rolduc meeting. feel connected!
rolduc meeting. feel connected! The annual Genetics Retreat … aims to inform scientists about the current status of research, novel scientific developments and technological innovations in the fields of human and clinical genetics, molecular and translational genetics and epigenetics. offers an excellent scientific communication platform specifically for junior researchers to gain proficiency in discussing their ongoing research and to expand their presentation skills in an informal, professional setting. Besides scientific presentations, there is ample opportunity to discuss research projects among peers and with principal investigators in the field. is open to junior researchers (PhD students and postdocs), their supervisors and all other scientists from universities, clinical institutions and research centres in the Netherlands and neighboring countries. 2 Genetics Retreat 2015 25th edition 22 | 23 | 24 april 2015 kerkrade table of content feel connected!5 A short history6 25 years of Genetics Retreat7 From pencil sketch to logo9 venue10 Organization 11 sponsors12 programme wednesday16 programme thursday18 programme friday20 HONORARY GUEST SPEAKER22 HONORARY GUEST SPEAKER26 abstracts28 4 Genetics Retreat 2015 feel connected! There are many good reasons for the organizers to choose the theme: ‘feel connected’. First and foremost it symbolizes our aim to make all participants at the Retreat feel part of the ever expanding human genetics family. This family comprises ‘veterans’, but also many new PhD students from the different centres in the Netherlands and, this year for the first time, from across the borders in Belgium and Germany. In addition, we aim to bring the research communities in the lab and in the clinic closer to each other, i.e. to connect bench and bed side. Last but not least, the Rolduc Genetics Retreat is a place where the academic research community meets industry. To feel connected means to exchange novel scientific findings, to discuss novel ideas and outlooks, and, equally important, to meet peers in a friendly, constructive atmosphere. We wish you all a pleasant stay and a fruitful meeting and we hope that the Rolduc Genetics Retreat makes everyone feel connected to the common cause of furthering our understanding of human disease for the benefit of patient care. Genetics Retreat 2015 5 A short history of the Rolduc Genetics Retreat The Genetics Retreat at Rolduc was first held in 1990. In those days, most NWO grants supported PhD projects; the PhD students were required to provide yearly progress reports. In the late eighties, four human genetics research communities in the Netherlands decided to jointly organize these yearly sessions. Both the organization as well as the meeting venue alternated. In 1990 Ruud Schuurman, PhD (University Leiden) and professor Joep Geraedts (Maastricht University) opted for the newly established conference centre at the former Augustinian abbey of Rolduc (Kerkrade, the Netherlands). Because of its unique atmosphere the meeting was so successful that it was decided to return to this site from then on, and thus a tradition was born: the yearly Genetics Retreat at Rolduc. Genetica Retraite Genetica Retraite 19-20 March 2009 willem ROLDUCK church 6 Genetics Retreat 2015 FOUNDER JOEP porter SJANG 25 years of Genetics Retreat Somewhere during the past 25 years, the meeting was moved onward from autumn, the original Rolduc meeting season, to spring. The reason for this change was a practical one: overcrowding of November with meetings and conferences. For those readers that have been doing the math: this explains why only 25 meetings have been organized between 1990 and 2015. Over the years the NWO granting system has changed and the mandatory yearly progress meetings became facultative. The Rolduc Genetics Retreat, 6-8 March 2008 however, remained the meeting event in human genetics for Dutch PhD students and their supervisors. To celebrate the 25th Genetics Retreat anniversary, besides to the eight university medical centres, invitations have been extended to colleagues from centres relatively close to the Dutch borders in Germany and Belgium. Genetics Retreat 2015 7 8 Genetics Retreat 2015 From pencil sketch to logo The organization of the Genetics Retreat proudly presents the new logo, designed by Guus van Rooy. writing catchwords puzzle pieces green pea DNA double helix Mendel an Augustinian monk an association of ideas join together alterations deleting crossing out starting all over again covering with a paper focus on a detail old-fashioned sketching with a pencil transforming to Illustrator and Indesign further sketching on the monitor judge put aside sliding pinching staring gazing into the distance piddling hanging up a print asking family members feedback making some small changes finalizing Genetics Retreat 2015 9 Venue Conference centre Rolduc history Heyendahllaan 82 6464 EP Kerkrade T + 31 45 5466888 [email protected] 10 Genetics Retreat 2015 ROLDUC Organization Maastricht UMC+ in cooperation with NVHG NVHG Genetics & Cell Biology PO Box 5800 6202 AZ Maastricht T + 31 43 3875899 [email protected] organization MUMC+ Genetics Retreat 2015 11 We thank our sponsors for their generous contribution beckmancoulter QIAGEN eppendorf illumina perkinelmer ve to be esn’t ha o d is s o the often “Diagn fforts in mpaign e a c d l n a a n n o ease! entio ternati r your att a rare dis ed the in fo h m c g o n fr in u k g la s n a are sufferi ng, Shire ands we patients This spri ease are e Netherl nosis for th g ia in d t re Fabry dis c e h e H it rr . w o ” s. c re ts a n ra h for ymptom f patie pecific s ing searc ly 25% o -s n te o a n im f x o challeng nge Appro increase a wide ra difficult. ortant to eases is ts having is n e d 1,2 It is im p ti with rare a re p ra e is of patients to thes gnosis. to e ia y u d tl ct d c t Diagnos e c d ir e e os . 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Availa in Europ y 2015. ar nu Ja accessed rnati Shire Inte /C-A Ref.: NL NPROM /ELA/15/ 15 arch 20 0010 - M y EN.indd Brief Fabr 14 19-03-15 1 Genetics Retreat 2015 12:44 Genetics Retreat 2015 15 WEDNESDAY 22 APRIL 2015 10.00 - 10.50 Registration and Welc OPENING MEETING (conference room 2) 10.50 - 11.00 Willem Voncken GENOME ANALYSIS (conference room 2) 11.00 - 11.15 11.15 - 11.30 11.30 - 11.45 11.45 - 12.00 Thomas Eggermann (Aachen) Yasmijn van Herwaarden Tessa van Dijk Joyce Gietel-Habets Simon Ardui Maastricht UMC+ Welcome and Openin Radboudumc Nijmegen AMC Amsterdam Maastricht UMC+ KU Leuven WES provides novel in Mosaic uniparental is Motives, consideratio Single Molecule Sequ 12.00 - 13.00 CLINICAL & TRANSLATIONAL GENETICS (conference room 2) 13.00 - 13.15 13.15 - 13.30 13.30 - 13.45 13.45 - 14.00 14.00 - 14.15 Lunch (Foyer) Frank Baas (Amsterdam) & Hilde Van Esch (Leuven) Bart Appelhof Minh Nguyen Tom Theunissen Mirjam de Pagter Sarah Hempenstall AMC Amsterdam Maastricht UMC+ Maastricht UMC+ UMC Utrecht LUMC Leiden 14.15 - 14.45 WES in a cohort of PC Whole exome sequen Unraveling the geneti Chromothripsis in hea A novel assay for prot Coffee / Tea Break (Fo 14.45 - 15.00 15.00 - 15.15 15.15 - 15.30 15.30 - 15.45 Daniel Kofink Sietske Kevelam Glen Monroe Elke Mersy UMC Utrecht VU Amsterdam UMC Utrecht Maastricht UMC+ 15.45 - 16.00 DISCOVERIES & FUNCTIONAL METHODS (conference room 2) 16.00 - 16.15 16.15 - 16.30 16.30 - 16.45 16.45 - 17.00 17.00 - 17.15 Loss of chromosome PLP1 mutations affect Monocarboxylate Tra Non-invasive detectio Short Break Joep Geraedts (Maastricht) & Annelies de Klein (Rotterdam) Nathalie Fieremans Robbert Weren Serdar Yavuzyigitoglu Romy Mesman Maarten Massink KU Leuven Radboudumc Nijmegen Erasmus MC Rotterdam LUMC Leiden UMC Utrecht 17.15 - 17.30 Identification of intel Identification of a nov BAP1 correlates with m Functional analysis of Proper genomic profi Short Break LECTURE, TIPS AND HINTS (conference room 2) For PhD students and young postdocs 17.30 - 18.00 Joris Veltman Radboudumc Nijmegen 18.00 - 19.30 How to obtain grants Dinner (Grote Eetzaa KEYNOTE LECTURE (Aula Minor) 19.30 - 20.30 20.30 - 24.00 16 Genetics Retreat 2015 Christiane Nüsslein-Volhard Tübingen, Germany The development of c Social Evening (Boere come with Limburgse vlaai in Foyer ng meeting nsights regarding germline aberrations in a family with an autosomal dominant inherited serrated polyposis phenotype sodisomy of chromosome 15q in an atypical PCH patient ons and experiences with preimplantation genetic diagnosis uencing of the FMR1 CGG Repeat CH patient: a PCH7 candidate gene ncing: discovering genetic causes of mitochondrial disorders ic and pathophysiological basis of mitochondrial disorders althy individuals affects multiple protein-coding genes and can result in severe congenital abnormalities in offspring teasomal activity as a marker of myogenic dysfunction oyer) Y and carotid atherosclerosis severity in men ting PLP1/DM20 alternative splicing causes Hypomyelination of Early Myelinating Structures ansporter 1 Deficiency and Ketone Utilization on of fetal sex by simultaneous amplification of Y chromosome DNA and trophoblast-specific RNA llectual disability genes in female patients with skewed X-inactivation vel adenomatous polyposis and colorectal cancer predisposing gene metastasis in polyploid uveal melanoma f variants of uncertain significance in BRCA2 iling of (BRCA1-mutated) basal-like breast carcinomas requires prior removal of tumor infiltrating lymphocytes s in a competitive research environment? al) colour patterns in fishes. Towards an understanding of the evolution of beauty en Kelder) Genetics Retreat 2015 17 THURSDAY 23 APRIL 2015 until 08.30 Breakfast (Grote Eetz CARDIOGENETICS & MOLECULAR CARDIOLOGY (conference room 2) 08.45 - 09.00 09.00 - 09.15 09.15 - 09.30 Joris Veltman (Nijmegen) Daiane Hemerich Robin Verjans Mark Hazebroek UMC Utrecht Maastricht UMC+ Maastricht UMC+ Integrating data sour Identification of Micr Genetics of Dilated ca MOLECULAR NEUROLOGY (conference room 2) 09.30 - 09.45 09.45 - 10.00 10.00 - 10.15 10.15 - 10.30 10.30 - 10.45 Jo Vanoevelen (Maastricht) & Dorien Peters (Leiden) Roeland Vanhauwaert Marijke Versteven Kiliana Bekelaar Liesbeth Zwarts Ivo Eijkenboom KU Leuven KU Leuven KU Leuven KU Leuven Maastricht UMC+ Generation of a nove Agonistic sound stim Pharmacogenetics of Glial Notch regulates A read-out panel refle 10.45 - 11.15 Coffee / Tea Break (Fo DEVELOPMENTAL & CANCER GENETICS (conference room 2) 11.15 - 11.30 11.30 - 11.45 11.45 - 12.00 12.00 - 12.15 12.15 - 12.30 Alexander Hoischen (Nijmegen) & Gerard te Meerman (Groningen) Hester Happé Alessio Marcozzi Mike Jeurissen Mandy Steinbusch Machteld Oud LUMC Leiden UMC Utrecht Maastricht UMC+ Maastricht UMC+ Radboudumc Nijmegen 12.30 - 13.15 Gene inactivation at d Engineering chromos Hematopoietic overe The RMRP snoRNA an Endocrine-cerebro-os Lunch (Foyer) DEBATE ACROSS BORDERS (conference room 1) Joep Geraedts (Maastricht), Guido de Wert (Maastricht), Lidewij Henneman (Amsterdam), Klaus Zerres (Aachen), Pascal Borry (Leuven) 13.15 - 15.15 Debate Genetics, Eth 15.30 - 17.30 Jubilee Programme 18.00 - 20.00 Walking dinner / Pitch KEYNOTE LECTURE (Aula Minor) 20.00 - 21.00 21.00 - 24.00 18 Genetics Retreat 2015 Johan Braeckman Ghent University, Belgium Why people are extre Social Evening; drinks zaal) rces in druggability analysis of genes implicated in dilated cardiomyopathy roRNAs Regulating Heart Failure: A Phenotypical High-Throughput Screening Approach ardiomyopathy el Drosophila melanogaster Parkinson’s disease model mulates Drosophila male- male aggression f lithium and valproate in Drosophila melanogaster s Drosophila behavior by maintaining adult brain homeostasis ecting small fiber neuropathy in zebrafish oyer) different ages in an inducible kidney specific Pkd1 Knockout model results in multiple models with different characteristics somal translocations using the CRISPR/Cas system expression of Cyp27a1 reduces hepatic inflammation independently of 27-hydroxycholesterol levels in LDL-r-/- mice. nd chondrogenic differentiation steodysplasia syndrome is a ciliary disorder hics & Societies hes / Poster Margo Eijck-Vievermans (Foyer) emely gullible s by Perkin Elmer (Verloren Zoon) Genetics Retreat 2015 19 FRIDAY 24 APRIL 2015 until 08.30 Breakfast (Grote Eetz 08.30 - 08.45 08.45 - 09.00 Peter Leegwater Lambert Dorssers Utrecht University Erasmus MC Rotterdam A nonsense mutation Deciphering malignan GENES & ENVIRONMENT (conference room 2) 09.00 - 09.15 09.15 - 09.30 09.30 - 09.45 09.45 - 10.00 Colin Logie (Nijmegen) & Dineke Verbeek (Groningen) Jolien Vanhove Xiaoqing Zhu Zuzanna Borek Tom Houben KU Leuven Maastricht UMC+ UMC Groningen Maastricht UMC+ Active and repressive Novel insights in AMP Dynamic expression p Storage solutions: tar 10.00 - 10.30 COMPLEX GENETICS (conference room 2) 11.00 - 11.15 11.15 - 11.30 11.30 - 11.45 11.45 - 12.00 12.00 - 12.15 12.15 - 12.30 Coffee / Tea Break (Fo Maurice Zeegers (Maastricht) Nadia Roumans Maria Magdalena Zorro Ivan Brankovic Martin Singer Vasiliki Matzaraki Jan Henk Dubbink Maastricht UMC+ UMC Groningen Maastricht UMC+ VU Amsterdam UMC Groningen VU Amsterdam 12.30 - 13.30 Lunch (Grote Eetzaal) 13.30 - 13.45 20 Sex-specific variation Celiac disease associa Do NOD1 and NOD2 Host genetic variation Systems approach to The role of polymorph Joep Geraedts Genetics Retreat 2015 Maastricht UMC+ Award Ceremony zaal) n in stillborn Friesian horses with hydrocephalus classifies the disorder as muscular dystrophy-dystroglycanopathy nt testicular germ cell cancer progression e histone marking during stem cell-derived hepatocytes in vitro PK function: AMPK-MKNK1 connection profile of lncRNA genes upon stimulation of human intestinal gluten-specific T-cells rgeting disturbed lysosomes to improve hepatic inflammation oyer) n in extracellular matrix genes is associated with weight regain and maintenance after weight loss ated genes are involved in intestinal barrier function functional polymorphisms influence susceptibility to Chlamydia trachomatis infection and the risk of tubal factor infertility? n in innate immunity genes cause both protective and risk enhancing effects on the outcome of Haemophilius ducreyi infections Candida infection hisms on the susceptibility and clinical manifestations of sexual transmitted infections in South African women ) Genetics Retreat 2015 21 Guest speaker Christiane Nüsslein-Volhard Christiane Nüsslein-Volhard studied biology, chemistry and physics in Frankfurt am Main from 1962 to 1964. At the university of Tübingen she received her diploma in biochemistry in 1968 and her PhD (genetics) in 1973. She worked as a postdoc subsequently in the laboratories of Prof. Dr. Walter Gehring at the Biozentrum in Basel (Switzerland) and of Prof. Dr. Klaus Sander at the university of Freiburg. Thereafter she was appointed group leader at the European Molecular Biology Laboratory (EMBL) at Heidelberg from 1978 to 1980 and at the Friedrich-Miescher-Labor (FML) of the Max-Planck-Gesellschaft in Tübingen from 1981 to 1985. Since 1985 she is scientific member of the Max-Planck-Society and director at the Max Planck Institute of Developmental Biology at Tübingen and since 1989 honorary professor at the university of Tübingen. Her research interests concern the molecular and genetic analysis of development. The research presently focuses on pattern formation, growth and cell migration in the zebrafish, a new vertebrate model organism. For the discovery of genes that control development in animals and humans, and the demonstration of morphogen gradients in the fly embryo she received a number of awards and honours, among others the Albert Lasker Medical Research Award (New York/USA), the Prix Louis Jeantet de Médecine (Geneva/Switzerland), the Ernst Schering Price (Berlin/Germany), and in 1995 the Nobel Price for Medicine or Physiology together with Eric Wieschaus and Edward Lewis. 22 Genetics Retreat 2015 She is recipient of honorary degrees of the Yale, Harvard, Princeton and Rockefeller Universities (USA), the University of Utrecht (the Netherlands), the University College London, the Universities of Oxford, Sheffield and St. Andrews (UK), the Ochanomizu University (Tokyo/Japan), the Universities of Freiburg and Munich (Germany), the University of Geneva (Switzerland) as well as the Weizmann Institute (Rehovot/Israel). She was secretary general of the EMBO until 2009, president of the Gesellschaft deutscher Naturforscher und Ärzte (GDNÄ) in 2008, member of the National Ethics Council of Germany from 2001 to 2007 and member of the Scientific Council of the European Research Council (ERC) (2007-2012). She is member of the Royal Society (London/UK), the National Academy of Sciences (Washington/USA), the Order Pour le Mérite (Germany), the Deutsche Akademie Leopoldina (Halle/Germany), the Berlin-Brandenburgische Akademie der Wissenschaften (Germany), the Kurie der Wissenschaft (Vienna/Austria), the Académie des Sciences (Paris/France), as well as of several advisory boards and committees. She is chancellor of the Order Pour le merite (2013-2017). In order to support women with children in science she founded the Christiane-Nüsslein-Volhard-Stiftung. She is interested in music (voice, flute), literature, gardening and cooking. Genetics Retreat 2015 23 Christiane Nüsslein-Volhard The development of colour patterns in fishes. Towards an understanding of the evolution of beauty METHODS Colour patterns are prominent features of many animals and have important functions in communication such as camouflage, kin recognition and mate selection. As targets for natural as well as sexual selection, they are of high evolutionary significance. The molecular mechanisms underlying colour pattern formation in vertebrates are not well understood, despite their amazing diversity between closely related species. Progress in the transgenic toolkit, in vivo imaging and the availability of a large collection of mutants make the zebrafish (Danio rerio) an attractive model to study vertebrate colouration. Zebrafish display golden and blue horizontal stripes composed during metamorphosis as mosaics of yellow xanthophores, silvery or blue iridophores and black melanophores in the hypodermis. Mutant analysis indicated that interactions between all three pigment cell types are required for the formation of the pattern, and a number of cell surface molecules and signalling systems have been identified as mediators of these interactions. Lineage tracing revealed the origin of the adult pigment cells 24 Genetics Retreat 2015 and their individual cellular behaviour during formation of the striped pattern during metamorphosis. The understanding of the mechanisms that underlie colour pattern formation is an important step towards comprehending the genetic basis of variation in the evolution of bio-diversity. Max-Planck-Institut für Entwicklungsbiologie, 72076 Tübingen, Germany Review Singh, A. P. and Nüsslein-Volhard, C. (2015): Zebrafish stripes as a Model for Vertebrate Colour Pattern Formation. Current Biology, 25, R81-R92 Foto: Appie Derks Genetics Retreat 2015 25 Guest speaker Johan Braeckman Johan Braeckman (°1965) has a Masters degree in Philosophy (Ghent University, 1989) and Human Ecology (Free University of Brussels, 1990). He also studied Environmental History and Human Ecology at the University of California, Santa Barbara. Braeckman has been affiliated as a researcher to the Department of Philosophy at Ghent University since 1990. In 1997, he obtained a PhD in Philosophy with a doctoral dissertation on philosophical aspects of Darwin’s evolutionary theory. In 1998, he was appointed as full time professor/lecturer of Philosophy. His main area’s of interest are ethical and philosopical aspects concerning science and technology, and pseudoscience and irrationalism. From 2003-2008 he was also ‘Socrates professor’ at Amsterdam University. Braeckman is (co-)author, contributor or editor of several books and articles, columns and reviews. He has published scientific articles and reviews in The Journal of Personality and Social Psychology, Journal of Medical Ethics, Journal of Cross-Cultural Psychology, Xenotransplantation, Personal Relationships, Journal of Theoretical Biology, a.o. For a list of his articles and books, and for more information about his research and scientific activities, see his website at: www.johanbraeckman.be. 26 Genetics Retreat 2015 Why people are extremely gullible Most people think they know how are vulnerable for mental infection to think rationally. Most of us believe with bad, plain stupid, pseudo-scien- we have good reasons and arguments tific and superstitious ideas. In this for our beliefs, and we tend to be lecture we discuss the psychology sceptical about belief systems that are behind our gullibility and naïvité. exotic or strange from our perspec- We discuss several examples to tive. In other words, we think that we illustrate the point that our brain are able to think critically and reason- can rapidly and easily be fooled (or able. It seems easy enough, from an rather: fool itself), and we offer ways intuitive point of view. Nevertheless, to enhance our critical thinking skills. research shows that clear and critical thinking is much harder than we presume. Some people are so critical that they reject everything that science has to offer. Clearly, something there went wrong. But all of us Genetics Retreat 2015 27 Yasmijn van Herwaarden Whole-exome sequencing provides novel insights regarding germline aberrations in a family with an autosomal dominant inherited serrated polyposis phenotype In serrated polyposis syndrome (SPS), characterized by the development of serrated polyps in the colon, an association with heritable germline aberrations is suspected . However, high-penetrant germline aberrations which predispose to the development of SPS have not been identified yet. Identification of the genomic defects underlying this clinical phenotype is crucial for clinical management since SPS patients are at high risk to develop colorectal cancer. We describe a large family, showing autosomal dominant inheritance of SPS, and aim to identify the causative germline mutation underlying the SPS phenotype. 