12th Annual Meeting
Friday, March 2, 2012
Palais des Congrès, Liège
Next Generation Sequencing
and
Recent Advances in Genetics
Belgian Society of Human Genetics
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Next Generation Sequencing and Recent Advances in Genetics
Belgian Society of Human Genetics
A word from the president
On behalf of the board of the Belgian Society of Human Genetics, it is my great pleasure to
welcome you all to Liège for our 12th annual conference. These are exciting times for our
society. Genetics is invading all fields of medical sciences and human geneticists find
themselves in ever increasing interactions with all medical practitioners. The field of human
genetics is rapidly evolving, especially with the sequencing technologies that are now
becoming available to all of us. This technological change is a revolution not only for our
daily practice but also for mankind.
We, as clinical geneticists, scientists and as members of the society, still have a long way to
go to fully understand the human genome, and its use for the benefit of us all. New
sequencing technologies will have a major impact on diagnostic testing in the near future.
The great challenge is to integrate the wealth of information provided by the new
technologies into patient care in a harmonious and beneficial way. From a societal
perspective, concerns are raised that relevant guidelines are needed to ensure an ethically
and medically appropriate use of genetic testing. This meeting addresses the new science
and the challenges ahead.
Welcome and enjoy the meeting!
Hélène Antoine-Poirel
Board members
Hélène Antoine-Poirel, president
Thomy de Ravel de l’Argentière, secretary
Guy Van Camp, treasurer
Paul Coucke
Pascale Hilbert
Mauricette Jamar
Sonia Van Dooren
Catheline Vilain
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Belgian Society of Human Genetics
Local Organizing Committee
Vincent Bours
Michel Georges
Mauricette Jamar
Cécile Libioulle
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Next Generation Sequencing and Recent Advances in Genetics
Belgian Society of Human Genetics
Dear colleagues,
We are pleased to welcome you in Liège for the 12th meeting of the BeSHG.
As genetics has recently seen major technological changes that are deeply modifying our
research and diagnostic approaches, the local committee has decided to invite speakers who
have recently used Next Generation Sequencing techniques to open novel directions in the
understanding of monogenic or complex diseases as well as cancers. We hope that these
talks will illustrate the new era of genetic investigations and bring new perspectives and
hopes to researchers, clinicians as well as patients. Indeed, the future will tell us, probably
quite rapidly, how these technological and bio-informatics advances will be translated into
significant scientific and clinical progresses.
We hope that this meeting will generate enthusiasm within the attendees and that everyone
will take advantage of this yearly event to meet and discuss with colleagues from other
centers and to discover the work of Belgian scientists and international experts.
We wish you a very good time in Liège.
Best wishes.
On behalf of the local organizers:
Vincent Bours, ULg
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Belgian Society of Human Genetics
Next Generation Sequencing and Recent Advances in Genetics
08.00 – 08.45
Registration (Coffee and croissants)
08.45 – 09.00
Welcome address:
Hélène Antoine-Poirel, president BeSHG
Vincent Bours (for the local organizing committee)
09.00 – 10.30
Invited Presentations I
Chair: Vincent Bours - Hélène Antoine-Poirel
09.00 Interrogating the architecture of Cancer Genomes
Peter Campbell (Wellcome Trust Sanger Institute, Cambridge, UK)
09.30 Comparison of genomic rearrangements, DNA methylation and gene expression patterns
between different foci of multifocal breast cancers
Christine Desmedt (Institut J. Bordet - ULB, Brussels, B)
10.00 A mitochondrial view of microRNAs
Alexandra Henrion-Caude (Inserm U781, Hôpital Necker-enfants malades, Paris, F)
10.30 – 11.00
Coffee break, Poster session and Exhibition
11.00 – 12.00
Selected Oral Presentations by Young Investigators I
Chair: Sonia Van Dooren – Mauricette Jamar
Beata Nowakowska
Targeted re-sequencing of the remaining 22q11.2 region in atypical DiGeorge patients
Isabelle Cleynen
Genetic and functional evidence for a role of CYLD in Crohn’s Disease: results from a European
consortium
Alejandro Sifrim
Computational annotation and interpretation of single nucleotide variation to identify diseasecausing variants by next-generation sequencing
Damien Lederer
Deletion of KDM6A, a histone demethylase interacting with MLL2, in three patients with
Kabuki syndrome
Lindsey Van Haute
Multiple large deletions in the mitochondrial DNA of human embryonic stem cells
12.00 – 13.00
General Assembly for all BeSHG members
13.00 – 14.00
Lunch, Poster session, Exhibition & Satellite meetings
13h30 - 13h40 satellite meeting Multiplicom
Prof. Jurgen Del-Favero: MASTR Assays in clinical diagnostics with NGS
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14.00 – 15.00
Selected Oral Presentations by Young Investigators II
Chair: Lucienne Michaux – Christian Herens
Lidia Ghisdal
Genome-wide association study identifies 13 loci associated with acute T-cell rejection in
Caucasian renal transplant patients
Kaat Durinck
Unraveling a NOTCH1-lncRNA-miRNA regulatory network in acute T-cell lymphoblastic
leukemia and normal T-cell development
Pascal Brouillard
Mutations in KIF11 cause Microcephaly-Lymphedema-Chorioretinal dysplasia syndrome
(MLCRD)
Fjoralba Zeka
Outcome prediction of neuroblastoma patients using microRNA gene expression profiling in
both fresh frozen and archived tumor samples
Gert Van Peer
An unbiased high-throughput miRNA library screen identifies novel miRNA regulators of key
oncogenes and tumor suppressor genes implicated in different cancer types
15.00 – 15.30
Coffee break, Poster session and Exhibition
15.30 – 17.00
Invited Presentations II
Chair: Michel Georges - Paul Coucke
15.30 New genetic approaches to cardiac diseases
Richard Redon (L’institut du thorax, University & Hospital of Nantes, F)
16.00 Disease gene identification by exome sequencing
Alexander Hoischen (Dpt of Human Genetics, Radboud University, Nijmegen, NL)
16.30 From Galton to GWAS (and beyond): what we have learned about the genetics of quantitative
traits in human population?
Peter Visscher (Queensland Brain Institute, Aus)
17.00 – 17.15
Conclusions and Summary
17.15 – 18.30
Reception and Prizes
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Belgian Society of Human Genetics
The BeSHG thanks the sponsors of this meeting:
Gold Sponsors
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Silver Sponsors
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Next Generation Sequencing and Recent Advances in Genetics
Belgian Society of Human Genetics
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Invited Speakers
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Interrogating the Architecture of Cancer Genomes
Peter Campbell
Cancer Genome Project, Wellcome Trust Sanger Institute
Cancer is driven by mutation. Using massively parallel sequencing technology, we can now
sequence the entire genome of cancer samples, allowing the generation of comprehensive
catalogues of somatic mutations of all classes. Bespoke algorithms have been developed to
identify somatically acquired point mutations, copy number changes and genomic
rearrangements, which require extensive validation by confirmatory testing. The findings
from our first handful of genomes illustrate the potential for next-generation sequencing to
provide unprecedented insights into mutational processes, cellular repair pathways and
gene networks associated with cancer development. I will also review possible applications
of these technologies in a diagnostic and clinical setting, and the potential routes for
translation.
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Comparison of genomic rearrangements, DNA methylation and gene expression patterns
between different foci of multifocal breast cancers
Christine Desmedt
Institut J. Bordet - ULB, Bruxelles
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Belgian Society of Human Genetics
A mitochondrial view of microRNAs
Alexandra Henrion-Caude
Inserm U781, Hôpital Necker-enfants malades, Paris
Recent results show that many transcripts undergo post-transcriptional regulation through
microRNA (miRNA) that associate with Argonaute proteins. This regulation, which is part of
the RNA interference (RNAi) pathway typically results in translational repression of the
transcripts. Evidence is accumulating that many components of the miRNA machinery and
the RNAi process itself may not be localized in the cytosol but that they occur in association
with different cellular organelles or structures. From a mitochondrial point of view, we will
review the different aspects of miRNA biology, targeting, involvement in human disease and
localization. The novel localization of RNA interference components in human mitochondria
that we recently identified paves the way of the molecular bases for a novel layer of
crosstalk between nucleus and mitochondria.
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New genetic approaches to cardiac diseases
Richard Redon
L’institut du thorax (Inserm 1087/ CNRS 6291), University & Hospital of Nantes
With the development of massively parallel sequencing, genome-wide screening for rare
genetic variants has become possible, allowing the direct detection of allelic variants that
have not been described yet in human populations. At the institut du thorax, we are
developing new genetic screening strategies based on Whole Exome Sequencing and
Identity-by-Descent analysis on extended pedigrees. We aim at identifying rare genetic
variants associated with cardiac sudden death and cardiac valve disease in order to better
understand the molecular bases of those cardiac defects for which genetic investigations
have been hardly successful so far.
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Disease gene identification by exome sequencing
Alexander Hoischen
Department of Human Genetics, Radboud University Medical Centre Nijmegen, Nijmegen,
The Netherlands
Next generation sequencing can be used to search for Mendelian disease genes in an
unbiased manner by sequencing the entire protein-coding sequence, known as the exome.
Identifying the pathogenic mutation amongst thousands of genomic variants is a major
challenge, and novel variant prioritization strategies are required. The choice of these
strategies depends on the availability of well-phenotyped patients and family members, the
mode of inheritance, the severity of the disease and its population frequency. In this review
we discuss the current strategies for Mendelian disease gene identification by exome
resequencing and we describe the lessons learned from the first exome sequencing studies.
Exome sequencing is likely to become the most commonly used tool for Mendelian disease
gene identification for the coming years and bears a great diagnostic potential as well.
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From Galton to GWAS (and beyond): what we have learned about the genetics of
quantitative traits in human population?
Peter M. Visscher
University of Queensland, Brisbane, Australia
Quantitative traits and common complex disease are caused by a combination of multiple
genes and environmental effects. Traditionally, since Galton in the late 1800s, the genetics
of complex traits has been studied using concepts that refer to the combined effect of all
genes (e.g., heritability or sibling risk). Genome-wide association studies (GWAS) facilitate
the dissection of heritability into individual locus effect. They have been successful in finding
many SNPs associated with complex traits and have greatly increased the number of genes
where variation is known to affect the trait. However, GWAS have been criticized for not
explaining more of the genetic variation that we know exists in the population and many
hypotheses have been put forward to explain the missing heritability. The most plausible
explanations are that (i) causal effects are too small to be detected with statistical
significance and (ii) causal variants are not well tagged by the SNPs on the commercial
arrays, for example because their heterozygosity is lower than that of genotyped SNPs. The
use of all GWAS data simultaneously in an estimation rather than hypothesis testing
framework can capture much more variation than in gene discovery approaches, and allows
the partitioning of variation across chromosomes and chromosome segments. We show
how such whole genome methods can be used to better understand the genetic
architecture of complex traits, with applications in height, BMI, schizophrenia and other
traits. The results demonstrate that for all traits studied, a substantial proportion of additive
genetic variation is tagged by common SNPs and that genetic variation is smeared out over
the entire genome. We conclude that these traits are highly polygenic, that variation
explained by causal variants is small on average and that GWAS with increasing sample size
will discover more variants.
