ANNUAL SYMPOSIUM 2019 19 & 20 September VELDHOVEN
Dutch Society for Human Genetics (www.nvhg-nav.nl)
The NVHG encompasses two professional associations: the Dutch Association of Clinical Geneticists (Vereniging Klinische Genetica Nederland, VKGN), which unites medical specialists in clinical genetics, the Association of Clinical Genetic Diagnostic Laboratories (Vereniging Klinisch Genetische Laboratoriumdiagnostiek, VKGL) and the NACGG (Nederlandse Associatie voor Community Genetics en Public Health Genomics) which unites recognised clinical genetics laboratory specialists.
Belgian Society for Human Genetics (www.beshg.be)
The BeSHG represents the community of Belgian geneticists towards the other national and international Societies of Genetics, and provides a discussion forum on scientific, professional, social and ethical issues linked to the practice of human genetics.
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Message from Dr. Mieke van Haelst, president NVHG and Prof. Dr. Gert Matthijs, president BeSHG
Dear colleagues,
Welcome to the second joint meeting of the Belgian and Dutch Society for Human Genetics. It is a pleasure to welcome you in Veldhoven!
Both societies are characterized by a genuine interest in research in human genetics in combination with a strong clinical and societal commitment. Implementation of the scientific results to improve human health is an everyday goal for the majority of genetic researchers present at this meeting. Many of us have combine research and clinical activities and obligations, and our PhD students and post-docs are often implicated in research that stems from clinical observations or deals with patients’ material.
Human genetics is a broad field, with a myriad of research opportunities and clinical applications. Our professional societies play a role in promoting scientific research in genetics and genomics, in its broadest sense. A lot is going on, and clearly, by your attendance, you witness that you wish to be part of these great times for human genetics.
We are pleased to offer you a star-studded line-up of plenary speakers covering exiting developments in our field. John van der Oost will teach us about CRISPR-Cas, and guide us through a plethora of applications that have changed the world of genetic research and are finding their way to clinical application. Douglas Easton is a global leader in breast cancer genetics research and will focus on the use of polygenic risk score (PRS) in daily clinical practice. Michel Georges is a well-known leader in the field of animal genetics, but his work also reaches into the realms of human genetics, where he made important contributions to better understand GWAS loci
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in Inflammatory Bowel Disease. Monogenic diseases – or what we believe are monogenic diseases- are discussed by Elfride De Baere who studies inherited retinal diseases and will show how integrated omics lead to better diagnosis and therapeutic applications for specific types of blindness. Joris Veltman, whom we know from pioneering novel genomic technologies, will focus on clinical outcomes that provide more insight into the success of assisted reproductive technologies, as well as the health of future offspring. Malte Spielmann studies genetics from yet another angle: structural variation in 3D. In his lecture, he will show us how the field is revolutionized with the study of genomes at the single cell level. Lisanne Vervoort is our youngest invited speaker. She will bring a wonderful story about the optical mapping of structural variation. Finally, we are very happy to have Peter de Knijff to entertain us about what he has learned during his 25 year journey in forensic genetic research.
The program also focusses on the challenges we all face. In the interactive panel-discussion on ‘Hurdles for genetic research – what can we do about it?’ we will collectively explore specific issues that either affect or impede genetic research and clinical services, including diagnostics. We are all familiar with GDPR (in Dutch: AVG), ethical review procedures, and FAIR data, but really how does it affect our work? We look forward to interacting with the audience and learn also from your experiences.
Finally, we are proud to announce that Danielle Posthuma has been awarded the Lodewijk Sandkuijl price for her ground-breaking work in Complex Genetics and she will enlighten us about her work in her lecture “From GWAS to Function”.
We sincerely thank all the participants for their contribution. From the abstracts, the Scientific Committee has been able to select high quality presentations. Unfortunately we have not been able to offer all abstract submitters a slot in the program, so please also visit the posters.
We also thank the sponsors, not only for their essential and generous financial contribution for the organization of the meeting, but also because they join us to present their latest innovations. This interaction between users and providers is very valuable, for all, but mostly for young people and trainees in the labs.
Importantly, we wish to sincerely thank all the members that actively engage, in the organization and function of our Societies. We are happy we can count on you to make our societies thrive! We hope that this year’s meeting will bring you many new ideas and inspiring new collaborations.
Mieke van Haelst, president NVHG Gert Matthijs, president BeSHG
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General information
Venue Hotel NH Eindhoven Conference Centre Koningshof Locht 117, 5504 RM Veldhoven Eindhoven - Nederland Tel.: +31 40 253 7475 www.nh-hotels.nl/hotel/nh-eindhoven-conference-centre-koningshof [email protected]
Registration Holland Foyer: open on Thursday September 19, 2019 09:30 - 10:30hrs
Reception and catering Holland foyer
Networking event Brabantzaal
Abstracts Abstracts guest speakers G 01 to G 08 Abstracts talks: T 01 to T 16 Abstracts posters : P 01 to P 39 Abstracts T 01 - T 16 and P 01 - P 31 are available as download through the NVHG website For abstracts of the NACGG, VKGN and VKGL sessions we refer to these societies and the NVHG website
Posters Poster boards have a size of 200 cm (height) and 100 cm (width) Please put up your poster immediately after arrival. Do not forget to remove it at the end of the meeting
Language The official language of the annual meeting will be English
Accreditation Accreditation forms are available at the registration desk (GAIA ID number: 370535)
Photos Photographs will be taken during the meeting which can be used by the NVHG for PR purposes. Anybody objecting should list their name on the form available at the NVHG registration desk.
Badges You are requested to hand in your badge at the end of the symposium
Presentations You are requested to timely hand in an USB stick with your presentation to the chairperson of your symposium session 4
Organization
Scientific organization
NVHG board Dr. Mieke van Haelst (President) Prof. dr. Johan den Dunnen (Secretary) Dr. ir. Aimée Paulussen (Treasurer) Dr. Terry Vrijenhoek Dr. Lidewij Henneman Dr. Roland Kuiper Prof. dr. Richard Sinke Prof. dr. André Uitterlinden Dr. Lisenka Vissers Dr. Gijs Santen
BeSHG board
Prof. dr. Gert Matthijs (President) Prof. Dr. Nisha Limaye (Secretary) Dr. Lut Van Laer (Treasurer) Dr. Saskia Bulk Prof. Dr. Bert Callewaert Prof. Dr. Kathelijn Keymolen Dr. Damien Lederer Dr. Julie Soblet
Administrative organization
Julia Lopez Hernandez ([email protected]) Henny Schurmann ([email protected])
www.nvhg-nav.n 5
Sponsors
We are very grateful for the financial support from Simons Fund Foundation
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We thank the following companies for their support:
Golden Sponsors
Agilent Technologies Moon www.agilent.com www.diploid.com/moon
Illumina Takara Bio www.illumina.com www.takarabio.com
Sophia Genetics SA PerkinElmer www.sophiagenetics.com www.perkinelmer.com/lifesciences
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Silver Sponsors
Sanofi Nimagen B.V. www.sanofigenzyme.nl www.nimagen.com
Pacific Biosciences GE Healthcare www.pacb.com www3.gehealthcare.nl
BIOKÉ B.V. BGI Global Genomic Services www.bioke.com www.bgi.com
Twist Bioscience Sanbio www.twistbioscience.com www.sanbio.nl
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Thermo Fisher Macrogen www.fishersci.nl dna.macrogen.com
Westburg Hettich www.westburg.eu www.hettich.com
Regular Sponsors
Novogene Bionano Genomics en.novogene.com bionanogenomics.com
Alnylan www.alnylam.com
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Program
Time Thursday 19th September
09:30 - 10:30 Registration Room: Holland Foyer
10:30 - 12:40 Opening & Plenary session 1 Room: Auditorium Chair: Mieke van Haelst (NVHG) / Saskia Bulk (BeSHG)
10:30 Opening by Dr. Mieke van Haelst president NVHG / Dr. Saskia Bulk board member BeSHG 10:35 Prof. Dr. John van der Oost (Wageningen, NL) CRISPR-Cas – from biology to applications
11:15 DNA Day Contest Award
11:20 Prof. Dr. Douglas Easton (Cambridge, UK) Predicting Breast Cancer Risk using Rare and Common Variants
12:00 Lodewijk Sandkuijl Lecture: Danielle Posthuma (Amsterdam, NL) From GWAS to Function
12:40 - 14:00 Lunch, room: Holland Foyer
13:00 Business/private meetings VKGN Room: Parkzaal 13:00 Business/private meetings VKGL Room: Auditorium
14:00 - 16:00 Plenary session 2 Room: Auditorium Chair: Terry Vrijenhoek (NVHG) / Bert Callewaert (BeSHG)
Hurdles for genetic research - what can we do about them? Panel session to explore how genetic research could be further facilitated 14:00 Introduction session 14:15 Panel presentation and hurdle identification 15:00 Power Break 15:15 Plenary discussion - how to overcome the hurdles 15:45 Conclusion in images
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16:00 - 17:30 Posters presentation with coffee & tea Room: 65 / Holland Foyer
17:30 - 18:30 Plenary session 3 Room: Auditorium Chair: Johan den Dunnen (NVHG)
17:30 Prof. Dr. Peter de Knijff (Leiden, NL) A result is only a result if it results in a result
18:30 - 21:00 Social Evening Program Room: Brabantzaal
18:30 Pre-dinner drinks 19:00 Dinner 21:00 Networking event
Time Friday 20th September
08:30 - 09:30 Registration Room: Holland Foyer
08:45 - 10:05 Plenary session 4 Room: Auditorium Chair: André Uitterlinden (NVHG) / Saskia Bulk (BeSHG)
8:45 Prof. Dr. Michel Georges (Liege, BE) Identifying causative variants and genes in GWAS-identified IBD risk loci 9:25 Prof. Dr. Elfride De Baere (Ghent, BE) Integrated omics to accelerate diagnosis and therapy in inherited retinal diseases causing blindness
10:05 - 11:15 Parallel sessions A/B
A. Fertility and Pregnancy Room: Auditorium Chair: Lidewij Henneman (NVHG) / Björn Menten (BeSHG)
10:15 Martine De Rycke Preimplantation Genetic Testing with HLA matching: from counselling to birth and beyond 10:30 Karuna van der Meij TRIDENT-2: National Implementation of Genome-Wide Non-Invasive Prenatal Testing as a First-Tier Screening Test in the Netherlands
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10:45 Kris Van Den Bogaert The landscape of pathogenic copy number variations in healthy, reproducing females 11:00 Masoud Zamani Esteki In vitro fertilization does not increase the incidence of de novo copy number alterations in fetal and placental lineages
B. Personalized Genomics Room: Parkzaal Chair: Gijs Santen (NVHG) / Damien Lederer (BeSHG)
10:15 Inge Lakeman Longitudinal assessment of the 313-SNP based Polygenic Risk Score for breast cancer risk prediction in a Dutch prospective cohort 10:30 Helen Roessler Towards the treatment of Cantú syndrome 10:45 Remco Hack NOTCH3 cysteine altering variants and their phenotypes in 92,456 whole exome sequenced participants of the Geisinger DiscovEHR initiative 11:00 Eline van Hugte Towards personalized treatment of genetically classified refractory epilepsies using human induced pluripotent stem Cells (hIPSCs) as an ex-vivo tool
11:15 - 11:45 Poster viewing with coffee & tea Room 65 / Holland Foyer
11:45 - 12:45 Parallel sessions C/D
C. Diagnostic Opportunities Room: Auditorium Chair: Richard Sinke (NVHG) / Kris Van Den Bogaert (BeSHG)
11:45 Jeroen van Rooij Genotyping On ALL patients (GOALL); clinical implementation of high-throughput genotyping arrays. 12:00 Kornelia Neveling The added value of long-read amplicon sequencing for clinical applications 12:15 Peter Henneman Reliable application of DNA-methylation signatures in genetic diagnostic testing 12:30 Helga Westers What if we would use a diagnostic multi-cancer gene panel for opportunistic screening? A study in 2,090 Dutch familial cancer patients
D. Biological Insight into Rare Disease Room: Parkzaal Chair: Lisenka Vissers (NVHG) / Arvid Suls (BeSHG)
11:45 Margot Reijnders Neurodevelopmental disorders: a next generation 12:00 Lore Pottie 12
Transcriptome and protein analysis highlight the endosomal pathway in disease pathogenesis of metabolic CL syndrome 12:15 Eva D'haene Noncoding structural variants disrupt the regulatory architecture of Rett genes 12:30 Daphne Smits Loss of neutral sphingomyelinase-3 (SMPD4) links neurodevelopmental disorders to cell cycle and nuclear envelope anomalies
12:45 - 14:00 Lunch and poster viewing
13:00 NVHG General Members' Meeting Room: Auditorium
14:00 - 16:30 Plenary session 5 Room: Auditorium Chair: Roland Kuiper (NVHG) / Bert Callewaert (BeSHG)
14:15 Prof. Dr. Joris Veltman (Newcastle, UK) De novo mutations affecting male reproductive health 14:55 PD. Dr. Malte Spielmann (Berlin, DE) Human genetics at single cell resolution 15:35 Winner Genetics Retreat 2019: Lisanne Vervoort (Leuven, BE) Optical Mapping of 22q11.2 Low Copy Repeats reveals structural hypervariability
16:00 Awards 2019 Chair: Mieke van Haelst (NVHG) / Bert Callewaert (BeSHG) Young Investigator Award (for best thesis) Annual Award (for best oral presentation) Poster Award (for best poster) Puzzle prize
16:15 Closing Remarks 16:30 Meeting ends
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Floor plan
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Special Guest speaker Lodewijk Sandkuijl Lecture
From GWAS to Function
Danielle Posthuma, Professor Complex Trait Genetics, Vrije Universiteit Amsterdam
Thursday September 19, 2019 Time: 12.00 – 12.40 Room: Auditorium
Genome-wide association studies (GWAS) have successfully identified many novel loci for neuropsychiatric traits. At the same time the results of GWAS showed that these traits are highly polygenic, mostly influenced by large numbers of weakly associated variants. Interpreting such polygenic results is challenging. Recent large-scale initiatives, such as those from the Allen Brain Institute and the PyschEncode consortium provide fine-scaled atlases of functional genetic elements at cellular level. This novel information can be used to interpret results from GWAS studies and facilitate biological understanding of complex traits. We developed two tools that aid in biological interpretation of GWAS findings and use biological information at cellular resolution: FUMA, for functional annotation and gene prioritization, and MAGMA, for gene-based and gene-set analysis. I will discuss recent GWAS findings for several brain related traits, the biological implications of these findings and possible routes for functional follow-up, ultimately leading to a mechanistic understanding of complex traits.
Email: [email protected]
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Special Guest speaker
A result is only a result if it results in a result Peter de Knijff, Professor Population and Evolutionary Genetics, Leiden University Thursday September 19, 2019 Time: 17.30 – 18.30 Room: Auditorium
Peter de Knijff is professor of population and evolutionary genetics at the department of human genetics of the LUMC. There, he is also, since 1994, head of the forensic laboratory for DNA research (FLDO).
The research of Peter de Knijff is besides population genetics, it is also targeted at developing new forensic genetic diagnostic research tools. He was, with two German colleagues, responsible for the introduction and widespread use of polymorphic Y-chromosome loci in forensics. More recently, he pioneered in the forensic use of massively parallel sequencing. He started, as research technician, his scientific career at Leiden University in January 1980. There he obtained his PhD on June 4, 1992. He was appointed as full professor in 2005. His inaugural lecture was spoken on June 23 2006 and was titled "Crying with the wolves”.
Email: [email protected]
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Abstracts Guest Speakers (G)
G 01
CRISPR-Cas – from biology to applications
John van der Oost Laboratory of Microbiology, Wageningen University
Ten years ago major discoveries at the level of comparative genomics [1], molecular microbiology [2] and biochemistry [3] have revealed that CRISPR-Cas is a heritable adaptive immune system of bacteria and archaea, based on RNA-guided DNA interference. These key findings have initiated a revolution, including exploration the natural diversity of CRISPR-Cas classes and types [4], characterizing structure-function relations of CRISPR-associated proteins and RNA guides [5], and development of a range of applications in biotechnology and medicine [6,7]. Apart from comparing multi-subunit Cascade-like systems (class-1) and single-protein Cas9/Cas12-like systems (class-2) systems, both natural and synthetic features will be discussed that are relevant for applications [8,9].
[1] Mojica et al. (2005) J Mol Evol 60, 174-182 [2] Barrangou et al. (2007) Science 315, 1709-1712 [3] Brouns et al. (2008) Science 321, 960-964 [4] Makarova et al. (2015) Nat Rev Microbiol 13, 722-736 [5] Mohanraju et al. (2016) Science 353, aad5147/1-12 [6] Charpentier & Doudna (2013) Nature 495, 50-51 [7] Hsu et al. (2014) Cell 157, 1262-1278 [8] Zetsche et al. (2017) Nat Biotechnol 35, 31-34 [9] Wu et al. (2018) Nat Chem Biol. 14, 642-651
Email: [email protected]
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G 02
From GWAS to Function
Prof. dr. Danielle Posthuma Vu university, dept complex trait genetics & vumc dept clinical genetics
Genome-wide association studies (GWAS) have successfully identified many novel loci for neuropsychiatric traits. At the same time the results of GWAS showed that these traits are highly polygenic, mostly influenced by large numbers of weakly associated variants. Interpreting such polygenic results is challenging. Recent large-scale initiatives, such as those from the Allen Brain Institute and the PyschEncode consortium provide fine-scaled atlases of functional genetic elements at cellular level. This novel information can be used to interpret results from GWAS studies and facilitate biological understanding of complex traits. We developed two tools that aid in biological interpretation of GWAS findings and use biological information at cellular resolution: FUMA, for functional annotation and gene prioritization, and MAGMA, for gene-based and gene-set analysis. I will discuss recent GWAS findings for several brain related traits, the biological implications of these findings and possible routes for functional follow- up, ultimately leading to a mechanistic understanding of complex traits.
Email: [email protected]
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G 03
Predicting Breast Cancer Risk using Rare and Common Variants
Prof.dr. Douglas Easton Centre for Cancer Genetic Epidemiology, Department of Public Health and Primary Care and Department of Oncology, University of Cambridge, Cambridge, UK.
The risks of breast cancer are associated with germline genetic variants, lifestyle or hormonal factors and breast density. Genetic factors include rare coding variants in at least ten susceptibility genes, and common polymorphisms (mostly SNPs), identified through genome-wide association studies, that confer more moderate risks1,2. The associations for common variants appear to be largely independent and can be summarised by a polygenic risk score (PRS). A recently published PRS, comprising 313 SNPs, confers an estimated a relative risk per 1 standard deviation of 1.61(95%CI:1.57-1.65); women in the top 1% of the risk distribution have an approximately 4-fold risk of ER-positive disease and 3-fold risk of ER-negative disease3. The risks also appear largely independent (on a log-scale) of the risks of associated with lifestyle risk factors, breast density and, approximately, high/moderate risk gene variants. The BOADICEA model, which incorporates all these components, is being made available for clinical use4. Despite their now widespread use, the evidence for association with cancer for coding variants in many genes on genetic testing panels is often weak, and many of the underlying risk estimates are very imprecise5. However, recent results from the large population studies are providing more reliable estimates for both truncating and missense variants. The current version of BOADICEA incorporates five genes (BRCA1, BRCA2, ATM, CHEK2, PALB2) but recent results from the Breast Cancer Association Consortium BRIDGES project indicate that several other genes, including BARD1, RAD51C and RAD51D, could now be reliably included. 1. Michailidou, K. et al. Association analysis identifies 65 new breast cancer risk loci. Nature 551, 92- 94, doi:10.1038/nature24284 (2017). 2. Milne, R. L. et al. Identification of ten variants associated with risk of estrogen-receptor-negative breast cancer. Nat Genet, doi:10.1038/ng.3785 (2017). 3. Mavaddat, N. et al. Polygenic Risk Scores for Prediction of Breast Cancer and Breast Cancer Subtypes. Am J Hum Genet 104, 21-34, doi:10.1016/j.ajhg.2018.11.002 (2019). 4. Lee, A. et al. BOADICEA: a comprehensive breast cancer risk prediction model incorporating genetic and nongenetic risk factors. Genet Med, doi:10.1038/s41436-018-0406-9 (2019). 5. Easton, D. F. et al. Gene-panel sequencing and the prediction of breast-cancer risk. N Engl J Med 372, 2243-2257, doi:10.1056/NEJMsr1501341 (2015).
Email: [email protected]
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G 03
A result is only a result if it results in a result
Prof.dr. Peter de Knijff Department of Human Genetics, Leiden University Medical Center, The Netherlands.
Performing forensic genetic research essentially boils down to catching criminals and secure a conviction by an independent Court. During this “hunt” for justice, the forensic DNA expert must face a complex landscape of adversaries who will have their own (often ill-informed) opinion about your work and its relevance. Furthermore, despite its extremely high societal relevance, forensic genetic research is very low - if at all - on the agenda of important scientific organizations such as the KNAW (not a single forensic-sciences member), NWO (no funding possibilities), de Nationale Wetenschapsagenda (no interest), and editorial boards of high ranking journals such as Nature, Science or Cell (unless something scandalous happened). Even, one of the former Deans of my own organization told me very early in my career “when people see our logo they must think: we are here to treat patients, not to catch criminals, keep that in mind”. It is in this, sometimes unhospitable, landscape that I have been travelling the past 25 Years. I will use one recent criminal case to illustrate how, by being patient, persistent, and creative, such journeys can still be very rewarding.
Email: [email protected]
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G 04
Identifying causative variants and genes in GWAS-identified IBD risk loci
Prof.dr. Michel Georges Unit of Animal Genomics, GIGA Institute, University of Liège, Belgium.
More than 250 IBD risk loci have been identified by GWAS accounting for > 50 % of inherited risk. GWAS identified risk loci typically span 250Kb encompassing thousands of variants and ~5 genes (range: 0 -100). Identifying the causative variants and genes amongst those is essential to reap the full benefits of GWAS towards precision medicine and intelligent drug design. For approximately 10 IBD risk loci the causative variants are coding hence allowing effective identification of causative variants and genes “in one shot”. However, the majority of risk variants underlying common complex disease are regulatory. Identifying these and the genes they control requires the application of sophisticated fine-mapping methods and integration of expression QTL information. The effectiveness of these approaches is limited by LD-structure and tissue heterogeneity, respectively. We will describe our results in applying these methods as well as our attempts to overcome their limitations.
Email: [email protected]
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G 05
Integrated omics to accelerate diagnosis and therapy in inherited retinal diseases causing blindness Prof.dr. Elfride De Baere Center for Medical Genetics Ghent (CMGG), Ghent University Hospital, Ghent, Belgium
Integrated genomics and transcriptomics reveal an increasing number of non-coding mutations in Mendelian disorders including inherited retinal diseases (IRD), representing a major cause of early- onset blindness in 2 million people worldwide. Of these the majority are deep-intronic splicing mutations, typically leading to pseudo-exon inclusion and amenable to antisense oligonucleotide- mediated rescue, already implemented in the clinic. Non-coding mutations in cis-regulatory elements (CREs) are more scarce. Paradigms for regulatory variants are IRD subtypes with recognizable phenotypes and without or - in case of autosomal recessive disease - monoallelic coding variants in the presumed disease genes. Examples are Stargardt disease (ABCA4), choroideremia (CHM), Leber congenital amaurosis 9 (NMNAT1) and North Carolina Macular Dystrophy (PRDM13 and IRX1). Particularly interesting CREs are ultraconserved non-coding elements (UCNEs) that are clustered in genomic regulatory blocks (GRBs) and that may act as distant enhancers. A search for IRD genes in GRBs revealed genes in 12 GRBs, containing 306 UCNEs. In three of these genes (CHM, PRDM13, USH2A), non-coding mutations have already been reported. In a set of genes under control of the retinal transcription factor CRX, 138 genes were found in GRBs that harbor 3,424 UCNEs. Only four of these are known IRD genes. These GRBs strongly coincide with topologically associating domains or TADs, playing key roles in gene regulation and determined by chromosome conformation capture techniques. Integration of UCNEs with epigenomic datasets generated in relevant cell types contribute to functional genome annotations in retina and accelerate diagnosis and therapy in IRD.