28 METHODS RESULTS We applied whole-exome sequencing (WES) on the DNA of six affected family members. Variants which are frequently encountered in controls (minor allele frequency (MAF) >0.01) or in non-coding genomic regions were excluded. We screened for pathogenic germline variants in six genes which previously have been associated with the development of multiple sessile serrated adenomas (SSA/P) (1) and we focused on all rare (MAF <0.01) germline variants shared between the six affected family members. Co-segregation analyses were performed for the remaining variants and RNA expression levels as well as possible allele specific expression bias were determined using in EBV-transformed B-lymphocytes derived from affected and unaffected family members. In our index family we encountered truncating variants in two genes previously described; a nonsense and frameshift mutation in PIF1 and RBL1, respectively. Both variants did not co-segregate with the development of serrated polyps in the family and the frequency of the germline variant in PIF1 was high in our control dataset (N=2,329; MAF 0.0167). Thirty variants were shared between all six exomes of which twenty-nine variants were excluded because of a high MAF (>0,01) in 3 additional control databases (n=16) or because they did not cosegregate with the polyposis phenotype in the family (n=13). One missense variant (p.E333K) in the BCAT1 gene remained, but was predicted to be benign based on five in silico prediction tools, did not Genetics Retreat 2015 [email protected] www.radboudumc.nl/Informatievoorverwijzers affect RNA expression levels, and allele specific expression differences were not observed in vitro. The BCAT1 gene is located approximately 250kb upstream of the KRAS gene and all affected family members may share a common haplotype encompassing this locus . However, RNA expression levels of KRAS did not differ between EBV cell lines derived from affected and unaffected family members. Authors van Herwaarden,Y.J.*(1), Weren, R.D.A.*(2), Kets, C.M. (2), Kamping, E.J. (2), Dura, P. (1), Voorendt, M. (2), Nagengast, F.M. (1), Ligtenberg, M.J.L. (2,3), Nagtegaal, I.D. (3), Bisseling, T.M. (1), Hoogerbrugge, N.**(2) and Kuiper, R.P.**(2). *Authors share co-first authorship **Authors share co-last authorship Affiliations 1. Department of Gastroenterology and Hepatology, Radboud university medical CONCLUSIONS We present a family which shows that SPS can be inherited in an autosomal dominant manner. We did not identify the causative germline aberration using our WES-based approach. Our data show that two truncating germline variants in RBL1 and PIF1, which were associated with multiple SSA/P (1), are unlikely to explain this phenotype because of incomplete cosegregation and frequent occurrence in the normal population. Considering the strong autosomal dominant inheritance pattern in this family, we anticipate that the causative alteration might be located outside the regions captured by the exome sequencing approach, and required whole genome sequencing to be identified. center, Nijmegen, The Netherlands 2. Department of Human Genetics, Radboud university medical center, Nijmegen, The Netherlands 3. Department of Pathology, Radboud university medical center, Nijmegen, The Netherlands Abstract body text (max of 1500 characters per box) References Gala MK, Mizukami Y, Le LP, Moriichi K, Austin T, Yamamoto M, et al. Germline mutations in oncogene-induced senescence pathways are associated with multiple sessile serrated adenomas. Gastroenterology. 2014;146(2):520-9. Epub 2014/02/12. Genetics Retreat 2015 29 Tessa van Dijk Mosaic uniparental isodisomy of chromosome 15q in an atypical PCH patient Pontocerebellar hypoplasia (PCH) describes a heterogeneous group of neurodegenerative disorders with a prenatal onset. Common features are hypoplasia/atrophy of the cerebellum and ventral pons, progressive microcephaly, severe mental and motor retardation and dyskinesia/chorea. So far, ten subtypes of PCH are described, based on differences in phenotypes and/or genotype. In about 40% of patients a mutation in one of the PCH related genes is identified, in 60% the genetic cause remains unknown. One of the goals of our research is to identify new candidate genes involved in PCH. Here we describe an atypical PCH patient with a neuronal migration disorder. The patient has a mosaic paternal uniparental disomy (UPD) of chromosome 15q24.2 – 15qter, which probably is very rare, and a deleterious heterozygous mutation in the POLG gene (also located on 15q). We hypothesize a relation between these findings and the phenotype. 30 METHODS RESULTS A SNP array was preformed to detect copy number variations and regions of homozygosity. Whole Exome Sequencing was done in the patient and his parents and variants were analyzed with Ingenuity Variant Analysis (QIAGEN). A single cell PCR of the informative markers in the 15q region of 40 white blood cells was done. No pathogenic copy number variations or mutations were identified with the SNP array and exome sequencing. However, the BAF profile and read number analysis in the exome data revealed an unusual pattern at chromosome 15q that was suggestive of a mosaic paternal uniparental isodisomy (UPD). Single cell analysis confirmed this finding, as 34% of the cells were homozygous for the paternal allele in this region and 66% of cells were heterozygous. No cells homozygous for the maternal allele were identified. The supposed mechanism is a somatic recombination. Genetics Retreat 2015 [email protected] www.amc.nl/web/Research/Who-is-Who Upon re-examination of the exome variants, a deleterious heterozygous mutation in the POLG gene was identified on the maternal allele. POLG is essential for the replication of mitochondrial DNA and homozygosity for this mutation is supposed to be lethal. This probably explains the absence of cells homozygous for the maternal 15q allele. Tentatively we like to hypothesize that this cell line existed and died at some point during development, leading to the brain disorder in this boy. However, this is very difficult to prove. Authors Tessa van Dijk (1), Bart Appelhof (1), Veerle R.C. Eggens (1), Anneloor L.M.A ten Asbroek (1), Marian A.J. Weterman (1), Aimee D.C. Paulussen (3), Jos C.F.M. Dreesen (3), P.G. Barth (2), Bwee Tien Poll-The (2), Frank Baas (1) Affiliations 1. Department of Genome Analysis, Amsterdam Medical Centre, Amsterdam 2. Department of Pediatric Neurology, Amsterdam Medical Centre, Amsterdam 3. Department of Clinical Genetics, Maastricht University Medical Centre CONCLUSIONS We identified a mosaic paternal uniparental isodisomy of chromosome 15q in an atypical PCH patient. Moreover, this patient is heterozygous for a deleterious mutation in the POLG gene on the maternal allele. This explains the absence of cells homozygous for the maternal 15q allele. However, the relation to the PCH-like phenotype remains to be elucidated. Genetics Retreat 2015 31 Joyce Gietel-Habets Motives, considerations and experiences with preimplantation genetic diagnosis Preimplantation genetic diagnosis (PGD) is a reproductive option to prevent transmission of a serious genetic disorder to the offspring. Embryo selection takes place by means of in vitro fertilization (IVF) after which an embryo without the genetic predisposition is transferred into the woman’s uterus. Since many advantages and disadvantages are involved and alternative options exist, decision making on this topic is complex. Motives and considerations to opt for or refrain from PGD have been mapped to a certain degree, mostly among couples with genetic cancer syndromes but they diverge between indications. The literature shows that this reproductive decision making process can result in a negative psychological impact, such as feelings of doubt, guilt and regret, especially among couples who refrain from options to prevent transmission of the genetic disorder. Experiences and ultimate satisfaction with PGD have not been thoroughly investigated. 32 METHODS RESULTS This exploratory qualitative study investigated decision-making, experiences and level of ultimate satisfaction concerning PGD. Semi-structured interviews were conducted among 15 couples of reproductive age with different genetic disorders. The interview guide was developed based on previous data we collected regarding PGD decision making. Duration of the interviews was between 31 and 85 minutes. The study was based on the grounded theory approach. Data was collected between May and September 2014. All couples had received extensive reproductive counselling and had made a decision regarding PGD, whereas 11 couples actually experienced one or more PGD treatment(s). Seven couples had a child after PGD, two after prenatal diagnosis (PND) and seven after a natural pregnancy. Reproductive decision making was based on perceived seriousness of the genetic condition which was highly related to personal experiences with the condition. Couples with a high perceived seriousness opted for PGD or PND as opposed to couples with a lower perceived seriousness who opted for a natural pregnancy. Genetics Retreat 2015 [email protected] www.mumc.nl PND was often chosen for practical reasons, whereas PGD was preferred for moral reasons. Experiences with a PGD treatment differed between couples and interpersonally. The emphasis with PGD experience was on physical and psychological aspects. Ultimate satisfaction with the choice for PGD was expected to be strongly related to the outcome of the PGD treatment. This appeared to be true when the outcome was successful, in which case levels of ultimate satisfaction were high. However, levels of ultimate satisfaction were also high among most couples for whom PGD treatment had not been successful. These couples underlined that they were put at ease by knowing that they had tried everything. experience are the psychological and physical facets of such a treatment. Nearly all couples that opted for PGD remained satisfied with their choice up to years after completing this chapter of their lives, irrespectively of the treatment’s outcome. It seems that PGD is one of the technologies that applies to the technological imperative: its mere existence affects reproductive decision making and its psychological aftermath, also for couples who refrain from this option. Optimal information, guidance and psychological support in this field is therefore essential. Authors Habets J.J.G.1,2, Reumkens K1,2., Arens Y.H.J.M.1,2, de Die-Smulders C.E.M1,2. CONCLUSIONS Motives and considerations in PGD decision making and experiences with PGD are affected by many factors such as perceived seriousness of the genetic condition, former experiences, personality traits and perspective. Therefore, it may vary highly between and sometimes within couples. The principal aspects described with PGD Affiliations 1. GROW - School for Oncology and Developmental Biology, Maastricht University Medical Centre+, Maastricht, the Netherlands 2. Department of Clinical Genetics, Maastricht University Medical Centre+, Maastricht, the Netherlands Genetics Retreat 2015 33 SIMON ARDUI Single Molecule Sequencing of the FMR1 CGG Repeat The fragile X mental retardation gene (FMR1) is an X-linked gene which contains a CGG repeat in the 5’ untranslated region (5’UTR). When this CGG repeat expands to more than 200 units, the region will become methylated which will silence the gene and cause the fragile X syndrome (FXS). The syndrome is the most common form of inherited mental retardation and among others associated with autism spectrum disorder (ASD), behavioural problems and Attention Deficit Hyperactivity Disorder (ADHD). Carriers of alleles with 55-200 CGG repeats are at risk for fragile X- associated tremor/ataxia syndrome (FXTAS; mainly males) or fragile x-associated premature ovarian insufficiency (POF; only females). To date, we still lack a complete understanding of the molecular basis of the disease. Firstly, FXS is a complex disease whereby the penetrance of the disease is influenced by the size of the CGG repeat, the percentage of methylated alleles and intra- and inter tissue mosaicism. In addition, researchers are limited by the current state of the art techniques (southern blot and PCR) which only generate a moderate size resolution and fail to detect minor alleles. Sequencing the repeat would break through those limitations, but was not possible using Massively Parallel Sequencing (MPS) technologies. 34 METHODS RESULTS Similar to Loomis et al. (Genome Research 2013), we performed longread amplicon sequencing of the CGG repeat using the Single Molecule Real Time (SMRT) technology developed by Pacific Biosciences. The result was analysed using the Long Amplicon Analysis pipeline (SMRT analysis) for de novo assembly. The accuracy of the technology was assessed by aligning the assembly to the expected reference. We sequenced the CGG repeat of a normal, premutation and full mutation allele. We show that we are able to accurately size the normal and premutation allele. Sequencing of the full mutation shows we are able to sequence through a 100% GC rich region > 1 kb. However, sizing of the full mutation is more complicated due to extensive variability of the large repeat in the amplicon product. Long read sequencing also permits the detection of AGG units interspersed in the CGG repeat. Genetics Retreat 2015 [email protected] www.gbiomed.kuleuven.be CONCLUSIONS We show that long read sequencing is extremely suited to sequence long, heavily repeated and GC-rich regions. The technology allows to accurately categorize different CGG alleles, which can potentially be an aid in the diagnosis of FXS. Interestingly, this approach also has the means for an easy detection of AGG interruptions which can be used to predict the likelihood of expansions of the repeat between generations. Consequently, in genetic counselling this information can be used for guiding woman who are carrying a premutation and are weighing the risk of having a child with FXS. Finally, sequencing of the full mutation shows a lot of variability in the amplicon product, which indicates that long read sequencing would greatly benefit from upstream improvements to be able to sequence chromosomal DNA directly. Authors Ardui S. (1), Hestand M. (1), Vermeesch J. (1) Affiliations Center for Human Genetics, KU Leuven, Belgium Genetics Retreat 2015 35 Bart Appelhof WES in a cohort of PCH patient: a PCH7 candidate gene Pontocerebellar hypoplasia (PCH) represents a heterogeneous group of neurodegenerative disorders with a prenatal onset. So far, ten subtypes are described (PCH1-10), based on genetic and clinical features. Coinciding symptoms are hypoplasia and/or atrophy of the pons and cerebellum and patients suffer from severe cognitive and motor defects. Currently, only symptomatic treatment is available and most patients die before adulthood. Despite the large number of genes involved in PCH, still a significant number of cases without a genetic cause. Therefore, whole exome sequencing was performed on a cohort of PCH patients with an unknown genetic cause. Here we describe mutations that were identified, including a new gene causing PCH7, i.e. PCH with genital abnormalities. No genes are associated before with the PCH7 subtype. 36 METHODS RESULTS Whole exome sequence data of 31 PCH patients, including and 6 trios’, was analyzed. With the resulting, most viable candidate genes a morpholino knockdown experiment was performed in zebrafish. The zebrafish embryos were morphologically assessed and studied using whole mount in situ hybridization. Screening for mutations in these candidate genes in a larger cohort of PCH patients was done by standard Sanger sequencing. In 9 of the 30 patients we could identify a mutation in known disease genes in CASK, PEX7 and PMM2 and ITPR1. Unknown candidate genes included CLK2 and TOE1 in one PCH7 patient. CLK2 knockdown in zebrafish resulted in cell death at the mid-hindbrain boundary, however the phenotype could not be rescued by coinjection of human or zebrafish CLK2 mRNA. TOE1 knockdown did not give a phenotype. Screening a cohort of PCH patients resulted in 12 patients in 10 families with mutations in TOE1 and none in CLK2. All the patients have the PCH7 subtype. Genetics Retreat 2015 [email protected] www.amc.nl nl.linkedin.com/pub/dir/Bart/Appelhof CONCLUSIONS Exome sequencing of a cohort of 30 PCH patient revealed likely pathogenic mutations in 9 patients. Most genes were previously associated with other disorders and include CASK, PEX7, PMM2 and ITPR1. We identified two candidate genes for PCH7: CLK2 and TOE1. CLK2 morpholino knockdown in zebrafish resulted in cell death at the mid-hindbrain boundary and other phenotypes. However, this can be considered as morpholino induced side effects since the phenotype cannot be rescued. In contrast, TOE1, which does not give a phenotype in the zebrafish knockdown model, has a high coincidence of rare compound heterozygous and homozygous mutations in a group of 12 PCH7 patients form 10 families. Therefore we propose TOE1 as the gene involved in the PCH7 subtype. Authors Appelhof B. (1), Eggens V.R.C (1), van Dijk T. (1), ten Asbroek A.L.M.A. (1), Weterman M.A.J. (1), Poll-The B.T. (2), Barth P.G. (2), Baas F. (1) Affiliations 1. Department of Genome Analysis, Amsterdam Medical Centre, Amsterdam 2. Department of Pediatric Neurology, Amsterdam Medical Centre, Amsterdam Genetics Retreat 2015 37 Minh Nguyen Whole exome sequencing: discovering genetic causes of mitochondrial disorders Mitochondrial disorders are the most common group of metabolic disorders mainly associated with oxidative phosphorylation (OXPHOS) abnormalities. Since mitochondria are under the dual genetic control of both the mitochondrial and nuclear genomes, mutations within either DNA molecule may result in a OXPHOS deficiency. Mitochondrial disease manifestations can occur at any age and affect any tissue although mostly the tissues with the highest energy demand, like brain, muscle, heart and liver are most severely affected. Diagnosis is complex due to an extreme clinical and genetic heterogeneity. Traditionally, biochemical and genetic investigations were time consuming, expensive, and highly specialized, often involving an invasive biopsy of the affected tissue or organ. However, recent development of sequencing strategies enables us to sequence all genes in a short period of time, facilitating faster genetic diagnosis for more patients compared to traditional methods. 