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Selected Oral Presentations
by Young Investigators
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O1: Targeted re-sequencing of the remaining 22q11.2 region in atypical DiGeorge patients.
Beata A. Nowakowska1, Jeroen J. Van Houdt1, Ann Swillen1, Koenraad Devriendt1, Sonia
Bouquillon2, Francesca Novara3, Cedric Le Caignec4, Lucjusz Jakubowski5, Wanda Hawuła5,
Anna Kutkowska-Kazmierczak6, Ewa Obersztyn6 & Joris R. Vermeesch1
1
Center for Human Genetics, KU Leuven, Belgium
Hôpital Jeanne de Flandre, CHRU de Lille, France
3
Genetica Medica, Universita` di Pavia, Italy
4
CHU Nantes, Service de Génétique Médicale, Nantes, France
5
Instytut Centrum Zdrowia Matki Polki, Łódź, Poland
6
Institute of Mother and Child, Warsaw, Poland
2
The 22q11.2 deletion syndrome, also called the DiGeorge syndrome (OMIM 188400) is the
most common chromosomal deletion syndrome in humans with an incidence of 1 in 2-4000
live births. Although the clinical presentation of 22q11.2 syndrome is variable, the major
clinical characteristics of the syndrome are intellectual disability, congenital heart
anomalies, velopharyngeal abnormalities and characteristic facial appearance. In addition,
to the variability amongst patients with the “typical” phenotypic features, occasionally
(about 1/100) 22q11.2 deletion carriers present atypical malformations. Despite intensive
studies it remains unclear what causes this phenotypic variability of patients with the same
deletion. We hypothesize that the distinct rare features are caused by unmasking a
recessive allele which occurs at low frequency in the general population. In our study we
focused on patients with 22q11.2 deletion and one of the phenotypic features outside the
traditional 22q11.2 spectrum, like anorectal malformation, arthrogryposis, polymicrogyria,
eye anomalies, inner ear malformations and laryngeal web. For identification of the
functional role of genes within the common deletion we captured coding parts of the
remaining 22q11.2 region using custom designed Nimblegen capture arrays, and resequenced the enriched samples with 454 GS FLX Titanium chemistry. SNP analysis of the
remaining 3 Mb region, in 26 individuals with deletion and rare, atypical phenotypic feature,
showed surprising high diversity of genomic variants in re-sequenced genes. The recurrent
deleted region harbors approximately 50 genes. Each patient carried five to eighteen nonsynonymous mutations. Overall, mutations were identified in an astonishing 29 genes and
several patients carried stop mutations (which are thus nullisomic). For some of those, the
nullisomic mice were shown to be embryonic lethal. Further analysis performed based on
comparison of variants present in patients with the same phenotypic feature against
variants found in patients without that characteristic and candidate genes responsible for
particular feature, will be presented.
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O2: Genetic and functional evidence for a role of CYLD in Crohn’s Disease: results from a
European consortium
Isabelle Cleynen1, Emilie Vazeille2, Marta Artieda3, Magdalena Szczypiorska3, Marie-Agnès
Bringer2, Hein W. Verspaget4, Peter L. Lakatos5, Frank Seibold6, Kirstie Parnell7, Rinse K.
Weersma8, Jestinah M. Mahachie John9, Rebecca Morgan-Walsh10, Dominiek Staelens1,
Ingrid Arijs1, Stefan Müller11, Atilla Tordai12, Daniel W. Hommes4, Tariq Ahmad7, Cisca
Wijmenga13, Sylvia Pender10, Paul Rutgeerts1, Daniel Lottaz14, Kristel Van Steen9, Severine
Vermeire1 & Arlette Darfeuille-Michaud2
1
Department of Pathophysiology, KU Leuven, Leuven, Belgium
Inserm U1071, Université d’Auvergne, Clermont-Ferrand, France
3
Progenika Biopharma, S.A., Derio, Spain
4
Department of Gastroenterology and Hepatology, Leiden University Medical Center, Leiden, the
Netherlands
5
1st Department of Medicine, Semmelweis University, Hungary
6
Department of Gastroenterology, Spitalnetz Bern, Switzerland
7
Peninsula Medical School, University of Exeter & Plymouth, UK
8
Department of Gastroenterology and Hepatology, University Medical Center Groningen
9
Systems and Modeling Unit, Montefiore Institute, University of Liège, Belgium
10
Clinical and Experimental Sciences, Faculty of medicine, University of Southampton, UK
11
Department of Clinical Research, University of Bern, Switzerland
12
Hungarian National Blood Transfusion Service - Molecular Diagnostics
13
Department of Genetics, University Medical Center Groningen and the University of Groningen,
the Netherlands
14
Department of Rheumatology, Clinical Immunology and Allergology, University Hospital of Bern,
Switzerland
2
Background
Data from both humans and experimental animals underscore the critical role of intestinal
bacteria in the establishment and maintenance of inflammatory bowel disease (IBD). Host
defense to counteract bacterial colonization and maintaining mucosal integrity involves
intestinal proteases and protease inhibitors.
Methods
We performed a genetic association study of all top-ranked protease (and inhibitor) genes,
in a previously published systematic review (Cleynen I and Jüni P et al, PLoS One 2011). 185
haplotype tagging SNPs in 23 genes were genotyped in an exploratory dataset of 650
Crohn’s disease (CD) patients, and 542 healthy controls (HC). Validation was performed in
1670 CD and 1254 HC. Statistical analysis was performed using SVS v7.5.2 (crude association
analysis, additive genetic model), and plink v1.0.7 (meta-analysis of results in the
exploration and validation datasets, interaction analysis). A corrected p<0.05 was
considered statistically significant. The T84 epithelial cell line was used for functional
assessment of CYLD.
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Belgian Society of Human Genetics
Results
10 markers were found to be significantly associated with CD in the meta-analysis: 4
in USP40, 1 in APEH, 1 in USP3, and 4 in CYLD. The top signals were in CYLD, a cytoplasmic
deubiquitinating enzyme located adjacent to NOD2 on 16q12: rs12324931 (p=1.64e-18),
rs17314544 (p=1.06e-9), rs7205423 (p=1.89e-8), and rs1861762 (p=1.07e-5). A significant
interaction between ‘NOD2 overall’ and rs12324931 was found. In patients without
any NOD2 risk alleles, a significant CD-risk association with rs12324931 was present
(p=0.001, OR=4.05 [1.68-9.73]). Upon infection of T84 intestinal epithelial cells with the
adherent-invasive Escherichia coli (AIEC) strain LF82, the prototype strain of AIEC associated
with ileal CD, decreased CYLD expression was observed, leading to an increased ability of
LF82 AIEC to replicate within T84 cells (through CYLD siRNA transfection). Together with the
AIEC LF82-induced CYLD decrease, we observed proteasome-dependent degradation of the
NFκB inhibitor, IkB-α, in AIEC LF82 infected T84 cells, and an increased translocation of the
NFκB p65 subunit into the nucleus.
Conclusion
Our data provide strong genetic and functional evidence for a role for CYLD in CD
pathogenesis. We show that AIEC bacteria are able to take advantage of decreased CYLD to
replicate within host epithelial cells, and that CYLD acts as a negative, NFκB-mediated
regulator for E. coli colonization.
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O3: Computational annotation and interpretation of single nucleotide variation to identify
disease-causing variants by next-generation sequencing
Alejandro Sifrim1, Zeynep Kalender2, Jeroen Vanhoudt3, Georgios Pavlopoulos1, Stein Aerts2,
Joris Vermeesch3, Yves Moreau1& Jan Aerts1
1
ESAT/SCD, IBBT Future Health Department, University of Leuven
Laboratory for Computational Biology, Center for Human Genetics/VIB, University of
Leuven
3
Center for Human Genetics, University of Leuven
2
Background: Interpreting exome and genome sequencing data from patients to discover
disease-causing variants requires new computational and statistical analysis methods
because of the size and complexity of such data. Bottlenecks towards identifying the
variation underlying rare Mendelian diseases currently include poor cross-sample querying,
difficulty in setting cutoffs for data filtering, and computationally intensive and constantly
changing functional annotation.
Methods: We describe a methodology (embodied in our web application Annotate-it) that
makes it possible for geneticists to explore and analyze patient sequencing data towards the
identification of causal variants under several underlying genetic hypothese (recessive,
dominant, and de novo inheritance). As a novelty, Annotate-it offers interactive visual
analytics to effectively determine appropriate filtering criteria. We also propose a novel
pathway analysis technique based on regression models to discover significantly
overmutated pathways in groups of sequenced exomes, this allows for the discovery of
oligogenic etiologies. Results: We demonstrate the effectiveness of our strategy on two case
studies: Schinzel-Giedion syndrome and a semi-synthetic Miller syndrome data set. In these
datasets we show that simple analysis strategies show significant power when trying to
discover the cause of rare Mendelian disease.
Conclusions: Here we describe Annotate-it, a versatile framework for the analysis of
multisample SNV data generated by NGS. Annotation of samples is performed on the server
side, eliminating the need for the installation of complex tools and annotation sources by
the end-user and automatically keeping those annotations up to date. Thanks to novel
interactive visual analytics techniques, we facilitate the selection of optimal thresholds for
filtering through exploration of the underlying characteristics of the data. The query and
filtering interface enables the geneticist to quickly test different genetic hypotheses
(recessive, dominant, de novo) in multisample setups and aggregates available information
at the gene and variant level, facilitating the manual revision of candidate gene lists.
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O4: Deletion of KDM6A, a histone demethylase interacting with MLL2, in three patients
with Kabuki syndrome
Damien Lederer1, Bernard Grisart1, Maria C. Digilio2, Valérie Benoit1, Marianne Crespin1,
Sophie C. Ghariani3, Isabelle Maystadt1, Dallapiccola Bruno2 & Christine Verellen-Dumoulin1
1
Centre de Génétique Humaine, Institut de Pathologie et Génétique, Charleroi (Gosselies),
6041, Belgium
2
Medical Genetics, Bambino Gesù Pediatric Hospital, IRCCS, Rome, 00165, Italy
3
Centre Neurologique William Lennox, Neuropédiatrie, Ottignies, 1340, Belgium
Kabuki syndrome (KS) is a rare genetic cause of developmental delay and congenital
anomalies. Since the identification of MLL2 as the major causal gene in KS syndrome, MLL2
mutations have been identified in 56–76% of affected individuals, suggesting that there may
be additional KS genes. Using microarray CGH, we identified in two KS female patients a de
novo deletion of KDM6A (lysine-specific demethylase 6a), a gene located on the X
chromosome that encodes a histone demethylase that interacts with MLL2. Subsequently,
we looked for mutation of KDM6A and UTY (its Y chromosome paralogue) in 22 KS
individuals tested negative for MLL2 mutation and found an exonic deletion of KDM6A in
one male patient. Although KDM6A escapes X-inactivation, we found in female patients a
skewed X-inactivation pattern, with the deleted X chromosome inactivated in the majority
of cells. This study identified KDM6A as a second KS causal gene and highlights the growing
role of histone methylases/demethylases in multiple congenital anomaly/intellectual
disability syndromes.