Email: [email protected]
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G 06
De novo mutations affecting male reproductive health Prof.dr. Joris A Veltman Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands
During spermatogenesis and oogenesis the 6 billion letters of our diploid genome need to be accurately copied in order for the gametes to successfully pass on our genetic information. By studying the genomes of children and their parents, we now know that each human is born with 50-100 de novo mutations. These de novo mutations, by affecting important genes during development, can result in severe neurodevelopmental disorders such as intellectual disability or epilepsy. In this presentation, I will discuss our new research focused on studying the role of de novo mutations in severe forms of male infertility. There is very little known about the role of genetics of male infertility, unfortunately genomics approaches are not routinely applied in research and/or diagnostics. I will present our first studies in which we studied the DNA of patients with azoospermia or severe oligospermia and compared this to that of their unaffected parents. We identified not only de novo point mutations but also de novo deletions in their genomes, determined the parent of origin and are currently replicating this work as well as performing functional studies within the framework of the International Male Infertility Genomics Consortium (www.IMIGC.org). This work is aimed at furthering our understanding of the genetic causes of male infertility with the goal to improve diagnostics, provide more insight into the success of assisted reproductive technologies, as well as the health of future offspring.
Email: [email protected]
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G 07
Human genetics at single cell resolution
PD.dr. Malte Spielmann Max Planck Institute for Molecular Genetics, Germany
Mammalian embryonic development is an astonishing process. In mice most major internal and external organs develop within a short window of time, termed organogenesis. The key regulators of developmental defects can be studied during this critical window, but conventional approaches lack the throughput and resolution to obtain a global view of the molecular states and trajectories of a rapidly diversifying and expanding number of cell types. We have recently developed a three-step combinatorial barcoding method to profile single-cell transcriptomes ('sci-RNA-seq3') without requiringphysical isolation of each cell. We have used this new method to profile whole mouse embryos staged between 9.5 and 13.5 days of gestation withsci-RNA-seq3, and created a transcriptional atlas of mouse organogenesis at single cell resolution. We identify hundreds of expanding, contracting and transient cell types, many of which are only detected because of the depth of cellular coverage obtained here, and define the corresponding sets of cell type-specific marker genes, several of which we validate by whole mount in situ hybridization. We also delineate and annotate 56 single cell developmental trajectories of mouse organogenesis. We explore the dynamics of proliferation and gene expression within cell types over time, including focused analyses of the apical ectodermal ridge, limb mesenchyme and skeletal muscle. Our single cell atlas of the development of wild-type mice represents a first step towards understanding pleiotropic developmental disorders at the organismal scale. We are currently performing detailed single-cell investigations in mutant mice and patients samples of subtle roles for genes and regulatory sequences involved in developmental defects.
Email: [email protected]
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G 08
Optical Mapping of 22q11.2 Low Copy Repeats reveals structural hypervariability Lisanne Vervoort1, Wolfram Demaerel1, Yulia Mostovoy2, Feyza Yilmaz3,4, Steven Pastor5,6, Matthew S. Hestand7,8, Ann Swillen1, Elfi Vergaelen1, Elizabeth A. Geiger4, Curtis R. Coughlin4, Stephen K. Chow2, Donna McDonald-McGinn5,6, Bernice E. Morrow9, Pui-Yan Kwok2, Ming Xiao10, Beverly S. Emmanuel5,6, Tamim H. Shaikh4, Joris R. Vermeesch1
1Department of Human Genetics, KU Leuven, Leuven, Belgium, 2Cardiovascular Research Institute, UCSF School of Medicine, San Francisco, CA, United States, 3Department of Integrative Biology, University of Colorado Denver, Denver, CO, United States, 4Department of Pediatrics, Section of Clinical Genetics and Metabolism, University of Colorado Denver, Aurora, CO, United States, 5Division of Human Genetics, Children's Hospital of Philadelphia, Philadelphia, PA, United States, 6Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States, 7Division of Human Genetics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, United States, 8Department of Human Genetics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, United States, 9Department of Genetics, Albert Einstein College of Medicine, Bronx, NY, United States, 10School of Biomedical Engineering, Drexel University, Philadelphia, PA, United States.
Introduction: The 22q11.2 locus is structurally one of the most complex areas of the human genome due to the presence of low copy repeats (LCR22s). Despite the newest sequencing technologies, the human reference genome hg38 still comprises three unresolved sequence gaps in LCR22-A. The recurrent deletion/duplication breakpoints of 22q11.2 deletion syndrome (22q11.2DS) are embedded within these repeats, but the exact location remains unclarified. Materials and Methods: We performed an LCR22 de novo assembly using fiber-FISH. Long DNA molecules were extracted from cells, stretched onto coverslips, and hybridized with LCR22 specific probes. Following manual signal screening, alleles were de novo assembled by tiling fibers based on matching colors and distances between the probes. Results: LCR22 haplotyping in 24 individuals uncovered the presence of 26 different alleles for LCR22-A. Subunits cluster in larger substructures, which vary in orientation, copy number, and presence. The rearranged alleles of 22q11.2DS patients were mapped in nine families and differences in the location of the deletion breakpoints were identified. In addition, LCR22-A alleles were assembled for the Great Apes to uncover insights in the evolutionary context. Conclusions: For the first time, the overall architecture of the 22q11.2 locus was successfully unraveled. The LCR22 hypervariability implicates interindividual gene dosage differences. As a consequence, copy number variations could influence gene expression profiles. Additionally, mapped rearrangement breakpoints vary among patients. We hypothesize this variability could provide a genetic explanation for some of the phenotypic variability characterizing the 22q11.2DS.
Email: [email protected]
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Abstracts Talks (T)
No. Name Titel E-mail A. Fertility and Pregnancy T 01 Martine De Rycke Preimplantation Genetic Testing with HLA [email protected] matching: from counselling to birth and beyond T 02 Karuna van der TRIDENT-2: National Implementation of [email protected] Meij* Genome-Wide Non-Invasive Prenatal Testing as a First-Tier Screening Test in the Netherlands T 03 Kris Van Den The landscape of pathogenic copy number [email protected] Bogaert variations in healthy, reproducing females T 04 Masoud Zamani In vitro fertilization does not increase the [email protected] Esteki incidence of de novo copy number alterations in fetal and placental lineages
B. Personalized Genomics
T 05 Inge Lakeman* Longitudinal assessment of the 313-SNP based [email protected] Polygenic Risk Score for breast cancer risk prediction in a Dutch prospective cohort T 06 Helen Roessler* Towards the treatment of Cantú syndrome [email protected] T 07 Remco Hack* NOTCH3 cysteine altering variants and their [email protected] phenotypes in 92,456 whole exome sequenced participants of the Geisinger DiscovEHR initiative T 08 Eline van Hugte* Towards personalized treatment of genetically [email protected] classified refractory epilepsies using human induced pluripotent stem Cells (hIPSCs) as an ex-vivo tool
C. Diagnostic Opportunities
T 09 Jeroen van Rooij* Genotyping On ALL patients (GOALL); clinical [email protected] implementation of high-throughput genotyping arrays. T 10 Kornelia Neveling The added value of long-read amplicon [email protected] sequencing for clinical applications l T 11 Peter Henneman Reliable application of DNA-methylation [email protected] signatures in genetic diagnostic testing T 12 Helga Westers What if we would use a diagnostic multi-cancer [email protected] gene panel for opportunistic screening? A study in 2,090 Dutch familial cancer patients
D. Biological Insight into Rare Disease
T 13 Margot Reijnders Neurodevelopmental disorders: a next [email protected] generation T 14 Lore Pottie* Transcriptome and protein analysis highlight [email protected] the endosomal pathway in disease pathogenesis of metabolic CL syndrome T 15 Eva D'haene* Noncoding structural variants disrupt the [email protected] regulatory architecture of Rett genes T 16 Daphne Smits* Loss of neutral sphingomyelinase-3 (SMPD4) [email protected] links neurodevelopmental disorders to cell cycle and nuclear envelope anomalies
* Eligible for Annual Award
26
T 01
Preimplantation Genetic Testing with HLA matching: from counseling to birth and beyond
M. De Rycke1, A. De Vos, F. Belva, V. Berckmoes, M. Bonduelle, A. Buysse, K. Keymolen, I. Liebaers, J. Nekkebroeck, P. Verdyck, W. Verpoest
Centre for Medical Genetics and Centre for Reproductive Medicine, Universitair Ziekenhuis Brussel, Laarbeeklaan 101 1090 Brussels, Belgium. Vrije Universiteit Brussel (VUB), Reproduction and Genetics, Laarbeeklaan 101, 1090 Brussels, Belgium.
Introduction Preimplantation genetic testing with HLA typing (PGT-HLA) involves HLA typing of a single or a few cells biopsied from in vitro fertilized preimplantation embryos with the aim of obtaining a pregnancy where the fetus is HLA compatible with an affected sibling in need of a hematopoietic stem cell transplantation (HSCT). During PGT-M-HLA the identification of a HLA compatible embryo is combined with the detection of mutations underlying immunodeficiencies and hemoglobinopathies. Materials and methods This is a combined retrospective and prospective cohort analysis of PGT-(M)-HLA procedures from 1998 until 2017 with follow-up of transplantations until 2019. Data on patient intake and dropout, preclinical workup, cycle outcome, pregnancy, baby and transplantation follow-up were collected from our patient files, with informed consent from the couples. Results During the study period, 234 couples from 22 countries were invited for a multidisciplinary intake. Psychological counselling showed that couples had high hopes on saving their ill child. Two couples were rejected (no HLA indication) and 70 couples refrained from PGT before or after preclinical workup (various causes), leaving 162 couples for which 414 clinical cycles were carried out. The majority of cycles (81%) involved PGT-M-HLA (51% for sickle cell anemia). Cleavage stage biopsy followed by single cell multiplex PCR for STR-based haplotyping was mostly applied (98.7%). The diagnostic efficiency was high (94.8%) but genetic selection left only 16.5% of embryos as genetically suitable for transfer. Fresh embryo transfer cycles mainly included HLA compatible embryos (91.3%) whereas the majority of cryopreserved embryos were surplus HLA non-compatible embryos (89.9%). Fresh and frozen-thawed embryo transfer resulted in 67 clinical pregnancies and 74 liveborn children (52 singletons, 22 twins) of which 60 children were HLA compatible. This yielded a live birth delivery rate of 30.3% per transfer. Information on neonatal characteristics showed reassuring outcomes. So far, HSCT was carried out successfully for 24 out of 25 cases. The most striking findings following psychological assessment of a small group of couples (21%) after unsuccessful PGT treatment were resilience and absence of any regrets. Conclusion Our data show that PGT-(M)-HLA is a valuable procedure: the high complexity and limited delivery rate are balanced by the successful HSCT outcome and the positive impact on families.
E-mail: [email protected]
Keywords: preimplantation genetic testing HLA matching hematopoietic stem cell transplantation sibling donor
27
T 02
TRIDENT-2: National Implementation of Genome-Wide Non-Invasive Prenatal Testing as a First-Tier Screening Test in the Netherlands
Karuna R.M. van der Meij1, Erik A. Sistermans1, Merryn V.E. Macville2, Servi J.C. Stevens2, Caroline J. Bax3, Mireille N. Bekker4, Caterina M. Bilardo5, Elles M. J. Boon1, Marjan Boter6, Karin E.M. Diderich6, Christine E.M. de Die-Smulders2, Leonie K. Duin7, Brigitte H. W. Faas8, Ilse Feenstra8, Monique C. Haak9, Mariëtte J.V. Hoffer10, Nicolette S. den Hollander10, Iris H.I.M. Hollink6, Fernanda S. Jehee6, Maarten F.C.M. Knapen11, Angelique J.A. Kooper12, Irene M. van Langen13, Klaske D. Lichtenbelt14, Ingeborg H. Linskens5, Merel C. van Maarle12, Dick Oepkes9, Mijntje J. Pieters15, G. Heleen Schuring-Blom14, Esther Sikkel16, Birgit Sikkema-Raddatz12, Dominique F.C.M. Smeets8, Malgorzata I. Srebniak6, Ron F. Suijkerbuijk12, Gita M. Tan-Sindhunata1, A. Jeanine E.M. van der Ven17, Shama L. van Zelderen-Bhola1, Lidewij Henneman1, Robert-Jan H. Galjaard6, Diane Van Opstal6 and Marjan M. Weiss1 for the Dutch NIPT Consortium
1 Department of Clinical Genetics, Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands. 2 Department of Clinical Genetics, GROW School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands. 3 Department of Obstetrics and Gynaecology, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands. 4 Department of Obstetrics and Gynaecology, Utrecht University Medical Center, Utrecht, The Netherlands. 5 Department of Obstetrics and Gynaecology, Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands. 6 Department of Clinical Genetics, Erasmus Medical Center, Rotterdam, The Netherlands. 7 Department of Obstetry and Gynaecology University Medical Centre Groningen, University of Groningen, Groningen, the Netherlands. 8 Department of Genetics, Radboud Institute for Molecular Life Sciences, Radboud university medical Center, Nijmegen, The Netherlands. 9 Department of Obstetrics, Leiden University Medical Center, Leiden, The Netherlands. 10 Department of Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands. 11 Department of Obstetrics and Fetal medicine, Erasmus Medical Center, Rotterdam, The Netherlands. 12 Department of Clinical Genetics, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands. 13 Department of Genetics, University Medical Centre Groningen, University of Groningen, Groningen, the Netherlands. 14 Department of Genetics, Utrecht University Medical Center, Utrecht, The Netherlands. 15 Department of Obstetrics and Gynecology, Maastricht University Medical Center, Maastricht, The Netherlands.
The Netherlands is the first country to perform a nationwide implementation study on non-invasive prenatal testing (NIPT) as a first-tier test in the screening for Down-, Edwards-, and Patau syndrome. This started April 1st 2017 as the so-called TRIDENT-2 study, licensed by the Dutch Ministry of Health. All pregnant women are offered prenatal screening and are counseled by certified counselors, generally midwives. A first-tier NIPT costs women € 175, comparable to the standard first-trimester combined test (FCT) (~€ 170). Women can choose to have a targeted report of chromosomes 21, 18, and 13 or a whole-genome report including findings other than trisomy 21, 13, 18 on the remaining autosomes, if over 10-20 Mb. The former applies a targeted filter in the bioinformatic analysis pipeline while the latter does not. Sex chromosomes are not analyzed. In the first year, NIPT was performed in 73,239 pregnancies (42% of all pregnancies), 7239 (4%) chose the FCT, and 54% did not participate. The number of trisomies 21 (239, 0.33%), 18 (49, 0.07%) and 13 (55, 0.08%) reported is in line with earlier studies. The Positive Predictive Values ((PPV), 96% for trisomy 21, 98% for trisomy 18 and 53% for trisomy 13) were higher than expected in this general obstetric population. Overall 78% of women chose to have the findings other that trisomy 21, 18 and 13 reported. The number of additional findings was 207 (0.36%), among which other trisomies (101, 0.18%, PPV 6%), structural chromosomal aberrations (95, 0.16%, PPV 32%) and complex abnormal profiles indicative of maternal malignancies (11, 0.02%, PPV 64%). The implementation of genome-wide NIPT is under debate, as the benefits of detecting other fetal chromosomal 28
aberrations need to be balanced against the risks of false positives, parental anxiety and a potential increase in (invasive) diagnostic procedures. The first year data provided here including clinical and laboratory follow-up will fuel this debate. Furthermore, we have shown that NIPT can successfully be embedded in a national screening program, with a single chain for prenatal care including counseling, testing and follow-up.
E-mail: [email protected]
Keywords: prenatal screening; non-invasive prenatal testing; cell-free DNA; pregnancy; aneuploidy
29
T 03
The landscape of pathogenic copy number variations in healthy, reproducing females
Brison Nathalie, Dehaspe Luc, Melotte Cindy, Dimitriadou Eftychia, Vancoillie Leen, Breckpot Jeroen, Peeters Hilde, Van Esch Hilde, Van Buggenhout Griet, Vogels Annick, de Ravel Thomy, Legius Eric, Devriendt Koen, Vermeesch Joris, Van Den Bogaert Kris
Center for Human Genetics, KU Leuven and UZ Leuven, Belgium
Since July 1, 2017, non-invasive prenatal testing (NIPT) is reimbursed by the Belgian Social Security for all pregnant women resulting in a high uptake. NIPT is an efficient means to detect fetal aneuploidies. However, the major fraction of cell-free DNA in maternal plasma is derived from maternal cells. Detection of clinically important variants in the maternal genome provides the opportunity to diagnose maternal constitutional and acquired disorders and optimize follow-up and management. We here report our one year experience on reported maternal secondary variants, detected in a series of 26,123 NIP-tests performed in Leuven. Autosomal rearrangements with a dominant effect were observed in 21 pregnancies (incidence 1/1243). These included five microdeletions involving a tumor suppressor gene (NF1, BRIP1, MSH6, BRCA1 and DICER1). Four different genomics disorders were detected: the 3q29 microdeletion (n=1) , PWS/AS duplication (n=1), CMT1A (n=3) and HNPP (n=7). Two deletions included a gene causing a developmental disorder when deleted (ASXL3 (apparently non mosaic); DNMT3A (mosaic)). Two large deletions were reported: a mosaic 17Mb 9q21 deletion and one non-mosaic 9.5Mb 4qter deletion. With regard to autosomal rearrangements with a recessive effect, 9 recurrent GJB6 microdeletions were reported. Not unexpectedly, two thirds of actionable CNV´s were X-linked (65/95). The recurrent STS deletion was the most common (27 cases), followed by intragenic DMD deletions or duplications (n=11) and SHOX deletions (n= 4). Known XL-recessive disease genes that were found to be deleted include Factor 8, COL4A6/COL4A5, and four deletions involved a developmental disorder gene (IL1RAPL1, FRMPD4, ARSE, RPS6KA3 (n=2)). The recurrent int22h1/int22h2-mediated Xq28 duplication syndrome was observed 6 times; the reciprocal deletion 4 times. Six large X chromosomal deletions were detected. Our results show that 1/275 pregnant women carry a clinically significant secondary finding. Genome-wide NIPT has the potential of informing pregnant women on significant reproductive risks. However, this potential can only be fully reached on the condition that the interpretation is performed by an expert team and reporting is based on predefined guidelines with rapid access to follow-up genetic testing and high quality pre- and posttest counselling.
E-mail: [email protected]
Keywords: non-invasive prenatal testing, secondary findings, maternal copy number variations
30
T 04
In vitro fertilization does not increase the incidence of de novo copy number alterations in fetal and placental lineages
Masoud Zamani Esteki 1,2,3,#,*, Triin Viltrop 4,#, Olga TÅ¡uiko 3,4,#, Airi Tiirats 5, Mariann Koel 6, Margit Nõukas 7, Olga Žilina 7, Katre Teearu 7, Heidi Marjonen 8, Hanna Kahila 9, Jeroen Meekels 1, Viveca Söderström-Anttila 10, Anne-Maria Suikkari 10, Aila Tiitinen 9, Reedik Mägi 11,12, Sulev Kõks 13, Nina Kaminen-Ahola 8, Ants Kurg 7, Thierry Voet 3,14,*, Joris Robert Vermeesch 3,* & Andres Salumets 4,5,9,15,*
1 Department of Clinical Genetics, Maastricht University Medical Centre+, Maastricht, The Netherlands 2 Department of Genetics and Cell Biology, GROW School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands 3 Centre for Human Genetics, University Hospital Leuven, Department of Human Genetics, KU Leuven, Leuven, Belgium 4 Department of Biomedicine, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia 5 Department of Obstetrics and Gynaecology, Institute of Clinical Medicine, University of Tartu, Tartu, Estonia 6 Department of Cell Biology, Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia 7 Department of Biotechnology, Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia 8 Department of Medical and Clinical Genetics, Medicum, University of Helsinki, Helsinki, Finland 9 Department of Obstetrics and Gynecology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland 10 Väestöliitto Fertility Clinic, Helsinki, Finland 11 Estonian Genome Center, Institute of Genomics, University of Tartu, Tartu, Estonia 12 Department of Bioinformatics, Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia 13 Centre for Comparative Genomics, Murdoch University, Murdoch, Western Australia and The Perron Institute for Neurological and Translational Science, Nedlands, Western Australia 14 Sanger-EBI Single Cell Genomics Centre, Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom 15 Competence Centre on Health Technologies, Tartu, Estonia # Co-first author * Co-senior author
We profiled the genomic landscape of fetal and placental tissues postpartum from in vitro fertilized (IVF)- and naturally-conceived children to investigate the persistence of IVF-related CIN. To this end, we alnalyzed de novo DNA copy number alterations in DNA samples extracted from cord blood cells and placental tissue, which are derived from embryonic and extra-embryonic cell lineages, respectively, of both IVF and naturally conceived neonates using haplarithmisis and molecular karyotyping. Our innovative haplarithmisis-based approach can be used not only to estimate fetal and maternal contribution to placental tissue, but also facilitates identification of mosaic and non-mosaic aberrations in placental and fetal genomes in prenatal diagnostics. Importantly, the transferred embryos of IVF newborns were not screened nor counter selected for chromosomal aneuploidy status before intrauterine transfer. Following birth, we scrutinized genome-wide single nucleotide polymorphism (SNP) profiles in DNA from the father, mother, placenta and neonate umbilical cord blood of 111 families, of which 49 and 62 quartets were from IVF and natural pregnancies, respectively. We detected de novoCNVs in placental genomes of twelve families; of the total fifteen detected aberrations four were also present in newborns and eleven were exclusively present in the placenta. We did not detect any CNVs present only in umbilical cord blood DNA, which would indicate genomic alteration in the neonate only. The mosaic (n= 12) de novoCNVs were 5–70% mosaic and were together with de novo non-mosaic CNVs (n = 3) randomly located across the genome. Our analysis showed comparable prevalence (P= 0.327 odds ratio test or P= 0.775 Pearson´s chi square test) of de novo CNVs in both cohorts with AGA: six of the 50 families with naturally conceived neonates (12.0%) and in three of the 48 families with IVF neonates (6.3%). The high level of chromosomal mosaicism in IVF cleavage-stage embryos is not preserved at later stages of prenatal development. Although we found a low-level mosaic trisomy in one IVF placental genome, it was not present in the fetal lineage of the same pregnancy, suggesting a proliferative defect of trisomic cells. The sporadic de novo genomic aberrations found in approximately 10.8% of pregnancies were scattered across the genome and represented random 31
and rare events of embryonic CIN, without any observed functional consequence on placental biology or fetal health.
E-mail: [email protected]
Keywords: Embryo genetics, prenatal development, chromosome instability, haplarithmisis
32
T 05
Longitudinal assessment of the 313-SNP based Polygenic Risk Score for breast cancer risk prediction in a Dutch prospective cohort
Inge M.M. Lakeman (1), J. van Rooij (2), R. Ruiter (3), C.J. van Asperen (4), André G. Uitterlinden (2,3), Peter Devilee (1,5)
1. Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands 2. Department of internal Medicine, Erasmus Medical Center Rotterdam, the Netherlands 3. Department of Epidemiology, Erasmus Medical Center, Rotterdam, the Netherlands 4. Department of Clinical Genetics, Leiden University Medical Center, the Netherlands 5. Department of Pathology, Leiden University Medical Center, the Netherlands
Purpose Breast cancer associated SNPs, summarized in a Polygenic Risk Score (PRS) are presently not used to guide the appropriate starting age for breast cancer screening. At this moment the most predictive PRS for breast cancer is based on 313 SNPs. The aim of this study is to validate the performance of the 313-SNP PRS in a Dutch prospective cohort and explore the utility of risk stratified screening. Materials and Methods We included women from the Rotterdam study, a Dutch prospective cohort study started in 1989 with >70% response rate. Phenotypic information was available with 25 years of follow-up. Genotyping was performed with the Illumina 550k and 610k array for 6670 women aged 45 years or older. After imputation with the HRC1.1 and 1KG reference panels, the standardized 313-SNP-based PRS (sPRS), per 1 standard deviation (SD) in controls from the Breast Cancer Association Consortium (BCAC) was calculated. Cox regression analysis, adjusted for birth year and age at inclusion, was performed to estimate the association between the sPRS and incident breast cancer risk. Results Of all included women with an average age of 66, 145 had prevalent breast cancer and 323 developed incident breast cancer during follow-up. These women had a higher sPRS, mean 0.45 (SD=1.06) for cases with in situ carcinoma (N=37) and 0.51 (SD=1.06) for cases with invasive carcinoma (N=431) compared to 0 (SD=1.01) in all 6202 controls, and 0.05 (SD=0.98) in 842 of these controls with other tumors than breast cancer. Breast cancer risk increased by sPRS with an Hazard Ratio (HR) per SD=1.56, 95%CI [1.40-1.73], and compared with the middle sPRS quintile, the lowest quintile had an HR of 0.57, 95%CI[0.38-0.87] and the highest quintile an HR of 1.81, 95%CI[1.32-2.50]. We noticed a slightly smaller effect of the sPRS for in situ compared to invasive breast cancer risk only (HR=1.45, 95%CI[1.02-2.01], and 1.58, 95%CI[1.41-1.76], respectively). Discussion and conclusion: The 313-SNP PRS can be used for breast cancer risk stratification in the Dutch population and seems specific for breast tumors and not for other tumor types. Further analyses are being performed that include other breast cancer risk factors (i.e., reproductive and lifestyle factors), which are available for the majority of the included women. Furthermore, the added value of the 313-SNP PRS on long- and short-term breast cancer risk prediction and screening stratification will be explored.