38 METHODS RESULTS 12 Patients with suspected mitochondrial disorders were selected for whole exome sequencing. The inclusion criteria were based phenotypical, histochemical and/or biochemical diagnosis of mitochondrial disease in a clinically affected tissue (skeletal muscle, liver, or heart) and the absence of mutations in the mtDNA. Informed consent was obtained from all participants. Exome data was filtered for genes containing at least two alleles with a non-synonymous variant, splice variant or variant leading to a premature stop codon, which had an allele frequency of <0.01 or were absent in dbSNP132, the 1000 genomes data and our in house database and which were predicted to be damaging. Only genes meeting these criteria were considered for further studies. Exome sequencing revealed pathogenic mutation in known disease genes (CWF19L1 and RRM2B) in two patients, in candidate disease genes in four patients and no clear candidate was found for the 7 patients. Follow up experiments for one of the candidate disease genes (SLC25A46) revealed that the mutation in the patient resulted in the skipping of part of exon 1 resulting in an altered reading frame and premature stop codon, most likely resulting in nonsense mediated mRNA decay. The yeast homologue of SLC25A46, named Ugo1p, has been reported to be involved in mitochondrial fusion. We observed a fragmented mitochondrial network in the patients fibroblasts supporting a possible role of SLC25A46 in mitochondrial fusion. Genetics Retreat 2015 [email protected] www.gcb.mumc.nl However, additional experiments are warranted to elucidate the exact role of SLC25A46. Also for the other candidate disease genes further studies are needed to determine their function and role in disease pathology. CONCLUSIONS This heterogeneity is typical for mitochondrial diseases and supports the use of next-generation sequencing early in the diagnostic approach. However, we could not identify a potential candidate mutation in 7 patients (58%), possibly because the causative mutations lay in deep intronic regions or may involve deletions or duplications (copy number variations) missed by exome sequencing.. For these patients whole-genome sequencing and improved bioinformatic tools will assist in identifying the pathogenic variants. Authors Minh Nguyen1,2, Iris Boesten1, Debby M.E.I. Hellebrekers1, Jo Vanoevelen1, Radek Szklarczyk1, Rick Kamps1, Bart de Koning1, Irenaeus F.M. de Coo3, Mike Gerards1,2,* and Hubert J.M. Smeets1,2,* Affiliations 1. Department of Clinical Genetics, Unit Clinical Genomics, Maastricht University Medical Centre, Maastricht, The Netherlands 2. School for Oncology and Developmental Biology, Maastricht University Medical Centre, Maastricht, The Netherlands 3. Department of Neurology, Erasmus Medical Centre, Rotterdam, The Netherlands Genetics Retreat 2015 39 Tom Theunissen Unraveling the genetic and pathophysiological basis of mitochondrial disorders Mitochondrial disorders are a clinically heterogeneous group of disorders that have a prevalence of 1 in 8500 in the general population. Mitochondrial defects mainly affect high energy-demanding organs since energy production is often severely affected. Impairment of the oxidative phosphorylation (OXPHOS), which is underlying mitochondrial disease, therefore often leads to neuromuscular symptoms within patients suffering from mitochondrial defects. The components of the OXPHOS machinery are encoded by both the nuclear and mitochondrial DNA. Therefore mutations in the mitochondrial DNA or nuclear DNA can affect proper functioning of the OXPHOS, leading to mitochondrial disease. For a significant part of the nuclear DNA encoded mitochondrial disorders, the genetic cause has not been unraveled yet. Our lab performed WES analysis on a patient-population (around 60 patients) that has been diagnosed with mitochondrial disease. We aim to identify novel gene variants that are the underlying cause of the patient’s phenotype and to test these genetic variations using model systems (e.g. cell cultures, zebrafish model). METHODS During the last few years whole-exome sequencing (WES) has evolved rapidly, becoming an important tool for identification of novel diseaseassociated mutations in the human exome. Although, WES allows rapid detection of genetic variations in patient populations; candidate mutations require suitable read-out models in order to validate their association with a biochemical and clinical phenotype. Patient fibroblasts allow testing for defects in mitochondrial respiration by means of Blue-Native, Seahorse, Oroboros, Westernblot. Besides, availability of patient fibroblasts enables us to perform complementation studies in relatively fast manner. 40 Genetics Retreat 2015 At physiological/developmental level, the zebrafish is a suitable in vivo model for testing identified mutations by means of a morpholino injection approach. In this manner a knockdown model can be generated relatively fast and eventually used for testing specific mutations by coinjecting mutated mRNA. The zebrafish genome has relatively high homology with the human genome (82% of the human disease causing genes have at least one orthologue in zebrafish) and many physiological processes rely on the same pathways. For this reason zebrafish can be used as an animal model system to test pathophysiological effects of a specific genetic variation. [email protected] www.gcb.mumc.nl RESULTS CONCLUSIONS WES data analysis resulted in the identification of mutations (novel and described) that underlie pathogenicity within mitochondrial patients. Among others, we have identified pathogenic mutations in SERAC1, CLPP, MTFMT, FBXL4, AARS2, TPM1, SCN4A, RRM2B, ARHGAP19 and THEM19B. Biochemical measurements on mitochondrial respiration revealed affected mitochondrial translation and/or complex assembly in a significant number of patients. To confirm the pathogenic effects of mutations on OXPHOS, we aim to restore associated biochemical defects in patientfibroblasts by means of complementation testing. Besides, we have validated mitochondrial translational defects within a group of MTFMT patients. Intervention-experiments are currently performed on these patient-cells by testing the impact of different compounds (antibiotics and enzyme substrates) on mitochondrial respiration. For as far, we can state that in 40% of the patient cases a clear genetic cause has been identified which could explain the observed symptoms. However, for a majority of the detected candidate mutations, in vitro and in vivo studies are performed in order to prove their roles in pathogenicity. Performed read-out experiments contribute to our insight in molecular mechanisms involved in mitochondrial disease. Authors T.E.J. Theunissen, MSc; M. Gerards, PhD; R. Szklarczyk, PhD; Prof. H.M.J. Smeets, PhD Affiliations Department of Clinical Genomics, Maastricht University, Maastricht, Netherlands Genetics Retreat 2015 41 Mirjam de Pagter Chromothripsis in healthy individuals affects multiple protein-coding genes and can result in severe congenital abnormalities in offspring Chromothripsis represents an extreme class of complex chromosome rearrangements (CCRs) with major effects on chromosomal architecture. Although recent studies have associated chromothripsis with congenital abnormalities, the incidence and pathogenic effects of this phenomenon require further investigation. 42 METHODS RESULTS Here, we analyzed the genomes of three families in which chromothripsis rearrangements were transmitted from a female carrier to her child, by a combination of copy number analysis, karyotyping and nextgeneration mate-pair sequencing. The chromothripsis in the carriers resulted in completely balanced rearrangements involving 8-23 breakpoint junctions across 3-5 chromosomes. Two carriers did not show any phenotypic malformations, although 3-13 protein coding genes were affected by breakpoints. Unbalanced but stable transmission of a subset of the derivative chromosomes caused apparently de novo complex copy number changes in two children. This resulted in gene dosage changes, which are likely responsible for their severe congenital phenotypes. In contrast, one patient with severe congenital disease, carried all three chromothripsis chromosomes from his healthy mother, but one of the chromosomes acquired de novo rearrangements leading to copy number changes. Genetics Retreat 2015 [email protected] www.umcutrecht.nl www.linkedin.com/pub/mirjam-de-pagter CONCLUSIONS These results show that the human genome can tolerate extreme reshuffling of chromosomal architecture, including breakage of multiple protein coding genes, without noticeable phenotypic effects. The presence of chromothripsis in healthy carriers strongly affects reproduction and is expected to substantially increase the risk of spontaneous abortions and severe congenital disease. Authors de Pagter M.S. (1), Roosmalen M.J. (1), Baas A.F. (2), Renkens I. (1), Duran K.J. (1), van Binsbergen E. (2), Tavakoli-Yaraki M. (1), Hochstenbach R. (2), van der Veken L.T. (2), Cuppen E. (1,3) Affiliations 1. Department of Medical Genetics, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht, The Netherlands 2. Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands 3. Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, The Netherlands Genetics Retreat 2015 43 Sarah Hempenstall A novel assay for proteasomal activity as a marker of myogenic dysfunction Ageing muscle is characterised by loss of mass, fat infiltration and progressive muscle weakness. Little is currently known regarding the mechanisms underlying muscle ageing. Poly-A binding protein nuclear 1 (PABPN1), regulates mRNA processing via mediation of poly-A site (PAS)selection in the 3’UTR of immature transcripts, thus facilitating recruitment of the polyadenylation and cleavage machinary to yield mature mRNA. PABPN1 expression is preferentially decreased in muscle with ageing and a mutation in the n-terminus of PABPN1 is the genetic cause of oculopharyngeal muscular dystrophy, a late onset dystrophy and suggested paradigm for accelerated muscle ageing. The ubiquitin-proteasomal system(UPS) is the most significantly dysregulated pathway in OPMD and disruption of protein homeostasis is strongly implicated in the onset of muscle weakness in ageing. This study investigated the role of the UPS in myogenic function and developed a robust, high-throughput cell based assay for quantification of proteasomal activity. METHODS Cell culture and mouse tissue was generated and handled as previously reported(Raz et al., 2014). Myoblast differentiation was carried out as previously described(Raz et al., 2011). Affinity binding probes, specific to individual and all proteasomal beta-subunits were obatined from the lab of Dr. Bogdan Florea (Li et al., 2013) and optimised for use in FACS analysis and Cellomix scanning. Native activity-gels were also run to compare cell based proteasomal activity with in-gel, protein specific activity. 44 Genetics Retreat 2015 Gene expression of proteasomal genes was measured using RT-qPCR and protein levels of PABPN1, proteasomal subunits and deubiquitinating enzyme PSMD14 were measured using Western Blotting. RESULTS Proteasomal gene expression and protein levels are dysregulated under PABPN1 down-regulated conditions. PABPN1 downregulation results in impaired myoblast differentiation, impaired myogenic function and decreased mitochondrial efficiency. Affinity binding probes targeted to proteasomal beta-subunits are specific and suitable for use in FACS and Cellomix scanning. [email protected] www.humgen.nl www.linkedin.com/pub/sarah-hempenstall Proteasomal activity is significantly decreased in PABPN1 down-regulated conditions. Modulation of proteasomal beta-subunit activity results in accumulation of PABPN1. Authors Hempenstall, S., Raz, Y., Paniagua-Soriano, G., Raz., V. Affiliations 1. Department of Human Genetics, LUMC, Leiden, Netherlands CONCLUSIONS Proteasomal activity is severely affected under conditions of PABPN1 depletion and may represent a robust biomarker for myogenic dysfunction. Pharmacological and genetic therapies ( e.g antisense oligonucleotides targeted to alternative PAS in the 3’UTR of transcripts) focused on components of the UPS may be promising targets for restoration of PABPN1 levels and, by extension, improvement of muscle function. This would be relevent not only for OPMD patients, but also for progressve muscle weakness associated with ageing. 2. Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden, Netherlands Genetics Retreat 2015 45 Daniel Kofink Loss of chromosome Y and carotid atherosclerosis severity in men While chromosome Y has long been considered “genomic wasteland”, recent findings suggest that age-related loss of chromosome Y (LOY) in blood cells is associated with higher all-cause mortality and cancer risk. Animal studies have linked genetic variants on chromosome Y to altered gene expression in immune cells. Given the involvement of immune cells in atherosclerosis development, we hypothesize that LOY is associated with atherosclerosis severity in men. 46 METHODS RESULTS In 582 male patients with carotid atherosclerosis enrolled in the Athero-Express Study, LOY is quantified in carotid plaque and blood by calculating log R ratio values from genotyping intensity data. LOY will be associated with traditional cardiovascular risk factors as well as with severity of carotid plaque phenotypes, such as plaque hemorrhage. To investigate whether LOY affects gene expression in these men, we will analyze gene expression profiles of hematopoietic immune cells. Furthermore, we will study whether LOY is associated with poorer secondary outcomes during three years of follow-up. Preliminary results show a negative association of Y chromosome intensities with increasing age (Beta= -0.003, P=1.29e-12). When tissue type (blood or carotid plaque) was added to the regression model, there was no significant tissue type x age interaction (P=0.18), suggesting that both tissues are equally affected by LOY. Genetics Retreat 2015 [email protected] CONCLUSIONS LOY is age-related in a cohort of men with severe atherosclerotic disease. The degree of LOY appears to be similar in blood and plaque. Results on the association of LOY with carotid plaque phenotypes and outcomes in this cohort are soon to be expected. Authors Kofink D. (1), Haitjema S. (1), van Setten J. (1), van der Laan S.(1), de Jager S. (1), Hofer I. (1), de Bakker P.I.W. (2), Pasterkamp G. (1), Asselbergs F.W. (1), den Ruijter H. (1) Affiliations 1. Department of Experimental Cardiology, University Medical Center Utrecht, the Netherlands 2. Department of Biomedical Genetics, University Medical Center Utrecht, the Netherlands Genetics Retreat 2015 47 Sietske Kevelam PLP1 mutations affecting PLP1/DM20 alternative splicing causes Hypomyelination of Early Myelinating Structures Inherited leukodystrophies represent a diagnostic challenge, as many patients remain without definitive diagnosis. In this study we investigated the genetic etiology of the recently described X-linked childhood disorder ‘Hypomyelination of Early Myelinating Structures’ (HEMS). In HEMS, brain structures normally myelinating early, are hypomyelinated, which is in contrast to known hypomyelination disorders, like Pelizaeus-Merzbacher disease (PMD). METHODS We included patients diagnosed with HEMS by brain MRI criteria and their affected siblings. Exome sequencing was used to search for causal mutations in 16 patients of 10 families. In silico analysis of effects of the mutations on alternative splicing and secondary RNA folding was performed. Also, gene splicing was examined in RNA prepared from patients’ fibroblasts and an immortalized immature oligodendrocyte cell line after transfection with mutant minigene splicing constructs. RESULTS 48 All patients had unusual hemizygous mutations of PLP1, either in exon 3B, which is spliced out in isoform DM20, or in intron 3. Four identified mutations located deep in intron 3 were predicted to destabilize a long-distance interaction structure in the secondary PLP1 RNA fragment involved in regulating PLP1/DM20 alternative Genetics Retreat 2015 splicing and led to a decreased PLP1/ DM20 ratio. One missense, 2 silent, and 1 intronic mutation were predicted to alter PLP1/DM20 alternative splicing, either by affecting exonic splicing silencers motifs, creating a new splice donor site or by affecting the secondary RNA structure of the PLP1 splice donor site. A frameshift deletion led to truncated PLP1, but not DM20. In vitro studies showed a decreased PLP1/DM20 ratio in patients’ fibroblasts and transfected cells. CONCLUSIONS We show that specific mutations in the PLP1 gene cause a hypomyelination pattern which contrasts the pattern seen in PMD, also caused by PLP1 mutations. This indicates that PLP1/DM20 alternative splicing is important for early myelination, probably by an impact on the PLP1/DM20 ratio. Furthermore, our data show that intronic mutations affecting a second- [email protected] ary PLP1 RNA structure involved in regulation of PLP1/DM20 alternative splicing cause a HEMS phenotype and support the need to include intron 3 in diagnostic sequencing. 7. Child Neuropsychiatry Unit, Department of Brain and Behavioral Sciences, University of Pavia, Pavia, Italy 8. Departments of Pediatrics, Neurology and Neurosurgery, Division of Pediatric Neurology, Montreal Children’s Hospital, McGill University Health Center, Montreal, Canada Authors 9. Department of Pediatric Neurology, Erasmus Kevelam S.H. (1,2)*, Taube J.R. (3)*, van Spaendonk University Hospital - Sophia Children’s R.M.L. (4)*, Bertini E. (5), Sperle K. (3), Tarnopolsky Hospital, Rotterdam, The Netherlands M. (6), Tonduti D. (7), Valente E.M. (5), Travaglini 10. Division of Biochemical Diseases, Department L. (5), Sistermans E.A. (4), Bernard G. (8), of Pediatrics, BC Children’s Hospital, Centre Catsman-Berrevoets C.E. (9), van Karnbeek C.D.M. for Molecular Medicine and Therapeutics, (10), Ostergaard J.R. (11), Friederich R.L. (12), University of British Columbia, Vancouver, Elsaid F.M. (13), Schieving J.H. (14), Tarailo- Canada Graovac M. (15), Orcesi S. (16), Steenweg M.E. 11. Centre for Rare diseases, Department of (1,2), van Berkel C.G.M. (1), Waisfisz Q. (4), Paediatrics, Aarhus University Hospital, Abbink T.E.M. (1,2), van der Knaap M.S. (1,2,17), Aarhus, Denmark Hobson G.M. (3,18,19)+, Wolf N.I. (1,2)+. 12. Department of Child Neurology, Kaiser Permanente Pediatric Specialties, Roseville, CA, USA Affiliations 13. Department of Pediatric Neurology, Hamad 1. Department of Child Neurology, VU University Medical Center, Amsterdam, The Nether- Medical Corp, Doha, Qatar 14. Department of Child Neurology, Radboud lands University Medical Center, Nijmegen, 2. Neuroscience Campus Amsterdam, VU University, Amsterdam, The Netherlands The Netherlands 15. Department of Medical Genetics, University 3. Nemours Biomedical Research, Alfred I. duPont Hospital for Children, Wilmington, USA of British Colombia, Vancouver, Canada 16. Child Neurology and Psychiatry Unit, 4. Department of Clinical Genetics, C. Mondino National Neurological Institute, VU University Medical Center, Amsterdam, The Netherlands Pavia, Italy 17. Department of Functional Genomics, Center 5. Unit for Neuromuscular and Neurodegene- for Neurogenomics and Cognitive Research, rative Diseases, Laboratory of Molecular VU University, Amsterdam, the Netherlands Medicine, Bambino Gesu’ Children’s Research 18. Department of Biological Sciences, University Hospital, IRCCS, Rome, Italy 6. Department of Pediatrics, McMaster Chil- of Delaware, Newark, USA 19. Department of Pediatrics, Jefferson Medical dren’s Hospital, Hamilton, Ontario, Canada College, Thomas Jefferson University, Philadelphia, USA *These authors share first authorship. +These authors share senior authorship. Genetics Retreat 2015 49 Glen Monroe Monocarboxylate Transporter 1 Deficiency and Ketone Utilization Ketone bodies serve as the major circulating energy source during fasting. Ketoacidosis, a pathologic state, occurs when ketone formation exceeds ketone utilization and results in slightly acidic ketones accumulating in the blood. The clinical consequences of ketoacidosis are exemplified by diabetic ketoacidosis, a condition marked by vomiting, osmotic diuresis, dehydration,and Kussmaul breathing and that may progress to decreased consciousness and death. Only two genetic causes of recurrent ketoacidosis are currently known: SCOT deficiency and ACAT1 deficiency. We performed targeted exome sequencing of homozygous genomic regions in a patient of consanguineous descent who had recurrent, severe ketoacidosis. A homozygous mutation was detected in the gene encoding monocarboxylate transporter 1 (MCT1). Subsequently, we evaluated a series of 96 patients with recurrent ketoacidosis to identify other mutations and confirmed reduced protein production and transport using protein analysis and functional assays. METHODS 50 The index patient had repetitive episodes of profound metabolic acidosis, with a blood pH below 7.00 on three occasions. Metabolic analysis revealed massive urinary excretion of 3-hydroxybutyrate and acetoacetate. We performed targeted exome sequencing of homozygous genomic regions detected by HumanCytoSNP-12 array in the index patient and her family members. Coding parts of homozygous regions were then captured and sequenced using the SOLiD 5500. We sequenced the entire coding region of MCT1 in a series of 96 patients with ketoacidosis in whom known ketolytic defects had been ruled out. In addition, we performed Sanger sequencing of the related genes MCT2, MCT3, and MCT4, plus the ancillary gene BSG. Genetics Retreat 2015 A C-terminal antibody against MCT1 was used in patient and control fibroblasts to evaluate protein expression. An erythrocyte transporter assay with patient and control sample was used to assay the mutations’ effect upon small molecule transport. RESULTS Snp array analysis confirming consanguinity. NGS yielded nine rare variants; of these, a homozygous, novel single-nucleotide insertion in MCT1 was the best candidate. Subsequent sequencing in a larger cohort yielded nine patients with novel mutations. Six of these were heterozygous and three homozygous for the MCT1 mutation. All mutations were truncating except for one missense mutation in a highly conserved catalytic site. [email protected] cmm.umcutrecht.nl Immunoblot analysis with fibroblast homogenates from patients with a homozygous MCT1 mutation confirmed that predicted truncating mutations lead to an absence of the full-length protein, and fibroblasts from patients with a heterozygous truncating mutation showed significantly reduced levels of MCT1 protein relative to control levels. Erythrocyte lactate transport was significantly reduced in samples from homozygous patients compared to control samples. In erythrocytes from both symptomatic and asymptomatic heterozygous carriers, transport activity was significantly reduced as compared with control samples but was significantly higher than that in homozygous patients. CONCLUSIONS Reduction of MCT1 full length protein due to a mutation reduces the body’s ability to transport ketones out of the blood. The severity of the ketoacidosis episode and patient phenotype are