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O5: Multiple large deletions in the mitochondrial DNA of human embryonic stem cells
Lindsey Van Haute1, Claudia Spits1, Sara Seneca2 & Karen Sermon1
1
Research Group Reproduction & Genetics, Faculty of Medicine and Pharmacy, Vrije
Universiteit Brussel, Belgium
2
Centre for Medical Genetics, UZ Brussel, Belgium
Human embryonic stem cells (hESC) are pluripotent cell lines derived from the inner cell
mass of blastocyst stage embryos. These cells have the capacity to differentiate into all
three embryonic germ layers, which makes them interesting in developmental biology,
regenerative medicine and in in vitro pharmacological studies.
Mitochondria contain their own, maternally inherited, DNA (mtDNA) that has a very high
mutation rate. Despite the increasing number of reports on the high instability of the
nuclear genome of hESC and the clear role of mitochondria in maintaining the pluripotent
state, little is known about the integrity of the mtDNA.
In this study, we screened for mtDNA deletions at different passages of 15 hESC lines. The
mtDNA was amplified by a PCR that generated a fragment of 8.7Kb in wild-type mtDNA. The
PCR products were visualized by agarose gel electrophoresis. The specificity of the PCR was
controlled by Sanger sequencing. Eleven breakpoints were determined and deletions
between 7045 and 8487 bp were found. Five different breakpoints were found in one single
cell line. The mtDNA mutations do not share identical regions of deletion or breakpoints,
and are not restricted to the common region of deletion seen in vivo in patients.
Two hypotheses could explain the origin of these mutations. Since different mutations are
already present at very early passages, it is possible that they originate from the embryo
that was used for the derivation of the line. Another possibility is that the deletions appear
spontaneously in culture due to low expression levels of genes involved in the mtDNA
replication and repair. To test this hypothesis, we measured the gene expression of four of
these genes by quantitative real-time RT-PCR and found a significant downregulation of
thymidine phosphorylase in hESC compared to human fibroblasts.
This study shows that the mtDNA of hESC is highly unstable. This should be taken into
account when performing research on hESC. Further study on the mtDNA of hESC is
necessary to check whether these cells can safely be used for clinical purposes.
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O6: Genome-wide association study identifies 13 loci associated with acute T-cell rejection
in Caucasian renal transplant patients.
Lidia Ghisdal1, Wouter Coppieters2, Yvon Lebranchu3, Eric Alamartine4, Yann Le Meur5, Guy
Touchard3, Christian Noël6, Marie Essig7, Pierre Merville8, Zineb Ajarchouh9, Miriana Dinic4,
Annick Massart1, Michel Geogres2, Daniel Abramowicz1 & Marc J. Abramowicz9
1
Nephrology-Renal Transplantation Department, Hôpital Erasme-ULB, Brussels, Belgium
GIGA institute, University of Liège, Belgium
3
Nephrology-Renal Transplantation Department, CHU Tours, France
4
Nephrology-Renal Transplantation Department, CHU Saint-Etienne, France
5
Nephrology-Renal Transplantation Department, CHU Brest, France
6
Nephrology-Renal Transplantation Department, CHRU Lille, France
7
Nephrology-Renal Transplantation Department, CHU Limoges, France
8
Nephrology-Renal Transplantation Department, CHU Bordeaux, France
9
Medical Genetics Clinic, Hôpital Erasme-ULB, Brussels, Belgium
2
Background: Acute rejection (AR) of renal allograft still affects ±15% of patients and remains
a major risk factor for chronic allograft dysfunction and graft loss. Since now, no study has
evaluated the genetic susceptibility of AR by a hy pothesis-free approach. The aim of our
study was to find out susceptibility loci associated with AR in Caucasian HLA-unsensitized
first renal transplant patients by a genome-wide association study (GWAS). Methods: We
have set up a belgo-french consortium providing a database and DNA samples for a total of
4127 renal transplant patients. Cases (n=275) were patients having ≥1 biopsy-proven T-cell
AR episode during the 1st year and hypercontrols (n=503) as patients without AR and a
stable graft function without proteinuria despite HLA B and DR mismatches. After exclusion
of degraded DNA (electrophoresis) and quantification (picogreen) in triplicate, individual
DNAs from cases and hypercontrols respectively were pooled in triplicate. Each triplicate
was hybridized on Human Omni 2.5-4 v1 DNA BeadChip (Illumina). Analyses were
performed with Genome Studio software. Results: After filtering data, excluding SNPs with a
mean allelic frequency difference of <10% (between cases and hypercontrols) and SNPs with
a variance (across triplicates) >0.001, we found 13 regions defined by at least 4 adjacent
SNPs (within a maximum interval of 50000 bases), including 6 protein-coding genes and 7
intergenic regions. One gene tagged by 7 adjacent SNPs is directly involved in TCR signalling,
and three loci indicate genes expressed in a specific cellular organelle. The replication of
those signals in an independent cohort is ongoing. Conclusions: We have found several loci
associated with T-cell AR in Caucasian HLA-unsensitized first renal transplant patients by a
genome-wide association study (GWAS), using a pooling method.
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O7: Unraveling a NOTCH1-lncRNA-miRNA regulatory network in acute T-cell lymphoblastic
leukemia and normal T-cell development
Kaat Durinck1, Pieter Mestdagh1, Tom Taghon2, Joni Van der Meulen1, Filip Pattyn1, Nadine
Van Roy1, Yves Benoit1, Bruce Poppe1, Pieter Van Vlierberghe1, Björn Menten1, Jo
Vandesompele1, Pieter Rondou1 & Frank Speleman1
1
Center for Medical Genetics, Ghent University, Belgium
Department of Clinical Chemistry, Microbiology and Immunology, Ghent University
Hospital, Belgium
2
Background
NOTCH1 acts as a central player in oncogenesis of several cancer entities, in particular T-cell
acute lymphoblastic leukemia (T-ALL). T-ALL is a hematological malignancy, characterized by
uncontrolled proliferation and arrested differentiation of precursor T-cells. Activating
NOTCH1 mutations are present in more than 50% of all T-ALL patients. Although the protein
coding regulatory network governed by NOTCH1 is extensively characterized, its function in
non-coding networks remains largely unexplored. In this study, we aim 1) to identify long
non-coding RNAs (lncRNAs) and microRNAs (miRNAs) whose activities are controlled by
NOTCH1 in normal and malignant T-cell development and 2) functionally validate relevant
candidate lncRNAs and miRNAs in vitro.
Methods
Using a γ-secretase inhibitor (compound E) interfering with NOTCH1 signaling in four T-ALL
cell lines (JURKAT, HPB-ALL, DND-41 and T-ALL1), transcriptional response of all protein
coding genes and 8000 lncRNAs was assessed using gene expression microarrays (Agilent).
In parallel, lncRNA expression profiles were also established for two sorted normal T-cell
progenitor populations derived from either CD34+ cord blood or thymus cells cultured
either with or without NOTCH1 stimulation using OP9-DL1 or OP9-GFP feeder layers. Finally,
a top-50 ranked list of highest expressed lncRNAs in eight different T-ALL cell lines was
generated upon RT-qPCR profiling of 1250 lncRNAs (Biogazelle). Next to lncRNAs, the
miRNAome (756 miRNAs) for all experimental conditions as well as nine distinct sorted
subsets of normal developing T-cell populations and 20 T-ALL cell lines were profiled using
high-throughput stem-loop qPCR (Applied Biosystems).
Results
Using a unique integrated experimental approach, we were able for the first time to identify
a subset of lncRNAs and miRNAs acting downstream of the NOTCH1 signaling cascade with a
presumed function in both normal and malignant T-cell development. Differentially
expressed lncRNAs and miRNAs between untreated (DMSO) and treated (compound E)
conditions for each T-ALL cell line were scored in a time course experiment. Established
NOTCH1 protein coding target genes were evaluated to validate the procedure. Correlation
analysis of the lncRNA-miRNA-mRNA data followed by gene set enrichment analysis (GSEA)
provided information on putative functional annotation for the identified lncRNAs. Amongst
the highest expressed lncRNAs in T-ALL cells, interesting candidates as SUZ12P, PTENP1 and
MALAT1 were identified, the latter being previously demonstrated to be up-regulated in
various solid tumors and playing a role in metastasis.
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Belgian Society of Human Genetics
Conclusions
We applied a unique experimental approach that enabled the first landscaping of a
comprehensive integrated network of lncRNAs and miRNAs acting downstream of the
NOTCH1 signaling pathway in normal T-cells and T-ALL. Given the central role of NOTCH1 in
T-ALL oncogenesis, these data pave the way towards development of novel therapeutic
strategies impacting on hyperactive NOTCH1 signaling.
30
Next Generation Sequencing and Recent Advances in Genetics
Belgian Society of Human Genetics
O8: Mutations in KIF11 cause Microcephaly-Lymphedema-Chorioretinal dysplasia
syndrome (MLCRD)
Pascal Brouillard1, Matthieu Schlögel1, Antonella Mendola1, Pia Ostergaard2, Arash
Ghalamkarpour1, Pradeep Vasudevan3, Bart L Loeys4, Koen Devriendt5, Thomy de Ravel de
l’Argentière5, Laurence Boon6, Nicole Revencu6, Jules G Leroy7, Lut van Laer4, Amihood
Singer8, Martin G Bialer9, Taija Makinen10, Peter S Mortimer11, Sahar Mansour12, Steve
Jeffery2, Miikka Vikkula1 & The Lymphedema Research Group13
1
Laboratory of Human Molecular Genetics, de Duve Institute, Université catholique de
Louvain, B-1200
2
Medical Genetics Unit, Biomedical Sciences, St George's University of London, London
SW17 0RE, UK
3
Clinical Genetics,University Hospitals of Leicester NHS Trust, Leicester Royal Infirmary,
Leicester, UK
4
Center for Medical Genetics, Antwerp University Hospital and University of Antwerp, B2650 Antwerp, Belgium
5
Katholieke Universiteit Leuven, Leuven, Belgium
6
Center for Vascular Anomalies, Cliniques Universitaires St-Luc, Brussels, Belgium
7
C. Hooft Children’s Hospital, Ghent University Hospital, B-9000 Ghent, Belgium
8
Pediatrics and Medical Genetics, Barzilai Medical Center, 78306 Ashkelon, Israel
9
Division of Medical Genetics, North Shore LIJ Health System, Manhasset, NY 11030, USA
10
Lymphatic Development Laboratory, Cancer Research UK London Research Institute,
London WC2A 3PX, UK
11
Department of Cardiac and Vascular Sciences, St George's University of London, London
SW17 0RE, UK
12
South West Thames Regional Genetics Service, St. George's University of London, London
SW17 0RE, UK
Microcephaly-Lymphedema-Chorioretinal dysplasia syndrome (MLCRD, MIM 152950) and
chorioretinal dysplasia, microcephaly and mental retardation syndrome (CDMMR, MIM
156590) have significant phenotypic overlap. They can be observed as an autosomal
dominant disorder with variable expressivity; mainly characterized by mild to severe
microcephaly, often associated with developmental delay, ocular defects and lymphedema,
essentially on the dorsum of the feet.