E-mail: [email protected]
Keywords: Polygenic Risk Score, Breast Cancer, Genetic Epidemiology, Prospective cohort, Breast screening
33
T 06
Towards the treatment of Cantú syndrome
Helen I. Roessler(1), Conor McClenaghan(2), Yan Huang(2), Carmen M. Halabi(2,3), Theresa Harter(2), Sanne Savelberg(1), Federico Tessadori(1,4), Jeroen Bakkers(4,5), Attila Kovacs(6), Maria S. Remedi(2,6), Edwin P. Kirk(7,8), Ingrid Scurr(9), Sarah F. Smithson(9), Dorothy K. Grange(2,3), Colin G. Nichols(2), Mieke M. van Haelst(10,11) and Gijs van Haaften(1)
1 Department of Genetics, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands. 2 Center for the Investigation of Membrane Excitability Diseases (CIMED), and Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, Missouri, USA. 3 Center for the Investigation of Membrane Excitability Diseases (CIMED), and Departments of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, USA. 4 Hubrecht Institute-KNAW and University Medical Centre Utrecht, Utrecht, the Netherlands. 5 Department of Medical Physiology, Division of Heart and Lungs, University Medical Center Utrecht, Utrecht, the Netherlands. 6 Center for the Investigation of Membrane Excitability Diseases (CIMED), Medicine, Washington University School of Medicine, St. Louis, Missouri, USA. 7 Dept of Medical Genetics, Sydney Children's Hospital, Randwick NSW, Australia. 8 School of Women's and Children's Health, University of New South Wales, Randwick NSW, Australia. 9 Department of Clinical Genetics, St Michael's Hospital, Bristol, UK. 10 Department of Clinical Genetics, Amsterdam Medical Center, University of Amsterdam, Amsterdam, The Netherlands. 11 Department of Clinical Genetics, VU Medical Center, VU University Amsterdam, Amsterdam, The Netherlands.
Cantú Syndrome (CS), first described in 1982, is a rare genetic disorder caused by gain-of-function (GOF) mutations in genes encoding the pore-forming (Kir6.1, KCNJ8) and regulatory (SUR2, ABCC9) subunits of ATP-sensitive potassium (KATP) channels. Multiple case reports of individual patients have described the various clinical features of CS, but systematic studies have been lacking. To generate a critical mass of CS patients to find further clinical characteristics of the disease as well as to ensure a rapid progression towards future interventional studies we developed a standardized REDCap-based registry. We report phenotypic and associated genotypes on 75 CS patients, with confirmed ABCC9 variants in 72 of the cases. Clinical data is obtained during annual Cantú research clinics. Polyhydramnios during fetal life, lymphedema, patent ductus arteriosus (PDA), cardiomegaly, dilated aortic root, vascular tortuosity of thoracic and cerebral arteries and migraine headaches are common features. CS is debilitating with no targeted therapy available. Clinical management currently involves symptomatic treatments to address second-order complications such as heart failure and edema. The off-patent KATP inhibitor glibenclamide which is already widely applied in clinic to block GOF KATP channels involved in Type 2 diabetes holds promise as a potential treatment for CS. In order to perform therapeutic drug screening, we have developed novel CS animal models in which disease-causing mutations were knocked-in to the endogenous ABCC9 loci in zebrafish and mice using CRISPR/Cas9 genome engineering. Both models recapitulate key cardiovascular features of CS which significantly reverse after glibenclamide administration. Hypercontractility and abnormally high-output were reversed in zebrafish larvae whilst cardiac hypertrophy in mice was similarly attenuated. No sustained hypoglycemia was observed after chronic exposure to glibenclamide in mice. These results provide key pre-clinical evidence for the in vivo efficacy of glibenclamide for the treatment of CS. Hence, we intend to perform a clinical trial to test glibenclamide in CS patients.
E-mail: [email protected]
Keywords: Cantu syndrome, KCNJ8, ABCC9, Polyhydramnios; PDA; Hypertrichosis; Cardiomegaly; CRISPR/Cas9; zebrafish; cardiovascular system
34
T 07
NOTCH3 cysteine altering variants and their phenotypes in 92,456 whole exome sequenced participants of the Geisinger DiscovEHR initiative
R.J. Hack1, J.W. Rutten1*, N. Pearson3*, , J.Li3, A. Khan2, M.A. Iqbal2, J. Hornak2, V. Abedi3, Y. Zhang4, M.T.M. Lee4, C. Griessenauer2, Regeneron Genetics Center5, R. Zand2†, S.A.J. Lesnik Oberstein1†* Shared second authors †Shared last authors
1. Department of Clinical Genetics, Leiden University Medical Center, Leiden, the Netherlands 2. Neuroscience institute, Geisinger Health System, Danville, Pennsylvania, USA 3. Biomedical and Translational Informatics Institute, Geisinger, Danville, Pennsylvania, USA 4. Geisinger Genomic Medicine Institute, Danville, Pennsylvania, USA 5. Regeneron Genetics Center, Tarrytown, New York, USA
Background: Cysteine altering missense variants in NOTCH3 have been exclusively associated with CADASIL, a hereditary cerebral small vessel disease (SVD). Recently, we discovered that these variants, especially those located in the NOTCH3 protein´s epidermal growth factor-like repeat (EGFr) domains 7-34, have an unexpectedly high frequency in the general population worldwide, namely 1:300. The goal of this study was to investigate the phenotype of participants with NOTCH3 cysteine altering variants in the Geisinger DiscovEHR initiative. Methods: We utilized whole exome sequence data from 92,456 Geisinger-Regeneron DiscovEHR participants. We selected individuals with a cysteine altering variant in one of the NOTCH3 EGFr domains. The control group, matched for age and sex, had only synonymous variants in NOTCH3. We reviewed and recorded all the patients´ demographic and clinical information, as well as neuroimaging characteristics. Group comparisons were done using the ?2 test for categorical variables, and unpaired t-test for normally distributed continuous variables. SPSS 24.0 was used for all statistical analyses. Results: 135 individuals with a NOTCH3 cysteine altering missense variant were identified (frequency 1:685), of which 134 had a variant in one of EGFr domains 7-34. Medical records were available for 118 cases, with a mean age of 58.1 ± 16.9 years. The control group consisted of 184 individuals, mean age 57.9 years ± 16.6. In the case group, 12.7% had a history of stroke, compared to 4.9% of controls (p=0.014). Age at onset of stroke did not differ between cases and controls: 67,8 years ± 20.4 vs 65,0 years ± 11.72. There was no significant difference in the frequency of migraine headache, depression, and dementia. Twenty-nine (25%) cases and 45 (24%) controls had an interpretable MRI. Compared to controls, cases more frequently had large areas of confluent white matter hyperintensities (Fazekas 3) (p=0.025), and there was a trend to a higher lacune count (p=0.055). There was no significant difference in the number of microbleeds (p=0.550). Conclusion: NOTCH3 cysteine altering variants in the population, almost exclusively located in EGFr domains 7-34, are associated with an increased prevalence of stroke and SVD markers on MRI. This suggests that NOTCH3 EGFr 7-34 variants are a hitherto unknown risk factor for SVD in the general population, but are rarely associated with a classical CADASIL phenotype.
E-mail: [email protected]
Keywords: CADASIL, cerebral small vessel disease, whole exome sequencing, NOTCH3, biobank
35
T 08
Towards personalized treatment of genetically classified refractory epilepsies using human induced pluripotent stem Cells (hIPSCs) as an ex-vivo tool
E.J.H. van Hugte1,2,3, J. M. Keller1,2, E. Lewerissa1,2, M. Frega2,3, J. Nicolai1, H.J.M. Majoie1, H.J. Schelhaas1, J.S. Verhoeven1, J.H.L.M van Bokhoven2, N. Nadif Kasri2,3
1 Department of Neurology, Academic Center for Epileptology, Heeze, The Netherlands. 2 Department of Human Genetics, Radboudumc, Nijmegen, The Netherlands. 3 Donders Institute for Brain, Cognition, and Behaviour, Centre for Neuroscience, Nijmegen, The Netherlands.
Dravet syndrome (DS) is a severe epileptic encephalopathy (EE) manifesting in the first year of life. DS is characterized by recurrent and prolonged seizures, behavioral problems and developmental delay. ln addition, EEs are associated with substantial cognitive and neuropsychological decline as a result of seizures during brain development. lt is of crucial importance to timely control the seizure activity. Although numerous antiepileptic drugs are available, it remains a challenge for clinicians to select drugs with high efficacy but low side effects for each patient individually. Choosing treatment is based on a rational trial and error approach, but an effective predictive tool to investigate treatment options is lacking. ln 70-80% of the cases, DS results from de novo mutations in the SCN1A gene, encoding the voltage gated nav1.1 channel. Conversely, not every patient with a mutation in SCN1A develops the same symptoms and other genes have been associated with a DS phenotype, in addition to SCN1A. Both this genetic and phenotypic heterogeneity add to the difficulties of finding an appropriate treatment for DS. Animal models can neither recapitulate mutation-specific effects nor genetic background factors, and are not suitable for devising personalized medicine interventions. Hence, there is a strong need for patient-specific disease models. The development of human induced pluripotent stem cells that are differentiated into induced neurons (iNeurons) provides a solution. Here, we show that DS patient iNeurons exhibit an hyperexitable phenotype on a single cell level and describe how iNeurons, cultured on micro- electrode arrays (MEA) develop into neuronal networks. Specifically we generate and evaluate a patient specific neuronal network (PNN) as a disease model for DS by co-culturing both excitatory and inhibitory iNeurons. By assessing developing and mature PNNs, the MEAs allow us to study the patient specific network fingerprint, providing a predictive tool for drug efficacy and side effects.
E-mail: [email protected]
Keywords: Epilepsy, human induced Pluripotent Stem Cells, brain-on-a-chip, neuronal networks, Micro-electrode arrays, anti-epileptic drugs, Dravet Syndrome
36
T 09
Genotyping On ALL patients (GOALL); clinical implementation of high-throughput genotyping arrays.
J.G.J. van Rooij1, J.M.H. Verkerk1, B. Sedaghati-khayat1, L. Broer1, J.H. de Vries1, G.M. van Dijk1, J.B.J. van Meurs1, A.G. Uitterlinden1.
1 Department of Internal Medicine, Erasmus Medical Center, Rotterdam, the Netherlands
Introduction: Genetic testing is increasingly used in standard clinical practice for most clinical disciplines. However, genetic tests are expensive and/or must be rerun when additional clinically relevant variants/genes are identified. Dense genotyping arrays (such as GSA or PMDA) cover a large portion of genetic variation and provide a cost-effective, high-throughput and highly standardized alternative. In the GOALL project, we are determining the clinical utility and applications of genotyping arrays. Methods: We investigate the clinical utility of the GSAMD_v2 and PMDA arrays by: 1) technical validation of common (vs. WES) and rare variants (vs. clinically determined genotypes by different methods), 2) prospectively offering polygenic risk scores (PRS) as primary utility in daily clinical practice, 3) prospectively counseling and testing eligibility of feeding back secondary findings (pharmacogenomics, ACMG mutations) using array based genotyping. We are piloting these applications in collaboration with the departments of Oncology (breast cancer), Ophthalmology (age-related macular degeneration), Clinical Chemistry (pharmacogenomics), Clinical Genetics (counseling) and Public Health within the Erasmus Medical Centre. Results: The GSAMD_v2 array contains 12,755 clinical variants from the ClinVar database. Our preliminary results suggest that >90% of the mutations routinely determined in our hospital are covered by the array design (e.g. APOE, MTFHR, F5 etcera). We are currently selecting carriers of clinically relevant mutations to check genotyping concordance. Comparison of array vs. WES of 197 population samples shows an overall non-reference concordance of 98%, and 95% for singletons among ~30,000 directly genotyped variants and respectively 97% and 93% among ~150,000 imputed variants (QUAL>0.8). Discussion: Our preliminary results suggest that a portion of current hospital-wide clinical genetic testing can cost-effectively be replaced by array-based genotyping. In addition to reporting highly concordant results, array-based genotyping allows additional reporting of several PRS and pharmacogenomics. This data can easily be stored and re- analyzed when additional genetic loci or PRS are identified, or unblinded when a patient needs further genetic testing not part of the initial primary clinical question.
E-mail: [email protected]
Keywords: arrays, personalized medicine, pharmacogenomics, polygenic risk scores
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T 10
The added value of long-read amplicon sequencing for clinical applications
Kornelia Neveling, Dorien Lugtenberg, Arjen Mensenkamp, Ronny Derks, Amber den Ouden, Michael Kwint, Simone van der Heuvel, Menno Tjon-Pon-Fong, Maartje van de Vorst, Linske de Bruijn, Evelien Hoenselaar, Marjolijn Ligtenberg, Helger Yntema, Marcel Nelen, Lisenka Vissers, Lonneke Haer-Wigman, Richarda de Voer
Department of Human Genetics, Radboud university medical center, Nijmegen, the Netherlands
Next generation sequencing has revolutionized the field of human genetics by offering new possibilities to unravel human diseases. Due to limitations of short read sequencing however, various additional tests including single gene sequencing, MLPA or genescan are still needed for the analyses of certain difficult genomic regions. Long read high fidelity amplicon sequencing can provide very accurate, efficient, high throughput sequences from single molecules, with read lengths largely limited by PCR. The data is much easier to interpret, phased variants and breakpoints are present within high fidelity individual reads, and additional tests can largely be eliminated. We here demonstrate how we implemented long read SMRT sequencing in our diagnostic routine. As examples, we will concentrate on the PMS2 and OPN1 genes. In both cases, homologous regions make short read sequencing results very difficult to interpret. For PMS2, three amplicons ranging in size from 11.4 kb to 16.8 kb were designed using primers unique to this gene, covering the entire 36 kb of sequence. SMRT sequencing produced HiFi reads with coverages ranging from 200x to 1500x. We will show examples of various types of aberrations detected by long-range sequencing, including two deletions >1000kb with precise breakpoint mapping. For the homologous OPN1LW and OPN1MW, specific 14.0 kb and 15.6 kb amplicons were designed covering the regions of interest. Long read sequencing results will be shown for several patients in which gene conversions and hybrid genes made short read sequencing data nearly impossible to analyze. For all cases, PacBio sequencing confirmed the mutations originally identified by orthogonal technologies. Moreover, it added further detailed information such as the ability to distinguish between genes and pseudogenes and included phasing and precise breakpoint mapping. We feel that long-range sequencing is an attractive alternative strategy to accurately analyze homologous genes, based on the added value to reduce interpretation complexity and the need for additional confirmatory tests.
E-mail: [email protected]
Keywords: PacBio, long read sequencing, amplicons, PMS2, OPN1
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T 11
Reliable application of DNA-methylation signatures in genetic diagnostic testing
Peter Henneman1,2, Marielle Alders1, Erfan Aref-Eshghi3,4, Jennifer Kerkhof4, Andrea Venema1, Jet Biek1, Sonna Stolk1, Ruud van den Boogaard1, Bekim Sadikovic3,4 and Marcel Mannens1
1Amsterdam University Medical Centers, department of Clinical genetics, Genome diagnostics; 2Amsterdam University Medical Centers, department of Clinical genetics, Core Facility Genomics; 3Department of Pathology and Laboratory Medicine, Western University, London, ON N6A 3K7, Canada;4Molecular Genetics Laboratory, Molecular Di-agnostics Division, London Health Sciences Centre, London, ON N6A 5W9, Canada.
Current genetic diagnostics of individuals suffering from rare syndromes that include impaired neurodevelopmental and congenital anomalies, generally involves WES and CNV analysis. A substantial proportion of such analyses however leaves the phenotype unexplained. Many of these syndromes have been shown to result from a variant or mutation annotated to chromatin modifying enzymes or chromatin binding protein complexes. Here, we have validated the clinical diagnostic utility of genome-wide DNA methylation analysis of peripheral blood in unresolved cases (Kabuki, SOTOS, CHARGE and others - VUS carriers) using the previously reported methodology and in collaboration with Aref-Eshghi and Sadikovic et al (AJHG, 2019). We applied a computational model enabling concurrent detection of 20 syndromes using DNA methylation data with full accuracy. We were able to fully resolve the diagnosis of individuals with uncertain clinical and genetic diagnosis for the following disorders: ADNP-related syndromes, BAFopathies, Cornelia de Lange syndrome, CHARGE syndrome, Kabuki syndrome, SOTOS syndrome, Claes Jensen Syndrome, BWS, SRS and Angelman syndrome. This study confirms that genomic DNA methylation analysis can facilitate the molecular diagnosis of unresolved clinical cases and highlights the potential value of genome-wide DNA-methylation testing in the routine clinical assessment of unresolved cases with rare disorders involving neurodevelopmental and congenital anomalies. Upcoming research will extend the number of detectable syndromes up to 40 and the panel will be updated shortly. Moreover, the number chromatin modifying proteins is estimated to involve approximately 200 genes, which potentially are candidates for further research and that may be included in future versions of our panel. Our test (EpiSign) has now been fully implemented in collaboration with, London, Canada, and is for European requests exclusively available at the AUMC, clinical genetics, genome diagnostics department (genomediagnostics.amsterdamumc.nl).
E-mail: [email protected]
Keywords: DNA-methylation signature, genetic diagnostics, rare syndrome
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T 12
What if we would use a diagnostic multi-cancer gene panel for opportunistic screening? A study in 2,090 Dutch familial cancer patients
L.F. Johansson, K.K. van Dijk-Bos, A.H. van der Hout, A.P. Knopperts, B. Leegte, P.C. van den Akker, K. Kok, I.M. van Langen, M.A. Swertz, R. K. Weersma, R.J. Sinke, B. Sikkema-Raddatz, R.H. Sijmons, H. Westers
University of Groningen, University Medical Center Groningen, Department of Genetics, Groningen, the Netherlands University of Groningen, University Medical Center Groningen, Department of Gastroenterology, Groningen, the Netherlands
Purpose In familial cancer (FC) diagnostics, analysis of next-generation sequencing data typically focuses on genes known to be associated with the cancer type that prompted referral. Currently, however, it is debated whether opportunistic screening should be performed when sequence data is available for other genes. We aimed to determine how many secondary findings (SFs) would be detected in cancer-predisposing genes present in our FC gene panel if we offered opportunistic screening to patients within FC diagnostics. Methods We anonymously reanalyzed sequencing data of 2,090 FC patients for either 73 genes (original FC panel) or 85 genes (updated panel) for SNVs, indels and CNVs. To determine the background prevalence of pathogenic variants in FC genes, we screened 1,326 individuals from the general Dutch population. Results We detected SFs in 3.0% of patients (excluding heterozygous CHEK2 and MUTYH variants), and a (likely) pathogenic variant matching their family´s cancer type in 10.1% of patients. In the Dutch population cohort, 3.2% of individuals had a (likely) pathogenic variant in a cancer-predisposing gene. Conclusion Our results can assist in the design of future research programs on opportunistic screening. These programs are needed because there is not yet sufficient evidence to meet international screening program criteria.
E-mail: [email protected]
Keywords: Genetic screening, diagnostic yield, gene panel, next-generation sequencing, familial cancer
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T 13
Neurodevelopmental disorders: a next generation
Margot R.F. Reijnders1, Tjitske Kleefstra2, Lisenka E.L.M. Vissers2, Han G. Brunner1,2
1 Department of Clinical Genetics, Maastricht University Medical Center, Maastricht, The Netherlands. 2 Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands.
Since 2010 whole exome sequencing (WES) discovered a large numbers of novel genes and neurodevelopmental disorders (NDDs), denoted here as 'next-generation NDDs´. Yet, ~40% of NDD patients remain undiagnosed. Therefore, we previously established a cohort of 826 patients with unexplained ID, a subtype of NDD, for whom trio-based WES was performed. ID severity ranged from mild (27%) to severe (27%) and 32.2% of patients had other NDD-subtypes. We identified pathogenic variants in known NDD genes in 28.6% of patients and 386 patients (46.7%) had at least one de novo mutation in a gene not previously associated with NDDs. Between 2014 and 2018, we have so far confirmed seven of 586 genes as next-generation NDD-gene (SON,TLK2,RAC1,RHEB,USP9X,RAB11B,WAC). Matchmaking was successful for all but one (RHEB), resulting in a maximum of 35 additional patients (TLK2) and underscoring the need for data-sharing for next-generation NDDs. Additionally, mutations in these genes together only diagnosed Ëœ1% of patients of our initial cohort, illustrating the rarity of next-generation NDDs. Of note, for USP9X, WAC and TLK2, most patients were mildly affected. Further systematic analysis of our cohort showed that overall a similar percentage of de novo mutations in known NDD genes was present in patients with mild ID (27.9%) compared to more severe ID (27.2%), indicating that WES should not be restricted to patients with more severe phenotypes. Finally, we observed a challenge to translate the discovered next-generation NDDs to clinic. To improve patient care, we propose an organizational model, in which parents have a key role.
E-mail: [email protected]
Keywords: Rare neurodevelopmental disorders; mild intellectual disability; data sharing; clinical translation.
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T 14
Transcriptome and protein analysis highlight the endosomal pathway in disease pathogenesis of metabolic CL syndrome
Pottie L, Van Gool W, Van Hooydonck M, De Saffel H, Vermassen P, Coucke P, Sips P and Callewaert B
Center for Medical Genetics, Ghent University and Ghent University Hospital, Ghent, Belgium
We recently described a novel autosomal recessive cutis laxa (CL) syndrome, caused by missense mutations in subunit V1E1 of the vacuolar H+-ATPase (ATP6V1E1). The condition is clinically characterized by loose redundant skin folds, variable mental disability, typical facial characteristics, lipodystrophy, hypotonia, and cardiopulmonary involvement including pneumothorax, hypertrophic cardiomyopathy and aortic root dilatation. Similarly to ATP6V0A2-related cutis laxa, impaired retrograde Golgi transport, glycosylation abnormalities, and a dysfunctional secretory pathway have been implicated in the pathogenesis, but the pleiotropic manifestations remain largely unexplained. We report on two knock-out (KO) zebrafish models. The first model has a proviral insertion in the 5´UTR disrupting atp6v1e1b transcription. The second model has a premature stop in atp6v1e1b, generated by CRISPR-Cas9 mutagenesis. Both models are embryonically lethal, show decreased pigmentation, have reduced touch-evoked escape response and hatching, manifest altered craniofacial cartilage structures, and show abnormal blood vessel structure and cardiac dysfunction. Transcriptome analysis revealed downregulation of transferrin-a (tfa), hemoglobin alpha embryonic 1.1 and hemoglobin beta embryonic 1.3. KEGG pathway analysis indicates a decreased function of the oxidative phosphorylation pathway. These results suggest a disturbance in iron homeostasis. KO of atp6v1e1b in zebrafish disrupts the maturation from the early endosome into the late endosome. Protein analysis indicates upregulation of early endosome antigen 1 and small rab GTPases 5 while small rab GTPases 7 and lysosome associated membrane glycoprotein show a trend towards downregulation. We hypothesize that reduced acidification of the endosomes will block the release of iron and the recycling of tfa-tfa receptor complex to the cell surface implementing pathological manifestations. In conclusion, KO of atp6v1e1b in zebrafish faithfully recapitulates the human cutis laxa syndrome. The vacuolar H+-ATPase is important for proper functioning of the endosomal pathway opening avenues for therapeutic strategies.