dependent on the zygosity of the mutation and resulting different levels of protein. Patients with homozygous mutations in MCT1 tended to present at a younger age and had mild-to moderate developmental delay, whereas patients with milder deficiencies of MCT1 had normal development. We identified MCT1 deficiency as a disorder of ketone utilization, expanding the spectrum of disorders leading to ketoacidosis from those of ketolysis to those of ketone delivery. This finding may aid in timely diagnosis of the disorder and allow for improved disease management. Authors Peter M. van Hasselt (1), Sacha Ferdinandusse (2), Glen R. Monroe (3), Jos P.N. Ruiter (2), Marjolein Turkenburg (2), Maartje J. Geerlings (3), Karen Duran (3), Magdalena Harakalova (3), Bert van der Zwaag (3), Ardeshir A. Monavari (4), Ilyas Okur (5), Mark J. Sharrard (6), Maureen Cleary (7), Nuala O’Connell (8), Valerie Walker (9), M. Estela Rubio-Gozalbo (10), Maaike C. de Vries (11), Gepke Visser (1), Roderick H.J. Houwen (12), Jasper J. van der Smagt (3), Nanda M. Verhoeven-Duif (3), Ronald J.A. Wanders (2) and Gijs van Haaften (3) Affiliations 1. Division of Pediatrics, Wilhelmina Children’s Hospital, Utrecht, Netherlands 2. Laboratory Genetic Metabolic Diseases, Academic Medical Center, Amsterdam 3. Center for Molecular Medicine, University Medical Center Utrecht, Utrecht, Netherlands 4. National Centre for Inherited Metabolic Disorders, Dublin, Ireland 5. Gazi University School of Medicine, Turkey 6. Sheffield Children’s Hospital, Sheffield, UK 7. Great Ormond Street Hospital, London, UK 8. Chemical Pathology, Salisbury, UK 9. Department of Clinical Biochemistry, Southampton General Hospital, UK 10. Maastricht University Medical Center 11. Radboud University Medical Center, Nijmegen 12. Wilhelmina Children’s Hospital, Utrecht Genetics Retreat 2015 51 Elke Mersy Non-invasive detection of fetal sex by simultaneous amplification of Y chromosome DNA and trophoblast-specific RNA Cell-free fetal (cff) nucleic acids in the plasma of pregnant women can be used for noninvasive prenatal testing (NIPT). NIPT of the fetal sex is performed if the fetus is at risk for an X-linked inherited disorder or for autosomal recessive inherited congenital adrenal hyperplasia, or if the fetus presents with ambiguous genitalia on ultrasound examination. If Y-chromosome-derived cff-DNA sequences are present in the maternal plasma, the fetus is presumed to be male. However, even in the case of a male fetus, failure of Y-detection can occur, e.g. due to insufficient levels of cff-DNA. As a consequence, the test requires an intrinsic marker for the presence of fetal nucleic acids. Still, the ideal “fetal marker” has yet to be developed. We hypothesized that the amplification of cff-RNA sequences of genes that are exclusively expressed in the trophoblast of the placenta, can be used as fetal marker throughout pregnancy. In this proof of principle study, we aim to develop a simple and reliably test for NIPT of the fetal sex, by simultaneous PCR amplification of Y-chromosome cff-DNA and trophoblast-specific cff-RNA sequences. METHODS The plasma samples of 75 pregnant women were tested in duplicate. In all cases, the fetal sex was known (in 72 samples from prenatal karyotyping, in 2 samples from postnatal array, and in 1 samples from another NIPT assay). In addition, the plasma samples of 20 males and 20 non-pregnant females were tested. After extraction of cff-DNA and cff-RNA, we carried out a multiplex PCR of three Y chromosome genes (AMELY, SRY, and UTY). In parallel, in a one-tube experiment, a multiplex specific reverse transcription and PCR were per- 52 Genetics Retreat 2015 formed sequentially, in which one Y chromosome gene (AMELY) and two trophoblast-specific gene groups - the genes CSH1 and CSH2, encoding placental lactogen, and the genes CGB, CGB1, CGB2, CGB5, CGB7 and CGB8, encoding human chorionic gonadotropin - were amplified. The PCR products were detected and separated by capillary electrophoresis. The results of the PCR amplification of Y-chromosome cffDNA and trophoblast-specific cff-RNA sequences were combined to determine the fetal sex. [email protected] www.gcb.mumc.nl https://www.linkedin.com/pub/elke-mersy RESULTS CONCLUSIONS The initial results of the first 40 tested maternal plasma samples (40/75) demonstrate that the fetal sex is correctly determined by our NIPT assay. We furthermore see fluctuations in relative cff-RNA peak height in the course of the pregnancy, in accordance with the expected level of expression in the trophoblast. Before 12 weeks of gestation, the CGB-peak is high and the CSH-peak is low to absent, while after 22 weeks, the CGBpeak is low to absent and the CSHpeak is high. Between 12 and 22 weeks of gestation both cffRNA peaks are present. All control samples (40/40) showed correct results: no RNA or DNA peaks were seen in non-pregnant females and in all male controls only the Y-product was detected. The remaining 35 plasma samples of pregnant women (35/75) are tested at the moment. Amplification of cffRNA sequences of genes that are exclusively expressed in the trophoblast, is a gestational age-dependent fetal marker that can be used for NIPT of fetal sex. Further validation experiments are pending before implementing this approach of fetal sex testing in maternal plasma in clinical diagnostics. Authors Mersy E. (1,2), Spierts S. (1), Macville M.V.E. (1), Frints S.G.M. (1,2), Faas B.H.W. (3), Paulussen A.D.C. (1), and Veltman J.A. (1,3) Affiliations 1. Department of Clinical Genetics, Maastricht University Medical Center+, Postbox 5800, 6202 AZ Maastricht, The Netherlands 2. GROW School for Oncology and Developmental Biology, Maastricht University Medical Center+, Postbox 616, 6200 MD Maastricht, The Netherlands 3. Department of Human Genetics, Donders Centre for Neuroscience, Radboud university medical center, Nijmegen, The Netherlands. Genetics Retreat 2015 53 Nathalie Fieremans Identification of intellectual disability genes in female patients with skewed X-inactivation Intellectual disability (ID) is a very heterogeneous disorder and the etiology remains unknown in many cases. Traditionally, X-linked ID studies focused on males due to the hemizygous state of the X chromosome. Healthy females can be carrier of the mutation but are generally not affected due to inactivation of the mutant X chromosome in most of their cells (skewing of X-inactivation). Previously we identified two female patients with RETT-like phenotypes caused by a de novo microduplication including MECP2. While carrier females of MECP2 duplications are generally unaffected due to skewing of X-inactivation of the X carrying the duplication, these 2 females show complete inactivation of the apparently normal X chromosome. We therefore expect a harmful mutation on the other X causing skewing in these patients, which then forces the MECP2 duplication to be expressed, resulting in the RETT-like phenotype (Fieremans et al. 2014). Since this two-hit mechanism could also occur in other female patients with sporadic ID and skewed X-inactivation we have searched for X-linked defects in sporadic female ID patients by using skewing of X-inactivation as a criterion for further analysis. METHODS We used the standard androgen receptor (AR) X-inactivation assay to screen for skewed X-inactivation in 291 female ID patients with sporadic and syndromic ID. To identify mutations on the X chromosome that could lead to ID or skewing of X-inactivation we performed high-resolution X chromosome-specific microarray-based comparative genome hybridization (X-array-CGH) on 19 patients, exome sequencing on 19 patients and XIST Sanger sequencing on 7 patients. 54 Genetics Retreat 2015 Candidate variants were then confirmed by qPCR and/or Sanger sequencing. Interesting variants were further analysed using online databases, literature reports, genotype-phenotype and tissue expression-endophenotype correlation studies. [email protected] www.med.kuleuven.be RESULTS We analyzed the X-inactivation patterns of 291 ID females and found that 7.6% had extreme skewing of X-inactivation which is much higher than in the general population (3.6%). X-array-CGH revealed the de novo microdeletion of the escape genes KDM5C and IQSEC2 in a girl with severe intellectual disability and autistic features (Fieremans et al. 2015). Exome sequencing of 19 female patients with skewing of X-inactivation revealed 5 de novo mutations that could explain their ID phenotypes. Authors Fieremans N. (1,2), Belet S. (1), Van Esch H. (1), Devriendt K. (1), Martinez F. (3), Marynen P. (1), Froyen G. (1) Affiliations 1. Center for Human Genetics, KU Leuven, Leuven, Belgium 2. VIB Department of the biology of disease, Leuven, Belgium 3. Genetics Unit, Hospital Universitario La Fe, Valencia, Spain CONCLUSIONS Our data thus demonstrate that skewing can be used to screen for (novel) genetic mutations resulting in X linked ID in females. Genetics Retreat 2015 55 Robbert Weren Identification of a novel adenomatous polyposis and colorectal cancer predisposing gene. Genetic germline aberrations in genes can underlie the constitutive development of colonic adenomas, the premalignant precursors of colorectal cancer. However, known predisposing genes only explain a subset of these patients, which indicates that novel predisposing genes await discovery. Identification of these genes will improve clinical management and enable the identification of individuals at increased risk to develop colonic adenomas and cancers. 56 METHODS RESULTS We applied whole-exome sequencing to 51 individuals, from 48 different families, with multiple colonic adenomas. Data analysis was performed to exclude common genomic polymorphisms and we selected for potential pathogenic and recurrent variants. After candidate gene selection, cosegregation analyses were performed. The somatic mutation spectrum of cancers and adenomas was determined by sequencing for mutations in 409 cancer-related genes and by targeted deep-sequencing of the APC gene, respectively. In four index patient from three families, we identified a homozygous germline nonsense mutation in the base excision repair gene NTHL1. This mutation was exclusively found in a heterozygous state in 2,329 in-house controls (MAF 0.0036). Co-segregation analysis revealed that this variant was only present in a homozygous state in family members affected with multiple colonic adenomas (n=3) and pedigree analysis showed that the polyposis phenotype was inherited in a recessive manner. Genetic analysis of three carcinomas and five adenomas derived from homozygous carriers (n=5) revealed a non-hypermutated profile enriched for C-to-T transitions, in line with a germline base excision repair defect. Genetics Retreat 2015 [email protected] CONCLUSIONS We showed for the first time that heritable germline mutations in the NTHL1 gene predispose to the development of colonic adenomas and carcinomas in an autosomal recessive manner. Affiliations 1.Department of Human Genetics, Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, The Netherlands 2.Department of Pathology, Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, The Netherlands Authors Robbert D.A. Weren (1), Marjolijn J.L. Ligtenberg (1,2), C. Marleen Kets (1), Richarda M. de Voer (1), 3.Department of Genome Sciences, University of Washington, Seattle WA 98115 4.Department of Gastroenterology and Eugène T. P. Verwiel (1), Liesbeth Spruijt (1), Hepatology, Radboud university medical Wendy A.G. van Zelst-Stams (1), Marjolijn C. center, Nijmegen, The Netherlands Jongmans (1), Christian Gilissen (1), Jayne Y. Hehir-Kwa (1), Alexander Hoischen (1), 5.These authors contributed equally to this study. Jay Shendure (3), Evan A. Boyle (3), Eveline J. Kamping (1), Iris D. Nagtegaal (2), Bastiaan B.J. Tops (2), Fokko M. Nagengast (4), Ad Geurts van Kessel (1), J. Han J.M. van Krieken (2), Roland P. Kuiper (1,5), and Nicoline Hoogerbrugge (1,5) Genetics Retreat 2015 57 Serdar Yavuzyigitoglu BAP1 correlates with metastasis in polyploid uveal melanoma Most of the Uvea Melanoma (UM) display a near-diploid karyotype with only a few chromosomal changes. In contrast to these simple aberrations 10% of the UM samples show a polyploid character (> 58 chromosomes) and were associated with unfavorable prognosis. This study attempts to gain insight in polyploidy in UM and supplement the old data with the current knowledge on mutations in UM specific genes. 58 METHODS RESULTS Fluorescence-In-Situ-Hybridization (FISH) and/or Single-Nucleotide-Polymorphism (SNP) array was used to determine the ploidy status. Tumors showing signs of polyploidy (range tritetraploidy) were further investigated. Immune-histochemistry was used to determine the BAP1 expression and mutation analyses of BAP1 (coding regions) or the hotspots for the GNAQ, GNA11, SF3B1 and EIF1AX genes was carried out using Sanger Sequencing. Polyploidy, resembling triploidy or tetraploidy, was seen in 18 tumor samples. Ten of the UM patients developed metastases with a median follow-up of 35 months. Twelve tumors showed loss of BAP1 expression and all 18 polyploid tumors harbored a GNAQ or GNA11 mutation. SF3B1 mutations were found in three UM specimens and none of the tumors harbored EIF1AX mutations. Univariate analyses showed a significant association with decreased survival for chromosome 1, 3 and 8 aberrations, SF3B1 wild type and a loss of BAP1 expression. In the multivariate analyses, BAP1 expression was the only independent prognostic marker within the polyploid tumors (HR 10.1; p=0.008). Genetics Retreat 2015 [email protected] nl.linkedin.com/in/serdaryayo CONCLUSIONS Also for the tumors displaying polyploidy loss of BAP1 expression is associated with an increased risk of metastatic disease. Authors S. Yavuzyigitoglu (1, 2), H. Mensink (3), J. Vaarwater (1, 2), N.C. Naus (1), H.T. Brüggenwirth (2), D. Paridaens (3), A. de Klein (2), E. Kilic (1) Affiliations Department of Ophthalmology and 2. Department of Clinical Genetics, Erasmus University Medical Center, Rotterdam, the Netherlands; 3. The Rotterdam Eye Hospital, Rotterdam, the Netherlands. Genetics Retreat 2015 59 Romy Mesman Functional analysis of variants of uncertain significance in BRCA2 Members of suspected high breast cancer risk families are eligible for genetic testing for mutations in BRCA1 and BRCA2. In 5-10% of the families a sequence variant is identified for which there is currently insufficient information available on the consequence for protein function. Since the cancer risk associated with these variants of uncertain significance (VUS) is unknown, they represent a challenge for genetic counseling and clinical management of the families. Already more than 2000 unique BRCA2 variants are identified worldwide, including missense and silent substitutions, small in frame insertions and deletions and intronic variants. Since these BRCA2 variants are in most cases very rare, there is often insufficient family data (e.g.co-segregation, tumor histopathology) to make inferences about their associated cancer risk. In silico analysis, a technique based on sequence conservation and physiochemical properties of the amino acid residue, is not reliable enough to make important clinical decisions. There is a strong demand to establish the clinical significance of BRCA2 VUS because this will enable reliable cancer risk assessment and thereby will determine the clinical management for carriers. This underlines the importance of functional assays that assess the impact of BRCA2 variants on protein function. We developed a mouse embryonic stem cell (mES) based model to functionally test all types of BRCA2 variants, including variants that may affect RNA splicing. METHODS Our test is based on the ability of human BRCA2 (hBRCA2) to rescue the lethality of BRCA2 deficiency in mES cells. To enable functional analysis of all types of variants, the entire human BRCA2 sequence, located on a bacterial artificial chromosome (BAC) is used. VUS introduced in the hBRCA2 gene that fail to rescue the lethal phenotype are considered to be deleterious for protein function. The impact of non-lethal variants is tested in a series of biological assays, based on the role 60 Genetics Retreat 2015 of BRCA2 in homologous recombination. These assays include the analysis of double strand break repair and the sensitivity towards DNA damaging agents such as MMS and PARPinhibitors. RESULTS To validate our assay we used a series of known pathogenic and neutral variants in BRCA2 for which the clinical significance has already been established on the basis of genetic data. When a hBRCA2 gene [email protected] containing a pathogenic variant was introduced into mES cells, the cells did not survive after disrupting endogenous mBrca2 expression. However, neutral variants were able to complement endogenous Brca2 deficient lethality. BRCA2 protein function was tested in these cells using several assays. The neutral variants did not show reduced HR efficiency or increased sensitivity towards DNA damaging agents compared to cells expressing Wild-type hBRCA2. These results show that our assay is able to discriminate between variants that affect BRCA2 function (e.g. pathogenic variants) and neutral variants that do not affect protein function. Subsequently, we have tested the functionality of several clinically relevant BRCA2 VUS (n=16). Although several variants complemented the lethal phenotype, in some cases we observed reduced HR efficiency and increased sensitivity towards MMS and PARP-inhibitor treatment. Clearly, these variants have an impact on BRCA2 function but apparently there is residual activity that is sufficient for cell survival. Whether the impaired function of BRCA2 related to these variants might be associated with an intermediate cancer risk has to be determined using clinical data. CONCLUSIONS Our mES cell based method enables rapid generation and functional analysis of BRCA2 VUS that are identified in the clinic. The effect of the variant on RNA splicing, protein stability and function can be rapidly assessed. Once our assay is sufficiently validated it will enable the classification of rare BRCA2 VUS, even in the absence of sufficient family data. Improving the classification of BRCA2 VUS will facilitate risk assessment and support decision making regarding risk reduction and surveillance options in carriers. Authors Mesman, Calleja, Morolli, Hendriks, Asperen, Vrieling, Vreeswijk Affiliations Department of Human Genetics, Leiden University Medical Center, the Netherlands Genetics Retreat 2015 61 Maarten Massink Proper genomic profiling of (BRCA1-mutated) basal-like breast carcinomas requires prior removal of tumor infiltrating lymphocytes BRCA1-mutated breast carcinomas may have distinct biological features, suggesting the involvement of specific oncogenic pathways in tumor development. The identification of genomic aberrations characteristic for BRCA1-mutated breast carcinomas could lead to a better understanding of BRCA1-associated oncogenic events and could prove valuable in clinical testing for BRCA1-involvement in patients. 62 METHODS RESULTS Integrated genomic and gene expression profiles of basal-like BRCA1-mutated breast tumors (n=27) were compared with basal-like familial BRCAX (non-BRCA1/2/CHEK2*1100delC) tumors (n=14) in a familial cohort of 120 breast carcinomas. To further substantiate our findings, we used flow cytometry to isolate cancer cells from formalin-fixed, paraffin-embedded, BRCA1-mutated triple negative breast carcinomas after which genomic profiling was performed with the use of shallow whole genome sequencing. Genome wide copy number profiles of the BRCA1-mutated breast carcinomas in our data appeared heterogeneous. Gene expression analyses identified varying amounts of tumor infiltrating lymphocytes (TILs) as a major cause for this heterogeneity. Indeed, selecting tumors with relative low amounts of TILs, resulted in the identification of three known but also five previously unrecognized BRCA1-associated copy number aberrations. Moreover, these aberrations occurred with high frequencies in the BRCA1-mutated tumor samples. Using these regions it was possible to discriminate BRCA1-mutated from BRCAX breast carcinomas, and they were validated in two independent cohorts. Genetics Retreat 2015 [email protected] www.cmm.umcutrecht.nl nl.linkedin.com/in/MaartenMassink To further substantiate our findings, we used flow cytometry to isolate cancer cells from formalin-fixed, paraffin-embedded, BRCA1-mutated triple negative breast carcinomas with estimated TIL percentages of 40% and higher. Genomic profiles of sorted and unsorted fractions were compared by shallow whole genome sequencing and confirm our findings. Authors Maarten P.G. Massink (a), Irsan E. Kooi (a), Saskia E. van Mil (a), Ekaterina S. Jordanova (b), Najim Ameziane (a), Josephine C. Dorsman (a), Daphne M. van Beek (a), J. Patrick van der Voorn (c), Daoud Sie (c), Bauke Ylstra (c), Carolien H.M. van Deurzen (d), John W. Martens (e), Marcel Smid (e), Anieta M. Sieuwerts (e), Vanja de Weerd (e), John A. Foekens (e), Ans M.W. van den Ouweland (f), Ewald van Dyk (g), Petra M Nederlof (h), Quinten Waisfisz (a), Hanne CONCLUSIONS This study shows that genomic profiling of in particular basal-like, and thus BRCA1-mutated, breast carcinomas is severely affected by the presence of high numbers of TILs. Previous reports on genomic profiling of BRCA1-mutated breast carcinomas have largely neglected this. Therefore, our findings have direct consequences on the interpretation of published genomic data. Also, these findings could prove valuable in light of currently used genomic tools for assessing BRCA1 involvement in breast cancer patients and pathogenicity assessment of BRCA1 variants of unknown significance. The BRCA1-associated genomic aberrations identified in this study provide possible leads to a better understanding of BRCA1-associated oncogenesis. Meijers-Heijboer (a) Affiliations 1.Departments of (a)Clinical Genetics, (b) Obstetrics and Gynaecology; Center for Gynaecologic Oncology, and (c) Pathology, VU University Medical Center, Amsterdam, The Netherlands. 