Using whole exome sequencing on the London platform, we discovered heterozygous KIF11
mutations in numerous individuals with MLCRD and CDMMR. All variants were either
nonsense, splice site, missense, or indels causing frameshifts, all predicted to have
deleterious impact on the protein function. KIF11 encodes the protein EG5, a homotetramer
kinesin motor. Members of this protein family are known to be involved in establishing a
bipolar spindle during cell mitosis, in chromosome positioning and in centrosome
separation. Blocking of KIF11 prevents centrosome migration and arrest cells in mitosis.
Thus, identification of KIF11 mutations in most patients with MLCRD suggests a central role
of EG5 in the development of retinal, cranial and lymphatic structures.
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Belgian Society of Human Genetics
O9: Outcome prediction of neuroblastoma patients using microRNA gene expression
profiling in both fresh frozen and archived tumor samples
Fjoralba Zeka1, Katleen De Preter1, Pieter Mestdagh1, Joëlle Vermeulen1, Arlene Naranjo2,
Isabella Bray3, Victoria Castel4, Caifu Chen5, Elzbieta Drozynska6, Angelika Eggert7, Michael
D. Hogarty8, Ewa Iżycka6, Wendy B. London9, Rosa Noguera10, Marta Piqueras10, Kenneth
Bryan3, Benjamin Schowe11, Peter van Sluis12, Jan J. Molenaar12, Alexander Schramm7,
Johannes H. Schulte7, Raymond L. Stallings3, Rogier Versteeg12, Geneviève Laureys13, Nadine
Van Roy1, Frank Speleman1 & Jo Vandesompele1
1
Center for Medical Genetics, Ghent University, Ghent, Belgium
Children's Oncology Group, University of Florida, Gainesville, FL, USA
3
Department of Cancer Genetics, Royal College of Surgeons in Ireland, Dublin, Ireland
4
Pediatric Oncology Unit, Hospital La Fe, Valencia, Spain
5
Applied Biosystems, Foster City, 94404 CA, USA
6
Department of Pediatric Haematology, Oncology and Endocrinology, Medical University Gdansk, Poland
7
Department of Pediatric Oncology and Haematology, University Children's Hospital Essen, Germany
8
Division of Oncology, The Children's Hospital of Philadelphia, Philadelphia, USA
9
Children’s Oncology Group, Children’s Hospital Boston/Dana-Farber Harvard Cancer Center, Boston, US
10
Department of Pathology, Medical School, University of Valencia, Spain
11
Department of 2 Computer Science, Technische Universität Dortmund, Dortmund, Germany
12
Department of Human Genetics, Academic Medical Center, Amsterdam, the Netherlands
13
Department of Paediatric Hematology and Oncology, Ghent University Hospital, Ghent, Belgium
2
Current risk classification criteria for neuroblastoma patients may result in suboptimal
classification, contributing to less effective cancer therapy. By developing a miRNA
prognostic signature we aim at achieving higher risk stratification accuracy and thus better
neuroblastoma survival rates.
480 mature human microRNAs were profiled in 51 fresh frozen tumor samples upon which a
set of 25 prognostic miRNAs was identified. The signature was tested in 179 fresh frozen
tumor samples and validated in another 304 fresh frozen samples and 75 formalin-fixed
paraffin-embedded (FFPE) samples.
The 25-gene miRNA signature could accurately predict progression-free survival (PFS) and
overall survival (OS) (p<0.0001) in the test cohort, independently from currently used risk
predictors. Patients with increased risk for shorter PFS and OS could also be identified within
the high-risk subgroups from the test cohort and the validation cohort. Remarkably, the
signature could also predict OS and PFS in the FFPE sample set (p<0.01).
In this study we present the largest neuroblastoma miRNA expression study so far, including
more than 500 neuroblastoma samples originating from fresh frozen primary tumor biopsies
and 75 FFPE samples. We established and validated a robust miRNA classifier, able to
identify patients with higher risk for adverse outcome within the current high-risk group.
We could also clearly demonstrate that miRNA expression patterns in FFPE samples can be
used for patient outcome prediction. We are currently collecting larger FFPE sample cohorts
to evaluate the miRNA signature performance in terms of substratification of the current
risk-groups.
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Next Generation Sequencing and Recent Advances in Genetics
Belgian Society of Human Genetics
O10: An unbiased high-throughput miRNA library screen identifies novel miRNA
regulators of key oncogenes and tumor suppressor genes implicated in different
cancer types
Gert Van Peer, Evelien Mets, Pieter Mestdagh, Annelies Fieuw, Pieter Rondou, Frank
Speleman & Jo Vandesompele
Center for Medical Genetics, Ghent University, Ghent, Belgium
MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate the expression of
protein coding genes at the post-transcriptional level, most often through interaction with
the 3'UTR. They have been implicated in oncogenesis in a wide variety of cancers,
deregulating the expression of protein coding oncogenes and tumor suppressor genes.
Although bioinformatic analyses suggest that individual genes can be regulated by multiple
miRNAs, most often only single miRNA interactions are validated, making it difficult to
appreciate the true complexity of miRNA-cancer gene interactions. Furthermore, validated
interactions are biased towards miRNAs that are predicted to target a given gene by
algorithms that often fail to reveal all true targets and thus generate false negative results.
Hence, biologically interesting miRNAs are potentially ignored. To address this issue we have
developed an unbiased high-throughput miRNA library screen to identify miRNA regulators
of a dozen key oncogenes and tumor suppressor genes implicated in a variety of cancers
including neuroblastoma, leukemia, breast cancer, colon cancer and lung cancer. To explore
interaction of individual miRNAs with the 3’UTR of a selected cancer gene, reporter gene
activity was measured upon either overexpression or silencing of 470 different miRNAs.
Validated positive control interactions generally came out among the strongest hits in the
screen. Furthermore, a significant enrichment for algorithm predicted miRNA regulators as
well as for miRNAs harboring seed sequences present in the respective 3‘UTRs could be
observed among the strongest hits for various genes, indicating the sensitivity and
robustness of the screen. Interestingly, we were able to identify miRNA-target interactions
that were not predicted, as well as interactions that function in a cooperative manner. Our
work significantly contributes to the identification of new miRNAs and cooperating networks
controlling oncogene and tumor suppressor gene expression and provides benchmark data
for improving current target prediction algorithms.
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Next Generation Sequencing and Recent Advances in Genetics
Belgian Society of Human Genetics
Poster Presentations
12th ANNUAL MEETING 2012
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Belgian Society of Human Genetics
P1: COX15 gene mutations causing COX deficiency and Leigh syndrome
Kim Vancampenhout1, Linda De Meirleir2, Sonia Van Dooren1, Joël Smet3, Rudy Van Coster3,
Urielle Ullmann1, Willy Lissens4, Maryse Bonduelle4 & Sara Seneca4
1
UZ Brussel, Brussels, Belgium
Vrije universiteit Brussel, Brussels, Belgium;UZ Brussel, division of Paediatric Neurology,
Brussels
3
University Hospital Ghent, division of Paediatric neurology and metabolism, Ghent,
Belgium
4
UZ Brussel, Brussels, Belgium; Vrije universiteit Brussel; Brussels, Belgium
2
Cytochrome c oxidase (COX) deficiency is one of the most frequently observed
abnormalities of the OXPHOS system. Different clinical phenotypes are seen in patients with
COX deficiencies. COX is the terminal complex of the mitochondrial respiratory chain and
catalyses the reduction of molecular oxygen to water. The complex is heterodimeric and
consists of 13 subunits, 3 encoded by the mitochondrial DNA and 10 encoded by nuclear
genes. Mutations in structural genes encoded by the nuclear or mitochondrial DNA are rare.
The majority of the COX defects are caused by mutations in nuclear genes encoding COX
related assembly factors (SURF1, SCO1, SCO2, COX10, COX15 and LRPPRC).
Here we describe a patient born to healthy parents, who presented with clinical and
neurological traits of Leigh syndrome as a result of an isolated COX deficiency. The patient
died at 5 months of age. Sequencing analysis revealed that the index patient is compound
heterozygous for 3 single base substitutions in the COX 15 gene i.e. a c.452C>G, a c.905C>T
and a c.1120T>C substitution. This gene encodes a heme a synthase, an enzyme involved in
the mitochondrial heme biosynthetic pathway. The c.452C>G substitution is a heterozygous
nonsense substitution that changes a serine into a premature stopcodon. The second
variant (c.905C>T) is a heterozygous missense substitution and changes the strong
conserved proline into a leucine at site 302. A final homozygous missense substitution
(c.1120T>C) was found in only one of the 2 splice variants of the gene. This substitution
concerns a change at a weakly conserved site and is, most probably, a polymorphism. These
variants were also detected in the family members of the index patient. The father and the
brother both carry the c.905C>T substitution and the c.1120T>C substitution. The c.452C>G
substitution and the c.1120T>C substitution were present in the COX15 gene of the mother.
To study the effect of these variants on the mRNA expression level we are optimizing a
relative real-time PCR assay. We expect to see a decrease in the mRNA expression of COX15
in the patient and hypothesize a less pronounced decrease in the expression level of the
mother.
Until this moment only four other cases with COX15 mutations have been described in
literature. The c.452G>C mutation has already been reported twice before. In all cases
symptoms appear at an early age and for 4 out of 5 cases (including our patient) have led to
a fatal outcome. In summary we can say that COX15 mutations are rare and have a very
poor prognosis.
36
Next Generation Sequencing and Recent Advances in Genetics
Belgian Society of Human Genetics
P2: Determining a role for germline mutations in intermediate risk breast and ovarian
cancer susceptibility genes in the Belgian population
Kim De Leeneer, Annelies De Jaegher, Ilse Coene, Brecht Crombez, Anne De Paepe, Bruce
Poppe & Kathleen Claes
Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium
Germline mutations in the coding and splice regions of BRCA1&2 are identified in
approximately 15% of patients selected for genetic testing in the Belgian population. It is
estimated that germline mutations in intermediate risk genes like PALB2, ATM, CHEK2,
BRIP1 and BARD1 account for an increased breast and ovarian cancer risk in approximately
5% of familial breast cancer. In this study we evaluated the mutation prevalence of PALB2
mutations in 285 Belgian patients from 256 independent BRCA1/2 mutation negative
families with a young age at onset and/or family history of breast and/or ovarian cancer.