E-mail: [email protected]
Keywords: cutis laxa syndrome ATP6V1E1 zebrafish transcriptoom endosomal pathway iron homeostasis
42
T 15
Noncoding structural variants disrupt the regulatory architecture of Rett genes
Eva D´haene, Reut Bar-Yaacov, Inbar Bariah, Lies Vantomme, Sien Van Loo, Francisco Avila Cobos, Karen Verboom, Reut Eshel, Rawan Alatawna, Björn Menten, Ramon Y Birnbaum, Sarah Vergult
Center for Medical Genetics, Department of Biomolecular Medicine, Ghent University, Belgium Department of Life Sciences, Faculty of Natural Sciences, Ben-Gurion University of the Negev, Israel Center of Evolutionary Genomics and Medicine, Ben- Gurion University of the Negev, Israel Cancer Research Institute Ghent (CRIG), Ghent, Belgium Bioinformatics Institute Ghent from Nucleotides to Networks (BIG N2N), Ghent, Belgium
Rett syndrome is a progressive neurodevelopmental disorder, characterized by a severe developmental delay, absence of speech, seizures, hypotonia and stereotypic movements. It is typically caused by mutations in the MECP2 gene, but several other genes, including the transcription factors MEF2C and FOXG1, have been associated with a Rett-like phenotype as well. Recently, we and others identified several noncoding structural variants (SVs) in patients with Rett- like characteristics. All SVs are located proximal to the coding sequence of MEF2C or FOXG1, suggesting disruption of the regulatory structure governing these genes. Using Circularized Chromosome Conformation Capture (4C) sequencing in a neuronal cell line, we identified a complex regulatory interaction network in the MEF2C region. We found that the MEF2C promoter physically contacts multiple distal enhancer regions upstream of its coding sequence. Based on epigenetic enhancer marks and sequence conservation, we delineated 16 putative enhancer elements, of which 14 were active in in vitro luciferase assays and 8 displayed in vivo neuronal activity during zebrafish development. For FOXG1 as well, 4C-seq experiments have shown that the promoter interacts with at least three in vivo validated brain enhancers, all situated in a region affected by deletions or translocations in multiple Rett-like patients. In summary, Rett genes MEF2C and FOXG1 are part of complex regulatory networks involving multiple distal enhancers. Disruption of these regulatory structures by noncoding SVs could form the genetic basis of the Rett-like phenotype observed in some patients.
E-mail: [email protected]
Keywords: Rett syndrome, noncoding structural variation, gene regulation, chromosome conformation
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T 16
Loss of neutral sphingomyelinase-3 (SMPD4) links neurodevelopmental disorders to cell cycle and nuclear envelope anomalies
Pamela Magini(1,38), Daphne J. Smits(2,38), Laura Vandervore(2,3), Rachel Schot(2), Marta Columbaro(4), Esmee Kasteleijn(2), Mees van der Ent(5), Flavia Palombo(6), Maarten H. Lequin(7), Marjolein Dremmen(8), Marie Claire Y. de Wit(9), Mariasavina Severino(10), Maria Teresa Divizia(11), Pasquale Striano(12,13), Natalia Ordonez-Herrera(14), Amal Alhashem(15,16), Ahmed Al Fares(15,16), Malak Al Ghamdi(17), Arndt Rolfs(14), Peter Bauer(14), Jeroen Demmers(18), Frans W. Verheijen(2), Martina Wilke(2), Marjon van Slegtenhorst(2), Peter J. van der Spek(19), Marco Seri(20), Anna C. Jansen(3,21), Rolf W. Stottmann(22), Robert B. Hufnagel(23), Robert J. Hopkin(22,24), Deema Aljeaid(25), Wojciech Wiszniewski(26,27), Pawel Gawlinski(27), Milena Laure- Kamionowska(28), Fowzan S. Alkuraya(29), Hanah Akleh(30), Valentina Stanley(31), Damir Musaev(31), Joseph G Gleeson(31), Maha S. Zaki(32), Nicola Brunetti Pierri(33) , Gerarda Cappuccio(34), Bella Davidov(35), Lina Basel-Vanagaite(35), Lily Bazak(35), Noa Ruhrman Shahar(35), Aida Bertoli Avella(12), Ghayda M. Mirzaa(36,37), William B. Dobyns(36), Tommaso Pippucci(1), Maarten Fornerod(5,39), Grazia M. S. Mancini(2,39)
1- Medical Genetics Unit, S.Orsola-Malpighi Hospital, via Massarenti 9, 40138, Bologna, Italy 2- Department of Clinical Genetics, ErasmusMC University Medical Center Rotterdam, Wytemaweg 80, 3015 CN, Rotterdam, The Netherlands. 3- Vrije Universiteit Brussel, Centrum Medische Genetica, Laarbeeklaan 101, 1090 Brussel 4- Laboratory of Musculoskeletal Cell Biology, IRCCS Istituto Ortopedico Rizzoli, 40136 Bologna, Italy 5- Department of Cell Biology, ErasmusMC UnMeesiversity Medical Center Rotterdam, Dr. Molewaterplein 40, 3015 GD, Rotterdam, The Netherlands 6- IRCCS Institute of Neurological Sciences of Bologna, Bellaria Hospital, 40139 Bologna, Italy 7- Department of Radiology, University medical center Utrecht, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands 8- Department of Radiology, Sophia Children´s hospital, ErasmusMC University Medical Center Rotterdam, Dr. Molewaterplein 40, 3015 GD, Rotterdam, The Netherlands 9- Department of Child Neurology, Sophia Children´s hospital, ErasmusMC University Medical Center Rotterdam, Dr. Molewaterplein 40, 3015 GD, Rotterdam, Rotterdam, NL 10- Neuroradiology Unit, IRCCS Istituto Giannina Gaslini, 16147 Genova, Italy 11- UOC Genetica Medica, IRCCS Istituto Giannina Gaslini, 16147 Genova, Italy 12- Pediatric Neurology and Muscular diseases Unit, IRCCS Istituto Giannina Gaslini, 16147 Genova, Italy 13- Department of neursciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, University of Genova, 16126 Genova, Italy 14- CENTOGENE AG. Am Strande 7. 18055 Rostock, Germany 15- Department of Pediatrics, Prince Sultan Military Medical City, Riyadh 12233, Saudi Arabia 16- Department of Pediatrics, College of Medicine, Qassim University, Qassim 52571, Saudi Arabia 17- Department of Pediatrics, College of Medicine, King Saud University, Riyadh 11451, Saudi Arabia, 18- Proteomics Center, Room Ee-679A, Erasmus University Medical Center. Wytemaweg 80, 3015 CN Rotterdam 19- Department of Pathology, Clinical Bioinformatics, ErasmusMC University Medical Center PO Box, 3000CA Rotterdam, The Netherlands. 20- Medical Genetics Unit, Department of Medical and Surgical Sciences, University of Bologna, via Massarenti 9, 40138, Bologna, Italy 21 - Pediatric Neurology Unit, UZ Univeristy of Brussel, Laarbeeklaan 101, 1090 Brussel, Belgium 22- Department of Human Genetics, Division of Developmental Biology Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, MLC 7016. Cincinnati, OH 45229 23- Medical Genetics and Ophthalmic Genomics Unit, National Eye Institute, National Institutes of Health, 10 Center Drive, Building 10, Room 10N109, Bethesda, MD 20892 24- Department of Pediatrics, University of Cincinnati, College of Medicine, 3333 Burnet Ave. ML 4006 Cincinnati OH 4522933. 25- Department of Human Genetics, Faculty of Medicine, King Abdulaziz University, Jeddah 21589, Saudi Arabia 26- Oregon Health & Sciences University 3181 SW Sam Jackson Park Road L103. Portland, Oregon, U.S.A 27- Institute of Mother and Child, Kasprzaka 17a, 02-211 Warsaw, Poland 28- Department of Experimental and Clinical Neuropathology, Mossakowski Medical Research Centre Polish Academy of Sciences, Pawinskiego 5, 02-106 Warsaw, Poland 29- Department of Genetics, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia 30- Department of Obstetrics and Gynecology, King Faisal Specialist Hospital and Research Center, Riyadh 12713, Saudi Arabia 31- Laboratory for Pediatric Brain Disease, Department of Neurosciences, Howard Hughes Medical Institute, University of California, San Diego, California, USA 92093. 32 - Human Genetics and Genome Research Division, Clinical Genetics Department, National Research Centre, Cairo 12622, Egypt. 33 - Brunetti- Department of Translational Medicine, University of Naples "Federico II", Naples 80131 Italy 34 - Cappuccio - Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli 80078, Italy 35 - Department of Medical Genetics, Recanati Genetic Institute Rabin 44
Medical Center, Beilinson Campus, Petah Tikva, 4941492, Tel Aviv, Israel 36- Department of Pediatrics, University of Washington, Seattle, WA 98195, Washington, USA 37- Center for Integrative Brain Research, Seattle Children´s Research Institute, Seattle, WA 98101, Washington, USA. 38- These authors contributed equally 39 - These authors contributed equally
Introduction: Several neurodevelopmental processes including neuronal survival, migration and differentiation are controlled by sphingolipid metabolism. Sphingomyelinases generate ceramide from sphingomyelin as a second messenger in intracellular signaling pathways involved in cell proliferation, differentiation, or apoptosis. While the role of acid sphingomyelinase is well established, the role of neutral sphingomyelinases in human neurodevelopment has remained elusive. Methods: Twenty-nine children from 12 families presented with microcephaly with simplified gyral pattern, cerebellar hypoplasia, severe developmental encephalopathy, congenital arthrogryposis, diabetes mellitus and early fetal-postnatal demise. All index cases have bi-allelic loss of function variants in the SMPD4 gene, coding for neutral sphingomyelinase-3.We performed additional studies to elucidate the mechanisms contributing to disease pathogenesis. Results: Fibroblasts from affected individuals showed morphologic endoplasmic reticulum (ER) cisternae abnormalities, consistent with a previously suggested function of SMPD4 in the ER. Overexpression of human Myc-tagged SMPD4 in HEK392T cells showed localization of the protein to both the nuclear envelope and the ER. Previous studies localized SMPD4 to the outer nuclear membrane. Mass spectrometry of SMPD4-associated proteins detected peptides belonging to nuclear pore complex proteins. We also observed delayed cell cycle progression after downregulation of SMPD4 by siRNA. These data are similar to former studies in HeLa cells showing mitotic abnormalities after siSMPD4 treatment. Conclusions: Our study describes a novel microcephaly syndrome characterized by severe neurologic abnormalities. Our results suggest a major function of neutral sphingomyelinase-3 in regulation of mitosis through modulation of nuclear membrane lipids, pointing to a novel mechanism and pathway in the pathogenesis of microcephaly.
E-mail: [email protected]
Keywords: microcephaly, arthrogryposis, neutral-sphingomyelinase, SMPD4
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Abstracts Posters (P)
No. Name Titel E-mail
P 01 Hanka Venselaar Lost in translation - Experiences from a [email protected] Structural Biologist P 02 Nika Schuermans* NPC2 c.441+1G>A is a likely pathogenic [email protected] variant associated with an adult-onset Alzheimer-like phenotype P 03 Aude Beyens* A clinical flowchart to classify cutis laxa [email protected] patients, supported by electron microscopy and pathophysiology P 04 Eline Verberne* Genetic service for birth defects at the [email protected] Dutch Caribbean Islands P 05 Marielle van Gijn Dutch genome diagnostic laboratories [email protected] improved variant interpretation quality and speed by building and implementing a data sharing platform P 06 Marian Stevens- Next-generation cytogenetics: high- marian.stevens- Kroef resolution optical mapping to replace [email protected] FISH, karyotyping and CNA-microarrays P 07 Lennart Raman PREFACE: in silico pipeline for accurate [email protected] cell-free fetal DNA fraction prediction P 08 Jo Vanoevelen DTYMK deficiency is the cause of a [email protected] severe congenital neurodegenerative disease P 09 Eva Jacobs* A novel neurodevelopmental syndrome [email protected] caused by loss-of-function of the Zinc Finger Homeobox 3 gene (ZFHX3) P 10 Isabelle Bar Clinical, histopathological and molecular [email protected] characterization of a rare case of adult pilocytic astrocytoma with differentiation to pilomyxoid astrocytoma: case report P 11 Erika Souche Validation of whole genome sequencing [email protected] for the diagnosis of rare diseases P 12 Li-Yen Wong Investigation of the pathogenicity of [email protected] LMNA variants in human pluripotent stem cell-cardiomyocytes using CRISPR/Cas9 technology P 13 Mirjam Wamelink Transketolase (TKT) deficiency in 3 [email protected] patients with short stature, developmental delay and abnormal polyols P 14 Linda Zuurbier Diagnostic yield of Next-Generation [email protected] Sequence analysis of a 29 gene panel related to genetic dyslipidemias P 15 Machteld Oud Towards a novel diagnostic strategy [email protected] using patient-derived cells to diagnose ciliopathies P 16 Floor Postema The validation of a clinical screening [email protected] instrument for tumor predisposition syndromes in childhood cancer patients (TUPS): a prospective, observational, multi-center study
46
P 17 Hannie Kremer An in-frame deletion in RIPOR2 is an [email protected] important cause of adult-onset hearing impairment P 18 Erik-Jan Kamsteeg KIF1A variants are a frequent cause of [email protected] autosomal dominant hereditary spastic paraplegia P 19 Laura van Zutven 19q13.32 microdeletion syndrome: [email protected] further delineation of the clinical phenotype P 20 Gido Gravesteijn* NOTCH3 exon skipping as a rational [email protected] therapeutic approach for CADASIL: lessons from a family with naturally occurring exon 9 skipping P 21 Zeinab Fadaie* Identification of splice defects due to [email protected] non-canonical splice site or deep- intronic variants in ABCA4 P 22 Eva Sammels A novel missense PLP1 mutation causing [email protected] PLP1-related spastic paraplegia: a Case Report P 23 Ivo Fokkema* LOVD - supporting DNA variant [email protected] interpretation P 24 Joost Kummeling* SETD1A Loss of function causes a [email protected] distinct neurodevelopmental disorder in human and impaired learning & memory in Drosophila. P 25 Mubeen Khan* Towards comprehensive identification [email protected] and functional characterization of deep- intronic ABCA4 variants in 1000 Stargardt disease cases P 26 Mahdi Compound heterozygosity for missense [email protected] Motazacker and null variants in CEP55 lead to a novel viable phenotype P 27 Jessica Bos Clinical characteristics of patients with [email protected] an STXBP1 gene mutation: preliminary results of a cross-sectional and retrospective multilevel cohort study P 28 Vyne van der The impact of incidental findings in [email protected] Schoot* clinical exome sequencing, a qualitative interview study P 29 Shuang Li* CAPICE - a variant prioritization tool for [email protected] Consequence-Agnostic Prioritization of Clinical Exomes variations P 30 Matthias Van Gils* Targeted compound screening for PXE [email protected] using a CRISPR/Cas9 abcc6a mutant zebrafish model. P 31 Tamara Whole-exome sequencing identifies [email protected] Koopmann heterozygous stop-loss mutations in HBB resulting in an elongated β-globin chain in two children with severe anemia. P 32 Valerie A second family with homozygous [email protected] Jacquemin* mutation in KIDINS220 presenting as congenital hydrocephalus and limb contractures P 33 Rosanne Ausems* Intrinsic myogenic potential of skeletal [email protected] muscle-derived pericytes from patients with myotonic dystrophy type 1 P 34 Renée Development and Characterization of [email protected] Raaijmakers* Pericyte-Derived iPSCs and PiPs from DM1 patients 47
P 35 Marieke Klein Intellectual Disability-related genes [email protected] increase ADHD risk and locomotor activity in Drosophila melanogaster P 36 Mala Misra-Isrie An integrative approach to STXBP1- [email protected] Encephalopathy: in vitro, in vivo and patient clinics. P 37 Diantha Terlouw* The complexities of analysis for APC [email protected] mosaicism P 38 Serwet Demirdas The MAP3K7 gene: further elaboration [email protected] on phenotypes, and evidence for interaction with the RAS pathway/clinical overlap with Noonan syndrome P 39 Lianne Krab Further delineation of phenotypes [email protected] related to integral cohesion structural protein RAD21
* Eligible for Poster Award
48
P 01
Lost in translation - Experiences from a Structural Biologist
Venselaar
Radboudumc
Structural bioinformatics is of essential importance to understand the molecular effects of genetic variants. Over the last years, the CMBI at Radboudumc has been involved in many collaborative projects with (bio-)medical researchers. In these projects, the use of information obtained from 3D- structures often provided important information to understand genotype-phenotype relations and often, this information was a used as a lead for therapy or drug-development, or simply as a starting point for new experiments. We have seen an enormous rise in technological possibilities and the subsequent growth in data. In contrast, studying a protein in detail remains a laborious task that requires special skills, time, and experience. Fortunately, many tasks can be automated and we, and many others, have created automatic tools and software solutions to open up the world of protein structures to the bio-medical field. However, these tools rely on the completeness and correctness of the underlying data, which isn't always the case. Besides that, the result as presented by software isn't always clear for the user without a structural bioinformatics background, and doesn't always take the inter-human variation into account. In this lecture, we will discuss experiences, challenges and results from a day with a structural biologist.
E-mail: [email protected]
Keywords: protein structures mutations variant effect prediction
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P 02
NPC2 c.441+1G>A is a likely pathogenic variant associated with an adult-onset Alzheimer-like phenotype
Nika Schuermans1,2, Giulia Ascari1,2, Alexander Gheldof3, Céline Bellenguez4,5,6, Frans W. Verheijen7, Ariane Van Tongerloo1,2, Florence Pasquier6,8, Gaël Nicolas9, Jean-Charles Lambert4,5,6, Thorsten Marquardt10, Elfride De Baere1,2, Sandra Janssens1,2, Bart Dermaut1,2
1. Center for Medical Genetics, Ghent University Hospital, Belgium. 2. Department of Biomolecular Medicine, Faculty of Medicine and Health Sciences, Ghent University, Belgium. 3. Center for Medical Genetics, UZ Brussel, Brussels, Belgium. 4. Inserm U1167, Molecular Determinants of Aging-Related Diseases, Lille, France. 5. Institut Pasteur de Lille, Lille, France. 6. Excellence Laboratory LabEx DISTALZ, Lille, University of Lille, France. 7. Department of Clinical Genetics, Erasmus University Medical Center, Rotterdam, the Netherlands. 8. Department of Neurology, University Hospital of Lille, Lille, France. 9. Department of Genetics and CNR-MAJ, Normandie Université, UNIROUEN, Inserm U1245 and Rouen University Hospital, F 76000, Normandy Centre for Genomic and Personalized Medicine, Rouen, France. 10. Klinik für Kinder- und Jugendmedizin, Münster, Germany.
Background: Niemann-Pick type C disease (NPC) is an autosomal recessive neurodegenerative lipid storage disorder caused by mutations in the NPC1 and NPC2 genes. Due to a heterogeneous phenotype with a variety of clinical symptoms and highly variable ages of onset diagnosis is difficult. Although the clinical incidence is estimated at ~1/100.000, carrier frequencies of predicted pathogenic NPC1/2 variants in NGS-based population databases suggest a higher incidence. This could be due to an important clinical underascertainment of adult-onset neurodegenerative phenotypes (reduced or age-related penetrance) and/or an overestimation of the pathogenicity of certain variants (assessed by in silico prediction software). Here we studied the pathogenic role of NPC2 c.441+1G>A, a canonical splice-site variant with a minor allele frequency of ~1/150 and conflicting evidence of pathogenicity. Results: We identified a fetal and two adult cases homozygous for NPC2 c.441+1G>A. The 12-weeks-old fetal case was identified via chorion villus sampling in an at-risk pregnancy. After pregnancy termination filipin staining of cultured fetal fibroblasts showed increased levels of unesterified cholesterol confirming the diagnosis of NPC. Transcript analysis by means of RT-PCR revealed the presence of three aberrant transcripts, whereas no wild type transcript was present. Western Blotting showed strongly decreased NPC2 protein levels. One adult case was identified in the French ADES study, consisting of exome data from 1779 Alzheimer´s disease (AD) patients and 1273 controls. This patient was diagnosed with clinical AD at the age of 54 years, had a cerebrospinal fluid biomarker profile compatible with AD, a negative family history and was APOE ?4 negative. The other adult patient, homozygous for the NPC2 c.441+1G>A mutation, is a German female with a severe early-onset dystonia who is a also a carrier of an expanded CAG trinucleotide repeat in the ATXN3 gene, causing Machado-Joseph disease. Conclusions: Together our results suggest a likely pathogenic nature for NPC2 c.441+1G>A possibly leading to a late-onset neurodegenerative AD-like phenotype. These findings might have implications for diagnostics and management of patients with a neurodegenerative clinical presentation and the genetic counseling of carriers of this mutation.
E-mail: [email protected] Keywords: Niemann-Pick disease type C, NPC2, rare neurodegenerative diseases, next-generation sequencing, variant classification, diagnostics
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P 03
A clinical flowchart to classify cutis laxa patients, supported by electron microscopy and pathophysiology
A. Beyens1,2, H. Syryn1, L. Pottie1, R. De Rycke3, B. Fischer-Zirnsak4, T. Van Damme1, I. Hausser5, M. De Bruyne3, F. Baeke3, M. Morroni6, S. Nampoothiri7, K. Mahesh8, U. Kornak4, Z. Urban9, S. Hadj-Rabia10, C. Bodemer11, S. De Schepper2, E. C. Davis12, B. Callewaert1
1 Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium 2 Department of Dermatology, Ghent University Hospital, Ghent, Belgium 3 Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium 4 Institute of Medical Genetics and Human Genetics, Charité-Universitätsmedizin Berlin, Berlin, Germany 5 Institute of Pathology, Universitätsklinikum Heidelberg, Heidelberg, Germany 6 Department of Experimental and Clinical Medicine, Section of Neuroscience and Cell Biology, School of Medicine, Università Politecnica delle Marche and Electron Microscopy Unit, United Hospitals, Ancona, Italy 7 Department of Pediatric Genetics, Amrita Institute of Medical Sciences and Research Center, Kochi, India 8 Department of Pediatric Cardiology, School of Medicine, Kochi, India 9 Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA, United States 10 Service de Dermatologie, CHU Paris - Hôpital Necker-Enfants Malades, Paris, France 11 Centre MAGEC (Maladies rares Génétiques a Expression Cutanée), Service de Dermatologie, CHU Paris - Hôpital Necker-Enfants Malades, Paris, France 12 Department of Anatomy and Cell Biology, McGill University, Montréal, QC, Canada.
Introduction Cutis laxa (CL) syndromes are a heterogeneous group of rare, multisystem connective tissue disorders that share a loose, redundant skin as a common feature reflecting elastic fiber deficiency. The pathogenesis of each of the CL subtypes is different but affects elastogenesis. Light microscopy of the dermis is non-discriminative and the recently observed vast molecular heterogeneity mitigates the clinical validity and practicality of the current classification, based on the mode of inheritance and systemic involvement. We aim to classify the CL subtypes by means of a simple flowchart and to evaluate correlations between elastic fiber ultrastructural morphology and clinical presentation. Materials and Methods Transmission electron microscopy was used to evaluate all CL subtypes. We conducted a systematic search of all existing literature on CL patients with confirmed molecular defects in one of the known CL genes (ELN, FBLN4, FBLN5, LTBP4, SLC2A10, ATP6V0A2, ATP6V1E1, ATP6V1A, ALDH18A1, PYCR1, RIN2, GORAB and ATP7A). We established databases with the clinical data of our in-house cohort and all patients reported in literature. Results? We developed a clinical flowchart to classify the CL subtypes. As a proof of principle, we systematically evaluated 67 CL patients from our in-house database and could allocate 95% of patients to the right gene. To confirm final validation of our classification, we evaluated all 495 patients reported in literature and successfully classified more than 90% in a retrospective manner. Transmission electron microscopy in skin biopsies of all CL subtypes showed discriminative and specific findings that correlate with the main presenting symptoms (emphysema, arterial tortuosity, skeletal defects/mental disability with or without glycosylation defects/intrauterine growth retardation/cataract). Moreover, elastic fiber ultrastuctural morphology reflects the involved molecular pathogenesis and provides new insights in elastic fiber biogenesis.? Conclusions? Our novel nosology of the CL syndromes provides a practical approach to the broad differential diagnosis of CL syndromes. The classification forms a basis to integrate the clinical presentation with the pathogenesis and ultrastructural elastic fiber defects. We expect this nosology to ease the diagnostic work-up of CL patients, to be relevant for the interpretation of genotype-phenotype correlations, and to bode for new management guidelines and therapeutic approaches.