2..Departments of (d) Pathology, (e) Medical Oncology, and (f) Clinical Genetics, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands. 3..Departments of (g) Pathology and (h) Molecular Carcinogenesis, Netherlands Cancer Institute, Amsterdam, The Netherlands. Genetics Retreat 2015 63 Daiane Hemerich Integrating data sources in druggability analysis of genes implicated in dilated cardiomyopathy Dilated cardiomyopathy (DCM) is characterized by systolic dysfunction and dilation of the left ventricle of the heart, with a 5-year mortality estimated in 50%, and is the main indication for heart transplantation. Current treatments range from medication to device therapy, but these therapies primarily work by slowing down disease progression. Hence, unless patients get transplanted or receive a support heart (left ventricular assist device, LVAD), DCM ultimately leads to death. Treatments targeting genetic factors may potentially prolong survival and reduce side effects. A systematic literature search identified 110 genes implicated in DCM. We analyzed the druggability properties of these implicated genes and related genes in pathways. A list of gene-drug interactions was generated based on bioinformatics techniques, and we aim at identifying drugs that can be repurposed for DCM treatment, using information on indications and side effects of these drugs. 64 METHODS RESULTS We successfully identified 314 drugs that act on DCM-related genes, using DGIdb [1], chEMBL [2] and DrugBank [3]. We developed a Python script to identify synonyms of each drug, based on definitions from STITCH [4], and integrate these drugs with indications and side effects from SIDER [5], a project for data mining of medication labels. From the 314 drugs mapped for the genes of interest, 274 were found by the algorithm in the aliases database. The search returned 23.889 synonyms for the original names, a list of side effects for each drug listed (15.970 in total) as well as their indications (1.832 in total). Genetics Retreat 2015 [email protected] www.umcutrecht.nl/en/Research CONCLUSIONS Integrating data sources is a necessary step towards more complex analyses, and these may lead to better and faster translation to health care. Authors Hemerich, D. (1, 2), Kummeling, G. (1), Tragante, V. (1), Asselbergs, F.W. (1) Affiliations 1.Department of Cardiology, Division Heart and Lungs, University Medical Center Utrecht, Utrecht, The Netherlands 2. CAPES Foundation, Ministry of Education of Brazil, Brasília – DF 70040-020, Brazil References 1. http://dgidb.genome.wustl.edu/ 2. https://www.ebi.ac.uk/chembl/ 3. http://www.drugbank.ca/ 4. http://stitch3.embl.de/ 5. http://sideeffects.embl.de/ Genetics Retreat 2015 65 Robin Verjans Identification of MicroRNAs Regulating Heart Failure: A Phenotypical High-Throughput Screening Approach Heart failure (HF) is often caused by injury or stress to the myocardium. Subsequently to this stress the heart undergoes detrimental changes, such as: adaptive enlargement of the myocardium in response to pressure or volume overload (cardiac hypertrophy), increased collagen production by cardiac fibroblasts, resulting in excessive fibrosis and inflammation of the myocardium accompanied with proinflammatory activation of resident immune cells and infiltration of leukocytes. microRNAs (miRNAs) are small non-coding regulatory RNAs (20-23 nucleotides), which negatively regulate gene expression at post-transcriptional level. Recent studies in mice have revealed that miRNAs play important regulatory roles in different HF disease processes. miRNA targets identified in the past are often studied in one cell type only or in whole heart biopsies, making clarification of the exact heart failure regulating mechanism challenging. Our aim is therefore to identify miRNAs involved in hypertrophy, fibrosis and inflammation and to clarify the direction of this influence. miRNA identification using a high-throughput and high content phenotypic screening of a miRNA library in not one, but multiple relevant primary cell types allows better understanding of the miRNA regulatory function. METHODS miRNAs selection for screening was based on miRNA array data from three rodent heart failure models: 1. The ZFS1 model of obese, diabetic and hypertensive rats 2. the mouse model of cardiac transplant and 3. mouse CVB3 induced viral myocarditis model. To be able to identify/validate miRNAs the screen was performed in relevant primary cells types: neonatal rat cardiac myocytes (nRCMs) to study the hypertrophic response, neonatal rat cardiac fibroblasts (nRCFs) to determine the effect on fibrosis, and bone marrow 66 Genetics Retreat 2015 derived macro-phages (BMDM) to study the effect on inflammation. Overexpression of target miRNAs was achieved via miRNA mimic transfection. Cardiac myocyte hypertrophy was induced using phenylephrine (PE). As readout for nRCM hypertrophy, cells were immunofluorescently stained for a-Actinin and a-Tubulin, allowing automated quantification of myocyte cell size. A fibrotic response in nRCFs was induced using TGF-beta stimulation. In nRCFs, immunofluorescence staining for a-Tubulin and Collagen1a1 was performed to quantify collagen levels. BMDMs [email protected] http://www.carimmaastricht.nl/ were polarized using IFNy (M1) and IL-4 (M2) and activated using LPS. Immunofluorescence staining for a-Tubulin was used to determine polarisation via morphological features and activation was quantified using a NFkB (p65) nuclear trans-location assay. RESULTS Analysis of the array data of different rodent HF models revealed 194 differentially expressed miRNAs that were selected for cell-based validation. miRNA overexpression was achieved with high efficiency in all studied cell types. Phenotypically relevant cardiac disease alterations were induced in all three primary cell types. Cardiac myocyte cell size increased significantly after PE stimulation. In nRCFs, TGFbeta treatment lead to an increased collagen content in comparison with negative control. LPS stimulation of BMDMs induced a translocation of NFkB (p65) towards the nucleus. Polarisation with IFNy resulted in a significant decrease in the elongation of BMDMs whilst IL4 induced a more elongated phenotype. Most interestingly, we have determined for all 194 miRNAs what exact role they play in the different disease processes of heart failure. In addition, several (novel) miRNAs were found to be involved in more than one heart failure disease parameter and are therefore interesting therapeutic targets for heart failure. CONCLUSIONS We have performed a high throughput/content screen in three relevant primary (cardiac) cell types, thereby identifying novel miRNAs involved in hypertrophy, fibrosis and inflammation. In addition, more insight was gained in possible regulatory mechanism of already identified miRNAs via confirmation and exclusion of involved cell types. miRNA overexpression can be achieved with high efficiency in all studied cell types. Stimulation with appropriate stimulus resulted in significant morphological changes that are relevant in heart failure. High-throughput screening using primary (cardiac) cell types resulted in the identification of miRNAs able to influence cardiac hypertrophy, fibrosis and inflammation. Indicating that these miRNAs play a very important role in the regulation of different components of heart failure. These miRNAs form potential therapeutic targets. Authors Verjans R. (1,3) Derks W. (1,3), Korn K. (3), Soennichsen B. (3), Van Bilsen M. (2), Schroen B. (1), Heymans, S. (1) Affiliations 1.Department of Cardiology, Cardiovascular Research Institute Maastricht (CARIM) 2.Department of Physiology, Cardiovascular Research Institute Maastricht (CARIM) 3. Cenix BioScience, GmbH, Dresden, Germany Genetics Retreat 2015 67 mark Hazebroek MOGES classification in dilated cardiomyopathy The multifactorial pathogenesis leading to dilated cardiomyopathy (DCM) makes stratification difficult. The recent MOGES classification (Morphofunctional; Organ involvement; Genetic or familial; Etiology; Stage) addresses this issue. To investigate the applicability and prognostic relevance of the MOGES classification in DCM patients. 68 METHODS RESULTS Within the Maastricht Cardiomyopathy Registry patients with DCM were enrolled. We excluded patients with ischemic, valvular, hypertensive and congenital heart disease. All patients underwent complete diagnostic workup, including genetic evaluation and endomyocardial biopsy. Long-term outcome used a combined endpoint of life-threatening arrhythmia, heart transplantation, and death. Additionally, left ventricular reversed remodeling (LVRR) at 12 months was assessed. A total of 213 consecutive DCM patients were included, demonstrating organ involvement in 35 (16%) and genetic/familial DCM in 70 (33%) patients, including 16 (8%) with a pathogenic mutation. At least one etiology was found in 155 (73%) patients, of whom 48 (23%) had more than 1 possible etiology. LVRR was more common in patients with non-genetic/familial than with genetic/familial DCM (40% vs 25%; p=0.04). After a median follow-up of 47 [30-67] months, organ involvement and higher NYHA-class were associated with adverse outcome (Log rank P<0.001 and Log rank P=0.02, respectively). Genetic/familial DCM per se was of no prognostic significance, but when accompanied with additional etiological-environmental factor(s) i.e. significant viral load, immune-mediated, rhythm disturbances and/or toxic triggers, worse outcome was revealed (Log rank P=0.03). Higher presence of MOGES attributes (≥2 vs ≤1 attributes) showed an adverse outcome (Log rank P=0.007). Genetics Retreat 2015 CONCLUSIONS The MOGES classification in DCM is applicable and each attribute and/ or the gene-environment interaction (Organ involvement (O), Geneenvironmental Etiology (G+E), NYHA class (S)) is associated with outcome. Importantly, presence of multiple attributes was a strong predictor of adverse outcome. Finally, adaptation of the MOGES involving multiple possible etiologies is recommended. Authors M.R. Hazebroek*, S. Moors*, R. Dennert*, A. van den Wijngaard†, I. Krapels†, M. Hoos*, J. Verdonschot*, J.J. Merken*, B. de Vries‡, P. Wolffs§, H.J.G.M. Crijns*, HP. BrunnerLa Rocca*, S. Heymans* Affiliations 1.Department of Cardiology*, Department of Clinical Genetics†, Department of Pathology‡ and 2.Department of Medical Microbiology§, Maastricht University Medical Centre, Maastricht, The Netherlands Genetics Retreat 2015 69 Roeland Vanhauwaert Generation of a novel Drosophila melanogaster Parkinson’s disease model A homozygous missense mutation in Synaptojanin 1 (SYNJ1) has been shown to cause autosomal recessive early-onset Parkinson’s disease (PD). SYNJ1 is a lipid phosphatase that is highly expressed in the nervous system and especially enriched at synapses. Besides its proline rich domain, that is responsible for protein-protein interactions with other synaptic players, it consists of two phosphatase domains that dephosphorylate different phosphoinositides in membranes. The PD-causing point mutation resides in one of these phosphatase domains, called the SAC1 domain. SYNJ1 and its Drosophila homologue Synaptojanin (Synj) have been shown to be essential for efficient recycling of synaptic vesicles (SV) in neurons, suggesting a connection between PD and synaptic endocytosis, however, the exact effect of how this new PD mutation affects Synj function in vivo and how it leads to neuronal degeneration remains to be investigated. 70 METHODS RESULTS To assess the functional consequences of the disease causing mutation in SYNJ1 at the level of the synapse, we generated fruit flies expressing this clinical PD mutation. We used both an overexpression approach as well as a novel in-house developed knockin technique. We are now using live imaging of synaptic vesicles, electron microscopy and electrophysiology to gain insight into the synaptic defects caused by the PD-causing mutation, and we are comparing the defects to those observed in other PD fly models. Flies expressing the clinical Synj PD mutation rescue the Synj null mutant lethality but the flies show neurological defects. Moreover we found exciting synaptic phenotypes that we are further investigating in relation to neuronal degeneration. Genetics Retreat 2015 [email protected] www.verstreken.vib.be be.linkedin.com/in/RoelandVanhauwaert CONCLUSIONS A homozygous mutation in SYNJ1 is recently identified in PD. We generated a Drosophila PD model and are currently assessing the effects of this mutation on synaptic function and neuronal degeneration. Authors Roeland Vanhauwaert1,2,, Sven Vilain1,2, Sandra Soukup1,2, Nils Schoovaerts1,2, Jef Swerts1,2, and Patrik Verstreken1,2 Affiliations 1.VIB Center for the Biology of Disease, 3000 Leuven, Belgium 2.KU Leuven, Department of Human Genetics and Leuven Research Institute for Neuroscience and Disease (LIND), 3000 Leuven, Belgium Genetics Retreat 2015 71 Marijke Versteven Agonistic sound stimulates Drosophila male- male aggression Aggressive behavior is a universal trait essential for species survival and reproduction. It is a determining factor in the acquisition of territory, food or mates and is a defense mechanism against predators. We are combining genome-wide with single gene approaches to unravel the genetic complexity of aggression in Drosophila. Whole genome analysis revealed correlations with aggression of several clusters of functionally related genes. One cluster consists of genes associated with the auditory system in Drosophila. Acoustic communication is a component of aggressive behavior in various species. In Drosophila, wing threats generate specific sound patterns during male-male agonistic interactions. It remained an open question whether these aggression songs function as acoustic signals in terms of modulating an opponent’s behavior during aggressive interactions. We therefore analyzed in a systematic way whether auditory information affects agonistic behavior and have found that acoustic signals generated by male flies play a prominent role. 72 METHODS RESULTS - Importance of sound detection in aggression: aristectomy & antennal immobilization - Hearing impaired mutants - Requirement of auditory information transfer by the Johnston’s organ: targeted RNAi mediated knockdown of auditory genes & silencing neurotransmission - Presenting male flies with agonistic sound We analyzed in a systematic way whether aggression songs generated by male flies affect agonistic behavior. We show that impairing the mechanics of the fly’s antennal sound receiver reduces male-male aggression. Accordingly, silencing the auditory receptors but not the wind or gravity receptors in the antenna also reduces aggressive encounters. In addition, mutant flies with specific alterations of auditory receptor functions display specific alterations, either reduced or enhanced, of aggressive behaviors. Finally, whereas agonistic sounds increase aggression, courtship songs were found to reduce aggression. Genetics Retreat 2015 [email protected] CONCLUSIONS Our work reveals that male fruitflies detect and interpret intraspecific acoustic signals and adapt their behavioral response in a context-dependent manner. Taken together, we propose that hearing is an important sensory modality in Drosophila aggressive behavior. Authors Marijke Versteven1, Martin Göpfert², Ralf Heinrich², Patrick Callaerts1 Affiliations 1.VIB - Laboratory of Behavioral and Developmental Genetics, KU Leuven, Belgium 2.Department of Cellular Neurobiology, University of Göttingen, Germany Genetics Retreat 2015 73 Kiliana Bekelaar Pharmacogenetics of lithium and valproate in Drosophila melanogaster Lithium and valproate are mood-stabilizing drugs commonly used in the psychiatric clinic. However, important variations in therapeutic response to these drugs is observed. Responsiveness to lithium appears to cluster in families and is linked to occurrence of psychiatric disorders in relatives. This suggests that a genetic disposition is not limited to susceptibility to the disorder, but is also important in treatment responsiveness. Elucidation of the genetic and environmental factors involved in this differential drug response can help in diagnosis and treatment selection, in unraveling the mechanism of action of these drugs and in elucidating the etiology and pathophysiology of bipolar disorder. Therefore the pharmacogenetics of response to mood stabilizers has become a growing field of research. In a study on the genetic complexity of Drosophila aggressive behavior [Zwarts et al, 2011], our group observed genetic background-specific effects of lithium and valproate on Drosophila aggression. This observation prompted us to investigate the feasibility of using Drosophila melanogaster aggression as a model system to identify the genetic factors that contribute to differential drug response. In our study we performed a genome wide association (GWA) study in Drosophila melanogaster to investigate the pharmacogenetics of lithium and valproate. METHODS RESULTS We made use of the Drosophila Genetics Reference Panel of inbred stocks to identify SNPs associated with differential drug response. Flies were fed lithium or valproate for short (2 days) or longer (7 days) periods of time and aggression was quantified to assess behavioral response to treatment. The DGRP exhibits natural differences in aggression, and a significant variation in aggressive drug response is present among the lines within each drug and treatment duration environment. GWA analysis identified SNPs in 850 genes that are associated with differential drug response. From this list of SNPs we selected genes for further validation based on pathway based analysis, gene ontology, literature, and the presence of human orthologs. Using single gene mutants the role of these genes in differential drug response was confirmed. GWA analysis was performed to identify SNPs associated with differential drug response. Next we assessed the role of a number of the genes where those SNPs were located using single gene mutants. 74 Genetics Retreat 2015 [email protected] www.gbiomed.kuleuven.be We are currently evaluating whether the genetic basis of response to mood stabilizers is conserved between D. melanogaster and human. CONCLUSIONS These results demonstrate that Drosophila can be used successfully as a model to dissect the complex genetic basis of differential drug response. Authors Bekelaar K.(1,2), Herteleer L.(1,2), Zwarts L.(1,2), Magwire M.M.(3), Laenen A.(4), Lyman R.F. (3), Mackay T.F. (3), Callaerts P.(1,2) Affiliations 1.Laboratory of Behavioral and Developmental Genetics, Department of Human Genetics, KU Leuven, B-3000 Leuven, Belgium 2.Laboratory of Behavioral and Developmental Genetics, VIB Center for the Biology of Disease, B-3000 Leuven, Belgium 3.Department of Biological Sciences, North Carolina State University, Raleigh, North Carolina 27595, USA 4.Leuvens Biostatistiek en Statistische Bioinformatica Centrum, KU Leuven, B-3000 Leuven, Belgium Genetics Retreat 2015 75 Liesbeth Zwarts Glial Notch regulates Drosophila behavior by maintaining adult brain homeostasis A connection between inflammation and psychiatric disorders has been suggested from the 19th century onwards. However, despite numerous reports of immune abnormalities in various psychiatric disorders and the intensified interest in unraveling the etiology of this link over the past decades, very little is known on its mechanistic basis or causality. The Notch pathway has been shown to be an important regulator of both innate and adaptive immunity in vertebrates. In this context it has been linked to the pathogenesis of autoimmune disorders such as multiple sclerosis and diabetes type 1. Interestingly, Notch genes have been associated with bipolar disorder, schizophrenia, down syndrome and Alzheimer’s disease. While Notch is mostly known for its role during development, it is also expressed in the adult brain and has been shown to modulate behaviors in different species. We previously demonstrated that neuralized (neur) and Gp150, two genes involved in the modulation of the Notch pathway, modulate adult aggressive behavior in Drosophila. This suggests that Notch signaling may be directly involved in modulating aggressive behavior. 