The complete UTR, coding and splice site regions of PALB2 were analyzed with High
resolution melting followed by Sanger sequencing of the aberrant melting curves. In silico
predictions of variants with an unclear clinical significance was performed with the Alamut
software. In total we identified 20 unique sequence variants in PALB2 of which 6 are
previously unreported. Three novel unequivocal mono allelic mutations (PALB2
c.2834+1G>T c.2888delC and c.3423del4) were detected and shown to segregate with the
disease in the families. PALB2 c.2834+1G>T is a splice site mutation which undoubtedly will
lead to aberrant splicing; c.2888delC and c.3423del4 are both frame shift mutations in the
WD40 protein domain, responsible for the interaction with BRCA2. Furthermore, we
detected in nine patients 4 novel sequence variants (PALB2 c.-158G>C, c.498T>C, c.995T>A
and c.1520C>G) of which the clinical significance is currently unknown. Several in silico
analyses predict that PALB2 c.995T>A (p.Leu332His) could affect protein function. However,
segregation analysis revealed that the mother of the patient, diagnosed with breast cancer
at the age of 45 years did not carry the sequence variant. Deleterious PALB2 mutations were
only identified in familial breast cancer patients (3/208=1.4%) and not in 48 sporadic
patients with early onset or bilateral breast cancer. Interestingly, the average age at
diagnosis for the mutation carriers (62y; range 48-71) was higher compared to the average
age at diagnosis (39y, range: 25-78) of the non carriers. This limited role for PALB2 germline
mutations in the Belgian population, suggests locus heterogeneity and further investigations
in other intermediate risk breast and ovarian cancer susceptibility genes (ATM, CHEK2,
BRIP1 and BARD1) will be undertaken.
12th ANNUAL MEETING 2012
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Belgian Society of Human Genetics
P3: Screening for the involvement of genetic variants in BDNF in the pathogenesis of
childhood obesity
Doreen Zegers1, Sigri Beckers1, Ilse L. Mertens2, Kim Van Hoorenbeeck3, Stijn L. Verhulst3,
Raoul P. Rooman3, Kristine N. Desager3, Guy Massa4, Luc F. Van Gaal2 & Wim Van Hul1
1
Department of Medical Genetics, University of Antwerp
Department of Endocrinology, Diabetology and Metabolism, Antwerp University Hospital
3
Department of Paediatrics, Antwerp University Hospital
4
Department of Paediatrics, Virga Jesse Hospital, Hasselt
2
There is ample evidence that BDNF (brain-derived neurotrophic factor), a member of the
nerve growth factor family, has a role in the regulation of food intake and control of body
weight. Study of various animal models gave a clear indication that BDNF deficiency leads to
the development of obesity. Furthermore, it was stated that BDNF is a player in the leptinmelanocortin pathway and that expression of BDNF in the ventromedial hypothalamus
turned out to be dependent of MC4R signalling, which indicates an important role for BDNF
as a downstream effector of MC4R. Functional loss of one copy of the BDNF gene, due to
chromosomal rearrangements or microdeletions can cause an obesity phenotype in human
subjects. Therefore, we wanted to investigate whether point mutations in the gene also
result in a comparable phenotype.
We screened 554 severely overweight and obese children and adolescents and 561 lean
adults for mutations in the coding region of the BDNF gene. Mutation screening in all
patients and lean individuals was performed by high resolution melting curve analysis, using
the LightCycler® 480 System. When melting curves deviated from wild type, direct
sequencing was performed.
Screening of the obese patients led to the identification of 2 synonymous variations
(111g>a, V37V and 195c>t, H65H) and 2 heterozygous, non-synonymous, coding mutations
(T2I and V46M) in the BDNF gene. When we subsequently screened our extensive control
population for confirmation of these results, we found the T2I variant with comparable
frequency and confirmed that this is a rare and non-pathogenic variant in the BDNF gene. In
addition, we found another non-synonymous coding mutation (N187S) in the control
population.
In silico analysis of the V46M variant did not support a clear disease causing effect and no
family data were available in order to determine whether the mutation segregates with
obesity. However, we cannot rule out a possible pathogenic effect for this variant. In
general, we tend to conclude that mutations in the coding region of the BDNF gene are very
rare in obese patients and are therefore not likely to play an essential role in the
pathogenesis of childhood obesity.
38
Next Generation Sequencing and Recent Advances in Genetics
Belgian Society of Human Genetics
P4: Spondyloperipheral dysplasia as the mosaic form of platyspondylic lethal skeletal
dyplasia Torrance type in mother and fetus with the same COL2A1 mutation
Julie Désir1, Marie Cassart2, Catherine Donner3, Paul Coucke4, Marc Abramowicz1 & Geert
Mortier5
1
Medical Genetics Department, Hôpital Erasme-ULB, Route de Lennik 808, 1070 Brussels,
Belgium
2
Radiology Department, Hôpital Erasme-ULB, Route de Lennik 808, 1070 Brussels, Belgium
3
Fetal Medicine Department, Hôpital Erasme-ULB, Route de Lennik 808, 1070 Brussels,
Belgium
4
Center of Medical Genetics, Ghent University Hospital – UZGent, 9000 Gent, Belgium
5
Center of Medical Genetics, University Hospital of Antwerp – UZA, 2650 AntwerpenEdegem, Belgium
We describe a fetus with platyspondylic lethal skeletal dysplasia, Torrance type (PLSD-T), a
rare skeletal dysplasia characterized by platyspondyly, extremely short limbs, and mild
brachydactyly. Mutation analysis of COL2A1 identified a novel in-frame deletion
c.4458_4460delCTT (p.Phe1486del) in the C-propeptide region of the molecule, confirming
the clinical diagnosis. The phenotype in the mother was compatible with mild
spondyloperipheral dysplasia (SPPD). Molecular studies documented somatic mosaicism for
the same mutation in the mother. This observation further highlights the causal relationship
between PLSD-T and SPPD and emphasizes the importance of evaluating parents when
confronted with a skeletal dysplasia in a prenatal setting.
12th ANNUAL MEETING 2012
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Belgian Society of Human Genetics
P5: Mutation analysis of WNT10B in a Belgian population of obese children, adolescents
and adults
Jasmijn K. Van Camp1, Sigri Beckers1, Doreen Zegers1, Stijn L. Verhulst2, Kim Van
Hoorenbeeck2, Guy Massa3, An Verrijken4, Kristine N. Desager2, Luc F. Van Gaal4 & Wim Van
Hul1
1
Department of Medical Genetics, University of Antwerp, Universiteitsplein 1, 2610
Antwerp, Belgium
2
Department of Paediatrics, Antwerp University Hospital, Wilrijkstraat 10, 2650 Antwerp,
Belgium
3
Department of Paediatrics, Jessa Hospital, Stadsomvaart 11, 3500 Hasselt, Belgium
4
Department of Endocrinology, Diabetology and Metabolism, Antwerp University Hospital,
Wilrijkstraat 10, 2650 Antwerp, Belgium
Introduction: Wingless- type MMTV integration site family, member 10B (WNT10B) is an
activator of the Wnt pathway. The Wnt pathway is known to play an important role in
maintenance and differentiation of stem cells and has been implicated in the origination of
obesity. To evaluate the role of genetic variation in WNT10B in obesity further, we
performed a mutation analysis on Belgian obese patients and control subjects.
Materials: A mutation analysis of WNT10B by means of high-resolution melting curve
analysis and direct sequencing was performed on 546 obese children and adolescents
(mean Z-score of 2.6 and 2.5 respectively), 86 morbidly obese adults (mean BMI of 48.0 ±
0.4 kg/m²) and 399 lean, healthy controls (mean BMI of 22.1 ± 0.7 kg/m²).
Results: A total of six novel coding variants (S12S, A96A, G181D, G181G, R228Q and N335N)
and three intronic variants (g.-15C>A, IVS2+7C>T and IVS2+42G>C) were discovered in our
obese population. Additionally, seven coding variants and one intronic variant were found in
healthy individuals (of which R25R, A108P, S187R and P315S were found uniquely in control
samples). R228Q was the only coding, non-synonymous variant that was exclusively found in
patients, but the variant did not segregate in the family.
Conclusion: Coding variant frequency in lean individuals (2.0%) was higher than in patients
(1.1%) and familial segregation of the most promising variant in patients could not be
demonstrated. Therefore, we conclude that variations in WNT10B in our population are not
damaging for protein function and do not contribute to human monogenic obesity.
40
Next Generation Sequencing and Recent Advances in Genetics
Belgian Society of Human Genetics
P6: Starvation and overcrowding: culture conditions and DNA damage in embryonic stem
cells.
Kurt Jacobs1, Claudia Spits1, Mieke Geens1, Ilse J. Smolders2 & Karen D. Sermon1
1
2
Research group Reproduction and Genetics, Vrije Universiteit Brussel
Center for Neuroscience, Vrije Universiteit Brussel
Human embryonic stem cells (hESCs) are characterised by their self-renewal capacity and
pluripotency, and therefore offer huge possibilities for regenerative medicine and drug
testing amongst other. However, we (Spits et al., Nature biotech 26:1361, 2008) and others
(Maitra et al., Nature genetics 37:1099, 2005) have uncovered genomic instability during
long-term culture in hESCs, casting doubt on the safety of these cells in cell therapy. An
improved understanding of the effect of culture conditions on hESCs might contribute to the
preservation of genomic integrity, and thus safeguard their scientific and clinical value.
In high-density cultures, fewer nutrients are present per cell, possibly leading to a shortage
of substrates for nucleotide synthesis. A lack of nucleotides could then lead to stalled
replication forks, causing an increase in double-stranded DNA breaks (DSBs) and intra- and
inter-chromosomal rearrangements. To confirm this hypothesis, we plated 3 different hESC
lines (VUB07, VUB14 and VUB31) in 4 different densities and found a significant increase
(p<0.05) in the number of γH2AX foci, a marker for DSBs, in the more dense conditions. We
also noted a larger decrease in aspartate and glutamine (both essential for nucleotide
synthesis) concentration in dense cultures, together with a 110% increased DNA
fragmentation as shown by a single cell gel electrophoresis (COMET) assay. Moreover, we
suspect an increase of over 50% of the incidence of segmental chromosomal aberrations as
shown by FISH on chromosome 18. .
Our preliminary data indicate a correlation between culture density and the occurrence of
DNA breaks and segmental aberrations in hESC in culture. Bearing in mind that we find
these differences after 1 passage of 5 days, the long-term effect of culture density could
have a strong impact on the genetic stability of hESC cultures.
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Belgian Society of Human Genetics
P7: Developmental history of human embryos: How chromosomal chaos arises
Afroditi Mertzanidou1, Claudia Spits1, Hilde Van De Velde2 & Karen Sermon1
1
Research Group of Reproduction and Genetics, Vrije Universiteit Brussel, 1090 Brussels,
Belgium
2
Centre for Reproductive Medicine (CRG), UZ Brussel, 1090 Brussels, Belgium
Human oocytes and preimplantation embryos show high levels of aneuploidy and
mosaicism. The high incidence of chromosome abnormalities in preimplantation embryos
might explain the low success rate of IVF treatment cycles. Studies on polar bodies,
cleavage-stage embryos and blastocysts report high percentages of chromosomal
abnormalities in cleavage-stage embryos and a decrease at the blastocyst stage. Thus, the
third cleavage is considered as developmental stage at which a high number of
abnormalities arise, followed by a hypothetical elimination of abnormal embryos around the
morula stage. However, little is known about the day-4 of development to prove this
hypothesis. The aim of this study was to investigate the incidence of aneuploidy and
mosaicism in day-4 preimplantation embryos. For that reason, we analysed all the cells of
nine top-quality day-4 preimplantation embryos frozen at day 3 of development, by using
single cell array-CGH. Of the 145 analysed blastomeres, 24.1% were diploid and 75.9% had
chromosomal abnormalities. More in detail, 29% of the blastomeres had a single
monosomy, 4.8% had a single trisomy, 29.7% of the blastomeres exhibited complex
aneuploidy, 12.4% of the blastomeres carried only segmental aberrations and in 13 more
blastomeres segmental aberrations co-existed with whole chromosome abnormalities.