E-mail: [email protected] Keywords: cutis laxa, flowchart, classification, electron microscopy, elastic fiber 51
P 04
Genetic service for birth defects at the Dutch Caribbean Islands
E.A. Verberne, S. Faries, M. Manshande, S. Lo-a-Njoe, P. Philippi, H. Veenhuis, F. Falix, G. Ecury, I. Angelista, E. Arends, L. Rafael-Croes, W. Ponson, P. Thorsen, A. Oudshoorn, F. Croes, M. de Vroomen, P. Offringa, M. Tilanus, M.M.A.M. Mannens, M.M. van Haelst.
Amsterdam UMC, University of Amsterdam. St. Elisabeth Hospital, Curacao. Dr. Horacio E. Oduber Hospital, Aruba. Fundashon Mariadal, Bonaire. St. Maarten Medical Center, St. Maarten.
Introduction: Curacao, Aruba, Bonaire and St. Maarten are islands located in the Caribbean Sea and part of the Dutch Kingdom. Because of their small size and relative remoteness, they face several challenges, including limited access to (specialized) health care like medical genetics. We established a genetic service for pediatric patients on these Dutch Caribbean Islands. The aim of this study is to evaluate the diagnostic and clinical outcome of 7-year genetic care service for this pediatric population. Methods: Twice per year the clinical geneticist evaluates patients with birth defects at the pediatric clinic. If indicated, blood samples are send to the Netherlands for genetic testing. For this study, we retrospectively included all children (age <18 years) that had a genetic consultation between November 2011 and November 2018. We excluded children that already received a definitive diagnosis previously and healthy children that were referred for carrier testing. Because of the relatively isolated population we hypothesized that specific conditions (caused by autosomal recessive or founder mutations) could be more frequently identified amongst these children. Results: In total 479 children were included in this study. In 24% we were able to establish a definitive clinical and/or molecular diagnosis. Molecular diagnoses included autosomal dominant conditions (40%), autosomal recessive conditions (10%), microdeletion/-duplication syndromes (32%), X-linked conditions (7%), aneuploidy (6%) and other (5%). Conclusion: We present our strategy to provide genetic care in the Dutch Caribbean by establishing a local genetic service and show the results of the first 7-years. Since genetic results are still pending for part of the cohort, a final diagnosis could be higher than 24% of the children. Although the population structure of small islands could suggest a higher level of recessive conditions, we have not (yet) been able to show this for these Dutch Caribbean islands. A genetic diagnosis helps in ending the diagnostic odyssey for patients and parents and enables them to make informed reproductive choices. We suggest that this strategy with a visiting clinical geneticist or via teleconferencing can be used to provide genetic testing for birth defects in other small island developing states (SIDS).
E-mail: [email protected] Keywords: Genetic service, diagnostic strategy, birth defects, Dutch Caribbean
52
P 05
Dutch genome diagnostic laboratories improved variant interpretation quality and speed by building and implementing a datasharing platform
Ivo F.A.C. Fokkema, K. Joeri van der Velde, Mariska K. Slofstra, Claudia A.L. Ruivenkamp, Maartje J. Vogel, Rolph Pfundt, Marinus J. Blok, Ronald H. Lekanne Deprez, Quinten Waisfisz, Kristin M. Abbott, Richard J. Sinke, Rubayte Rahman, Isaäc J. Nijman, Hanneke W.M. van Deutekom, Bart de Koning, Gert Thijs, Nienke Wieskamp, Ruben J.G. Moritz, Bart Charbon, Jasper J. Saris, Johan T. den Dunnen, Jeroen F.J. Laros, Morris A. Swertz, Marielle E. van Gijn
Department of Human Genetics & Department of Clinical Genetics, Leiden University Medical Center, Leiden, the Netherlands; Genomics Coordination Center & Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands; Department of Clinical Genetics, Erasmus Medical Center, Rotterdam, the Netherlands; Department of Pathology, Netherlands Cancer Institute, Amsterdam, the Netherlands; Department of Human Genetics, Radboud University Medical Center, Nijmegen, the Netherlands; Department of Clinical Genetics, Maastricht University Medical Center+, Maastricht, the Netherlands;Department of Clinical Genetics, Academic Medical Center & Department of Clinical Genetics, Vrije Universiteit Amsterdam, Amsterdam UMC, Amsterdam, the Netherlands; DGG Genomics Software Solutions, Agilent Technologies, Leuven, Belgium; Department of Genetics, University Medical Center Utrecht, Utrecht, the Netherlands
Each year diagnostic laboratories in the Netherlands test thousands of individuals for heritable disease using next generation sequencing (NGS). This requires pathogenicity classification of millions of DNA variants on the standard 5-tier scale. To reduce time spent on data interpretation and increase data quality and reliability, the nine Dutch labs decided to publicly share their classifications. Variant classifications of nearly 145,000 unique variants were catalogued and compared in a centralized MOLGENIS database. Variants classified by more than one center are labeled as "consensus" when classifications agree, and shared internationally with LOVD and ClinVar. When classifications oppose (LB/B versus LP/P), they are labeled "conflicting", while other non-consensus observations are labeled "no consensus". We assessed our classifications using InterVar to compare to ACMG 2015 guidelines, showing a large overall consistency with only 0.3% discrepancies. Differences in classifications between Dutch labs or between Dutch labs and ACMG were mainly present in genes with low penetrance or for late onset disorders and highlight limitations of the current 5-tier classification system. To address these differences and diminish confusion a clinical arm could be added to the current classification system, to complement the ACMG system. Such a system is currently being developed in an ESHG initiative. The data sharing was implemented successfully in the laboratories in their daily practice and evaluation showed it boosted the quality of DNA diagnostics in Dutch labs. Publically sharing the data resulted a disease diagnosis in at least one positive match with a case from outside our consortium. This initiative proves that data sharing is warranted for good clinical practice in genome diagnostics
E-mail: [email protected] Keywords: Data sharing, variant pathogenicity classification, VKGL, ACMG classification system
53
P 06
Next-generation cytogenetics: high-resolution optical mapping to replace FISH, karyotyping and CNA-microarrays
Marian Stevens-Kroef1*; Tuomo Mantere1*; Kornelia Neveling1*; Marian Stevens-Kroef1*; Daniel Olde Weghuis1; Marc Pauper1, Guillaume van de Zande1, Susan Vermeulen1, Ellen Kater-Baats1, Michiel Oorsprong1, Ronald van Beek1, Dominique Smeets1; Alex Hastie2; Alexander Hoischen 1,3,4 * Equal contribution
1 Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands. 2 Bionano Genomics, San Diego, California, USA 3 Radboud Institute of Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands. 4 Department of Internal Medicine and Radboud Center for Infectious Diseases (RCI), Radboud University Medical Center, Nijmegen, The Netherlands.
Structural variants (SVs) are an important source of genetic variation in the human genome and they are involved in a multitude of human diseases. Somatic SVs are important for cancer development and progression while constitutional SVs are involved in many developmental disorders. In a diagnostic set-up for leukemia samples, comprehensive analysis of all cytogenetic aberrations in a given sample still requires a combination of techniques, such as CNA-microarrays, karyotyping and fluorescence in situ hybridization. We hypothesize that the combination of classical approaches could be largely replaced by high-resolution optical mapping. Leukemia bone marrow aspirates and blood samples were analyzed using standard of care workflow and the residual samples were processed for Bionano Saphyr optical mapping to detect chromosomal aberrations. We generated up to 400-fold genome coverage by imaging long chromosomal fragments and detected chromosomal aberrations by comparing optical maps to a reference and control dataset. In the first leukemia samples we compared the results with those obtained using standard methods. In each sample, all previously known clinically relevant aberrations were identified. This held true for deletions, insertions, inversions and translocations, including a 3-way Philadelphia chromosome (t(9;22;14)(q34;q11;q11)) and even chromothripsis structures were resolved. We were also able to identify aberrations in samples with cancer cell content less than 40% using Bionano´s latest single- molecule SV detection tool. Importantly, optical mapping additionally identified multiple novel events e.g. an inversion of chromosome 11 (chr11:24,875,044-26,299,641) and a translocation (t(5;14)(q35.2;q32.2)), both validated afterwards. These novel events are now followed-up to identify possible fusion genes as novel leukemia drivers. We are now conducting a study to systematically compare the sensitivity and specificity of optical mapping in 100 leukemia samples and 50 samples with known germline cytogenetic aberrations against the standard of care workflow. Of these, 17 with constitutional aberrations and 19 leukemia samples have been processed successfully so far. Optical mapping may have the potential to replace most classical cytogenetic tests. In addition this holds the potential to map new fusion genes as novel leukemia drivers rapidly.
E-mail: [email protected] Keywords: high-resolution optical mapping, FISH, karyotyping, CNA-microarrays.
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P 07
PREFACE: in silico pipeline for accurate cell-free fetal DNA fraction prediction
Raman Lennart, Baetens Machteld, De Smet Matthias, Dheedene Annelies, Van Dorpe Jo, Menten Björn
1 Department of Pathology, Ghent University, Ghent University Hospital, Ghent, Belgium. 2 Center for Medical Genetics, Department for Biomolecular Medicine, Ghent University, Ghent University Hospital, Ghent, Belgium.
OBJECTIVE: During routine non-invasive prenatal testing (NIPT), cell-free fetal DNA fraction is ideally derived from shallow-depth whole-genome sequencing data, preventing the need for additional experimental assays. The fraction of aligned reads to chromosome Y enables proper quantification for male fetuses, unlike for females, where advanced predictive procedures are required. This study introduces PREFACE (PREdict FetAl ComponEnt), a novel bioinformatics pipeline to establish fetal fraction in a gender-independent manner. METHODS: PREFACE combines the strengths of principal component analysis and neural networks to model copy number profiles. RESULTS: For sets of roughly 1100 male NIPT samples, a cross-validated Pearson correlation of 0.9 between predictions and fetal fractions according to Y chromosomal read counts was noted. PREFACE enables training with both male and unlabeled female fetuses. Using our complete cohort (nfemale =2468, nmale =2723), the correlation metric reached 0.94. CONCLUSIONS: Allowing individual institutions to generate optimized models sidelines between-laboratory bias, as PREFACE enables user-friendly training with a limited amount of retrospective data. In addition, our software provides the fetal fraction based on the copy number state of chromosome X. We show that these measures can predict mixed multiple pregnancies, sex chromosomal aneuploidies and the source of observed aberrations.
E-mail: [email protected]
55
P 08
DTYMK deficiency is the cause of a severe congenital neurodegenerative disease
Vanoevelen JM1,2*, Bierau J1*, Kamsteeg EJ3, Wevers RA4, Bok LA5, Abdel-Salam GMH6, van der Knaap M7, Bugiani M8, O´Driscoll M9, van den Wijngaard A1, Ferreira CR10, Brunner HG1, Wang L11, Stumpel CTRM1,2
1.Department of Clinical Genetics, Maastricht University Medical Centre+, Maastricht, The Netherlands; 2. GROW - School for Oncology and Developmental Biology, Maastricht, The Netherlands; 3. Department of Human Genetics, Radboud UMC, Nijmegen, The Netherlands; 4. Translational Metabolic Laboratory, Radboud UMC, Nijmegen, The Netherlands; 5. Department of Pediatrics, Màxima Medical Center, Veldhoven, The Netherlands; 6. Department of Clinical Genetics, Human Genetics and Genome Research Division, National Research Centre, Cairo, Egypt; 7. Department of Child Neurology, Amsterdam UMC, Amsterdam, The Netherlands; 8. Department of Neuropathology, Amsterdam UMC, Amsterdam, The Netherlands; 9. Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom; 10. National Human Genome Research Institute, National Institutes of Health, Bethesda, USA; 11. Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden; * shared first authors.
Human nucleotide metabolism is a complex, tightly controlled pathway regulating numerous cellular processes such as nucleic acid synthesis and repair, cellular proliferation and differentiation. In this study we describe DTYMK (dTMPK) deficiency as the cause of a severe neurodegenerative disease in two unrelated families. DTYMK encodes the dTMPK (deoxythymidylate monophosphate kinase) enzyme which catalyzes the penultimate step in the biosynthesis pathway of dTTP. We describe two unrelated children showing severe postnatal microcephaly and postnatal growth retardation. Fascinatingly, in both cases postnatal neurodevelopment was minimal. Imaging of the brain revealed extensive atrophy of the cerebral cortex with secondary white matter loss and atrophy of the basal ganglia. Two predicted pathogenic variants in DTYMK were identified; compound heterozygous variants in one patient and one of the former variants in a homozygous state in the other patient. No significant dTMPK enzyme activity could be detected in the patients´ fibroblasts, indicating a loss-of-function effect of the variants. Additionally, EdU labelling in fibroblasts confirmed a marked proliferation defect in these cells. To further substantiate a causal relationship between DTYMK disruption and the patient´s phenotype, we generated a dtymk loss- of-function allele in zebrafish. Homozygous dtymk mutant zebrafish are not viable beyond 5dpf (days post fertilization) and show microcephaly, small eyes, developmental delay, cardiac edema and prominent edema of the brain. Biochemical analysis of dTMPK activity in mutant zebrafish larvae confirmed that the allele represents a loss-of-function allele leading to undetectable enzyme activity. Furthermore, impairment of proliferation was also detected in the brain of mutant zebrafish larvae, as well as genome instability due to ribonucleotide incorporation and defects in the DNA- damage response repair mechanism. The striking similarities between the human and zebrafish phenotype strongly suggest a causal link between dTMPK deficiency and the neurodegenerative phenotype, observed in both patients. In summary, by combining genetic and biochemical approaches in a cellular and a zebrafish model we identified loss-of-function in DTYMK as the cause of a severe neurodegenerative disease. These cases highlight the importance of dTTP synthesis in the survival of neurons.
E-mail: [email protected] Keywords: Brain atrophy Nucleotide metabolism zebrafish
56
P 09
A novel neurodevelopmental syndrome caused by loss-of- function of the Zinc Finger Homeobox 3 gene (ZFHX3)
Eva Jacobs (1), Sarah Vergult (1), Maria Palomares Braro (2), Sixto Garcia-Minaur (2), Santos Simarro Fernando (2), Tina Duelund Hjortshøj (3), Marion Gérard (4), Arnaud Molin (4) , Pablo Villavicencio- Lorini (5), Jorgen Köhlhase (5), Katrin Hoffmann (5), Mareike Bauer (5), Hilde Peeters (6), Peter Aerssens (7), Nicole Revencu (8), Philippe Lysy (8), Pernille Torring (9), Kristina Sorensen (9), Carlo Marcelies (10), Lot Snijders Blok (10), Annelies Dheedene (1), Björn Menten (1), Bert Callewaert (1)
(1) Ghent University Hospital, Ghent, Belgium, (2) Hospital Universitario La Paz, Madrid, Spain, (3) Rigshospitalet, Glostrup, Danemark, (4) Centre Hospitalier Universitaire de Caen, Caen, France, (5) Universitätsklinikum Halle, Halle, Germany, (6) University Hospital Leuven, Leuven, Belgium, (7) Virga-Jesse Hospital, Hasselt, Belgium, (8) Cliniques Universitaires Saint- Luc, Brussels, Belgium, (9) Odense University Hospital, Odense, Denmark, (10) Radboud University Medical center, Nijmegen, the Netherlands
We describe the neurodevelopmental phenotype caused by de novo loss-of-function mutations in the Zinc Finger Homeobox 3 gene (ZFHX3). In an international collaborative effort, using the Decipher and Genematcher databases, we identified de novo 16q22.2 microdeletions in 6 patients with a minimal region of overlap only containing the ZFHX3 gene. Three additional patients harbored a de novo premature truncating mutation in this gene. All patients show a similar phenotype, namely intellectual disability and craniofacial malformations. Next to this, ZFHX3 has a pLI score of 1, which indicates this gene is intolerant for loss-of-function mutations. Therefore, we hypothesize that ZFHX3 is the causal gene for the observed syndromal intellectual disability. ZFHX3 belongs to the family of zinc-finger homeodomain transcription factors and encodes the ATBF1 protein. This transcription factor plays a role in multiple biological processes including tumorigenesis and cell differentiation. Expression of ZFHX3 increases during development of the human cortex and in zebrafish, expression of zfhx3 peaks three days after fertilization. In this period, the process of brain development takes place in zebrafish. In addition, localization of ZFHX3 to the nucleus has been shown to induce cell cycle arrest associated with neuronal differentiation. These data support a role for ZFHX3 in neural differentiation. So far, no human phenotype has been correlated with molecular defects in ZFHX3. Functional assays to determine how loss-of-function of ZFHX3 give rise to syndromal intellectual disability are ongoing.
E-mail: [email protected] Keywords: neurodevelopmental disorder
57
P 10
Clinical, histopathological and molecular characterization of a rare case of adult pilocytic astrocytoma with differentiation to pilomyxoid astrocytoma: case report
I Bar1, A Merhi 2, S Toffoli 3, C DeRop 3 , C Gilliard 4, O Fouard 4, JN Comps 4, M Di Santo 4, T Gustin 4, P Delree 5
1 Laboratory of Translational Oncology, Institute of Pathology and Genetics/ Grand Hôpital de Charleroi, Gosselies, Belgium. 2 IPG BioBank, Institute of Pathology and Genetics, Gosselies, Belgium. 3 Center for Human Genetics, Institute of Pathology and Genetics, Gosselies, Belgium. 4 Department of Neurosurgery, CHU UCL Namur, Yvoir, Belgium 5 Department of Anatomopathology, Institute of Pathology and Genetics, Gosselies, Belgium
Pilocytic astrocytomas (PAs) are the most common pediatric brain tumors. They are grade I tumors with a good prognosis following surgical resection. Hereby, we describe a rare example of PA in adult showing a rapid regrowth within 6 months with differentiation to grade III pilomyxoid astrocytoma (PMA). A 32 years old man was diagnosed with a cystic tumor in the right temporal region. Histological examination of the tumor was consistent with the diagnosis of pilocytic astrocytoma (PA). The proliferation index was low, with positive staining for GFAP, Vimentin and S100. Numerous eosinophilic granular bodies and Rosenthal fibers were observed. The tumor was resected by surgery. Six months later, the patient was diagnosed with recurrence of the primary PA into a grade III pilomyxoid astrocytoma (PMA). Rosenthal fibers were absent. The Ki-67 index and mitotic index were strongly increased. Apoptosis and necrosis areas were present. Comparative genomic hybridization was performed on both tumor samples. Monosomy 1p and trisomy 19 p were detected in the primary tumor. The second lesion revealed a more complex genomic profile, with genomic instability evidenced by an increased number of large copy number aberrations: chromosomes 2, 4, 5, 10, 11, 13 and 22 monosomy, co-deletion 1p/19q, 19p trisomy and 9p21 homozygous deletion (involving both CDKN2A and CDKN2B). Targeted sequencing of 409 oncogenes and tumor suppressor genes was performed on both tumors and blood leucocytes DNA (Ion AmpliSeq™ Comprehensive Cancer Panel). Both primary and secondary tumors had comparable mutation profiles, with no new somatic mutations in the grade III PMA compared to grade I PA. Somatic mutations in exon 4 of IDH1 and IDH2 were not detected, but a somatic point mutation in BRAF ( p.(V600E) – c.1799T>A – NM_004333.5) was present both in primary and secondary tumor. This was confirmed by BRAF V600E immunostaining. Methylation of MGMT promoter was also detected. Conclusion: In the present case, tumor progression seems to be mediated by chromosomal instability rather than acquisition of new somatic mutations. Very few studies have carefully analyzed the molecular parameters leading to the transformation of a low grade to a high grade pilocytic astrocytoma in adult. It is important to accumulate data to improve our understanding of the biology of these tumors, which could help to orientate therapeutics options.
E-mail: [email protected] Keywords: Pilocytic astrocytoma, pilomyxoid astrocytoma, adult patient, histopathology, molecular characterization
58
P 11
Validation of whole genome sequencing for the diagnosis of rare diseases
Erika Souche, Luc Dehaspe, Kris Van Den Bogaert, Marijke Bauters, Valérie Race, Anniek Corveleyn, Anneleen Boogaerts, Jens Van Bouwel, Stephanie Deman, Céline Helsmoortel, Wouter Bossuyt, Gert Matthijs, Joris Vermeesch
Center for Human Genetics
Whole Genome Sequencing (WGS) is quickly becoming the standard of care to diagnose rare hereditary diseases. In our project we aimed to implement WGS in a diagnostic setting because it promises to deliver more information than the current standard of care, namely, a 2-step approach of arrayCGH and whole exome sequencing (WES). Although these approaches are effective, there is still room for improvement. Indeed, copy neutral rearrangements cannot be detected by arrayCGH. Structural variants such as inversions and translocations are difficult to detect using WES as the breakpoints might not be exonic. Also, WES suffers from capture and PCR biases and some variants might be missed. WGS allows the detection of CNVs, complex structural variants but also single nucleotide variants in a single test. Its implementation in a diagnostic setting will homogenize the wet lab workflow, offer a single test for all conditions, and ultimately decrease cost and time to diagnosis. However, the capability of WGS to replace arrayCGH and WES has to be proven before moving forward. Well characterized cell lines have been sequenced on a NovaSeq (PE150) after shearing and library preparation according to KAPA Hyper PCR free protocol. WGS data has been compared to both arrayCGH and Platinum and Genome in a Bottle calls. Additionally, clinical exome data has been compared to WGS data for some real cases.
E-mail: [email protected] Keywords: Whole genome sequencing diagnostics
59
P 12
Investigation of the pathogenicity of LMNA variants in human pluripotent stem cell-cardiomyocytes using CRISPR/Cas9 technology
Wong, L.Y., van Dijk, W., Veltrop, R., Akbulut, C., van Tienen, F., Antoons, G., Nagy, M., Manders, E., Helmes, M., Broers, J., Schurgers, L., Heemskerk, J., Glatz, J., van den Wijngaard, A., Brunner, H.G.
Department of Clinical Genetics, MUMC+, Maastricht University, Maastricht, Netherlands Department of Genetics and Cell Biology, Maastricht University, Maastricht, Netherlands Department of Biochemistry, CARIM, Maastricht University, Maastricht, Netherlands Department of Physiology, CARIM, Maastricht University, Maastricht, Netherlands Cytocypher, VUMC, Amsterdam, Netherlands Department of Human Genetics, RadboudUMC, Nijmegen, Netherlands
Dilated cardiomyopathy (DCM), which is characterized by dilation of left- or both ventricles and systolic dysfunction, comprises many hospitalized cardiomyopathy cases and has major impact on public health. About 48% of DCM cases are familial, with over 50 DCM genes identified to date. Among them, mutations in LMNA gene (encoding nuclear protein Lamin A/C) account for 5-8% of families with familial DCM. Modern sequencing technologies, e.g. whole-exome-sequencing (WES), has identified numerous genetic variants in LMNA gene, however the pathogenicity of these variants and the molecular mechanisms leading to DCM remain unknown. This project aims to utilize induced pluripotent stem cells (iPSC) as a model system (i) to phenotypically characterize LMNA mutations causing DCM, and (ii) to design novel genome editing strategies to recapitulate the disease phenotype by applying the state-of-the-art CRISPR/Cas9 technology. This allows the interrogation of genetic mutations to decipher the genotype-phenotype relationship in a more precise and measurable manner. CRISPR/Cas9 genome editing of healthy wild-type IPSCs were performed to create mutant isogenic IPSC lines by introducing an LMNA variant identified in DCM patients in order to recapitulate the disease phenotype in comparison with patient-derived IPSCs. CRISPR-edited and patient-specific IPSCs, together with the relevant control IPSCs were subsequently differentiated to cardiomyocytes over a period of 2 weeks under feeder-free conditions. Metabolic selection of IPSC-CMs were performed to yield a high population of purified IPSC-CMs. Phenotypic and functional analyses such as gene expression, immunocytochemistry, contractility, and calcium handling will be assessed to distinguish the similarities/differences between patient-derived IPSCs and CRISPR-edited IPSCs, as compared to wild-type IPSCs. The powerful combination of IPSC-based disease modeling and CRISPR-Cas9 genome editing will provide insight into the contribution of these LMNA genetic variants to the pathogenesis of DCM and to study VUS functional phenotypes in a patient-specific manner. These findings can then be used for early disease detection, diagnosis and clinical management of patients, leading to an improvement in patient healthcare. This platform represents a promising tool for assessing DCM- associated VUS specifically and can significantly contribute to precision medicine for the study of genetic diseases in the near future.