76 METHODS RESULTS We make use of Drosophila melanogaster to gain insight into the function of the Notch pathway in the adult brain. We performed behavioral test to determine the cell types in which the different key players of this pathway exert their effect on behavior. Next we performed RNAsequencing to gain insight into the genetic mechanism underlying the abnormal behavior. We further validated our findings by using qRT-PR and fluorescent confocal microscopy on reporter lines for the identified genes. Finally, we perform genetic rescue experiments to revert the behavioral phenotypes. We demonstrate that neuronal Dl in the lateral neurons ventral (LNvs), the main pacemaker neurons of the Drosophila clock, interacts with glial Notch in the adult Drosophila brain to modulate behavior. Transcriptome analysis reveals that alterations in Notch signaling induce inflammatory alterations in the adult brain. Further analyses show that modulation in the LNv’s is sufficient to elicit a comparable change in inflammation. We show that the observed inflammatory alterations are causal for the observed behavioral changes. Our data Genetics Retreat 2015 [email protected] www.gbiomed.kuleuven.be shows a role for the IMD pathway in the LNvs and a role for JAK/ STAT in both the LNv’s and the glia. Both pathways rescue the effect of Notch in their respective cell types. Authors Liesbeth Zwarts (1), Veerle Vulsteke (1), Jason Clements (1), Wouter Van Delm (2), Patrick Callaerts (1) Affiliations CONCLUSIONS We provide insight into the genetic mechanisms that form the link between immune deregulation and abnormal behavior. We show that knock down of Notch in glia results in a profound deregulation of two main immune pathways in Drosophila. This immune deregulation is mainly present in neurons and is causal for the observed behavioral changes. 1.VIB, Laboratory of Behavioral and Developmental Genetics, Leuven, Belgium, and K.U.Leuven, Department of Human Genetics, Herestraat 49, bus 602, B-3000 Leuven, Belgium 2.VIB, Nucleomics Core, Leuven, Belgium. Herestraat 49, box 816, B-3000, Leuven, Belgium Genetics Retreat 2015 77 Ivo Eijkenboom A read-out panel reflecting small fiber neuropathy in zebrafish Neuropathic pain is a symptom of small-fiber neuropathy causing significant impact on patients’ quality of life and health care costs. Recently, researchers from our consortium identified several mutations in voltage-gated sodium channels, SCN9A and SCN10A, underlying small-fiber neuropathy. To further unravel the genetic aspects of small-fiber neuropathy we apply unbiased sequencing approaches to identify genetic variants in a large patient population with small-fiber neuropathy. Since electrophysiological studies are expensive and extremely time consuming and the numbers of possible pathogenic mutations detected are expected to be high, there is a need for a medium throughput screening model. The zebrafish (Danio rerio) meets these criteria and will be used to screen candidate pain-related mutations. Before we can test the pathogenicity of the identified variants a panel of read-out parameters reflecting neuropathic pain in zebrafish must be set up and validated. This panel is based on clinical hallmarks of patients with small fiber neuropathy like, an abnormal response to thermal stimuli and a reduced density of sensory neurons in a skin biopsy. Validation of this read-out panel will be performed using a knockdown/knock-out approach and by overexpressing human mutations causing small-fiber neuropathy. METHODS Our read-out panel is based on clinical-diagnostic tests to diagnose small-fiber neuropathy and exists of behavioral tests like, touch-evoked response and reaction to temperature change. Touch response will be assessed using a stereomicroscope, whereas the other responses will be recorded using a customized Zebrabox device (Viewpoint). In addition to behavioral parameters, a morphological characteristic will also be assessed; sensory neuron axon density in the skin using a transgenic line (sensory:GFP), marking all sensory neurons 78 Genetics Retreat 2015 followed by confocal microscopy. The read-out panel will be validated using knockdown and knock-out strategies for the zebrafish sodium channels and by overexpressing human mutations causing small-fiber neuropathy. After validation of the read-out panel, novel variants identified by partners will be tested with this panel. These results will provide information whether these variants are causative for small-fiber neuropathy. [email protected] nl.linkedin.com/pub/ivo-eijkenboom RESULTS CONCLUSIONS We have customized a ZebraBox system which allows us to assess and quantify in a medium through-put manner (48 wells plate) touch-evoked response and reaction to temperature change. Validation of this system has been performed using a published Scn8aa morpholino. These published results showed the absence of the touch response after knockdown of scn8aa and could be confirmed in our hands with the touch response assay. Furthermore, we showed that scn8aa knockdown resulted in a diminished behavioral response to increasing temperatures which was also confirmed with the Nebula line (scn8aa knock-out). The next step is to determine the effects of scn8aa knock down/Knockout on the axon density of sensory neurons and the behavioral response to irritating compounds like, mustard oil. Both tests, the quantification of sensory neurons and the behavioral response to irritating compounds, are already optimized for wildtype zebrafish. Additionally, we will also validate our panel by overexpressing 2 pathogenic mutations in the human sodium channel (SCN9A) that play an important role in the development of small fiber neuropathy. We confirmed published results that a loss-of-function of scn8aa shows the absence of a touch response. Besides, we showed that a loss-offunction of scn8aa results in a diminished temperature response, which is not yet described. We can conclude that our panel; which exists of touch-evoked response, reaction to temperature change works optimal with wildtype and Scn8aa knockdown/ knock-out zebrafish. The other 2 test of our panel, reaction to irritating compounds and density of sensory neurons are in progress and already optimized for wildtype zebrafish. Currently, we are validating our panel by overexpressing 2 pathogenic mutations in the human sodium channel (SCN9A) that play an important role in the development of small-fiber neuropathy. Authors Eijkenboom I. (1), Vanoevelen J.M. (1) Faber C.G. (2), Smeets H.J.M. (1) Affiliations 1.Department of Clinical Genetics, Maastricht University, Maastricht, the Netherlands 2. Department of Neurology, Maastricht University Medical Center, Maastricht, the Netherlands Genetics Retreat 2015 79 hester happé Gene inactivation at different ages in an inducible kidney specific Pkd1 knockout model results in multiple models with different characteristics To study Autosomal Dominant Polycystic Kidney Disease (ADPKD) and perform preclinical studies, we previously developed a kidney specific tamoxifen inducible Pkd1 k nockout mouse model. We showed that gene disruption in young adult mice, at postnatal day 40 (PN40), results in end-stage PKD 16-18 weeks after gene disruption. In cystic kidneys of these mice the majority of cysts are from proximal tubular origin and to lesser extend derived from distal tubules and collecting duct. In contrast, PKD progression is much faster when Pkd1 is disrupted at PN4 and the cysts in these mice are primarily derived from distal tubules and to limited extend from proximal tubules. A more suitable model that forms cysts mainly derived from distal tubules and collecting duct, or even all segments, is desired. Additionally, the rate of disease progression should enable a sufficient time window for therapeutic intervention. In this study we aimed to optimize our Pkd1 knockout model in order to obtain a model that shows these characteristics. METHODS Gene disruption in kidney specific Pkd1 knockout mice was induced by administration of tamoxifen in neonatal mice before and after a reported developmental switch in gene expression at PN12-13 (Piontek et al 2007), i.e. at PN10 and PN18 (PN10 and PN18 model respectively). To monitor renal function and disease progression, blood urea concentration was measured. The cystic burden and segmental origin of the cysts was studied using (immuno)histochemistry using PAS 80 Genetics Retreat 2015 staining and staining for megalin, uromodulin (Tamm-Horsfall protein) and aquaporin-2 for proximal tubules, distal tubule/thick ascending limb of the loop of Henle and collecting duct, respectively. RESULTS Pkd1 gene disruption at PN10, results in large polycystic kidneys within 3 to 4 weeks, characterized by large cysts derived from distal tubules and collecting duct. These cysts show the largest growth between 1 and 2 weeks [email protected] after gene disruption, while proximal tubules are only minimally involved. Kidneys from the PN18 model reveal cortical as well as medullary cysts that contribute equally to the phenotype, and develop end-stage PKD 11-14 weeks after gene disruption. Authors H. Happé(1), W.N. Leonhard(1), K. Veraar(2), E. De Heer(2), D.J.M. Peters(1), on behalf of the DIPAK-consortium Affiliations 1.Dept. of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands CONCLUSIONS 2.Dept. of Pathology, Leiden University Medical Center, Leiden, the Netherlands The PN18 Pkd1 deletion model may prove a valuable additional model for basic and pre-clinical research in ADPKD as it shows cyst formation in proximal tubules as well as distal tubules and collecting duct, in addition to a rate of disease progression that allows a sufficient therapeutic time window. Further study into the time course of cyst formation in the different nephron segments is ongoing. These results show that moment of gene inactivation, prior or after the developmental switch, greatly determines the characteristics of our mouse model for ADPKD with regard to the involved nephron segment and progression rate, although less sharp as proposed previously (Piontek et al, 2007). It is expected that this can also be applicable for models of other renal (tubular) diseases. Genetics Retreat 2015 81 Alessio Marcozzi Engineering chromosomal translocations using the CRISPR/Cas system Balanced chromosomal translocations can lead to severe congenital disease, such mental retardation and developmental delay. Although the genes disrupted by the translocation breakpoints can occasionally be linked to the disease phenotype, the effects of balanced translocations on genome-wide gene regulation and expression levels is unknown. Such information would be particularly relevant for patients who carry translocations with breakpoints that do not directly affect genes. The sporadic nature of most balanced translocations has major limitations for studying their cellular and phenotypic consequences, because these are all n = 1 studies. In addition, there is a lack of matching control samples that can serve as a baseline reference. Family members are not appropriate because of the large differences in genetic background. To overcome these limitations, we have leveraged the CRISPR/Cas system to engineer specific translocations into different human cell lines. 82 METHODS RESULTS A plasmid-mediated CRISPR/Cas system has been used to induce double strand breaks in different chromosomes of two different human cell lines, the HEK293 and the RPE-1. Recombination events among broken chomosomes have been detected by PCR and confirmed by sequencing. We succeeded to generate reciprocal translocations that mimic the situation in the patient. Clonal lines with translocations will be characterised at the molecular level and compared to the same cells without the translocation. Integration of data from multiple cell lines with different types Genetics Retreat 2015 [email protected] nl.linkedin.com/pub/alessio-marcozzi of engineered translocations sheds light on the effects of balanced translocations with respect to local and long-distance changes in gene expression and the mechanisms by which these effects are mediated. In the long term, these experiments will help to explain the phenotypic malformations in patients with balanced translocations. Authors Marcozzi A.(1) Kloosterman W.(1) Affiliations 1.Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands Genetics Retreat 2015 83 Mike Jeurissen Hematopoietic overexpression of Cyp27a1 reduces hepatic inflammation independently of 27-hydroxycholesterol levels in LDL-r-/- mice Non-alcoholic steatohepatitis (NASH) is characterized by hepatic lipid accumulation and inflammation. Currently, the underling mechanisms leading to hepatic inflammation are still unknown. The breakdown of free cholesterol inside Kupffer cells (KCs) by the mitochrondial enzyme CYP27A1 produces 27-hydroxycholesterol (27HC). We recently demonstrated that administration of 27HC to hyperlipidemic mice reduced hepatic inflammation. In line, hematopoietic deletion of CYP27A1 resulted in increased hepatic inflammation. In the current manuscript, the effect of hematopoietic overexpression of CYP27A1 on the development of NASH and cholesterol trafficking was investigated. We hypothesized that CYP27A1 overexpression in KCs will lead to reduced hepatic inflammation. 84 METHODS RESULTS Irradiated Ldlr-/- mice were transplanted (tp) with bone marrow from mice overexpressing CYP27A1 (Cyp27a1over) and wild-type (Wt) mice and fed either chow or high-fat, high-cholesterol (HFC) diet for 3 months. Additionally, gene expression was assessed in bone-marrow derived macrophages (BMDM) from Cyp27a1over and Wt mic. In line with our hypothesis, hepatic inflammation in HFC-fed Cyp27a1overtp mice was reduced and KCs were less foamy compared to Wt-tp mice. Remarkably, these changes occurred even though plasma and liver levels of 27HC did not differ between both groups. BMDM from Cyp27a1over mice revealed reduced inflammatory gene expression and increased expression of cholesterol transporters compared to Wt BMDM after LPS stimulation. Genetics Retreat 2015 [email protected] CONCLUSIONS Our data suggest that overexpression of Cyp27a1 in KCs reduces hepatic inflammation independently of 27HC levels in plasma and liver, further pointing towards KCs as specific target for improving therapy of NASH. Authors Mike L.J. Jeurissen (1), Tim Hendrikx (1), Veerle Bieghs (1), Sofie MA Walenbergh (1), Patrick J van Gorp (1), Fons Verheyen (1), Tom Houben (1), Jieyi Li (1), Yasmin Dias Guichot (1), Marion JJ Gijbels (1), Eran Leitersdorf (2), Marten Hofker (3), Dierter Lütjohann (4), Ronit Shiri-Sverdlov (1) Affiliations 1.Departments of Molecular Genetics, Molecular Cell Biology, ELMI unit (CRISP) and Pathology; Nutrition and Toxicology Research (NUTRIM) and Cardiovacsular Research (CARIM) institutes of Maastricht, University of Maastricht , Maastricht, the Netherlands 2.Department of Medicine, Hadassah-Hebrew University Medical Center, Jerusalem, Isreal 3.Department of Pathology & Laboratory Medicine, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands 4.Institute of Clinical Chemistry and Clinical Pharmacology, University of Bonn, Bonn, Germany Genetics Retreat 2015 85 Mandy Steinbusch The RMRP snoRNA and chondrogenic differentiation Mutations in the RMRP gene cause cartilage-hair hypoplasia (CHH). CHH patients present with a severe form of dwarfism, sparse hair, immunodeficiency and increased risk for malignancies. CHH is the first identified human disease caused by mutations in a nuclear encoded small nucleolar RNA (snoRNA) molecule. RMRP RNA is the noncoding RNA subunit of the RNase MRP complex, a macromolecular particle consisting of the RMRP snoRNA and at least 7 different protein subunits (Rpp20, Rpp25, Rpp30, Rpp38, Rpp40, Pop1 and Pop5) and with a major task as an endoribonuclease in pre-rRNA processing. CHH-associated mutations in the RMRP gene are thought to reduce the ribozyme activity of the RNase MRP complex or interfere with RMRP RNA expression. How this results in the development of CHH is unknown. The prominent dwarfism phenotype suggests that RNase MRP function is involved in chondrocyte development in the growth plate. We therefore hypothesized that RNase MRP is involved in chondrogenic differentiation of the growth plate during skeletal development. METHODS 86 To investigate the expression of RNase MRP during chondrogenic differentiation in vivo, RMRP protein subunits were detected in growth plates of 6 week-old mice by immunohistochemistry (IHC). To verify these results, expression of RMRP RNA, RNase MRP protein subunits and important chondrogenic differentiation markers (Sox9, Col2a1, Runx2, Col10a1) was also determined by RT-qPCR in chondrogenically differentiating ATDC5 cells, in the presence or absence of 1 nM hypertrophic inducer bone morphogenetic protein (BMP)-2. RMRP function during chondrogenic differentiation was targeted by transfection of specific siRNA duplexes. Transdifferentiation of adult human dermal fibroblasts to chondrocyte-like cells was carried out by hyperconfluGenetics Retreat 2015 ent plating of control and CHH patient fibroblasts on aggrecan coated wells. This allowed us to verify the chondrogenic capacity of CHH patient-derived primary cells. RESULTS To establish a role for RNase MRP during chondrogenic differentiation in vivo, mouse growth plate sections were stained for RNase MRP protein subunits by IHC. All proteins showed identical growth plate distribution patterns: hypertrophic zone chondrocytes express RNase MRP proteins, whereas no or only weak expression levels were observed in proliferative chondrocytes. These expression dynamics were confirmed in ATDC5 cells; RMRP RNA and Rpp40 expression was upregulated from day 14 onwards, simultaneously with markers [email protected] http://orthopedie.mumc.nl/onderzoeksteam nl.linkedin.com/pub/mandy-steinbusch for chondrocyte hypertrophy. Knockdown of RMRP RNA by RNAi resulted in an overall decreased chondrogenic marker expression, suggesting a role for RMRP in chondrogenic differentiation. These results prompted us to test whether ectopically inducing chondrocyte hypertrophy would affect RMRP RNA expression. Indeed, BMP2 increased the expression of chondrocyte hypertrophic markers, as well as RMRP RNA expression, harnessing the relation between chondrocyte hypertrophy and RMRP RNA expression. To investigate whether aberrant chondrocyte hypertrophy may be involved in CHH, we transdifferentiated CHH patient-derived fibroblasts and healthy controls to acquire a chondrocyte-like phenotype. Chondrogenic capacity of CHH patient fibroblasts was clearly disrupted and showed a more than 50% reduction of Col10a1 expression. CONCLUSIONS Our data show that expression of the snoRNA RMRP and its subunits are regulated during chondrogenic differentiation and indicate a predominant association with chondrocyte hypertrophy. These data for the first time show that expression levels of RNase MRP subunits can be influenced by cellular phenotypes and morphogenic differentiation cues and are the first to implicate a specific snoRNA in a developmental process. Importantly, these data may provide a rational for the CHH-associated skeletal phenotype. Our transdifferentiation experiments strongly suggest the existence of aberrant hypertrophic differentiation in CHH cells. However we do not know how RNase MRP function may regulate chondrocyte differentiation. In our ongoing research we are analyzing RNAseq data that were acquired from a transdifferentiation experiment (CHH versus healthy fibroblasts) that represent different time points (chondrocyte phenotypes) in differentiation. Data will be used to identify potential molecular pathways that may explain the CHH-related chondrocyte phenotype and ultimately its severe dwarfism. Importantly, this RNAseq experiment also allows for identification of the snoRNA networks that are involved in a chondrocyte differentiation. Authors Steinbusch M.M.F. (1), Caron M.M.J. (1), Eckmann F. (2), Lausch E. (2), van Rhijn L.W. (1), Zabel B. (2), Welting T.J.M. (1) Affiliations 1.Laboratory for Experimental Orthopaedics, Department of Orthopaedic Surgery, Maastricht University Medical Centre, Maastricht, the Netherlands 2Centre for Paediatrics and Adolescent Medicine, University Hospital Freiburg, Freiburg, Germany Genetics Retreat 2015 87 Machteld Oud Endocrine-cerebro-osteodysplasia syndrome is a ciliary disorder Endocrine-cerebro-osteodysplasia (ECO) syndrome (MIM#612651) is caused by mutations in ICK, which encodes a protein that was found to localize in cilia in previous Ick knockout studies. The primary clinical characteristics of ECO syndrome, being endocrine, cerebral and skeletal abnormalities, show significant overlap with other known ciliopathies such as Majewski, Mohr-Majewski and hydrolethalus syndromes. Our aim was to determine if ECO syndrome is a ciliopathy by investigating cilia in patient-derived cells. METHODS We cultured ECO patient-derived fibroblasts, harboring the homozygous p.R272Q mutation in ICK, and fibroblasts from two healthy controls and analyzed them for cilium presence, length and ciliary localization of intraflagellar transport (IFT) proteins by immunocytochemistry. RESULTS We found that ECO patientderived fibroblasts display ciliary abnormalities compared to control cells. The percentage of ECO patient-derived fibroblasts that formed cilia was significantly lower compared to both controls. Cilium length was not prominently altered. 