Additionally, analysis of all the blastomeres resulted in a complete set of karyotypes for
each cell in each embryo and enabled us to reconstitute the developmental history of each
embryo. We were able to determine the appearance of chromosomal abnormalities through
the consecutive cleavages in each cell line. The complex abnormalities found can only be
explained by combined events of meiotic errors, mitotic non-disjunction, anaphase lagging
and endoreduplication. Based on our results, it becomes apparent that good-quality frozenthawed well-developing day-4 embryos carry chromosomal abnormalities and exhibit high
rates of mosaicism. In these embryos at least, the hypothesis of correction at the morula
stage could not be proven. The data set we present is unique as it gives for the first time a
complete and detailed insight into the chromosomal status of the embryos at this stage of
preimplantation development.
42
Next Generation Sequencing and Recent Advances in Genetics
Belgian Society of Human Genetics
P8: Mutations in sFRP1 or sFRP4 are not a common cause of craniotubular hyperostoses
Eveline Boudin, Elke Piters, Igor Fijalkowski, Gino Stevenheydens & Wim Van Hul
Department of Medical Genetics, University of Antwerp, Belgium
Sclerosing bone dysplasias are a heterogeneous group of rare diseases marked by increased
BMD either caused by increased bone formation or by decreased bone resorption. In this
study we focused on the craniotubular hyperostosis mainly affecting the long bones and the
skull bones. Currently there are three causative genes identified namely LRP5, SOST and
LRP4 and they are all involved in the canonical Wnt signaling pathway. These findings
support the role of this pathway in regulating bone formation. Therefore we selected sFRP1
and 4, two members of the secreted Frizzled related protein (sFRP) family, as candidate
genes for mutation analysis in patients diagnosed with a disease form of craniotubular
hyperostosis. The sFRPs can modulate the Wnt signaling pathway by binding to Wnt ligands
or Frizzled receptors. Studies using mice models showed that both selected genes have an
important influence on bone formation. A sfrp1-/- mouse shows increased BMD values
especially after peak BMD was reached. On the contrary, transgenic sfrp4 overexpression
mice exhibit reduced BMD.
Using Sanger sequencing we screened the exons and intron/exon boundaries of sFRP1 and 4
in 58 patients. All patients were first tested for mutations in the three known genes. We
identified three unknown heterozygous variants, two in sFRP1 and one in sFRP4. The first
variant in sFRP1 is an intronic variant which, according to prediction programs, does not
affect the splicing of the gene. The second variant (p.Trp131Arg/-) was identified in a young
boy whose affected mother does not carry the variant. The third mutation we identified is a
p.Lys315Gln/- amino acid change in a female patient whose sister was diagnosed with the
same disease but does not carry the mutation.
In conclusion, based on our studies we can say that neither mutations in sFRP1 nor in sFRP4
are a common cause of craniotubular hyperostoses. Further research will be necessary to
identify the disease causing gene(s) in this group of patients.
12th ANNUAL MEETING 2012
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Belgian Society of Human Genetics
P9: Unraveling the selective advantage of the 20q11.21 amplification in human embryonic
stem cells
Ha Nguyen, Mieke Geens, Afroditi Mertzanidou, Kurt Jacobs, Karen D. Sermon & Claudia
Spits
Research Group Reproduction and Genetics, Vrije Universiteit Brussel
Owning to the capacities of unlimited self-renewal and differentiation into any cell types of
the human body, human embryonic stem cells (hESCs) are considered as a valuable
potential source of cells for regenerative medicine and as new models for developmental
biology research and drug discovery. However, maintaining and expanding hESCs
continuously in vitro can lead to culture adaptation and acquisition of chromosomal
abnormalities. Recently, amplification of 20q11.21 has been reported as a hot spot of
chromosomal abnormality in hESCs. This finding suggests that this mutation may provide
hESCs a selective advantage, but the mechanisms and pathways by which the 20q11.21
amplification affects the cells remain to be elucidated.
We hypothesize that the increase of gene dosage in the amplified region has an impact at
the transcriptional level, which in turn will affect the cellular pathway(s) that directly or
indirectly regulate cell survival, proliferation or self-renewal processes. In our lab, we have
identified four hESC lines carrying the 20q11.21 duplication. We have established the
smallest common region of duplication in these four lines by oligonucleotide-arrays based
comparative genomic hybridization and quantitative real-time PCR (qRT-PCR). We have
found that the minimal amplicon is approximately 1Mb and spans from the DEFB116 to
KIF3B genes. We performed gene-expression arrays on the four hESC lines with and without
duplication, and the results were validated using qRT-PCR. The gene expression results show
significant upregulation of the genes ID1 (inhibition of differentiation), TPX2, KIF3B (cell
cycle regulation) and POFUT1 (NOTCH signaling pathway) located in the region of
duplication, and deregulation of the genes CHCHD2, TRPC6 and RNF128 located elsewhere
in the genome. Several functional tests were carried out to characterize the phenotype of
the mutant cells. TUNEL testing indicated that hESCs lines with the 20q11.21 amplification
had a lower percentage of apoptotic cells, but the clonogenic efficiency experiments
showed no significant differences between the hESC lines. RHOA activation assays were
performed to evaluate the levels of the active RHOA, a down-stream factor of CHCHD2, but
no significant differences were detected. Further work is ongoing on the phenotypical
characterization of the cells, as well as knock-in experiments of ID1, TPX2, and POFUT1 in
normal hESCs to identify the gene that is the key driver of this mutation.
44
Next Generation Sequencing and Recent Advances in Genetics
Belgian Society of Human Genetics
P10: Identification of 15 novel mutations in Sotos syndrome
Sebastien Boulanger1, Melanie d'Amico1, Anne Destree1, Isabelle Maystadt1, Damien
Lederer1, Hilde Van Esch2, Jenneke van den Ende3, Nathalie Van der Aa3, Karin Segers4 &
Pascale Hilbert1
1
Human Genetics Center, Institute of Pathology and Genetics, Charleroi, Belgium
Human Genetics Center, K.U.Leuven, Belgium
3
Department of Medical Genetics, University of Antwerp, Belgium
4
Department of Human Genetics, CHU University of Liege, Belgium
2
Sotos syndrome (MIM 117550) is a genetic disorder characterized by three cardinal
features; distinctive facial appearance, overgrowth and learning disability. Moreover, major
features can also occur; behavioral problems, cardiac anomalies, renal anomalies, scoliosis
and seizures. This syndrome is inherited in an autosomal dominant manner but more than
95% of patients have a de novo mutation.
NSD1 (nuclear receptor SET domain1) is the only gene known to be associated with Sotos
syndrome and a mutation in that gene is found in about 80-90% of affected individuals. The
gene contains 23 exons encoding a 2696 amino acids protein.
Depending on the criteria defined for patient screening, 27-93% (among non-Japanese
individuals) of point mutations are found, approximately 5% of partial or total gene deletion
and about 10% have a 5q35 microdeletion that encompasses NSD1 gene.
The experience in our Human Genetics Center is based on a total of 86 patients presenting
at least the 3 cardinal features but referred by different physicians working anywhere in
Belgium.
The entire coding region of NSD1 gene was sequenced and large rearrangements were
investigated by MLPA (Multiple Ligation dependent Probe Amplification from MRC-Holland).
We here describe the clinical features for 18 patients (~21%) for whom an intragenic
mutation has been found. Among the mutations detected (6 nonsense, 7 missense and 5
frameshifts), 15 are novel. Up to now, no large rearrangements have been found in our
population.
12th ANNUAL MEETING 2012
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Belgian Society of Human Genetics
P11: Detection of inherited mutations for Alport syndrome using Next Generation
Sequencing.
Pascale Hilbert1, Marie-Line Lizon1, Dirk Goossens2, Jurgen Del-Favero2 & Karin Dahan1
1
2
IPG- 25 Av. G. Lemaitre, 6041Gosselies
Multiplicom NV, Waterfront Research Park, Galileilaan, B-2845 Niel
Alport Syndrome is a progressive renal disease with cochlear and ocular involvement. The
most common form (approximately 80%) is inherited in an X-linked pattern. The autosomal
recessive and dominant forms constitute about 15% of the cases. X-linked Alport Syndrome
is caused by mutations in the type IV collagen alpha chain 5 (COL4A5) whereas the type IV
collagen alpha chain 3 (COL4A3) and 4 (COL4A4) are responsible for recessive and dominant
Alport syndrome. Genetic testing for mutations in the 52, 48 and 51 exons of COL4A3,
COL4A4 and COL4A5 has become an integral part of clinical practice but the use of
conventional Sanger sequencing is time-consuming and expensive. To determine whether
amplicon based Next Generation Sequencing (NGS) would enable accurate, thorough, and
cost-effective identification of Alport syndrome, we set up a project with Multiplicom
(www.multiplicom.com) to design a two step multiplex PCR for the amplification of all the
coding regions of the three responsible genes. 149 amplicons ranging from 270 Bp to 510 Bp
were amplified in 4 multiplex PCR reactions. Genomic DNA from 24 patients with known
inherited mutations or polymorphisms were studied. To differentiate patients within a
single run, Multiple Identifiers (MIDs) were introduced during the PCR. The sequences were
run on a GS-FLX 454 (Roche) (titanium chemistry) and analyzed with the SeqNext module of
Sequence Pilot software (JSI medical). All the variants (21 mutations plus frequent SNPs)
were confirmed and there were zero false positive calls. Our data confirm that amplicon
based NGS will allow widespread genetic testing and genetic counseling for Alport syndrome
46
Next Generation Sequencing and Recent Advances in Genetics
Belgian Society of Human Genetics
P12: Bioinformatic pipeline for Next Generation Sequencing platform of UCL - de Duve
Institute
Raphael Helaers, Mustapha Amyere & Miikka Vikkula
UCL - de Duve Institute - GEHU
The field of human genetics is being revolutionized by exome and genome sequencing. A
massive amount of data is being produced, always faster. For example, targeted exome
sequencing can be completed in a few days using NGS, allowing new variant discovery in a
few weeks. To remain at the forefront of research, the Université catholique de Louvain has
recently acquired a next generation sequencing platform, installed in the Laboratory of
Human Genetics at de Duve Institute. The platform is composed of a Solid 5500XL (Life
technologies), a Personal Gene Machine (Ion Torrent, Life technologies) and a cluster for
bioinformatics processing. The platform should offer services like full exome sequencing and
RNA Seq, accessible to the Belgian research community.