E-mail: [email protected] Keywords: Induced pluripotent stem cells, dilated cardiomyopathy, CRISPR-Cas9 genome editing, cardiomyocyte differentiation
60
P 13
Transketolase (TKT) deficiency in 3 patients with short stature, developmental delay and abnormal polyols
Wamelink MMC1, Charles P2,3, Keren B3, Faudet A2, Buratti J3 Roos, B1, Jansen EEW1, Hay E4, Faravelli, F4
1VUmc, Dept of Clin Chem, Amsterdam, The Netherlands; 2 dept. of Neurology, Hôpital Pitié-Salpêtrière, APHP, Paris, France 3 Dept. Clinical Genetics,, Hôpital Pitié-Salpêtrière, APHP , Paris, France, 4 Clinical Genetics, Great Ormond Street, London, United Kingdom
BACKGROUND: Transketolase (TKT) deficiency is a rare autosomal recessive defect in the pentose phosphate pathway (PPP), previously only reported in 5 patients from 3 families. We describe 3 additional patients. CASE REPORT: WES/WGS identified biallelic variants in TKT in 3 females from 2 families, ranging in age from 5 to 32 years. Polyols in urine and plasma and sugar-P in urine were measured to confirm the pathogenicity. RESULTS: Patient 1, female 5 years old present significant short stature, microcephaly, severe developmental delay and bilateral frontal lobe polymicrogyria. Patient 2 and 3, sisters of 32 and 21 years old, present short stature (-2DS and -4DS) with growth hormone treatment for the oldest, microcephaly, developmental delay, behaviour and character disorders intellectual disability and morphological features of the face. The youngest presented tetralogy of fallot with surgical intervention at 8 months and developed bilateral cataract at 21 years with surgical treatment. The oldest presented dextrocardia and pyramidal signs. Three novel missense variants in TKT were identified: Patient 1: homozygous for c.362C>T, p.Ala121Val, patient 2 and 3: compound heterozygous for c.1421G>A; p.Arg474His and c.964G>A; p.Gly322Arg. In all 3 elevated urine excretion of erythritol, arabitol, ribitol, erythrose-4-P and pent(ul)ose-5-P and elevated plasma concentrations of erythritol, arabitol and ribitol, confirmed TKT deficiency. DISCUSSION: TKT deficiency is now diagnosed in 8 patients (7 female and 1 male). All patients were small for gestational age, had short stature, and had varying degrees of developmental delay and morphological features of the face. Congenital heart defects and juvenile cataract are common. Erythrose-4-P was elevated, indicating that there might be an alternative route. Our findings provide further support for the biochemical and phenotypic features reported in TKT deficiency. Given the lack of specific or distinctive diagnostic features in early years, we propose polyol testing in patients fitting this clinical phenotype.
E-mail: [email protected] Keywords: Transketolase, pentose phosphate pathway, polyols, TKT, short stature
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Diagnostic yield of Next-Generation Sequence analysis of a 29 gene panel related to genetic dyslipidemias
Linda C. Zuurbier, PhD, Olaf R.F. Mook, PhD, Martin A. Haagmans, Marcel M.A.M. Mannens, PhD, Joep. C. Defesche, PhD
Introduction: Genetic dyslipidemias, specifically those affecting the metabolism of low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and triglycerides, are extremely common in the general population and are strongly associated with premature atherosclerosis, presenting as myocardial infarctions and sudden death at young age. Because of this abundance, there is a need for rapid, reliable and comprehensive diagnostic tools. Advances in sequencing technology, such as Next-Generation Sequencing (NGS), and bioinformatics have made it possible to analyze large numbers of genes in many patients simultaneously. Method: On more than 4000 consecutive patients that were referred for confirmation of the clinical diagnosis of a genetic dyslipidemic disorder, NGS analysis of a dyslipidemia-associated 29-gene panel was applied. Results: Analysis of these data revealed that the clinical diagnosis frequently differed from the genetic diagnosis. For instance, one third of patients that was clinically diagnosed with hypercholesterolemia had a pathogenic variant that was causal for hypertriglyceridemia. On the other hand, compared to the conventional single gene sequencing approach, NGS supplied additional valuable results, as in more than 10% of cases a second or third pathogenic or pharmacogenetic variant was identified that was relevant for clinical decision making. Furthermore, by elaborating in more detail on pharmacogenetic variants, a statin intolerance variant was observed much more frequently than published in literature. Also other SLCO1B1 variants were identified that affected therapeutic response. Conclusion: For the detection of genetic dyslipidemias, NGS is a rapid and very efficient diagnostic tool that increases the yield and quality of diagnosis, compared to conventional Sanger sequencing. In this particular setting, the unsolicited findings not only explicate the clinical diagnosis, but also significantly optimize the medical management of genetic dyslipidemias.
E-mail: [email protected]
Keywords: dyslipidemia, hypercholesterolemia, cardiovascular, pharmacogenetics
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P 15
Towards a novel diagnostic strategy using patient-derived cells to diagnose ciliopathies
Machteld Oud1,2, Ronald van Beek1,2, Dorien Lugtenberg1, Ernie Bongers1, Ronald Roepman1, Helger Yntema1, Lisenka Vissers1,2
1Department of Human Genetics, Radboud University Medical Centre, Nijmegen, The Netherlands 2Donders Institute for Brain, Cognition and Behavior, Radboud University Medical Centre, Nijmegen, The Netherlands
Ciliopathies are rare recessive multiorgan disorders that are caused by dysfunction of the cilium. Cilia are small signaling organelles protruding from the cell membrane of nearly every vertebrate cell. Diagnostic exome analysis (WES) for ciliopathies leads to an inconclusive molecular diagnosis in about 30% due to the detection of variants of unknown significance. Functional tests evaluating the effect of genetic variants on cilium function could elucidate their pathogenicity. Our aim is therefore to develop a novel diagnostic strategy to improve accurate diagnosis of ciliopathies. We obtained skin-fibroblasts from 21 unrelated ciliopathy patients and controls, and cultured them in vitro. Immunocytochemistry techniques were applied to determine cilium phenotype parameters, including 1) ciliogenesis, based on presence or absence of a base and an axonemal marker, 2) cilium length, by measurement of the combined signal of an axonemal and a cilium membrane marker, and 3) intraflagellar transport (IFT), measured by the area of the ciliary tip based on the IFT88 signal. All experiments were performed in triplicate. We collected cilium phenotypic data from skin- fibroblasts of 21 (suspected) ciliopathy patients and 8 controls. The data of the eight control fibroblasts yielded comparable and reproducible results: >85% cells were ciliated, the cilium length was 2.97±0.381µm, and the IFT88 ciliary tip area was 0.14±0.03µm2. Significant differences in cilium length and IFT results were identified in nine short-rib thoracic dysplasia (SRTD) ciliopathy patients when compared to the controls. Interestingly, the data of the different subgroups within the SRTD patient cohort clustered together and were clearly distinguishable from the other clinical phenotypes and controls. The three parameters tested did however not result in identifiable clusters for the remaining seven ciliopathy and five suspected ciliopathy patients. In summary, we show that a standardized analysis of the cilium phenotype can lead to identifiable clusters for SRTD ciliopathies and possibly also for other ciliopathies. Based on these results, we believe that standardized tests based on cilium phenotypic data can provide an additional piece of the puzzle for conclusive diagnosis of ciliopathies, essential in routine diagnostic care.
E-mail: [email protected] Keywords: Ciliopathy, diagnostics, functional test
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P 16
***WITHROWN*** The validation of a clinical screening instrument for tumor predisposition syndromes in childhood cancer patients (TUPS): a prospective, observational, multi- center study
Floor A.M. Postema1,2, Saskia M.J. Hopman3, Corianne A.J.M. de Borgie4, Cora M. Aalfs3, Jakob K. Anninga2, Lieke P.V. Berger5, Charlotte J. Dommering6, Natasha K.A. van Eijkelenburg2, Peter Hammond7, Marry M. van den Heuvel-Eibrink2, Wijnanda A. Kors2, Tom G.W. Letteboer3, Jan L.C.M. Loeffen2, Lisethe Meijer2, Maran J.W. Olderode-Berends5, Anja Wagner8, Marjolijn C.J. Jongmans3, Raoul C. Hennekam1, Johannes H.M. Merks2
1. Department of Pediatrics, Emma Children´s Hospital, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands 2. Princess Máxima Center for Pediatric oncology, Utrecht, The Netherlands 3. Department of Genetics, University Medical Center Utrecht, Utrecht, The Netherlands 4. Clinical Research Unit, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands 5. Department of Genetics, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands 6. Department of Clinical Genetics, Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands 7. Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Oxford, United Kingdom 8. Department of Clinical Genetics, Erasmus Medical Center, Rotterdam, The Netherlands
BACKGROUND Up to 10% of children with cancer has a tumor predisposition syndrome (TPS). Identification of a TPS in a child with cancer is of clinical relevance. Previous studies have shown that half of TPSs are not recognized prior to treatment. To increase diagnostic accuracy and clinical efficiency we developed a screening instrument, ensuring evaluation for a TPS in all children with cancer. METHODS TuPS is a prospective, observational, multi-center study including children newly diagnosed with cancer in the Netherlands from 2016-2019 in whom no TPS was previously diagnosed. The screening instrument consists of a clinical assessment, 2D and 3D pictures, and integrated digital assessment by two independent clinical geneticists. If a TPS was suspected by at least one clinical geneticist (positive assessment), the patient was referred to previously uninvolved clinical geneticists for genetic counseling. In addition negative assessed patients were randomized for genetic consultation as control group. Primary outcome is the clinical value and acceptance of the instrument. PRELIMINARY RESULTS Results of 315 patients show a positive assessment rate of 57% (180/315) (25% by one assessor, 32% by both assessors). The reason for positive assessment was based mainly on type of cancer (54%) and family history (38%). In 17% (30/180) the clinical geneticist would not have assessed the child positive if pictures would not have been available. Hundred-seventy-one patients were evaluated by genetic consultation thus far. In the positive assessed group three (3/145) patients were diagnosed with a TPS and in four (4/145) patients a germline variant of non-conclusive significance was found. In 11 patients further analysis is still ongoing. Out of the 135 negative assessed patients, 26 patients were referred as a negative control group in which no TPS was diagnosed. CONCLUSION Preliminary results indicate the screening instrument seems feasible in routine care. No false negative results were identified in the negative control group, indicating that negative assessments by the screening instrument were justified.
E-mail: [email protected] Keywords: Pediatric cancer, morphology, dysmorphology, 3D photography, screening instrument, tumor predisposition syndrome
64
P 17
An in-frame deletion in RIPOR2 is an important cause of adult-onset hearing impairment
Suzanne E. de Bruijn,1,2,6 Jeroen J. Smits, 2,3,6 Chang Liu,4 Cornelis P. Lanting,3 Andy J. Beynon,3 Joëlle Blankevoort,1 Jaap Oostrik,2,3 Wouter Koole,1 Erik de Vrieze,2,3 DOOFNL Consortium, Cor W.R.J. Cremers,3 Frans P. M. Cremers,1,2 Susanne Roosing,1,2 Helger G. Yntema,1,2 Henricus P.M. Kunst,3,5 Bo Zhao,4 Ronald J.E. Pennings,2,3,7 Hannie Kremer1,2,3,7,*
1Department of Human Genetics, Radboud University Medical Center, 6500 HB, Nijmegen, The Netherlands; 2Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, 6500 HB, Nijmegen, The Netherlands; 3Hearing & Genes, Department of Otorhinolaryngology, Radboud University Medical Center, 6500 HB, Nijmegen, The Netherlands; 4Department of Otolaryngology-Head and Neck Surgery, Indiana University School of Medicine, IN 46202, Indianapolis, United States of America; 5Radboud Institute for Health Sciences, Radboud University Medical Center, 6500 HB, Nijmegen, 6,7 These authors contributed equally to this work
An in-frame deletion (c.1696_1707del) in RIPOR2 was identified to underlie dominantly inherited nonsyndromic hearing impairment (DFNA21) in twelve families of Dutch origin. The age of onset of the hearing impairment was in adulthood in the majority of the cases but ranged from childhood to 70 years. Also the configuration of audiograms displayed a high variability. Ex vivo experiments confirmed a functional effect of the variant as it resulted in aberrant localization of the mutant RIPOR2 protein throughout the stereocilia of mechanosensory hair cells whereas the wildtype protein was concentrated at the stereocilia base. Moreover, mutant RIPOR2 could not rescue the morphological defects observed in RIPOR2-deficient hair cells, in contrast to the wildtype protein. The variant is indicated to be a founder mutation and is present in one per 1,275 individuals of a cohort from the South-East of the Netherlands. This suggests that the deletion is the most important cause of monogenic hearing impairment in the Netherlands, with potentially ~8,600 affected individuals, and also a significant cause of hearing impairment in neighboring Western European countries. The penetrance of the condition in the studied families is >90%. As this is biased because of inclusion in the study of hearing impaired index cases, overall penetrance needs to be further addressed. For adult-onset nonsyndromic hearing impairment the diagnostic yield of genetic testing is low and decreases with an increasing onset age. RIPOR2 was already described to be associated with recessive congenital hearing impairment in a single family. Our study demonstrates that certain apparently 'mild´ variants in genes associated with recessive early-onset hearing impairment can be an important cause of (late) adult-onset hearing impairment.
E-mail: [email protected]
Keywords: Hearing loss, dominant inheritance, adult-onset
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KIF1A variants are a frequent cause of autosomal dominant hereditary spastic paraplegia
E. Kamsteeg1, M. Pennings1, M. Schouten1, R. Meyer1, J. van Gaalen1, S. T. de Bot2, M. Kriek2, C. G. J. Saris1, L. H. van den Berg3, M. A. van Es3, D. M. H. Zuidgeest4, M. W. Elting5, J. M. van de Kamp5, K. Y. van Spaendonck-Zwarts5, C. de Die-Smulders6, E. H. Brilstra3, C. C. Verschuuren7, B. B. A. de Vries1, J. Bruijn8, K. Sofou9, B. P. van de Warrenburg1;
1Radboud University Medical Centre, Nijmegen, Netherlands, 2Leiden University Medical Center, Leiden, Netherlands, 3Utrecht University Medical Centre, Utrecht, Netherlands, 4Ikazia Hospital, Rotterdam, Netherlands, 5Amsterdam University Medical Centre, Amsterdam, Netherlands, 6Maastricht University Medical Centre, Maastricht, Netherlands, 7Groningen University Medical Centre, Groningen, Netherlands, 8Skaraborg Hospital, Skövde, Sweden, 9The Queen Silvia Children's Hospital, Gotenborg, Sweden.
Background: Pathogenic variants in the KIF1A gene have been reported in autosomal recessive spastic paraplegia 30, autosomal recessive hereditary sensory neuropathy, and autosomal (de novo) dominant mental retardation type 9 . More recently, variants in KIF1A have also been described in a few cases with autosomal dominant spastic paraplegia. Methods: Clinical exome sequencing data of 315 unrelated patients with mostly 'pure´ spastic paraplegia were analysed for KIF1A variants. Clinical characteristics of patients with KIF1A variants were analysed, segregation analyses were performed, and types of variants were compared. Results: In 21 patients we identified 19 different KIF1A variants. Patients and affected family members showed a slowly progressive, mostly pure form of spastic paraplegia, but with a highly variable disease onset (0-57 years). Segregation analyses showed a de novo occurrence in five cases, and a dominant inheritance pattern in eight families. The motor domain of KIF1A seems to be the hotspot for pathogenic variants in autosomal dominant spastic paraplegia, similar to mental retardation type 9 and recessive spastic paraplegia type 30. Unlike these allelic disorders, however, dominant spastic paraplegia was also caused by loss-of-function variants outside this domain in six families. Three missense variants were identified outside the motor domain and need further characterization. Conclusions: KIF1A variants are a frequent cause of autosomal dominant spastic paraplegia in our cohort (5-7%), with a high de novo rate. The identification of KIF1A loss-of-function variants suggests haploinsufficiency as a possible mechanism in autosomal dominant spastic paraplegia.
E-mail: [email protected]
Keywords: spastic paraparesis loss-of-function kinesin
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19q13.32 microdeletion syndrome: further delineation of the clinical phenotype
L.J.C.M. van Zutven1, C.M.A. van Ravenswaaij-Arts2, T. Dijkhuizen2, M. McGregor-Schuerman3, L.H. Hoefsloot1, J.J. Saris1, Y. van Bever1, M.I. Srebniak1
1 Department of Clinical Genetics, Erasmus MC, Rotterdam, The Netherlands, 2 Department of Clinical Genetics, UMCG, Groningen, The Netherlands, 3 Department of Pediatrics, RKZ St. Vincentius, Paramaribo, Suriname
Background Interstitial 19q13.32 microdeletions are rare and have been reported in only five patients so far. Common features mentioned in the literature include intellectual disability/developmental delay, facial asymmetry, ptosis, oculomotor paralysis, orofacial clefting, micrognathia, kyphoscoliosis, cardiac abnormalities and constipation,. Since only a few patients have been reported, little is known about the phenotypic spectrum of these deletions. Methods To expand the knowledge on interstitial 19q13.32 microdeletions, we report two new patients with a de novo microdeletion in this region, and compare them with previously published patients. Results Evaluation of the phenotypic features in the five published and the two new patients showed that only developmental delay/intellectual disability was present in all patients. No cardiac abnormalities, facial asymmetry, ptosis or micrognathia were present in our patients. Patient 1, with a 1 Mb deletion 19q13.32q13.33, did not have oculomotor dysfunction, scoliosis or constipation, and patient 2, carrying a 3.2 Mb deletion 19q13.31q13.33, did not have a cleft palate. New features noted in patients with 19q13.32 microdeletions were ear abnormalities, including hearing loss, down slanting palpebral fissures and, in male patients, hypospadias. Conclusions Major clinical features seem to differ between the patients with 19q13.32 microdeletions reported so far. In addition, some of the previously presented key features for this microdeletion are absent in our patients. As a consequence, the syndrome may not be as recognizable as previously suggested. Identification of other patients is necessary to further delineate the clinical phenotype of the 19q13.32 microdeletion and to establish the critical region/genes responsible for intellectual disability in these patients.
E-mail: [email protected]
Keywords: 19q13.32 microdeletion
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NOTCH3 exon skipping as a rational therapeutic approach for CADASIL: lessons from a family with naturally occurring exon 9 skipping
Gido Gravesteijn, MD MSc1; Johannes G. Dauwerse2; Ingrid Hegeman3, Gwendolyn Brouwer, MSc2; Aat A. Mulder4; Carolina R. Jost, PhD4; Sjoerd van Duinen3; Annemieke Aartsma-Rus, PhD2; Julie W. Rutten, MD PhD1,2; Saskia A.J. Lesnik Oberstein, MD PhD1.
1 Department of Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands. 2 Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands. 3 Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands. 4 Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, The Netherlands.
Background CADASIL is a small vessel disease (SVD), caused by cysteine altering missense mutations in the NOTCH3 gene, which result in NOTCH3 protein aggregation in the (cerebro)vasculature. We recently described a rational therapeutic approach for CADASIL, aimed at counteracting NOTCH3 aggregation via antisense oligonucleotide (AON)-mediated skipping of mutated NOTCH3 exons. We demonstrated the feasibility of this approach in vitro, but in vivo experiments are so far hampered by inefficient delivery of the AONs. Here, we present clinical and molecular characteristics of a family with a NOTCH3 cysteine altering mutation located on a splice site, leading to natural exclusion of the mutation from the RNA via exon skipping. Methods All family members had brain MRI. Skin punch biopsies were taken and processed for fibroblast culture, immunohistochemistry and electron microscopy. Exon skipping was analysed using RT-PCR and Sanger sequencing, on RNA isolated from skin fibroblasts. Results The index (female, 63 years) was referred because of mild cognitive impairment and white matter hyperintensities on brain MRI suggestive of SVD. Gene panel testing revealed a novel NOTCH3 mutation in exon 9 (c.1492G>T, p.Gly498Cys). The mutation is located on the splice donor site of exon 9 and therefore was predicted to result in exon 9 skipping. Four additional family members with the mutation were identified, all with a relatively mild phenotype without lacunar strokes. RT-PCR and Sanger sequencing on fibroblast RNA showed abundant presence of exon 9 skip product, but also low levels of mutant NOTCH3 from which exon 9 was not skipped. Skin vessels showed only very minimal NOTCH3 aggregation and no granular osmiophilic material. Conclusions These individuals with naturally occurring NOTCH3 exon skipping have a late-onset SVD, likely attributable to their low levels of mutant NOTCH3 expression, which is consistent with the strikingly mild NOTCH3 aggregation in their skin vasculature. These findings also suggest that the abundant skipped NOTCH3 does not contribute to the SVD phenotype, supporting the potential feasibility of NOTCH3 exon skipping as a therapeutic strategy in CADASIL.
E-mail: [email protected] Keywords: CADASIL; NOTCH3; splice site variant; exon skipping; therapeutic strategy
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Identification of splice defects due to non-canonical splice site or deep-intronic variants in ABCA4
Zeinab Fadaie1, Mubeen Khan1, Marta Del Pozo-Valero1,2, Stéphanie S. Cornelis1, Carmen Ayuso2, [ABCA4 Studygroup: Rando Allikmets3, Miriam Bauwens4, Mohammad Ghofrani5, Michael B. Gorin6,7, Mohammad Keramatipur8, Francesca Simonelli9, Naeimeh Tayebi10, Andrea Vincent11, 12, Nicole Weisschuh13] Frans P.M. Cremers1, Susanne Roosing1
1 Department of Human Genetics, Donders Institute for Brain, Cognition and Behavior, Radboud University Medical Center, Nijmegen, the Netherlands. 2 Department of Genetics, Instituto de Investigación Sanitaria–Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), Madrid, Spain. 3 Department of Ophthalmology and Department of Pathology and Cell Biology, Columbia University, New York, NY, USA. 4 Center for Medical Genetics, Ghent University and Ghent University Hospital, Ghent, Belgium. 5 Cellular and Molecular Research Center, Qom University of Medical Sciences, Qom, Iran 6 Department of Ophthalmology, David Geffen School of Medicine, Stein Eye Institute, University of California, Los Angeles, Los Angeles, California, USA. 7 Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA. 8 Department of Medical Genetics, Tehran University of Medical Sciences, Tehran, Iran. 9 Eye Clinic, Multidisciplinary Department of Medical, Surgical and Dental Sciences, University of Campania Luigi Vanvitelli, Naples, Italy. 10 Program in Genetics and Genome Biology, The Hospital for Sick Children, Toronto, Canada 11 Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, The University of Auckland, Grafton, Auckland, New Zealand. 12 Eye Department, Greenlane Clinical Centre, Auckland District Health Board, Auckland, New Zealand. 13 Molecular Genetics Laboratory, Centre for Ophthalmology, Institute for Ophthalmic Research, University of Tuebingen, Tuebingen, Germany.
Pathogenic variants in the ATP-binding cassette transporter A4 (ABCA4) gene cause a continuum of retinal disease phenotypes, including Stargardt disease (STGD1). Non-canonical splice site (NCSS) and deep-intronic variants constitute a large fraction of disease-causing alleles, defining the functional consequences of which remains a challenge. We aimed to determine the effect on splicing of nine previously reported or unpublished NCSS variants, one near exon splice variant and nine deep-intronic variants in ABCA4, using in vitro splice assays in Human Embryonic Kidney (HEK293T) cells. Reverse transcription PCR and Sanger sequence analysis revealed splicing defects for 12 out of 19 variants. Four deep-intronic variants create pseudoexons or elongate the upstream exon. Furthermore, eight NCSS variants cause a partial deletion or skipping of one or more exons in mRNAs. Among the 12 variants, nine lead to premature stop codons and predicted truncated ABCA4 proteins. At least two deep-intronic variants are located in splice enhancers and silencers and, therefore, these conserved sequences should be carefully evaluated when predicting the outcome of NCSS and deep-intronic variants.