88 Genetics Retreat 2015 This latter finding is in contrast with the shorter cilium phenotype observed in Ick-deficient murine embryonic fibroblasts (MEFs). Our analysis of intraflagellar transport (IFT) proteins, IFT88 and IFT57, showed normal ciliary localization, however, we observed a decreased expression of both proteins in ECO patientderived fibroblasts. This is remarkable as cilia of Ick-deficient mice show anaccumulation of IFT proteins at the ciliary tip. The discrepancies in cilium length and IFT localization in cilia between human and murine fibroblasts could be explained by the likely hypomorphic nature of the homozygous ICK mutation in the patient. [email protected] CONCLUSIONS We show that ECO patient-derived fibroblasts display ciliogenesis defects and altered IFT expression, and thereby demonstrate that ECO syndrome is a ciliopathy. Affiliations 1.Dept. of Human Genetics, Radboud Institute for Molecular Life Sciences and Radboud Institute for Health Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands 2.Dept. of Biochemistry, University of Western Ontario, London, Ontario, Canada Authors 3.Medical Genetics Program, London Health Machteld M. Oud1*, Dorus A. Mans1*, C Anthony Sciences Centre, London, Ontario, Canada Rupar2-4, Nathalie P. de Wagenaar1, Piya Lahiry5, 4.Children’s Health Research Institute, London, Gregory J. Pazour6, Robert A. Hegele2,7, Ronald Roepman1, Victoria M. Siu**2-4, Heleen H. Arts**1,2,7 Ontario, Canada 5.Dept. of Paediatrics, The Hospital for Sick Children, Toronto, Ontario, Canada 6.Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA 7.Robarts Research Institute, London, Ontario, Canada. * First authors contributed equally ** Senior authors contributed equally Genetics Retreat 2015 89 Peter Leegwater A nonsense mutation in stillborn Friesian horses with hydrocephalus classifies the disorder as muscular dystrophy-dystroglycanopathy Hydrocephalus in Friesian horses is a developmental disorder that results in stillbirth of affected foals and often dystocia in the dams. The mode of inheritance is probably monogenic recessive and introduced by a single founder, but the pedigree structure of horse populations hampers proper segregation analysis. The aim of our study was to identify the causal mutation for hydrocephalus in Friesian horses. 90 METHODS RESULTS Diagnosis of cases of hydrocephalus in Friesian horses was performed by local veterinarians. A genome wide screen of 13 hydrocephalus cases and 69 controls using 29,720 SNPs was performed. The data was analyzed for association with GenABEL software and subsequently for regions of homozygosity by eyeball. Next generation DNA sequence analysis was performed of gene exons in the identified region from 4 cases and 6 controls. The association analysis indicated the involvement of a region on ECA1 (P < 10e-16). All cases, but none of the controls, carried 2 copies of a 0.58 Mb haplotype. Next generation sequencing of the exons in the region revealed a nonsense mutation that was identical to a mutation identified in a human case of muscular dystrophydystroglycanopathy with hydrocephalus. All 16 available cases and none of the controls were homozygous for the mutation. 32 controls were heterozygous of which 17 were dams of cases and 36 controls were homozygous for the normal allele. Genetics Retreat 2015 [email protected] CONCLUSIONS Hydrocephalus in Friesian horses is an autosomal recessive disease. Homozygosity of a nonsense mutation is responsible for the trait. The gene mutation classifies the phenotype as a muscular dystrophy-dystroglycanopathy. Application of a DNA test for the mutation in the breeding program will prevent animal suffering and reduce commercial loss. Affiliations 1.Animal Breeding and Genomics Centre, Wageningen University, Wageningen, The Netherlands 2.Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, The Netherlands 3.Department of Medical Genetics, University Medical Center Utrecht, The Netherlands 4.Koninklijke Vereniging “Het Friesch Paarden-Stamboek”, Drachten, The Netherlands 5.Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Authors The Netherlands Ducro B.J. (1), Schurink A. (1), Bastiaansen J.W.M. 6.Department of Surgery and Anaesthesiology (1), Boegheim I.J.M. (2), Van Steenbeek F.G. (2), of Domestic Animals, Faculty of Veterinary Vos-Loohuis M. (2), Nijman I.J. (3), Monroe, Medicine, Ghent University, Belgium G.R. (3), Hellinga I. (4), Dibbits B.W. (1), Back W. (5, 6), Leegwater P.A.J. (2) Genetics Retreat 2015 91 Lambert C J Dorssers Deciphering malignant testicular germ cell cancer progression Malignant testicular germ cell cancer (TGCC) is the predominant cancer in young males. In contrast to most cancers in adults, TGCC is extremely sensitive for conventional therapy and cure is achieved for most patients. Intrinsic resistance to therapy and poor outcome is observed in rare cases. TGCC are considered to be initiated very early in embryogenesis and resemble the omnipotent embryonic stem cells (EC, nonseminoma = NS) or the totipotent primordial germ cells (seminoma = SE). We have investigated a relatively small series of therapy-resistant cancers using various high throughput technologies and now integrate the results to resolve the early steps in TGCC development. 92 METHODS RESULTS Four NS cases with intrinsic therapy resistance have been studied for whole genome DNA alterations (WGS, Complete Genomics Inc.), genome methylation (Illumina Methylation 450 beadchip), and RNA expression (Illumina expression beadchip and Ion Proton). In addition, FISH was applied to tumor sections and targeted next generation sequencing (Ion Torrent, 197 designed targets) was performed on different histological and developmental components of the tumors after laser capture micro dissection. In particular we have focused on the nonmalignant precursor carcinoma in situ (CIS) cells and the intratubular noninvasive component, which represent the early stages of the cancer development. WGS identified relative low numbers of somatic mutations in these tumors. Interesting somatic variants have been confirmed using qPCR strategies and some were also recovered in the RNAseq data. Overlap in the mutation profile, explaining the resistant phenotype or tumor development, was however not observed. Read coverage and allele frequency data show that most chromosomes are present in 3 or more copies and that loss of heterozygosity is absent (i.e. both parental alleles are retained). All somatic variants were found in a low proportion of the reads and always accompanied by copies of the wild-type alleles. The DNA methylation profiles of these resistant cases were not different from the profiles of sensitive Genetics Retreat 2015 [email protected] www.erasmusmc.nl/pathologie/research/lepo/ cases with similar histology. Intensity analysis of these methylation assays showed similar chromosome copy number distributions for these tumors. In order to get detailed insight in the order of the changes during tumor progression, we isolated precursor CIS cells and the noninvasive intratubular components by laser capture and sequenced these samples for 197 regions selected across the genome (containing a somatic mutation or adjacent regions with multiple polymorphic sites). In agreement with FISH data for the X and Y centromeres, we observed tumor heterogeneity for specific regions and under-representation of the somatic variants. The analysis of the specific tumor populations is ongoing and will be reported. We acknowledge the support of many colleagues within the departments of Pathology (LEPO, Molecular Diagnostics), Bioinformatics, and the Cancer Computational Biology Center (CCBC). This study is supported in part by a grant from Complete Genomics, Inc. Authors Dorssers LCJ. (1), Rijlaarsdam MA. (1), Gillis AJM. (1), Stoop JH. (1), Bertovic D. (1), Heijsman D. (2), IJpma AS. (2), Van der Spek PJ. (2), Hiltemann SD. (2), Stubbs AP. (2), Van Marion R. (1), Dubbink E-J. (1), and Looijenga LHJ. (1) Affiliations 1.Department of Pathology, ErasmusMC, Rotterdam. 2. Department of Bioinformatics, ErasmusMC, Rotterdam. CONCLUSIONS In summary, these integrated analyses provide detailed information regarding the mutational and chromosomal changes and the order of events in the development of TGCC. Our data indicate that mutations and chromosomal aberrations may not represent the initiating event. Genetics Retreat 2015 93 Jolien Vanhove Active and repressive histone marking during stem cell-derived hepatocytes in vitro Cell fate decisions and lineage commitment are under epigenetic control. Insight in epigenetic regulatory mechanisms governing commitment of human embryonic stem cells (hESCs) to the hepatocyte lineage is pivotal to generate mature and functional hepatocytes that could be used as in vitro cell models for hepatitis infection, drug toxicity and safety studies. 94 METHODS RESULTS Specific histone modifications in promoter and enhancer regions of OCT4, HNF4A, AFP, ALB, AAT and CYP3A4 were monitored by chromatin immunoprecipitation-qPCR during the commitment of hESCs to hepatic endoderm and hepatocyte-like cells. Epigenetic profiles in promoter and enhancer regions of lineage-specific genes correlated with their gene inactivity/activity status, except for AFP, ALB and AAT in hepatocyte-like cells; these loci retained H3K27me3 marking while the genes were expressed. Even the hepatocyte-like cells obtained after 0.6% dimethyl sulfoxide (DMSO) treatment, which expressed higher levels of HNF4A, ALB, AAT and CYP3A4 transcripts continued to contain this repressive epigenetic marking in the regulatory regions of ALB, AAT and CYP3A4. Interestingly, the DMSO-treated hepatocyte-like cells displayed less compact chromatin and higher RNA polymerase II binding. Genetics Retreat 2015 [email protected] www.kuleuven.be/samenwerking/scil/ be.linkedin.com/pub/jolien-vanhove CONCLUSIONS We demonstrate that hepatocyte cell fate acquisition in vitro is accompanied by specific histone modifications on defined genomic loci. Our findings suggest that active but not repressive marks in enhancers and promoters of AFP, ALB, AAT and CYP3A4 in hESC-hepatocyte progeny appear to determine the transcriptional status of hESC-derived hepatocytes. Our observations provide novel insights into epigenetic processes contributing to hepatocyte development from pluripotent cells, insights that may be used to optimize hepatocyte differentiation protocols to model liver diseases. Affiliations 1.KU Leuven - Department Development and regeneration, Stem Cell Institute, Leuven, Belgium 2.Université Catholique de Louvain, Cliniques St-Luc – Institut de Recherche Expérimentale et Clinique (IREC), Laboratory of Pediatric Hepatology and Cell Therapy, Brussels, Belgium 3. University of Oslo - Department of Biochemistry, Institute of Basic Medical Sciences, Oslo, Norway 4. Maastricht University Medical Centre, Department of Molecular Genetics, Maastricht, The Netherlands * These authors equally contributed to this work Authors Vanhove J. (1*), Pistoni M. (1*), Welters M. (1), Eggermont K. (1), Vanslembrouck V. (1), Helsen N. (1), Ordovas L. (1), Boon R. (1), Najimi M. (2), Sokal E. (2), Collas P. (3), Voncken J.W. (4) and Verfaillie C.M. (1) Genetics Retreat 2015 95 Xiaoqing Zhu Novel insights in AMPK function: AMPK-MKNK1 connection As an energy sensor in cells, AMP-activated protein kinase (AMPK) responds to elevated cellular AMP and ADP levels and activated processes to maintain metabolic/energy homeostasis. In Metabolic syndrome and cancer, AMPK shows abnormal activity in different signalling pathways, involving in glucose and lipid metabolism, cell growth and autophagy. To delineate the molecular mechanisms in AMPK function, we tried to identify AMPK phosphorylation-targets using protein microarray technology. As a result, MAP kinase-interacting serine/threonine-protein kinase 1 (MKNK1) gave a high score, making it a promising target for AMPK. Currently, MKNK1 has been reported as a potential target for cancer therapy, as it regulates protein translation via phosphorylating eukaryotic translation initiation factor 4E (eIF4E). 96 RESULTS CONCLUSIONS In this study, we aimed to validate the AMPK-MKNK1 connection in vitro and in whole cells. MKNK1 as an AMPK phosphorylation target was validated by in-vitro kinase assay. Mass spectrometry identified the phosphorylation site on MKNK1 by AMPK; this was confirmed by mutation analysis. Furthermore, physical interaction of AMPK and MKNK1 was confirmed in different cell models. The biological relevance of AMPK-MKNK1 signalling will be explored for both protein translation and gene transcription. We identified MKNK1 as a phosphorylation target for AMPK. Our findings suggest that AMPK may regulate cellular responses to metabolic changes or an altered micro-environment via a MKNK1-dependent signalling pathway, especially in epigenetic regulation. These findings bear relevance in the context of metabolic syndrome as well as cancer. Genetics Retreat 2015 [email protected] www.gcb.mumc.nl/molecular-genetics Authors Xiaoqing Zhu, Milou Meeuse, Vivian van Leeuwen, Jan Willem Voncken, Dietbert Neumann Affiliations 1.Department of Molecular Genetics, CARIM, Maastricht University, Maastricht, the Netherlands Genetics Retreat 2015 97 Zuzanna Borek Dynamic expression profile of lncRNA genes upon stimulation of human intestinal gluten-specific T-cells Celiac disease (CeD) is an autoimmune disorder (AID) that can be triggered by gluten in subjects with the genetic susceptibility profile. Previously, we have uncovered SNPs in 39 non-HLA loci that are estimated, in combination with the correct HLA genotype, to explain 50% of the heritability of CeD. Most of the autoimmune disease associated SNPs (>95%) are located in non-protein coding regions. Interestingly, some of these SNPs locate within or near long non-coding RNA (lncRNA) genes suggesting that lncRNAs play a role in disease development. Here we describe lncRNA expression profiles in a cell type that is the lynch-pin in celiac disease - gluten-specific T-cells (gsT-cells) – at different time-points after activation. METHODS gsT-cells, obtained from intestinal biopsies taken from CeD patients, were stimulated with anti-CD3/CD28 for 0, 10, 30 and 180 minutes, respectively. Total RNA was isolated and interrogated by RNA sequencing (Illumina HiSeq2500). Alignment and quantification were performed by STAR and HT-Seq. Differential expression was assessed using DeSeq. The top 100 genes associated with the first two principal component analysis (PCA) were selected for enrichment and pathway analysis using WebGestalt. We analysed both the expression of protein-coding genes and lncRNA genes. We selected the top ten genes in each group by their fold change expression for each time 98 Genetics Retreat 2015 point and predicted functions for these genes using a guilt-byassociation approach (co-expression analysis). RESULTS We identified 1151 genes (207 lncRNAs) showing differential expression in time. 42 lncRNAs are encoded within loci associated either with CeD (10) or other AID (32). Genes associated with the first PCA show up-regulation at 180 min and lncRNAs constitute 7% of this set. However, transcripts associated with the second PCA are up regulated at 30 min and of this group lncRNAs constitute 31%. This implies that lncRNAs respond earlier to stimu- [email protected] www.rug.nl/research/genetics/ www.linkedin.com/pub/zuzanna-borek lation than protein-coding genes. Disease association analysis (WebGestalt) using both early and late genes resulted in overrepresentation of the terms: immune system disease, inflammation and genetic predisposition to the disease. Moreover, predicted functions for the top induced genes showed association with T-cell immunity. This analysis allowed us to prioritize candidates for follow-up knockdown experiments. Authors Borek Z. (1), Li Y. (1), Kooy-Winkelaar Y. (2), Kumar V. (1), van Bergen J. (2), Hrdlickova B. (1), Koning F. (2), Wijmenga C. (1), Withoff S. (1) Affiliations 1.Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands; 2.Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands CONCLUSIONS This is the first time that RNAseq has been performed to study coding and non-coding gene expression in a human T cell subset that is specific to an autoimmune disease. We were able to show the dynamic response of lncRNAs at early time points after stimulation, supporting the hypothesis of a regulatory role of lncRNAs in the expression of protein-coding genes. Currently, we are setting-up in vitro experiments to prove that the selected lncRNAs are important in activation of gsTs. Genetics Retreat 2015 99 Tom Houben Storage solutions: targeting disturbed lysosomes to improve hepatic inflammation Recently, the importance of lysosomes within the metabolic syndrome, including fatty liver disease, is gaining increasing attention. It has been suggested that macrophages during atherosclerosis as well as Kupffer cells (KCs) during hepatic inflammation demonstrate properties of an acquired lysosomal storage disorder. So far, it is unclear whether there is a causal relationship between lysosomal cholesterol accumulation (LCA) in KCs and hepatic inflammation. Additionally, the specific contribution of the oxidized LDL (oxLDL) fraction to LCA, its concomitant effect on lysosomal function, and hepatic inflammation is unexplored. METHODS RESULTS To induce lysosomal accumulation of total cholesterol inside KCs, irradiated low-density lipoprotein receptor knockout (Ldlr-/-) mice were transplanted (tp) with Niemann-Pick type C1 mutant (Npc1mut) bone marrow and fed a high-fat, high-cholesterol (HFC) diet for 3 months. In order to investigate the contribution of lysosomal accumulation of oxLDL, anti-oxLDL antibodies were increased by immunizing mice with heatinactivated pneumococci, thereby preventing the uptake of oxLDL by KCs. Hematopoietic deficiency of the Niemann-Pick type C1 protein, in the current study used as a tool to induce LCA in KCs of hyperlipidemic mice, caused severe hepatic inflammation and fibrosis. Thus, LCA in KCs is a trigger for hepatic inflammation. Next, by elevating the levels of anti-oxLDL antibodies in these mice, we showed that cholesterol metabolism, lysosomal disturbances and liver inflammation were improved. CONCLUSIONS This study provides evidence for a direct causal link between LCA and hepatic inflammation with a specific role for oxLDL. 100 Genetics Retreat 2015 [email protected] www.gcb.mumc.nl nl.linkedin.com/pub/tom-houben Authors Houben T. (1), Walenbergh S.M. (1), Hendrikx T. (1), van Gorp P.J. (1), Jeurissen M.L. (1), Verheyen F. (1), Gijbels M.J. (1), Binder C.J. (2,3), Koek G.H. (1), Hofker M.H. (4), Lütjohann D. (5), Shiri-Sverdlov R. (1) Affiliations 1.Departments of Molecular Genetics, Molecular Cell Biology and Electron Microscopy, Internal Medicine, Nutrition and Toxicology Research (NUTRIM), Maastricht University, Maastricht, The Netherlands 2.Center for Molecular Medicine (CeMM), Austrian Academy of Sciences, Vienna, Austria 3. Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria 4. Department of Pathology and Medical Biology, Molecular Genetics, Medical Biology Section, University of Groningen, Groningen, The Netherlands 5. Institute of Clinical Chemistry and Clinical Pharmacology, University of Bonn, Bonn, Germany. Genetics Retreat 2015 101 Nadia Roumans Sex-specific variation in extracellular matrix genes is associated with weight regain and maintenance after weight loss When people decrease their energy intake entering into a negative energy balance, mature adipocytes will decrease their fat content and will become smaller. The surrounding extracellular matrix (ECM) should adjust accordingly to changes in volume. However, shrinkage of the ECM is an energy-demanding process and may not readily occur in a state of negative energy balance. It has been proposed that this may lead to an improper fit between the cell and the surrounding ECM inducing tension and cellular stress. Reducing this cellular stress in adipocytes can be achieved by re-storing fat and increasing the cell volume, which would mean regain of weight for the host. If the ECM is able to adjust properly to the volume changes, less cellular stress is expected with lower risk for weight regain. Prior studies have noticed the importance of the ECM in relation to weight regulation and it can be hypothesized that variations in genes coding for components of the adipocyte ECM are candidates for determining the risk of weight regain after weight loss. In the present study, we examined if sex-specific genetic variation in ECM related genes is associated with weight regain among the participants of the European DiOGenes study. METHODS Overweight and obese subjects were started with an 8-week low calorie diet with a 6-month follow-up period. Body weight was measured before, after the diet and after follow-up. Weight maintenance scores (WMS) were calculated with the weight data. Buffy coats of EDTA-blood to extract DNA for genotyping were obtained before the diet. Genotype data was retrieved for single nucleotide 102 Genetics Retreat 2015 polymorphisms corresponding to 124 candidate genes related to ECM. Univariate linear regression analyses were carried out with SNP alleles as a predictor and WMS as outcome. The best genetic model of inheritance that describes the effect of the genotypes was determined by logistic regression analysis. Odds ratios and 95% confidence intervals were calculated to determine the effect of specific genotypes. [email protected] RESULTS Univariate linear regression analyses provided us with 6 significant SNPs in male population: 3 SNPs in the periostin gene (rs7323378, rs9315503, rs9547947) and the other SNPs in the laminin-1 (rs2158836), fibulin-5 (rs12589592) and collagen, Type XXIII, alpha1 (rs2672826) genes. For female subjects, 1 significant SNP was found: rs17516906 in the fibronectin 1 gene. The risk for weight regain increased with a C/C genotype for rs7323378 (OR=8.25, 95% CI=2.85-23.88); with a A/A genotype for rs2158836 (OR=18.43, 95% CI=2.35-144.63); with a A/A genotype for rs12589592 (OR=13.00, 95% CI=1.61-104.81); with a G/A genotype for rs2672826 (OR=3.94, 95% CI=1.28-12.10); and with a A/A genotype for rs17516906 (OR=2.81, 95% CI=1.40-5.63). Authors Roumans N.J.T. (1), Vink R.G. (1), Gielen M. (2), Zeegers M.P. (2), Holst C. (3), Wang P. (1, 4), Astrup A. (5), Saris W.H. (1), Hager J. (3), van Baak M.A. (1) and Mariman E.C.M. (1) Affiliations 1.Maastricht University, Department of Human Biology, Nutrition and Translational Research in Metabolism, Maastricht, The Netherlands 2. Maastricht University, Department of Complex Genetics, Nutrition and Translational Research in Metabolism, Maastricht, The Netherlands 3. Copenhagen University Hospital, Institute of Preventive Medicine, Centre for Health and Society, Copenhagen, Denmark. 