To analyze the huge amount of data produced by NGS, a bioinformatics pipeline was
established, using e.g. Lifescope (commercial package from Life technologies), a
combination of open source packages (BWA, GATK, snpEff), as well as home-made filters. In
this poster, we present the different stages of the pipeline and detail how to obtain data
manageable by researchers. The final output includes an Excel file containing the most likely
damaging variants with their potential effect and a series of filtering possibilities.
12th ANNUAL MEETING 2012
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Belgian Society of Human Genetics
P13: Genetic causes of primary lymphedema
A. Mendola1, M Schlögel 1, H.L. Nguyen1, A. Ghalamkarpour1, Y. Sznajer2, D. Thomas3, N.
Revencu2, K. Devriendt4, E. Legius4, G. Pierquin5, R. Hennekam6, G.A. Diaz7, J.B. Mulliken8,
L.M. Boon9, P. Brouillard 1, M. Vikkula1 & The Lymphedema Research Group10
1
Laboratory of Human Molecular Genetics, de Duve Institute, Université catholique de
Louvain
2
Center for Human Genetics, Cliniques Universitaires St Luc
3
Unité diagnostic anténatal, Hôpitaux Iris Sud
4
Center for Human Genetics, University Hospitals Leuven
5
Center for Human Genetics, University Hospital Center-CHU Sart-Tilman, University of
Liège
6
Department of Paediatrics, AMC, Amsterdam, The Netherlands and Clinical and Molecular
Genetics Unit
7
Departments of Genetics & Genomic Sciences, Mount Sinai School of Medicine, New York,
USA
8
Vascular Anomalies Center, Plastic Surgery, Children’s Hospital, Harvard Medical School,
NY, USA
9
Centre for Vascular Anomalies, Cliniques Universitaires St Luc
Lymphedema is caused by a dysfunction of the lymphatic system. It exhibits swelling of one
or more extremeties due to an abnormal lymphatic transport of protein-rich fluid.
Lymphedema can be primary and secondary. The hereditary lymphedema may occur in an
autosomal dominant or a recessive manner.
By dHPLC, high resolution melting and sequencing, we have screened different candidate
genes from a large panel of primary lymphedema samples (sporadic and familial, n>400).
These led us to discover several mutations in the three well-known lymphatic genes:
VEGFR3, FOXC2 and SOX18. The VEGFR3 gene, which codes for a receptor tyrosine kinase, is
mutated in patients with primary congenital lymphedema, or Nonne-Milroy disease.
Mutations in FOXC2 and SOX18, two transcription factors, cause, respectively lymphedemadistichiasis syndrome and hypotrichosis-lymphedema-telangiectasia syndrome. We
screened two more recently identified genes: CCBE1 and PTPN14. Mutations were identified
in the CCBE1 gene in only patients with Hennekam syndrome. The PTPN14 gene, which
encodes a non-receptor tyrosine phosphotase, seems only to be mutated in patients with
choanal atresia.
Independently, each known altered lymphangiogenic gene can be attributed to a specific
lymphatic disease. Therefore, the identification of the causative gene should help in more
precise diagnosis, follow-up and treatment.Nevertheless, we did not find a causative
mutation for a large number of patients (90%), suggesting that other genes should be
implicated. We are currently screening the recently implicated GJC2 and GATA2 genes in our
series. New technologies, such as massive parallel sequencing, should lead us to discover
new genes causing primary lymphedema.
48
Next Generation Sequencing and Recent Advances in Genetics
Belgian Society of Human Genetics
P14: Inactivation of glomulin causes glomuvenous malformations in man, and results in
early embryonic lethality in mouse.
P Brouillard1, M Amyere1, B McIntyre1, Y Achouri2, P Jacquemin3, F Lemaigre3, L Boon4 & M
Vikkula1
1
Human Molecular Genetics, de Duve Institute, Université catholique de Louvain, Brussels,
Belgium
2
Transgenesis core, Université catholique de Louvain, Brussels, Belgium
3
Liver and Pancreas Development, de Duve Institute, Université catholique de Louvain,
Brussels, Belgi
4
Center for Vascular Anomalies, Cliniques Universitaires St-Luc, Brussels, Belgium
Glomuvenous malformations (GVM, MIM #13800) are characterized by aberrantly
differentiated vSMC-like “glomus cells” around distended vein-like channels. We showed
that the lesions result from complete lack of glomulin due to the combination of a germline
and a somatic second-hit loss-of-function mutation in glomulin. To generate a model in
which to test potential therapies, and to understand the developmental role of glomulin, we
inactivated the murine gene by inserting the LacZ reporter gene at the start codon. Glmnheterozygotes are healthy for more than one-year-old, and show wide vascular expression
of LacZ. In contrast, no live knockouts are obtained, embryonic lethality occurring around
E8.5, with severe mesodermal defects.
To enable studies beyond the lethality time point, we generated transgenics with
conditionally inducible expression of two glomulin-specific RNAi. When down-regulation of
glomulin is induced ubiquitously, embryonic lethality ensues. However, knockdown embryos
develop further than the knockouts, and show vascular abnormalities, such as absent yolksac vasculature and hemorrhages. The early lethality in absence of glomulin suggests a
crucial role in embryogenesis, even before vascular development. Spatio-temporally
controlled induction is thus needed to elucidate the role(s) of glomulin during development,
and to mimic the double-hit mechanism underlying GVM, so as to obtain a model to study
novel GVM therapies.
12th ANNUAL MEETING 2012
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Belgian Society of Human Genetics
P15: Exome sequencing in syndromic patients with congenital heart disease performing
trio analysis
Jacoba J. Louw1, Yaojuan Jia2, Anniek Corveleyn2, Marc Gewillig3 & Koenraad Devriendt4
1
Centre for Human Genetics, KULeuven; Pediatric and Congenital Cardiology, UZLeuven
Centre for Human Genetics, KULeuven
3
Pediatric and Congenital Cardiology, UZLeuven
4
Centre for Human Genetics, KULeuven, UZ Leuven
2
Congenital heart defects (CHD) are a major cause of infant morbidity and mortality.
Reaching an etiological diagnosis in patients with a syndromic heart defect is important, not
only to gain insight into their pathogenesis and genetic counseling on recurrence risks, but
especially with regard to providing information on the future prospective, based on
knowledge on the natural course of the disorder.
In syndromic cases, an exact etiological diagnosis can be reached in an estimated 50-60%,
following careful clinical evaluation, complemented by various genetic tests, including arrayCGH. With the advent of exome sequencing, it is now feasible to perform a trio analysis, i.e.
sequencing of the coding parts of the genes in both parents and the child, where only the
child is affected, in order to identify a candidate gene.
For syndromic cases, we hypothesize that these patients have a thus far not recognized
monogenic condition, responsible for both the intelligence deficit and the heart defect.
Since the vast majority of syndromes featuring CHD are dominant, it is likely that at least in
a subset of these, a de novo dominant mutation is present.
In-solution capture (Nimblegen target enrichment system) and sequencing will be done on
the Illumina HiSeq2000 platform (Genomics Core KULeuven/ UZLeuven). Data analysis will
be done using commercial and in-house developed software. Afterward, the variants will be
annotated by Annovar to allow filtering of all found variants in order to identify potential
causal mutations.
Preliminary results on the exome sequencing of five sets of trio’s, with the child presenting
with congenital heart disease, dysmorphic features and mental retardation, will be
discussed.
50
Next Generation Sequencing and Recent Advances in Genetics
Belgian Society of Human Genetics
P16: The LMNA c. 1968+5G>C transversion leads to progerin generation and Hutchinson–
Gilford progeria syndrome.
V Benoit1, P Hilbert1, I Maystadt1, C Gaspard1, S Castedo 2 & T Kay2
1
2
Human Genetics Center, Inst. of Pathology and Genetics, Charleroi, Belgium
Lisboa Central-Hospital D.Estefânia, Lisboa, Portugal
Hutchinson–Gilford progeria syndrome (HGPS) is a very rare fatal genetic disorder
characterized by precocious ageing in children. Children with HGPS appear healthy at birth
and distinctive clinical features appear within the first years of life (including growth
retardation, alopecia, lipodystrophy, and atherosclerosis). Most of them die from coronary
artery disease at an average age of 13 years. Mutations in the Lamin A/C encoding gene
(LMNA) are described to be responsible for the disease, most HGPS patients carrying the
1824C>T mutation (G608G) in exon 11. This transversion activates a cryptic donor splice site.
Lamin A products in these patients then consist of mature Lamin A and progerin, a 50
amino-acids deleted Lamin A protein that exerts a dominant negative effect.
In a 6 year-old patient affected by HGPS, we have identified a 1968+5G>C variation in the
intron 11 of the LMNA gene. This mutation was not present in the DNA of the patient’s
parents, indicating that it arose de novo. This variant has already been evoked in the LOVD
database; the question of its implication in HGPS has been raised but let unsolved with
unknown pathogenicity. It seems that no further study has been undertaken as the question
of a possible modified splicing still remained.
We performed RNA studies and clearly showed that our patient’s RNA generated different
splicing products, including the normal one and at least one shorter form. By sequencing
these products, we were able to demonstrate that this shorter form corresponds to a
truncated RNA lacking 150 nucleotides and that the sequence perfectly matched the
sequence of progerin.
Taken together, our data unequivocally demonstrates that the c.1968+5G>C transversion is
a causal mutation of HGPS. Moreover this work reinforces the concept of progerin
involvement as a crucial actor in HGPS physiopathology.
12th ANNUAL MEETING 2012
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Belgian Society of Human Genetics
P17: Mitotic microhomology-mediated replication-based mechanisms underly nonrecurrent pathogenic microdeletions of the FOXL2 gene or its regulatory domain
Hannah Verdin1, Barbara D'haene1, Yana Novikova1, Diane Beysen2, Pablo Lapunzina3, Julian
Nevado3, Claudia Carvalho4, James R. Lupski4, Björn Menten1 & Elfride De Baere1
1
Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium
Department of Pediatrics, Ghent University Hospital, Ghent, Belgium
3
INGEMM, Universidad Autónoma de Madrid, Madrid, CIBERER, U783-ISCIII, Spain
4
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX,
USA
2
Genomic disorders are often caused by recurrent pathogenic copy number variations
(CNVs), with meiotic, non-allelic, homologous recombination as underlying mechanism. A
recent study by Vissers et al. 2009 suggested that rare pathogenic CNVs spread throughout
the genome are microhomology-mediated and stimulated by local genomic architecture.