E-mail: [email protected] Keywords: ABCA4, Non-canonical splice site variant; deep-intronic variants; splice enhancers; splice silencers; Stargardt disease
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A novel missense PLP1 mutation causing PLP1-related spastic paraplegia: a Case Report
Eva Sammels, Nathalie Fieremans, Annelies Fieuw, Elyssa Cannaerts, Ann Van den Bogaert, Elise Vantroys, Melissa Sys, Kathelijn Keymolen, Boyan Dimitrov
Center for Medical Genetics, UZ Brussels-Vrije Universiteit Brussel
A 12-year old boy presented with lower limb spasticity (first signs by the age of 4), mild facial dysmorphism (downslanting palpebral fissures) and learning difficulties. There was no particular familial history, besides a maternal niece with club foot. The patient underwent physiotherapy on a regular basis during infancy. Array CGH analysis was performed as a first-tier test, but it returned normal. Subsequently, a neurodevelopmental disorders gene panel (1160 genes) was sequenced in trio (parents and patient). We detected a novel, hemizygous, likely pathogenic PLP1 missense mutation (NM_000533.3(PLP1):c.263C>T, p.(Ala88Val)), inherited from the presumably asymptomatic mother, compatible with PLP1-related spastic paraplegia (X-linked recessive inheritance). Carrier females occasionally develop mild to moderate signs of the disease, especially in families with mildly affected males. Therefore, careful clinical follow-up is advised for the heterozygous mother (and if needed other maternal relatives). This missense mutation seems to reside within one of the mutational hotspots of the PLP1 gene. Of interest, a number of other neighbouring amino acid changes and another missense mutation affecting the same codon (p.(Ala88Asp)) have already been described as pathogenic. The highly conserved Ala88 residue is located in the transmembrane helix TM2. Mutations affecting this residue are predicted to affect the organisation of the four transmembrane helices of PLP1, thereby interfering with its biological function as the primary component of myelin. Patients carrying PLP1 mutations affecting the same and other nearby residues were reported to have clinical heterogenous phenotypes and different degrees of severity, making clear genotype-phenotype correlations for PLP1-related disorders still puzzling. Methodology: Array CGH analysis on DNA isolated from an EDTA blood sample was performed using a CytoSure Constitutional v3 60k array. Version (v)5 of the UZ Brussel neurodevelopmental disorders gene panel (NEUDGP) contains the coding regions and 11 basepair (bp) flanking intronic sequence of 1160 genes. The complete gene list is available on the BRIGHTcore website: http://www.brightcore.be/mendeliome-neurodev. Clinical follow-up was done in collaboration with the corresponding clinicians.
E-mail: [email protected] Keywords: PLP1 - Spastic paraplegia - Pelizaeus-Merzbacher disease - Neurodevelopmental disorders
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LOVD - supporting DNA variant interpretation
Ivo FAC Fokkema, Julia A Lopez Hernandez, Johan T den Dunnen
Leids Universitair Medisch Centrum
The simplest and cheapest answer to the possible consequences of a variant identified in a patient's DNA is history: has the variant been seen before, and what were the associated consequences? The "Global Variome shared LOVD" database builds on a >20 year history of collecting and sharing information on Individuals (patients), Phenotypes (disease/traits), Screenings and Variants. While it contains information from published literature, a significant fraction of all data has not yet been published and was submitted to LOVD only. When performing DNA diagnostics, it is essential to check LOVD, preventing easily available information to be missed. A growing number of human genetics journals has made submission of variant/phenotype data to LOVD or ClinVar mandatory before a manuscript will be accepted for publication. This brings additional advantages, like a free data quality check and the option to refer to the database as an alternative for supplementary files. All public LOVD databases together contain information of >6.5 million unique variants linked to >1,000,000 individuals, and can be queried in many ways including through the LOVD website, the API, or the GA4GH beacon network. For genes with active curators (incl. BRCA, colon cancer, CFTR, etc.) specific "Summary records" resume all supporting information whether the variant is or is not associated with a disease. "Classification records" show the classification regarding variant pathogenicity as shared by diagnostic labs (incl. data from the Dutch VKGL). Records labelled "In vitro (cloned)" show the results of assays performed to analyse the functional consequences of variants. Other features that distinguish LOVD from databases like ClinVar, include data collection on the geographic origin of the Individual, detailed Phenotype information and showing whether the variant has been analysed on RNA level and what the consequences were; essential information for the diagnosis in diseases like Duchenne/Becker muscular dystrophy. LOVD shows the parental origin of a variant and, when available, full haplotype information. The GV shared LOVD is a community driven initiative operating under the auspices of Global Variome, a UK charity. Each gene´s home page links to other resources and the major genome browsers. The data is shared with public repositories incl. UCSC, EBI and ClinVar, and copies can be downloaded from the gene home pages. URLs: LOVD.nl/shared, LOVD.nl, LOVD.nl/3.0/search
E-mail: [email protected] Keywords: LOVD, variant databases, diagnostics
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SETD1A Loss of function causes a distinct neurodevelopmental disorder in human and impaired learning & memory in Drosophila.
Joost Kummeling1* Diante E Stremmelaar1* Nicholas Raun2, 20 Margot RF Reijnders3 Marjolein H Willemsen1 Martina Ruiterkamp-Versteeg1 Marga Schepens1 Calvin CO Man1 Christian Gilissen1 Megan T Cho4 Kirsty McWalter4 Margje Sinnema3 James W Wheless5 Marleen EH Simon6 Casie A Genetti15,18 Alicia M Casey19 Paulien A Terhal6 Jasper J van der Smagt6 Koen. L. van Gassen6 Pascal Joset7 Angela Bahr7 Katharina Steindl7 Anita Rauch7 Elmar Keller8 Annick Raas-Rothschild9 David A Koolen1 Pankaj B Agrawal10, 15, 18 Trevor L Hoffman11 Nina N Powell-Hamilton12 Isabelle Thiffault13 Kendra Engleman14 Dihong Zhou14 Olaf Bodamer15 Dirk Schubert21 Rolph Pfundt1 Jamie M Kramer2, 16, 17 Tjitske Kleefstra1
1. Department of Human Genetics, Donders Institute for Brain, Cognition and Behavior, Radboud university medical center, P.O. Box 9101, 6500 HB, Nijmegen, the Netherlands. 2. Division of Genetics and Development, Children's Health Research Institute, London, Ontario, Canada. 3. Department of Clinical Genetics and School for Oncology & Developmental Biology (GROW), Maastricht University Medical Center, Maastricht 6229 ER, the Netherlands. 4. GeneDx, Gaithersburg, MD 20877, USA. 5. Division of Pediatric Neurology, University of Tennessee Health Science Center, , Memphis, TN, United States of America. . Neuroscience Institue & Le Bonheur Comprehensive Epilepsy Program, Le Bonheur Children's Hospital, Memphis, TN, United States of America. 6. Department of Genetics, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands. 7. Institute of Medical Genetics, University of Zurich, Schlieren-Zurich, 8952, Switzerland. 8. Division of Neuropediatrics, Cantonal Hospital Graubuenden, Chur, Switzerland. 9. Institute of Rare Disease, Danek Gertner Institute of Human Genetics, Sheba Medical Center, Tel Hashomer, Israel; Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Israel. 10. Division of Newborn Medicine, Boston Children's Hospital and Harvard Medical School, Boston, MA, 02115, USA. 11. Regional Department of Genetics, Southern California Kaiser Permanente Medical Group, 1188 N. Euclid Street, Anaheim, CA 92801, USA 12. Division of Medical Genetics, Alfred I. duPont Hospital for Children, Wilmington, DE 19803, USA. 13. Department of Pathology and Laboratory Medicine; Center for Pediatric Genomic Medicine, Children's Mercy Hospital; University of Missouri-Kansas City School of Medicine. 14. Division of Clinical Genetics, Children's Mercy Hospital; Department of Pediatrics, Children's Mercy Hospital; University of Missouri-Kansas City School of Medicine. 15. Division of Genetics and Genomics, Department of Medicine, Boston Children's Hospital/Harvard Medical School, Boston, Massachusetts, USA. 16. Department of Biology, Faculty of Science, Western University, London, Ontario, Canada. 17. Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada. 18. The Manton Center for Orphan Disease Research, Boston Children's Hospital and Harvard Medical School, Boston, MA, 02115, USA. 19. Division of Pulmonary and Respiratory Diseases, Boston Children's Hospital and Harvard Medical School, Boston, MA, 02115, USA. 20. Department of Biology, Faculty of Science, Western University, London, Ontario, Canada. 21. Department of Cognitive Neuroscience, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, P.O. Box 9101, 6500 HB, Nijmegen, the Netherlands
Defects in histone methyltransferases (HMTs) are a major contributing factor to Neurodevelopmental disorders (NDDs). Loss-of-function (LoF) variants of SETD1A (KMT2F), one of the genes involved in histone H3 lysine 4 (H3K4) methylation and thus in epigenetic modification, have been identified in several individuals from large schizophrenia cohort studies. Interestingly, dominant gene mutations were also identified in children with developmental delay. To provide further insight in the somatic and behavior profiles, we further characterized the SETD1A associated Mendelian syndrome by collecting the molecular and clinical features of 15 so far unpublished individuals with SETD1A variants via a GeneMatcher collaboration. Furthermore, to gain insight into the potential role of SETD1A in fundamental learning and memory processes, we studied the behavioral consequences of a Drosophila knock down of the orthologue Set1. The mutations identified in the patients mainly comprised of de novo variants that predict a loss of function with
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c.4582-2_4582delAG being a recurrent mutation resulting in intron retention instead of the presumed exon skipping. Haploinsufficiency of SETD1A leads to a set of symptoms comprising global developmental delay or intellectual disability, subtle facial dysmorphisms, behavior and psychiatric problems. In Drosophila, knockdown of Set1 in postmitotic memory neurons resulted in significantly reduced short- and long-term courtship memory. Together, this study defines a distinct neurodevelopmental disorder caused by dominant LoF mutations in the SETD1A and suggests further support for the important role of H3K4 methyltransferases in memory, developmental disability and neuron function in general.
E-mail: [email protected] Keywords: SETD1A; histone methylation; intellectual disability syndrome; behavioral phenotype; Drosophila Set1 orthologue
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Towards comprehensive identification and functional characterization of deep-intronic ABCA4 variants in 1000 Stargardt disease cases
Khan, M.,1,2 Cornelis, S.S.,1,2 del Pozo-Valero, M.,1,3 Khan, M.I.,1,2 Stöhr, H.,4 Grassmann, F.,5 Steehouwer, M.,1 Hoischen, A.,1 Ayuso, C.,3 Ramesar, R.,6 Meunier, I.,7 Defoort, S.,8 Weber, B.H.F.,4 Dhaenens, C-M.,9 and Cremers, F.P.M.1
1Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands 2Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands 3Department of Genetics, IIS-Fundación Jiménez Díaz, CIBERER, Madrid, Spain 4Institut für Humangenetik, Universität Regensburg, Regensburg, Germany 5Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm 6Division of Human Genetics, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa 7Institut des Neurosciences de Montpellier, INSERM, Université de Montpellier, Montpellier, France. 8Service des explorations de la fonction visuelle, CHRU de Lille, Lille, France. 9University Lille, Inserm UMR-S 1172, CHU Lille, Biochemistry and Molecular Biology Department - UF Génopathies, Lille, France
Purpose: Although the gene underlying Stargardt disease (STGD1), ABCA4, has been identified 22 years ago, for many cases the underlying mutations are unknown. This in part is due to the intronic location of many causal variants. In this study, we aim to develop a cost-effective whole ABCA4 gene sequencing technique to analyze >1000 genetically unsolved STGD1 probands. Methods: We designed 3866 single molecule molecular inversion probes (smMIPs), each capturing 110 nt of the 128-kb ABCA4 gene proper or ~40 kb of flanking sequences. Sense and antisense strands were targeted using overlapping smMIPs. Rebalancing of the smMIPs spanning the coding sequences was performed. We collected STGD1 probands from 21 collaborators worldwide. NextSeq 500 sequence analysis was performed for 20 test cases (16 STGD1, 4 healthy controls) and 200 probands in each run. Novel deep-intronic variants were tested for splice defects using in vitro splice assays employing ABCA4 midigenes. Results: smMIPs were designed for 99% of the ABCA4 gene proper and ~50% of flanking sequences as repeats outside the gene were refractory to smMIPs design. Sequence analysis of 16 previously genetically solved STGD1 patients revealed all known alleles with an average smMIP coverage of 933x. Single or double coverage (>100 reads) was achieved for 98.5% of the ABCA4 gene proper and all reported 17 causal deep-intronic variants were covered with smMIPs. We collected 1000 genetically unsolved STGD1 samples, ~900 of which previously were scanned for coding variants and ~100 probands lacked genotyping. In the first 200 STGD1 cases analyzed in a single run, the average smMIP coverage was 377x. We identified 7 different known deep-intronic variants in 23 alleles and 27 different novel deep-intronic variants in 55 alleles. The latter are being tested using midigene-based splice assays. Conclusions: Due to the paucity of sizeable repeats, the introns of ABCA4 could readily be captured using smMIPs. Apart from design and synthesis costs of smMIPs, the sequencing costs for the entire ABCA4 gene are USD25 per sample, rendering this the most cost-effective ABCA4 sequence analysis for coding and non-coding sequences. We will generate a comprehensive list of causal deep-intronic variants for ABCA4, which then can be used to design a minimal diagnostic set of smMIPs that capture 95% of causal ABCA4 alleles in each patient.
E-mail: [email protected] Keywords: ABCA4, Stargardt disease, smMIPs, deep-intronic variants and midigene
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Compound heterozygosity for missense and null variants in CEP55 lead to a novel viable phenotype
M. Mahdi Motazacker*, Eline Overwater*, M. Mieke van Haelst
1- Amsterdam UMC, University of Amsterdam, Laboratory of Genome Diagnostics, Meibergdreef 9, Amsterdam, Netherlands 2- Amsterdam UMC, University of Amsterdam, Department of Clinical Genetics, Meibergdreef 9, Amsterdam, The Netherlands. *These authors contributed equally to this work
The centrosomal protein-55 kDa (CEP55) gene encodes an essential regulator of the final stages of mitosis during cytokinesis. So far only a few cases with homozygosity for loss-of-function variants in CEP55 are reported in the literature. These cases present with an autosomal recessive perinatal or neonatal lethal phenotype similar to Meckel-like syndrome or MARCH syndrome (multinucleated neurons, anhydramnios, renal dysplasia, cerebellar hypoplasia and hydranencephaly and dysmorphic features; MIM 236500). Here we report a 40-year-old male born at a gestational age of 38 weeks with mild intellectual disability, developmental delay, microcephaly, bilateral toe syndactyly, bilateral hypoplastic fifth fingers with clinodactyly and left-sided congenital ptosis. Brain magnetic resonance imaging (MRI) at the age of 37 years showed no abnormalities except from nonspecific white matter changes. Whole exome sequencing (WES) revealed compound heterozygosity for a nonsense c.1274C>A p.(Ser425*) and a missense c.70G>A p.(Glu24Lys) variant in CEP55 (NM_018131.4). A subsequent enquiry through GeneMatcher network identified multiple cases with similar combination of variants and a similar non-lethal phenotype. We conclude that phenotypic spectrum of CEP55 pathogenic variants expands to viable phenotypes with multiple congenital anomalies. Exome sequencing is expanding the phenotypic spectrum of many disease causing genes. This should be considered especially when interpreting variants in genes for which only a few cases with specific class of pathogenic variants are reported.
E-mail: [email protected] Keywords: MARCH syndrome, CEP55, Genotype-phenotype correlation, WES
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Clinical characteristics of patients with an STXBP1 gene mutation: preliminary results of a cross-sectional and retrospective multilevel cohort study
Bos, J.1, Misra-Isrie, M.1, Van Berkel, A. 1,2, Lammertse, H.1,2, Engelen, M.3, Meijers-Heijboer, H.1, Van Haelst, M.1 and Verhage, M.1,2
1Amsterdam UMC, Vrije Universiteit Amsterdam, Department of Clinical Genetics, De Boelelaan 1117, Amsterdam, Netherlands 2Department of Functional Genomics, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, De Boelelaan 1078, Netherlands 3Amsterdam UMC, University of Amsterdam, Department of Pediatric Neurology/Emma Children's Hospital, Meibergdreef 9, Amsterdam, Netherlands
STXBP1-encephalopathy is caused by heterozygous mutations in the STXBP1 gene, which encodes Syntaxin binding protein 1 (STXBP1), also known as Munc18-1. STXBP1 regulates synaptic transmission by organizing the protein complex that drives synaptic vesicle exocytosis. Haploinsufficiency of STXBP1 results in a neurodevelopmental disorder, of which the first patients were reported in 2008, all diagnosed with Ohtahara syndrome. Since then, many case reports and small cohort studies were reported and the corresponding phenotype has expanded, including development delay and different forms of epilepsy. A systematic assessment of the phenotypes of STXBP1-encephalopathy has not been performed thus far and phenotypic features might have been underreported. The purpose of the current study is to provide a systematical and multilevel overview of characteristics of STXBP1-encephalopathy patients worldwide. Two clinic days were organized, during which a semi-structured interview and physical examination by two clinical geneticists were performed, a neurological examination was performed by a neurologist and a questionnaire was filled in by parents. Medical records were collected afterwards. The multilevel approach was complemented by EEG recordings, three-dimensional photographs and skin biopsies for generation of a cellular in vitro model of STXBP1-encephalopathy. Descriptive statistics were used to identify common characteristics. Statistical analysis was performed to assess the difference in prevalence of epilepsy in the current cohort and in literature. Eight of the 13 patients suffered from epilepsy; a significantly lower proportion than in literature (p 0.002). A high prevalence of neurological symptoms was observed; tremor and hypotonia were most frequently observed. Autistic features were observed in 60% of the patients. A characteristic leg cross was recognized by the parents as (possibly) a typical feature for STXBP1-encephalopathy. Common dysmorphic features included fetal finger pads and an abnormal shape of the upper lip. Impaired speech- and/or motor performance was observed in all patients. These results confirm that STXBP1- encephalopathy is a heterogeneous neurodevelopmental disorder in which epilepsy is not obligatory, but development delay is invariably observed and the presence of a characteristics leg cross might be typical. Subtle dysmorphic features and behavioral characteristics such as autistic features might have been underexposed thus far.
E-mail: [email protected] Keywords: STXBP1-encephalopathy, STXBP1, Munc18-1, neurodevelopmental disorder, development delay, intellectual disability, epilepsy, epileptic encephalopathy
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The impact of incidental findings in clinical exome sequencing, a qualitative interview study
Simone J. Viellevoije2, Femke Tammer3, Ilse Feenstra3, Lisenka E.L.M. Vissers3, Martine van Koolwijk3 , Han G. Brunner1,3, Yvonne H. J. M. Arens1, Helger G. Yntema3 and Anke J.M. Oerlemans2
1 Department of Clinical Genetics, Maastricht University Medical Centre, Maastricht, The Netherlands; 2 Sscientific Institute for Quality of Healthcare, Radboud Institute for Health Sciences, Radboud University Nijmegen Medical Centre, Nijmegen; 3 Department of Human Genetics, Radboud University Nijmegen Medical Centre Nijmegen, The Netherlands
Introduction Incidental findings in clinical exome sequencing are (likely) pathogenic variants in disease genes not related to the initial clinical question, but which could be of medical relevance to the patients and their families. The management and disclosure of IFs is an internationally recognised topic of debate. Current recommendations are based upon multiple perspectives (scientific, ethical, legal, clinical). However, little is known about the impact of IF disclosure on patients. Materials and methods We conducted ten (20 by October; study ongoing) semi-structured face-to-face interviews with patients and/or their relatives who underwent genetic testing and to whom an oncological (n=5) or a cardiological (n=5) IF was disclosed. Results Using thematic analysis we identified four relevant themes: psychological impact, actionability, comprehension and social context. Participants described the psychological impact being influenced by the patients´ prior health condition and the availability of preventive measures. They frequently mentioned the mitigating nature of actions taken (i.e. cardiological screening, prophylactic surgery) after the disclosure. Preventive measures and screening options for oncogenetic conditions seemed to assure a more definite answer to the frequently asked question 'am I sick or am I healthy?´. Difficulty of comprehending the actual health risk was a recurrent topic. Some participants took future health risks into account. Patients´ relationships with relatives and the lives of family members did not seem to be greatly influenced by disclosure of IF. Discussion and conclusion The prior health condition of the patient appears to influence the impact of incidental findings in clinical exome sequencing. For patients, the benefit of a diagnosis seems to outweigh the risk of the disclosure of an IF, noteworthy when preventive measures are available. In this context, we see a difference in impact between oncological IF and cardiological IF. These results contribute to the ongoing international debate about managing IFs in clinical DNA sequencing.
E-mail: [email protected] Keywords: incidental findings, impact, qualitative interview study
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CAPICE - a variant prioritization tool for Consequence- Agnostic PrIoritization of Clinical Exomes variations
KJ van der Velde, D. de Ridder, A.D.J. van Dijk, D. Soudis, L.R. Zwerwer , M.A. Swertz
1Genomics Coordination Center, Dept of Genetics, University Medical Center Groningen, Groningen, The Netherlands 2Bioinformatics Group, Wageningen University & Research, Wageningen, The Netherlands 3The Center for Information and Technology, University of Groningen, Groningen, The Netherlands
The past decades have seen rapid advances in sequencing techniques. However, current interpretation methods determining the pathogenicity or benigness of variants are often too time- consuming and of limited accuracy, even with the assistance of computation pathogenicity estimation tools. We here present CAPICE, that has 2x the precision of the next-best variant classification tool by equal recall of 95%. CAPICE overcomes issues that are common in the great number of predictors already built for this purpose, several issues exist. Firstly, a lot of predictors are built specifically for only a subset of SNVs (single nucleotide variants), in particular, non- synonymous variants. This creates difficulties in applications where variants of different molecular functions can be the causal variant. Secondly, the general predictors that do give predictions to all types of variations, such as CADD, often perform much worse compared to specific tools. Moreover, there is a lack of research examining the predictors´ performance in a general setting where variants of different molecular functions are considered and rare neutral variants are included. Therefore, to assist the existing clinical protocols in interpreting patients´ genetic profile the challenge is to extract all relevant information from different biological databases and integrate those into a robust classification algorithm to classify all benign and pathogenic variants. CAPICE uses an ensemble method for prioritizing pathogenic variants for Mendelian disorders in clinical exomes, including SNVs and short inserts and deletions (InDels). By training and fine-tuning a sophisticated machine learning model on a balanced large dataset collected from multiple public sources, we showed that the resulted model outperforms both the specific predictors and general predictors in distinguishing between pathogenic variants and rare neutral variants. Additionally, we showed that CAPICE performs consistently good for variants displaying different molecular consequences and allele frequency. Moreover, we provided suggestions for threshold selection when making pathogenicity predictions based on CAPICE scores, and showed that the prediction made based on CAPICE´s scores is of higher precision compared with existing tools for genes involved in different organ systems. The method for selection of thresholds for making predictions based on the CAPICE scores can be determined to suit different needs of user cases.
E-mail: [email protected] Keywords: Variant prioritization, machine learning, molecular consequence, allele frequency, SNVs, InDels
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Targeted compound screening for PXE using a CRISPR/Cas9 abcc6a mutant zebrafish model.
M. Van Gils; A. Willaert; P. Coucke; O.M. Vanakker
Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium Department of Biomolecular Medicine, Ghent University, Ghent, Belgium
Introduction: Pseudoxanthoma elasticum (PXE) is an ectopic mineralization disease due to biallelic ABCC6 mutations. As no curative therapy is available, disease models for rapid compound screening are necessary. We characterized a zebrafish abcc6a CRISPR/Cas9 knockout model (Cmg52), identifying spinal hypermineralization as an early, quantifiable phenotypic read-out. In this study, we evaluated 3 established anti-mineralization compounds - Vitamin K1 (VK1), Etidronate (Etid) and Sodium Thiosulfate (STS) - for their efficacy in PXE. Materials & Methods: Following Cmg52+/- incross, Cmg52+/+, Cmg52+/-and Cmg52-/- embryos were collected. At 3 days post-fertilization (dpf), embryos are randomly distributed per 20. From 3-10dpf, 60-100 embryos are treated with 8ml compound in E3-medium, refreshed daily. Based on literature we chose a dosage of 80 µM VK1 and 100µM Etid. Using LC50 analysis we opted for 30µM STS. After euthanasia at 10dpf, larvae are fixed, bleached and stained for mineralization. Mineralization is semi-quantified via ImageJ. After sequencing, mineralization values are linked to genotypes for statistical analysis (student t-test). Results: Analysis of Cmg52+/+ and Cmg52+/- data sets revealed no significant changes in spinal mineralization between treated and control cohorts. However, all STS-treated larvae had no spinal mineralization and in 33% ventral mineralization foci were observed, regardless of genotype, indicating putative toxicity. Reducing the dosage to 20µM STS abolished these effects. In contrast, all compounds significantly (P<0.05) reduced spinal mineralization in Cmg52-/- larvae. VK1 reduced mineralization by approximately 47% (Mean±SD: Control: 100±66; VK1: 53.5±28.5), while Etid reduced by 33% (Mean±SD: Control: 100±58.5; Etid: 67±42) and 20µM STS by 41% (Mean±SD: Control: 100±61; STS:59±35). Discussion & Conclusion: We demonstrate the validity of our compound screening assay, with Cmg52-/- spinal hypermineralization as a good read-out. Our data affirm Etid effects in PXE. Interestingly, contrary to the murine model but similar to PXE patients, it also highlights a role for VK1 in PXE. The significant effects of STS make it a potentially interesting topical treatment, e.g. for the skin mineralization in PXE. However, the STS data also underscore the importance of in-depth dosage analyses. Overall, our zebrafish chemical compound screening assay presents a powerful new tool for rapid investigation of putative therapeutic drugs for PXE..
