutations categorized as classic. The XCI pattern was skewed (i.e., ≥75:25% distribution) in 6/87 (7%) mouth epithelial cell samples, 31/88 (35%) blood samples, and 9/27 (33%) skin fibroblast samples. Clinical phenotype, α-galactosidase A (GAL) activity, and lyso-Gb3 levels did not show intergroup differences when stratified for X-chromosomal skewing and activity status of the mutated X-chromosome. Conclusions X-inactivation patterns alone do not reliably reflect the clinical phenotype of women with FD when investigated in biomaterial not directly affected by FD. However, while XCI patterns may vary between tissues, blood frequently shows skewing of XCI patterns. KW - Fabry disease KW - Fabry genotype KW - Fabry phenotype KW - female Fabry patients KW - X-chromosomal inactivation Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-312795 VL - 10 IS - 9 ER - TY - JOUR A1 - Weber-Lassalle, Nana A1 - Hauke, Jan A1 - Ramser, Juliane A1 - Richters, Lisa A1 - Groß, Eva A1 - Blümcke, Britta A1 - Gehrig, Andrea A1 - Kahlert, Anne-Karin A1 - Müller, Clemens R. A1 - Hackmann, Karl A1 - Honisch, Ellen A1 - Weber-Lassalle, Konstantin A1 - Niederacher, Dieter A1 - Borde, Julika A1 - Thiele, Holger A1 - Ernst, Corinna A1 - Altmüller, Janine A1 - Neidhardt, Guido A1 - Nürnberg, Peter A1 - Klaschik, Kristina A1 - Schroeder, Christopher A1 - Platzer, Konrad A1 - Volk, Alexander E. A1 - Wang-Gohrke, Shan A1 - Just, Walter A1 - Auber, Bernd A1 - Kubisch, Christian A1 - Schmidt, Gunnar A1 - Horvath, Judit A1 - Wappenschmidt, Barbara A1 - Engel, Christoph A1 - Arnold, Norbert A1 - Dworniczak, Bernd A1 - Rhiem, Kerstin A1 - Meindl, Alfons A1 - Schmutzler, Rita K. A1 - Hahnen, Eric T1 - BRIP1 loss-of-function mutations confer high risk for familial ovarian cancer, but not familial breast cancer JF - Breast Cancer Research N2 - Background Germline mutations in the BRIP1 gene have been described as conferring a moderate risk for ovarian cancer (OC), while the role of BRIP1 in breast cancer (BC) pathogenesis remains controversial. Methods To assess the role of deleterious BRIP1 germline mutations in BC/OC predisposition, 6341 well-characterized index patients with BC, 706 index patients with OC, and 2189 geographically matched female controls were screened for loss-of-function (LoF) mutations and potentially damaging missense variants. All index patients met the inclusion criteria of the German Consortium for Hereditary Breast and Ovarian Cancer for germline testing and tested negative for pathogenic BRCA1/2 variants. Results BRIP1 LoF mutations confer a high OC risk in familial index patients (odds ratio (OR) = 20.97, 95% confidence interval (CI) = 12.02–36.57, P < 0.0001) and in the subgroup of index patients with late-onset OC (OR = 29.91, 95% CI = 14.99–59.66, P < 0.0001). No significant association of BRIP1 LoF mutations with familial BC was observed (OR = 1.81 95% CI = 1.00–3.30, P = 0.0623). In the subgroup of familial BC index patients without a family history of OC there was also no apparent association (OR = 1.42, 95% CI = 0.70–2.90, P = 0.3030). In 1027 familial BC index patients with a family history of OC, the BRIP1 mutation prevalence was significantly higher than that observed in controls (OR = 3.59, 95% CI = 1.43–9.01; P = 0.0168). Based on the negative association between BRIP1 LoF mutations and familial BC in the absence of an OC family history, we conclude that the elevated mutation prevalence in the latter cohort was driven by the occurrence of OC in these families. Compared with controls, predicted damaging rare missense variants were significantly more prevalent in OC (P = 0.0014) but not in BC (P = 0.0693) patients. Conclusions To avoid ambiguous results, studies aimed at assessing the impact of candidate predisposition gene mutations on BC risk might differentiate between BC index patients with an OC family history and those without. In familial cases, we suggest that BRIP1 is a high-risk gene for late-onset OC but not a BC predisposition gene, though minor effects cannot be excluded. KW - breast cancer KW - ovarian cancer KW - BRIP1 gene KW - germline mutations Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-233433 VL - 20 ER - TY - JOUR A1 - König, Kirsten A1 - Pechmann, Astrid A1 - Thiele, Simone A1 - Walter, Maggie C. A1 - Schorling, David A1 - Tassoni, Adrian A1 - Lochmüller, Hanns A1 - Müller-Reible, Clemens A1 - Kirschner, Janbernd T1 - De-duplicating patient records from three independent data sources reveals the incidence of rare neuromuscular disorders in Germany JF - Orphanet Journal of Rare Diseases N2 - Background Estimation of incidence in rare diseases is often challenging due to unspecific and incomplete coding and recording systems. Patient- and health care provider-driven data collections are held with different organizations behind firewalls to protect the privacy of patients. They tend to be fragmented, incomplete and their aggregation leads to further inaccuracies, as the duplicated records cannot easily be identified. We here report about a novel approach to evaluate the incidences of Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA) in Germany. Methods We performed a retrospective epidemiological study collecting data from patients with dystrophinopathies (DMD and Becker muscular dystrophy) and SMA born between 1995 and 2018. We invited all neuromuscular centers, genetic institutes and the patient registries for DMD and SMA in Germany to participate in the data collection. A novel web-based application for data entry was developed converting patient identifying information into a hash code. Duplicate entries were reliably allocated to the distinct patient. Results We collected 5409 data entries in our web-based database representing 1955 distinct patients with dystrophinopathies and 1287 patients with SMA. 55.0% of distinct patients were found in one of the 3 data sources only, while 32.0% were found in 2, and 13.0% in all 3 data sources. The highest number of SMA patients was reported by genetic testing laboratories, while for DMD the highest number was reported by the clinical specialist centers. After the removal of duplicate records, the highest yearly incidence for DMD was calculated as 2.57:10,000 in 2001 and the highest incidence for SMA as 1.36:10,000 in 2014. Conclusion With our novel approach (compliant with data protection regulations), we were able to identify unique patient records and estimate the incidence of DMD and SMA in Germany combining and de-duplicating data from patient registries, genetic institutes, and clinical care centers. Although we combined three different data sources, an unknown number of patients might not have been reported by any of these sources. Therefore, our results reflect the minimal incidence of these diseases. KW - incidence KW - neuromuscular disease KW - spinal muscular atrophy KW - duchenne muscular dystrophy Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-222807 VL - 14 ER -