4.University Hospital Maastricht, Laboratory of Biochemical Genetics, Department of Clinical Genetics, Maastricht University Medical Centre, Maastricht, The Netherlands. 5.University of Copenhagen, Department of Nutrition, Exercise and Sports, Faculty of Science, Copenhagen, Denmark. CONCLUSIONS In conclusion, based on the model of adipocyte cellular stress as a driver for weight regain, we have investigated the role of genetic variation in ECM genes. The risk for weight regain after weight loss is increased by variation of the COL23A1, POSTN, LAMB1, and FBLN5 gene for males and the FN1 gene for females. Genetics Retreat 2015 103 Maria Magdalena Zorro Celiac disease associated genes are involved in intestinal barrier function Celiac disease (CeD) is an autoimmune disorder characterized by a strong intestinal inflammatory response to dietary gluten. Although the disease is most strongly associated with the HLA locus, 39 non-HLA loci have also been associated with CeD and of some of the genes in these loci the exact function is unknown. We have performed a systematic in silico analysis to prioritize causative genes in the CeD loci. Co-expression and pathway analyses have suggested that LPP and C1ORF106 could be involved in cell-cell interaction and intestinal barrier function, which is known to be impaired in CeD patients. To validate this hypothesis, we have evaluated the phenotypic implications of knocking down LPP and C1ORF106 in the human Caco-2 cell line, a model that is widely used to study epithelial barrier biology in vitro. 104 METHODS RESULTS Cis-eQTL and network analyses were applied to prioritize causative genes in all CeD loci. Gene knock-down was performed using plasmid-based shRNA targeting and confirmed by RTPCR and Western blot analysis. Cellcell interaction and barrier function were analyzed in 2D and 3D cultures by standard and fluorescent confocal microscopy. Barrier function is assessed by substrate transport assays and transepithelial electrical resistance measures. As expected our network analysis suggested that most of the prioritized genes play a role in immune function, but for LPP and C1ORF106 roles in “actin-cytoskeleton” network (p = 1.19x10-10) and “cell adhesion network” (p = 1.1x10-4) were predicted leading us to hypothesize that they play a role in epithelial barrier function. To prove this we generated LPP and C1ORF106-KD sub-lines. RT-PCR and Western blot analyses confirmed the knock-down (KD). The sub-lines grew more slowly than the parental Caco-2 cells. Moreover, in 3D cultures we observed that around 50% of the parental and scrambled-shRNA control sublines form typical Genetics Retreat 2015 [email protected] www.systemsgenetics.nl 80-100 µM hollow spheroids consisting of a single layer of columnar cells lining a central ‘lumen’. In these cells, the nuclei are positioned towards the basal side and a thin layer of actin is positioned at the luminal side. In the KD sub-lines we observed dramatic phenotypes. Most of the ‘spheroids’ derived from C1ORF106-KD cultures did not form hollow lumens, were fewer in number and smaller. In contrast, the number of spheroids in both LPP-KD sub-lines was similar to controls. Surprisingly, we found a remarkable difference between one of the LPP sub-lines when compared to three others where we found a subset of enlarged sheroids (“swollen”) with a flatter outer layer of cells while the other LPP-KD sublines were characterized mainly by the presence of speroids with filled lumen. conditions to evaluate cell-cell interaction protein complexes (tight junction and adherens junction proteins) by immuno-staining, transbarrier resistance, barrier permeability and transcriptome analysis under conditions that mimic the pro-inflammatory environment in CeD. Authors Zorro M.M.(1), Kumar V. (1), Jaroz L.M. (2), Medrano L.M.(1), Wijmenga C.(1), van IJzendoorn S.C.D. (2), Withoff S.(1) Affiliations 1.Departments of Genetics. University of Groningen, University Medical Center Groningen, Groningen, The Netherlands. 2.Cell Biology Department, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands. CONCLUSIONS The downregulation of LPP or C1orf106 has phenotypic consequences that support the hypothesis of a potential role for these genes in barrier function and CeD pathogenesis. We are setting up the Genetics Retreat 2015 105 Ivan Brankovic The role of NOD1 and NOD2 functional polymorphisms in susceptibility to Chlamydia trachomatis infection and risk of tubal factor infertility Genital Chlamydia trachomatis (CT) infections often have asymptomatic clinical course. This contributes to a high number of untreated cases and can ultimately lead to serious conditions such as tubal factor infertility (TFI). Recognition of bacterial wall structures by intracellular pattern-recognition receptors NOD1 and NOD2 can trigger specific biocascade resulting in inflammation and immune response to bacteria. However, whether functional polymorphisms in NOD1 and NOD2 impact CT infection and its clinical course has not been researched. We set out to determine if NOD1 +32656 G>TT and NOD2 1007fs polymorphisms affect susceptibility to (STD patients) and severity of (female patients visiting the Fertility clinic) CT infection in Dutch Caucasian women. METHODS Susceptibility cohort: From 1150 female patients that visited the STD outpatient clinic in Amsterdam, The Netherlands, we selected only Dutch Caucasian women (n=737, age 18-30). Questionnaires were collected from them in regards to urogenital complaints. Severity cohort: Our severity group comprised 490 Dutch Caucasian female patients (age 20-41) visiting the Fertility clinic in the University Medical Center Groningen. Laparoscopy was performed in order to grade the tubal pathology status as TFI grade 0-4. Controls were TFI grade 0 as assessed by either laparoscopy or hysterosalpingography (HSG). 106 Genetics Retreat 2015 Statistics: Susceptibility, severity (TFI) and increasing symptomatology were assessed in relation to polymorphisms and in relation to CT using 2x2 tables and trend analyses. RESULTS NOD2 1007fs was not found to be associated with the susceptibility to or severity of CT infection. Susceptibility: A significant association of the NOD1 +32656 GG insertion variant with protection against infection with CT has been detected [p: 0.0057; OR: 0.52]. Severity: Carriers of this variant were at a heightened risk of TFI [p: 0,038; OR: 2.25]. Presence of C. trachomatis [email protected] www.gcb.mumc.nl/public-health-genomics nl.linkedin.com/pub/ivan-brankovic IgG antibodies significantly increased in more severe TFI [p-trend <0.0001]. Symptomatology: When comparing CT positive women without symptoms, to CT positive women with symptoms, to CT positive women with TFI, we observed an increasing trend in carriage of the GG allele [p-trend: 0.0003]. Authors Brankovic I. (1, 2), van Ess E.F. (2), Noz M.P. (2), Wiericx W.J. (2), Spaargaren J. (2), Land J.A. (3), Ouburg S. (2), Morré S.A. (1, 2, 4) Affiliations 1.Institute for Public Health Genomics, Department of Genetics and Cell Biology, School for Oncology and Developmental Biology (GROW), Faculty of Health, Medicine and Life CONCLUSIONS The NOD1 +32656 GG insertion variant appears to protect against the infection with CT, while at the same time acting as a risk factor in developing TFI in women with a past CT infection. Also, the GG insertion leads to more symptoms in CT-positive women. From a biological perspective, carriage of the GG insertion might be enhancing successful clearing of CT infection, but acts excessive when expressed in the upper genital tract, leading to tubal pathology due to inflammation. The research is part of an ongoing effort of identifying key polymorphisms that determine the risk of TFI and effectively translating them into the clinical setting for the purpose of optimising diagnostic management of women at risk for developing TFI. Sciences, Maastricht University, Maastricht, The Netherlands 2.Laboratory of Immunogenetics, Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, The Netherlands 3.Department of Obstetrics and Gynaecology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands 4.Dutch Chlamydia trachomatis Reference Laboratory, Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, The Netherlands Genetics Retreat 2015 107 Martin Singer Host genetic variation in innate immunity genes cause both protective and risk enhancing effects on the outcome of Haemophilus ducreyi infections Haemophilus ducreyi is a sexually transmitted bacterial infection capable of causing a range of severe and less severe symptoms including chancroid and pustule formation. Host immunogenetic factors in this infection are yet to be studied, but have been shown to affect the outcome of infectious diseases in general. Studies have also shown that differences in cytokine and mRNA expression do affect the outcome of H. ducreyi infections significantly. This study examines the (possible) effects of single nucleotide polymorphisms (SNPs) in pathogen recognition pathways and cytokines: TLR2, TLR4, TLR9, SIGIRR, NOD1, NOD2, and IL-10 on the severity of H. ducreyi infections. 108 METHODS RESULTS DNA from 136 volunteers from the USA who were in triplo experimentally infected on their arm with H. ducreyi was genotyped for the various host targets using Real-time PCR. Groups were made according to pustule forming (3, 2, or 1 pustule) or clearance (0 pustules) on infection sites. Fischer exact tests, X2, and logistic regression analyses were performed on the produced data. We conducted a study that for the first time provides insight into effects of host immunogenetic factors on the severity of H. ducreyi infection. We found that severity of infection with H. ducreyi was associated with polymorphisms in the genes TLR9, and IL-10 which showed significant risk enhancing and protective effects based on their composition. Most significant associations in X2 tests for trends were found for a risk enhancing association of TLR9 TA haplotype (P=0,0057), and a protective genotype of TLR9 +2848 *A (P=0,0057). Logistic regression showed among other results a protective effect for IL-10 -2849 AA genotype (P=0,038, OR=0,195). Genetics Retreat 2015 [email protected] www.immunogenetics.nl nl.linkedin.com/in/MSinger2 CONCLUSIONS Our results show for the first time the relevance of host genetic variation in specifically TLR9 and IL-10 genes related to the severity of H. ducreyi infections. This is in line with previous research into cytokines expression. Authors Singer M. (1), Wei L. (2), Spinola S.M. (2), Ouburg S. (1), Morré S.A. (1, 3) Affiliations 1.Laboratory of Immunogenetics, Department of Medical Microbiology and Infection Control, VU University Medical Centre, Amsterdam, The Netherlands 2.Department of Microbiology and Immunology, Indiana University, School of Medicine, Indianapolis, Indiana, USA. 3.Institute of Public Health Genomics, Department of Genetics and Cell Biology, Research Institutes CAPHRI and GROW, Faculty of Health, Medicine & Life Sciences, University of Maastricht, The Netherlands Genetics Retreat 2015 109 Vasiliki Matzaraki Systems approach to Candida infection Candida albicans is an opportunistic fungal pathogen, which normally inhabits the human skin and mucosal surfaces. It is an important hospital-acquired pathogen and is ranked fourth among pathogens which cause systemic bloodstream infections (candidemia) when host defense is inadequate, with immunocompetent and immunocompromized patients being at increased risk. Noteworthy, not all individuals at risk will develop Candida infections, implying that host genes must influence disease susceptibility. Hence, insight into genetic susceptibility is the first step towards new treatment strategies. Since 2007, in an effort to identify genetic variants associated with susceptibility to infectious diseases, genome-wide association studies (GWAS) have revealed common genetic variants. However, an important challenge is that there is a need of large cohorts to obtain sufficient power to detect genetic variants. To overcome this limitation, here we applied systems genetics approach, to identify not only candidemia susceptibility variants, but also to gain biological insights into novel functional genes and molecular pathways implicated in host immune defense against C.albicans. This knowledge will, ultimately, help us identify new immunotherapeutic targets, improving the outcome of candidemia patients. METHODS We performed Immunochip-wide association analysis by using the largest candidemia cohort to date. In stage 1 (discovery), we performed an Immunochip-wide analysis by using cases of European ancestry and population-based controls. After filtering the data, the identified SNPs were analyzed for association with candidemia. Next, we tested whether these associations could be confirmed using a second diseasematched control group (validation). By using RNA sequencing data and genotype data from 629 blood samples, cisexpression quantitative trait loci 110 Genetics Retreat 2015 (cis-eQTL) mapping was performed to identify candidemia causal genes. The expression levels of the eQTL identified genes were further tested in immune cells that are stimulated with or without Candida stimulation. Identified SNPs were then correlated with cytokine levels in human Peripheral Blood Mononuclear Cells (PBMCs) stimulated by C.albicans. Lastly, pathway analysis on candidemia causal genes was performed by using the GeneNetwork database based on co-expression data. This analysis revealed novel biological pathways that underlie susceptibility to the fungal infection. [email protected] www.rug.nl/research/genetics/ RESULTS The Immunochip-wide association analysis revealed three GWAS significant loci associated with candidemia (P < 5x10-8). Out of 114.281 SNPs, 5.808 SNPs showed moderate association (<9.99x10-5) and we validated 28 independent associations (P < 0.05) by comparison with a disease-matched control group. By eQTL mapping, we observed that the identified SNPs affect the expression level of 67 genes in cis. We also found four SNPs affecting the expression of long non-coding RNAs. The eQTL implicated genes were further validated by using Candida stimulated gene-expression data . Out of 67 genes, 6 genes were significantly up-regulated at 4 and 24 hours as well and 5 and 11 genes were down-regulated at 4 and 24 hours correspondingly in response to Candida stimulation, suggesting the specific role of these genes in antiCandida mechanism. Subsequently, we also measured cytokine levels in response to Candida stimulation in PBMCs and correlated with genotypes at those 28 candidemia associated SNPs. We identified 4 SNPs to be correlated with cytokine expression, providing further evidence for their functional role in host immune defense against the fungus. Finally, by performing pathway analysis, we identified three key pathways im- portant for host defense against the fungus, including type I IFN signaling pathway. CONCLUSIONS By integrating many layers of functional data with candidemia-associated SNPs we identified novel genes and immune pathways. Our study also indicated lncRNAs as causal genes for candidemia. Apart from the wellknown role of type I IFN pathway for Candida infection, we also discovered two novel pathways. Thus, this “systems approach” seems to open new avenues to pinpoint novel genes and pathways implicated in the host immune defense against C.albicans. This knowledge could, ultimately, be used for identification of new immunotherapeutic targets, improving the outcome of candidemia patients. Authors Matzaraki V. (1), Ricano I. (1), Jonkers I. (1), Franke L. (1), Li Y. (1), Wijmenga C. (1), Netea M. (2), Kumar V. (1) Affiliations 1.Department of Genetics, University Medical Center Groningen (UMCG), Groningen, The Netherlands 2.Department of Medicine, Radbound University Nijmegen Medical Center, Nijmegen, The Netherlands Genetics Retreat 2015 111 Jan Henk Dubbink The role of polymorphisms on the susceptibility and clinical manifestations of sexual transmitted infections in South African women Urogenital Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis are the most prevalent bacterial sexually transmitted infections worldwide. Due to their often asymptomatic course of infection, C. trachomatis, N. gonorroeae, and T. vaginalis stay untreated. This could develop into late complications, such tubal pathology predisposing for infertility. Host genetic variations are described to play an important role in the course of both infections. Genetic variations in genes in relevant pathways are involved in the susceptibility to infections and can alter the course of infection in a way that symptoms can be present or completely absent. Twin studies demonstrate that 40% of the host resons is responsible for the clinical course of infection. We are currently analyzing the data of the detection of several SNPs on collected swabs of South African women. METHODS Samples were collected at primary healthcare facilities across the Mopani District, South Africa. 612 women, aged 18-49 years who reported to have been sexually active during the last 6 months were eligible and vaginal and rectal swabs were obtained. Swabs were frozen for storage without buffer (dry swabs). Patient information was provided and written consent obtained. STIs were detected by the Presto-plus CT-NGTV assay. Specific genetical markers are determined by KASP technology, including markers for pathogen recognition, inflammation induction, and negative regulators. Pattern 112 Genetics Retreat 2015 recognition receptors (PRRs), such as toll like receptors (TLRs), are important for recognition. Inflammation is possibly induced by MyD88, that is closely related to TLRs. Infections can be negatively regulated by anti-inflammatory interleukins as TRAIL-R1. Fisher exact and X2 tests were performed on the generated data. RESULTS We observed a cohort of 612 South African women. Prevalence in the Mopani Distrct, South Africa was earlier described by our group. Vaginal C. trachomatis, N. gonorrhoeae, T. vaginalis prevalence is 16%, 10%, [email protected] www.gcb.mumc.nl www.immunogenetics.nl and 17%, respectivley. Data analyses for SNPs are currently in progress. For instance, TLR2, TLR4, TLR9 are being analyzed. A SNP in TLR4 (-9799 T > C) is suggested to give increased odds for chlamydial infection. Authors de Waaij D.J. (1),Dubbink J(1,2), Struthers H.(3), McIntyre J.A. (3,4), Peters R.P.H. (3,5), Ouburg S.(1), Morré S.A.(1,2) Affiliations 1.VU University Medical Centre, Department of Medical Microbiology & Infection Control, CONCLUSIONS We detected an overall STI prevalence of 35% for all the three microorganisms at any site. In the current study, we are analyzing the data of the detected SNPs. Specific types of analyses are being done, such as multiple pathogen load, asymptomatic vs. symtomatic course of infection, and the possible influence of genetic variants for susceptibility of multiple infections. For instance, individuals are described to be more susceptible for HIV when infected with T. vaginalis. Laboratory of Immunogenetics, Amsterdam, The Netherlands. 2.Institute for Public Health Genomics (IPHG), Department of Genetics and Cell Biology, Research School GROW (School for Oncology & Developmental Biology), Faculty of Health, Medicine & Life Sciences, University of Maastricht, Maastricht, The Netherlands. 3.Anova Health Institute, Johannesburg and Tzaneen, South Africa. 4.School of Public Health & Family Medicine, University of Cape Town, Cape Town, South Africa. 5.Department of Medical Microbiology, University of Maastricht, Maastricht, The Netherlands. Genetics Retreat 2015 113 Colophon Editors Joep Geraedts Willem Voncken Judith Maszewski [email protected] www.gcb.mumc.nl Concept & Graphic design Suzanne van den Homberg [email protected] www.maasvdhomberg.nl
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