E-mail: [email protected] Keywords: PXE, Zebrafish, CRISPR/Cas9, Compound Screening
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Whole-exome sequencing identifies heterozygous stop-loss mutations in HBB resulting in an elongated β-globin chain in two children with severe anemia.
TT Koopmann, GWE Santen, Q Waisfisz, M Elting, FJ Smiers, JL Kerkhoffs, PJ de Pagter, CL Harteveld
1. Department of Clinical Genetics, Leiden University Medical Center, Leiden, Netherlands. 2. Department of Clinical Genetics, VU University Medical Center, Amsterdam, Netherlands. 3. Department of Pediatric Hematology, University Medical Center Leiden, Leiden, The Netherlands. 4. Haga Hospital, The Hague, The Netherlands.
Background: ß-Thalassemia is a genetic disease characterized by reduced or absence of ß-globin gene (HBB) expression. Some mutations in HBB may produce unstable, abnormal hemoglobins causing (haemolytic) anemia or dominantly inherited ß -thalassemia. Methods: Two unrelated children presented with macrocephaly and severe congenital anemia. They required stem cell transplantations at ages 2 and 4 without clear diagnosis. Since the parents of both probands had no family history of anemia or abnormal Hb, thalassemia was unsuspected. As genetic testing for variants in genes associated with congenital anemia did not result in an explanation for the phenotype, whole-exome sequencing was performed. Results: We identified two novel, de novo missense stop-loss mutations in HBB (c.442T>C and c.442T>A). The mutations identified caused the loss of a stop codon and an elongation of the translated beta-globin chain of 21 amino acids (167 a.a. instead of the usual 146) due to a new stop codon in the 3' untranslated region (3'UTR) of the HBB gene. The predicted ß-globin chains were p.(*148Glnext*21) or p.(*148Lysext*21); the latter variant was called HbMokum. Discussion: Using whole-exome sequencing, we identified a de novo mutation in two previously undiagnosed children with severe anemia. Both children carry an almost identical heterozygous stop-loss mutation in HBB resulting in an elongated ß-globin chain, which most likely produces an unstable hemoglobin. Frameshift mutations in the HBB gene resulting in elongated ß-globin chain have been described before, but resulted in shorter ß-chains (max. 157 a.a.) and milder phenotypes than the mutations described here.
E-mail: [email protected] Keywords: Thalassemia, stop-loss mutation, anemia, HBB gene, whole exome sequencing
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A second family with homozygous mutation in KIDINS220 presenting as congenital hydrocephalus and limb contractures
Jacquemin V., Antoine M., Duerinckx S., Perazzolo C., Cassart M., Thomas D., Segers V., Drielsma A., Desir J., Pirson I., Abramowicz M.
IRIBHM, Université Libre de Bruxelles; Centre Hospitalier Etterbeek-Ixelles, Université Libre de Bruxelles; CHU Brugmann ; Institut de Pathologie et de Génétique, Gosselies; Department of Genetic Medicine and Development, University of Geneva Medical School
Background Hydrocephalus is a devastating, potentially lethal neurological condition. A subgroup with prenatal onset and no apparent cause is called congenital primary hydrocephalus, affecting 1/10,000 newborns. A homozygous mutation in KIDINS220, a large scaffolding protein, was recently reported in a single family with enlarged cerebral ventricles and limb contractures in four fetuses (Mero et al 2017, Hum Mol Genet). Heterozygous, de novo mutations of KIDINS220 were also reported in SINO (spastic paraplegia-intellectual disability-nystagmus-obesity) syndrome. Case presentation We report a consanguineous family with 3 fetuses presenting with ventriculomegaly and limb contractures. Exome sequencing revealed as sole potentially disease-causing variant, a small homozygote in-frame deletion in KIDINS220 present in the three fetuses, resulting in the deletion of Glutamine 713, Valine 714, and Leucine 715; p.Gln713_Leu715del. This mutation was at heterozygous state in parents and unaffected siblings, they presented no symptoms of SINO syndrome. These amino acids are very conserved between species and lie in the most highly conserved stretch of the protein downstream of the fourth transmembrane domain. This region is implicated in the interaction with tyrosine receptor kinase TrkA. We co-expressed the mutated region and the TrkA receptor in a human cell line, and tested their interaction through immunoprecipitation experiments. The wild-type and mutated fragments produced the same results. We are now testing the full-length protein. Discussion and Conclusions The co-segregation in three consanguineous fetuses and the location of the mutation strongly suggest that we identified the second family with a homozygous, presumably loss-of-function mutation of KIDINS220, confirming its clinical presentation as a syndrome of congenital hydrocephalus and limb contractures.
E-mail: [email protected] Keywords: primary congenitale hydrocephalus, research, exome sequencing, KIDINS220
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Intrinsic myogenic potential of skeletal muscle-derived pericytes from patients with myotonic dystrophy type 1
Ausems Cornelia Rosanne Maria 1,2,3, Raaijmakers Renée Henrica Lamberta 1,2,3, Van Den Broek Walterus Johannes Antonius Adriana 3, Willemse Marieke 3, Van Engelen Baziel Gerardus Maria2*, Wansink Derick Gert3*, Van Bokhoven Hans1*
1. Radboud university medical center, Donders lnstitute for Brain Cognition and Behavior, Department of Human Genetics, 6500 HB, Nijmegen, The Netherlands 2. Radboud university medical center, Donders lnstitute for Brain Cognition and Behavior, Department of Neurology, 6500 HB, Nijmegen, The Netherlands 3. Radboud university medical center, Radboud Institute for Molecular Life Sciences, Department of Cell Biology, 6500 HB, Nijmegen, The Netherlands
Introduction: We are exploring the possibility of a cell-based therapy to combat muscle problems in DM1. Accordingly, we have successfully isolated a distinct class of myogenic progenitors, called pericytes, from skeletal muscle of DM1 patients and transgenic mice. Methods: Muscle biopsies from DMSXL mice and DM1 patients were cultured under conditions to promote outgrowth of pericytes, the muscle stem cells wrapped around blood vessels. ALP+/CD31- pericytes were isolated by flow cytometry and used for characterization of gene expression, cell growth and myogenic fusion characteristics. Results and Conclusions: Efficient isolation of ALP+/CD31- pericytes was confirmed by RT-PCR analysis and immunocytochemistry. Expression of expanded DMPK RNA matched the occurrence of nuclear MBNL foci. Interestingly, patient pericytes maintained myogenic potential and differentiated efficiently into multinucleated myotubes in vitro. These muscle progenitors will now be used in CRISPR/Cas9-mediated gene editing to excise the expanded (CTG)n repeat. Grant Support: Donders Institute, Radboud University Medical Centre.
E-mail: [email protected] Keywords: cell therapy; mesoangioblast; muscle stem cell; muscular dystrophy; myogenic progenitor cell; myotonic dystrophy; pericyte; RNA toxicity; triplet repeat expansion;
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Development and Characterization of Pericyte-Derived iPSCs and PiPs from DM1 patients
Renée H. L. Raaijmakers1,2,3, C. Rosanne M. Ausems1,2,3, Walther J. A. A. van den Broek3, Marieke Willemse3, Silvia Albert1, Baziel G. M. van Engelen2, Derick G. Wansink3, Hans van Bokhoven1
1 Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboud university medical center, Nijmegen, The Netherlands. 2 Department of Neurology, Donders Institute for Brain, Cognition and Behaviour, Radboud university medical center, Nijmegen, The Netherlands. 3 Department of Cell Biology, Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, The Netherlands.
Introduction: We aim to develop a muscle-focused iPSC-based cell therapy for DM1, using pericytes, the myogenic progenitors wrapped around blood vessels. To ensure an unlimited source of cells, we will generate pericyte-derived iPSCs. Subsequently, the pathogenic trinucleotide repeat will be excised from these cells via CRISPR/Cas9-mediated gene editing. Corrected pericyte-derived iPSCs can then be differentiated to PC-iPSC-derived pericyte-like cells (PiPs), which have shown to maintain their myogenic potential and have restorative potential. Methods: Skeletal muscle biopsies were taken from quadriceps muscle from DM1 patients and cultured for pericyte proliferation and isolation. Cells were sorted for ALP+/CD31- expression by flow cytometry, expanded in vitro, and transfected with non-integrating episomal vectors containing the Yamanaka factors. Differentiation of PC-iPSCs to PiPs has been performed following a protocol by Maffioletti et al. (2015, Nature Protocols) by the use of media components clear from transgene integration. Results: We successfully isolated ALP+/CD31- pericytes and were able to generated pericyte-derived iPSCs. Furthermore, preliminary results point to successful differentiation of PC-iPSCs to PiPs. Conclusions: We are currently applying CRISPR/Cas9-mediated repeat removal on PC-iPSCs and are optimizing the differentiation protocol for PiPs. Grant Support: Prinses Beatrix Spierfonds [grant W.OR18-18]
E-mail: [email protected]
Keywords: myotonic dystrophy type 1, pericytes, pericyte-derived induced pluripotent stem cells (PC-iPSCs), PC-iPSC derived pericytes (PiPs)
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Intellectual Disability-related genes increase ADHD risk and locomotor activity in Drosophila melanogaster
Marieke Klein1uginia Singgih1nne van Rens1, Ditte Demontis2,3,4, Anders D. Børglum2,3,4, Nina Roth Mota1,5 Anna Castells-Nobau1, Lambertus A. Kiemeney6, Han G. Brunner1, Alejandro Arias- Vasquez1,5, Annette Schenck1, Monique van der Voet1$, Barbara Franke1,5$*
1Radboud university medical center, Donders Institute for Brain, Cognition and Behaviour, Department of Human Genetics, Nijmegen, The Netherlands 2Department of Biomedicine, Aarhus University, Aarhus, Denmark 3Centre for Integrative Sequencing, iSEQ, Aarhus University, Aarhus, Denmark 4The Lundbeck Foundation Initiative for Integrative Psychiatric Research, iPSYCH, Denmark 5Radboud university medical center, Donders Institute for Brain, Cognition and Behaviour, Department of Psychiatry, Nijmegen, The Netherlands 6Radboud university medical center, Donders Institute for Brain, Cognition and Behaviour, Department for Health Evidence, Nijmegen, The Netherlands ual contribution $ shared final responsibility
Objective: Attention-Deficit/Hyperactivity Disorder (ADHD) is a common, highly heritable neuropsychiatric disorder. ADHD often co-occurs with Intellectual Disability (ID), and shared overlapping genetics have been suggested [1]. This study aimed to identify novel ADHD genes by investigating whether genes carrying rare mutations linked to ID contribute to ADHD risk through common genetic variants. Validation and characterization of candidates were performed using Drosophila melanogaster. Method: Common genetic variants in a diagnostic gene panel of 396 autosomal ID genes were tested for association with ADHD risk, through gene-set and gene-wide analyses [2, 3], using ADHD meta-analytic data of the Psychiatric Genomics Consortium (n=19,210) for discovery and iPSYCH ADHD data for replication (n=37,076) [4]. The significant genes were functionally validated and characterized in Drosophila by assessing locomotor activity and sleep upon knockdown of those genes in brain circuits [5]. Results: The ID gene-set was significantly associated with ADHD risk in the discovery and replication data-sets. The three genes most consistently associated were MEF2C, ST3GAL3, and TRAPPC9. Performing functional characterization of the two evolutionary conserved genes in Drosophila melanogaster, we found their knockdown in dopaminergic (dMEF2) and circadian neurons (dTRAPPC9) to result in increased locomotor activity and reduced sleep, concordant with the human phenotype. Conclusions: This study reveals that a large set of ID-related genes contributes to ADHD risk through effects of common alleles. Utilizing this continuity, we identified TRAPPC9, MEF2C, and ST3GAL3 as novel ADHD candidate genes. Characterization in Drosophila suggests that TRAPPC9 and MEF2C contribute to ADHD-related behavior through distinct neural substrates.
E-mail: [email protected]
Keywords: Intellectual disability, ADHD, genetics, Drosophila melanogaster, MEF2C, TRAPPC9
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An integrative approach to STXBP1-Encephalopathy: in vitro, in vivo and patient clinics.
Annemiek van Berkel*1,2, Hanna Lammertse*1,2, Jovana Kovacevic*1,2, Mala Misra-Isrie2, Jessica Bos2, Hanne Meijers-Heijboer2, Ruud Toonen1,2, Mieke van Haelst2 and Matthijs Verhage1,2
1Department of Functional Genomics, Center for Neurogenomics and Cognitive Resarch, Amsterdam Neuroscience, Vrije Universiteit (VU) Amsterdam, de Boelelaan 1078, 1081 HV Amsterdam, the Netherlands 2 Department of Clinical Genetics, Amsterdam University Medical Center, location VUmc, 1081 HV Amsterdam, The Netherlands * These authors contributed equally to this work.
STXBP1 is highly expressed in the brain and important for communication between neurons. Mutations in the STXBP1 gene cause early infantile encephalopathy (STXBP1-E). Following the first report of an STXBP1 microdeletion in a patient diagnosed with Ohtahara syndrome in 2008, more than 100 other cases have been reported of patients with heterozygous missense or nonsense mutations in the STXBP1 gene. The clinical features presented by patients with an STXBP1 mutation are highly diverse. All patients display a developmental delay and severe to profound intellectual disability. The majority of patients present with early-onset epilepsy. The seizure type and responsivity to anti-epileptic treatments is variable. Many other symptoms are often reported by families, but not systematically examined in this patient group, such as autism. To date, no therapy is known beyond seizure control. The pathological mechanisms underlying the symptoms remain to be elucidated and research has primarily focused on descriptions of individual cases using diverse methodology and criteria. In the present study, the aim is to generate a standardized, multilevel assessment of STXBP1-E in affected patients from clinics worldwide. We invite patients to our special STXBP1 clinics, where patients and their families/caregivers come to a central location for a multilevel assessment by a multidisciplinary team of researchers. Firstly, the phenotypic features of patients are described using standardized criteria, through questionnaires and a structured interview with the patient families/caregivers. Furthermore, patients are examined by a clinical geneticist (dysmorphologist) and a neurologist. Secondly, an EEG recording is performed. Thirdly, a skin biopsy is collected from a subgroup of patients. Induced pluripotent stem cell- technology is being used to generate a cellular in vitro model of STXBP1-E. This allows us to investigate the effects of STXBP1 mutations at the cellular and molecular level in patient neurons. Taken together, this multilevel cohort study will provide the first in depth systematic and quantitative assessment of STXBP1-E patients. Analysis of these data obtained will provide new insights into the possible disease mechanisms of STXBP1-Encephalopathy. In addition, this systematic assessment of the phenotypic characteristics can help achieving better diagnoses with the ultimate aim of providing the best treatment.
E-mail: [email protected]
Keywords: STXBP1-encephalopathy, STXBP1, neurodevelopmental disorder, developmental delay/intellectual disability, epilepsy, epileptic encephalopathy
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The complexities of analysis for APC mosaicism
D. Terlouw1*, M. Suerink1*, C. Tops1, A. Langers1, F. Hes2, S. ten Broeke3, L. Dams1, T. van Wezel1, H. Morreau1. Nielsen1
Leiden University Medical Center, Leiden, the Netherlands 2 University Hospital Brussels, Brussels, Belgium 3 University Medical Center Groningen, Groningen, the Netherlands */ese authors contributed equally to this work
Objectives Mosaic mutations in the APC gene have been identified as a common cause (25%) for polyposis in patients with >20 adenomas without germline mutation. The frequency remains unknown in patients with milder phenotypes. Methods The APC gene was sequenced in DNA isolated from 4 adenomas in our cohort (n=132) using Next Generation Sequencing. Patients were considered mosaic if an identical mutation was identified in all lesions. An identical variant in only a subset of the adenomas, was considered a so-called hybrid mosaicism. Detection rates and clinical characteristics were compared between different subgroups. Results The mosaicism detection rate was 17% in the entire cohort (22/132), 0% in patients with <10 adenomas (0/20), 7% in those with 10-20 adenomas (3/42) and 12% in patients over age 70 (2/17). Interestingly, 26 hybrid cases were identified. The mean age of diagnosis and number of adenomas was comparable in hybrid and non-mosaic cases (both ~61 years and ~23 adenomas) and differed from 'pure´ mosaic cases (45 years, 54 adenomas). Possible explanations for the occurrence of hybrid cases are; chance, field cancerization, contamination, spread of cells during colonoscopies and partial mosaicism with incidental adenomas. However, no universal explanation could be found. Conclusions Our results indicate that mosaic APC mutations also play a role in patients with <20 adenomas and/or older age at presentation. No universal explanation is identified to explain the occurrence of hybrid cases. So, case by case evaluation and expansion of the cohort is required.
E-mail: [email protected] Keywords: unexplained polyposis coli, APC mosaicism
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The MAP3K7 gene: further eloboration on phenotypes, and evidence for interaction with the RAS pathway/clinical overlap with Noonan syndrome
REM Senden1*, GM van Woerden2*, C de Konink2, R Avagliano Trezza2, JA Bassetti3, Y van Bever1, LM Bird4, BW van Bon5, AS Brooks1, I van der Burgt5, EW Klee6, Carlo Marcelis5, JA Morales- Rosado6, LA Schimmenti7, PA Terhal8, MW Wessels1, H van Wieringen9, M Nicetta10, Y. Elgersma2d S. Demirdas1
Department of Genetics, Erasmus Medical Center, Erasmus University, Rotterdam, the Netherlands 2 Department of Neuroscience and the ENCORE Expertise Center for Neurodevelopmental Disorders, Erasmus MC University Medical Center, 3015 CN Rotterdam, The Netherlands 3 Division of Medical Genetics, Department of Pediatrics, Weill Cornell Medical College, New York, N.Y, USA 4 Division of Genetics & Genomics, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts 5 Department of Human Genetics, Radboud University Medical Center, Radboud University, the Netherlands. 6 Department of Health Sciences Research, Division of Biomedical Statistics and Informatics, Mayo Clinic, Rochester, MN, USA. 7 Department of Otorhinolaryngology, Clinical Genomics and Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA. 8 Department of Genetics, University Medical Center Utrecht, Utrecht University, The Netherlands 9 Department of Pediatrics, St. Antonius Hospital, Nieuwegein, The Netherlands 10 Genetics and Rare Diseases Research Division, Bambino Gesù Children's Hospital, Rome, Italy.
Introduction The MAP3K7 gene is associated with two autosomal dominant inheriting syndromes; frontomethaphyseal dysplasia type 2 (FMD2, gain of function), and cardiospondylocarpofacial syndrome (CSCF, loss of function (LOF)). Whole exome sequencing in 5 Dutch patients suspected to have Noonan Syndrome (NS), showed novel variants in MAP3K7. Clinical assessment confirmed CSCF. The striking resemblance with NS suggested an upregulation of the RAS pathway caused by LOF variants in MAP3K7. Furthermore, some patients showed neuronal involvement, suggesting an effect on neuronal formation. Methods We studied 12 children and 2 adults with a novel variant in MAP3K7 (12 with CSCF; 2 with FMD2). Clinical assessments were made using a digital database containing specific clinical features. In order to test the effect of MAP3K7 on the RAS pathway, we measured levels of activated ERK (pERK) in transfected mice. To test the effect on neuronal migration (NM) in mice brain we used in vivo transfection via in utero electroporation. Laboratory technicians were blinded for patient's phenotype. Results The clinical features of CSCF patients overlapped with NS: short stature (n=7; 58%), congenital cardiac abnormalities (n=7, 58%), low-set posteriorly rotated ears (n=6; 50%), hypertelorism (n=8; 67%), deeply grooved philtrum (n=6; 50%), dental malocclusion (n=2, 16%) and epicanthic folds (n=6; 50%). Distinguishing features of CSCF are the wide nose with bulbous tip (n=6; 50%), peri-orbital fullness (n=4, 32%) and full cheeks (n=8; 67%). Neurological phenotype: ID (n=1), language delay (n=1) and autism (n=1). FMD2 patients (one with ID) showed phenotypes compatible with previously reported patients. We showed pERK to be upregulated in CSCF (pERK was stable in FMD2). MAP3K7 LOF variants therefore cause upregulation of the RAS pathway, explaining the Noonan-like phenotype. The brains of transfected mice with variants from patients with a neuronal phenotype showed disrupted NM. Conclusion We investigated the clinical features of patients with novel pathogenic variants in the MAP3K7 gene leading to either CSCF (n=12) or FMD2 (n=2). We conclude that CSCF should be considered in the differential diagnosis of NS because there is an overlap in phenotype and an upregulation of the RAS pathway. In vivo transfection of mice embryos effectively showed which MAP3K7 variants cause a neurological phenotype.
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E-mail: [email protected] Keywords: MAP3K7, Cardiospondylocarpofacial syndrome, frontometaphyseal dysplasia type 2, FMD2, RAS pathway, neuronal migration, in utero extrapolation, pERK, Noonan syndrome
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Further delineation of phenotypes related to integral cohesin structural protein RAD21
Lianne C Krab MD PhD, Asuman Zeynep Tümer MD PhD, Raoul C Hennekam MD PhD
Cordaan, Klinkerweg 75, 1033 PK Amsterdam, the Netherlands; [email protected] Department of Clinical Medicine, Rigshospitalet - Diagnostisk Center, Gamle Landevej 7-9, 2600 Glostrup; Department of Pediatrics, Amsterdam University Medical Centers, location Academic Medical Center, Meibergdreef 9, 1105AZ Amsterdam, the Netherlands.
Background: Aberrations in RAD21 (RAD21 Cohesin Complex Component) have been associated with clinical phenotypes that overlap with that of Cornelia de Lange Syndrome (CdLS). Although it has been stated that RAD21 variants lead to a relatively mild CdLS phenotype, the number of reported individuals on which this has been based is small, and insight into the cognitive and behavioral aspects is limited. We aimed to provide an overview of the RAD21 variants reported thus far, and to gather the associated phenotype data with a special focus on development, cognition and behavior. Method: Using international inquiry, literature and database search, we identified 54 cases with RAD21 variants (36 index; including 26 sequence variants and 10 CNV´s). With a dedicated questionnaire, we gathered extensive and updated data. This led to two patient cohorts: cohort A (29 patients, 22 index) with extensive clinical data, and cohort B (25 cases, 14 index) with limited clinical data. The phenotype of cohort A were compared to those of CdLS patients with NIPBL (n=67) and SMC1A (n=51) variants. With the help of in silico analyses of mutation effects, genotype-phenotype relationships were investigated. Results: All 29 patients in Cohort A had sufficient CdLS features to warrant molecular genetic testing for CdLS; 13 met clinical criteria for classical CdLS. Overall, somatic, cognitive and behavioral problems were markedly less prevalent and less severe in the RAD21 cohort compared to the NIPBL and SMC1A cohorts. Remarkable findings are a paucity of self-injurious behavior and apparent absence of major limb malformations. Cohort B included cases with phenotypes not previously associated with CdLS. These will be elaborated upon at the meeting. Two recurrent mutations were found each in 2 families, with large interfamilial variation. further genotype-phenotype analyses are in progress and will be presented at the meeting. At least 10 index cases had a familial variant, with part of the cases were only identified after diagnosis in a child or sib. The large intrafamilial variation has obvious consequences for counseling. Conclusion: This case series represents a comprehensive overview of RAD21 variants reported so far, including 18 new index cases. The data should be of benefit to clinical geneticists and physicians when counseling families with affected members with a RAD21 variation.
E-mail: [email protected] Keywords: RAD21 (RAD21 Cohesin Complex Component), Cornelia de Lange Syndrome, deep phenotyping, genotype phenotype correlations, cognitive functioning, behavior
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