Here, it was our aim to study whether such mitotic mechanisms and local genomic
architecture contribute to a series of non-recurrent, pathogenic, locus-specific CNVs. To this
end, we fine-mapped 44 rare pathogenic microdeletions encompassing the FOXL2 gene and
neighbouring region (34), or its regulatory domain (10) respectively, both leading to
blepharophimosis syndrome (BPES). For the breakpoint mapping we used targeted
arrayCGH, qPCR, long-range PCR and sequencing of the junction products. Microhomology,
ranging from 1 bp to 34 bp, was found in 86,7% of the breakpoint junctions, being
significantly enriched in comparison with a random control sample. This suggests that
microhomology-mediated repair mechanisms, such as fork stalling and template switching
(FoSTeS), microhomology-mediated break-induced replication (MMBIR), or alternative nonhomologous end-joining (MMEJ), underly these microdeletions. Moreover, genomic
architectural features, like sequence motifs, non-B DNA conformations and repetitive
elements, were found in all breakpoint regions. In conclusion, we propose that the majority
of these deletions is not caused by meiotic homology-based mechanisms but by
microhomology-mediated replication-based mechanisms like FoSTeS or MMBIR, and thus
have a mitotic origin instead. Finally the genomic architecture might stimulate the formation
of these rare deletions by increasing the susceptibility for DNA breakage or promote fork
stalling.
52
Next Generation Sequencing and Recent Advances in Genetics
Belgian Society of Human Genetics
P18: Targeted sequencing of non-syndromic familial CHD
Yaojuan Jia, Jacoba Louw, Jeroen Breckpot, Koen Devriendt & Anniek Corveleyn
Center for Human Genetics, K.U.Leuven, Belgium
Advances in genetic sequencing technology have the potential to enhance testing for genes
associated with genetically heterogeneous diseases, such as Congenital Heart Defects (CHD).
CHD refers to abnormalities in the structure or function of the heart and great vessels that
arise before birth, occurring in 5 to 10 per 1000 live births. The genetics of CHD is
heterogeneous: not only can different genes be involved, also different inheritance patterns
exist. CHD can be associated with various monogenic syndromes such as Holt-Oram
syndrome, Alagille syndrome and Noonan syndrome, and chromosomal syndromes such as
Down syndrome (trisomy 21), or microdeletion syndromes including Williams syndrome (del
7q11.2) or DiGeorge syndrome (22q11.2 deletion). However, the majority (~80%) of CHD
occurs as an isolated defect which is not associated with other manifestations (nonsyndromic CHD). Non-syndromic CHD that occur familial represent ~3% of all CHD cases.
Classical positional genetics approaches for identification of disease genes involved in nonsydromic CHD is difficult because often no DNA is available from deceased individuals from
previous generations. However, resequencing all genes using next generation sequencing
technology offers the unique opportunity to identify causative mutations, even when a
limited number of cases are available in the family.
The objective of this study is to investigate the performance characteristics of target
sequence capturing technology coupled with massively parallel sequencing, and to
determine the proportion of non-syndromic familial CHD cases that can be explained by the
currently known cardiac genes.
Therefore, we selected non-syndromic CHD families with 2 or more affected family
members sharing clear and specific phenotypes. The disorders in these families are assumed
to be monogenic. We captured all exons and the promoter regions of 43 CHD associated
genes (~300kb) using NimbleGen sequence capture arrays. With a stringent filter removing
repetitive sequences from the target region, the probe design covered over 94% of the
target bases. The target genes are implicated in non-syndromic CHD and were selected via
CHD Wiki (http://homes.esat.kuleuven.be/~bioiuser/chdwiki). The sequencing was
performed on the 454 FLX platform or the Illumina HiSeq 2000 platform. We initially
compared and evaluated the two different NGS platforms available in our center (GS-FLX
platform and Illumina HiSeq platform). With both platforms, we sequenced 2 control
samples with known SNPs in our target genes that were previously detected by either SNP
array or Sanger sequencing. Both platforms detected these SNPs with no false negatives.
The HiSeq platform provided much higher sequencing depth in comparison with the GS-FLX
system. We will also compare the differences in variant detection resulting from different
alignment and SNP calling pipelines (CASAVA/GATK) for the HiSeq platform.
12th ANNUAL MEETING 2012
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Belgian Society of Human Genetics
P19: Extension of Healthy Life Using Preventive Genomics
Raf Winand1, Pascal Borry2, Leen d'Haenens3, Jan Derboven3, Jos Dumortier4, David Geerts3,
Griet Verhenneman4, Joris Vermeesch5, Yves Moreau1 & Jan Aerts1
1
KU Leuven - ESAT - Bioinformatics
KU Leuven - CBMER
3
KU Leuven - CMC
4
KU Leuven - ICRI
5
KU Leuven - CME
2
The last decade has seen a tremendous evolution in the field of human genetics. Nextgeneration sequencing methods have made it possible to move research from a one-geneat-a-time approach to a whole-genome view. Furthermore, an always increasing number of
diseases has been associated with specific polymorphisms and mutations. We believe that
these developments will lead to a new field of preventive genomics, which leverages whole
genome DNA sequence information to extend the healthy life of people. Knowledge about
genetic predisposition for certain diseases can aid in detecting these at an early stage and
therefore improving prognosis. It could also inform individuals on the influence of changes
in behaviour or lifestyle to prevent or delay the onset of disease. Knowledge about carrier
status for rare diseases can also be used when planning for children. Finally, it can be used
within pharmacogenomics to inform physicians on the specific medication dosage at the
level of an individual patient. We will look at the technical, predictive and preventive use of
genomics within this context. Within this project, we will develop data analysis and
reporting tools that extract the relevant information from a genome sequence and present
it in a comprehensible manner to the end-user or general practitioner.
54
Next Generation Sequencing and Recent Advances in Genetics
Belgian Society of Human Genetics
P20: Structural variation of chromosomes in individuals with autism spectrum disorders
Sajid Iqbal1, Kris Van Den Bogaert1, Joris Vermeesch1, P De Cock2, K Ballon2, H Olivie2, J
Teyaert3, J-P Frijns1, K Devriendt1 & Hilde Peeters1
1
Center for Human Genetics, University Hospitals Leuven, Belgium
Department of Child Neurology, University Hospitals Leuven, Belgium
3
Department of Child and Adolescent Psychiatry, University Hospitals Leuven, Belgium
2
The genetic causes of autism spectrum disorders (ASDs) are heterogeneous and still
unknown in the majority of cases. Structural chromosomal variants were found in
sufficiently high frequency to suggest that cytogenetic and microarray analyses be
considered in routine clinical workup. However the number of aberrations found in ASD
patients is highly variable between different studies and depends on the inclusion criteria,
array resolution and differences in interpretation due to uncertainty on the true etiological
role of particular variants. Since the objective of most of these studies is the identification of
ASD risk variants, emphasis is often put on the number of patients rather than on
phenotypic data, family history and segregation. Nevertheless, these clinical data are
essential for the interpretation of the research findings into clinical diagnostics which is
complicated by ascertainment, penetrance, variable expressivity and multigenic inheritance.
We report on structural variations, phenotypic and relevant family data of a clinical cohort
of over 300 patients with ASD with or without multiple congenital anomaly/intellectual
disability syndromes. Patients with recognizable clinical syndromes were excluded. The ASD
diagnosis was made by a multidisciplinary team. With this report we aim to contribute to
the clinical validation of the current knowledge on ASD risk variants.
12th ANNUAL MEETING 2012
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Belgian Society of Human Genetics
P21: Unusual presentation of combined saggital-metopic synostosis may represent a novel
autosomal dominant craniosynostosis syndrome
Alexander Janssens1, Philippe Jeannin1, Paul J. Coucke2, Anne De Paepe2 & Olivier M.
Vanakker2
1
2
Department of Paediatrics, Jan Palfijn Hospital, Ghent, Belgium
Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium
Background
Craniosynostosis, caused by early fusion of one or more cranial sutures, can include
premature fusion of the saggital (scaphocephaly) or metopic suture (trigonocephaly).
Though often occuring as isolated findings, their co-existence in a craniosynostosis
syndrome is infrequent and mostly sporadic.
Case description
The male proband, born at term after an uneventful pregnancy, presented at birth with
premature fusion of the saggital and metopic suture. Antropometric measurements were
normal. Besides the scaphocephaly, a prominent boney mass on the forehead, several
centimeters in height and width, was noted, next to proptosis. Imaging suggested the
frontal mass to orgininate partially from the fused metopic suture, in addition to a
superimposed exostosis. Radiographs did not reveal other exostoses, though bilateral
agenesis of the middle phalanges in the feet was noted. Family history revealed the father,
his sister and half-sister, to have an isolated scaphocephaly with variable cutaneous
syndactyly. The paternal grandfather did not have craniofacial anomalies nor syndactyly,
though the great grandfather was said to have isolated syndactyly of both hands.
Conclusion
This four-generation family shows various expression of a craniosynostosis phenotype with
scaphocephaly and a particularly severe and unusual form of trigonocephaly. Several known
craniosynostosis genes (FGFR2, FGFR3, TWIST) have already been excluded. A saggitalmetopic synostosis together with agenesis of the middle phalanges has to our knowledge
not been reported before. Considering the family history, this is suggestive for a novel,
variable autosomal dominant craniosynostosis syndrome with possibility of gonadal
mosaicism or non-penetrance.
56
Next Generation Sequencing and Recent Advances in Genetics
Belgian Society of Human Genetics
P22: Computational identification of candidate genes for congenital heart defects,
congenital diaphragmatic hernia, and asthma
Léon-Charles Tranchevent1, Francisco Bonachela Capdevila2, Daniela Nitsch1, Bart de Moor1,
Patrick De Causmaecker2 & Yves Moreau1
1
Department of Electrical Engineering ESAT-SCD, IBBT- Future Health Department,
Katholieke Universite
2
CODeS Group, Department of Computer Science, IBBT- Future Health Department,
Katholieke Universiteit
Identifying disease associated genes is a major challenge in genetics. Linkage analysis,
association studies and more recently next generation sequencing are often used to
produce a manageable set of candidate genes among which only one or a few are
associated to the disease / phenotype of interest. In the last decade, several computational
methods have been developed to prioritize the candidate genes, i.e., to identify the most
promising genes among a large list of candidate genes - so as to maximize the yield and
biological relevance of further downstream validation experiments and functional studies.
Most of these computational methods integrate several sources of genomic data such as
gene expression data, functional annotations, and literature information.
In the current study, several gene prioritization tools are used in conjunction to study
several genetic diseases / phenotypes. A preliminary study based on literature data
indicates that 90% of the recently reported disease-gene associations are prioritized in the
first third of the candidate list. These results proved that computational methods can be
used to efficiently prioritize candidate genes.
We then focus on congenital heart defects (CHD), congenital diaphragmatic hernia (CDH),
and asthma to propose promising candidate genes. We identify promising candidate genes
that are located in disease relevant loci, that are involved in very similar disorders, or whose
expression profiles match the disease under study.
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P23: L914F, the VM-causative TIE2 mutation, strongly dysregulates endothelial cell gene
expression
Melanie Uebelhoer1, Marjut Nätynki2, Julie Soblet1, Antonella Mendola1, Laurence M. Boon3,
Lauri Eklund2, Nisha Limaye1 & Miikka Vikkula1
1
Laboratory of Human Molecular Genetics, de Duve Institute, UCL, Brussels, Belgium
Oulu Center for Cell-Matrix Research, University of Oulu, Oulu, Finland
3
Center for Vascular Anomalies, Cliniques Universitaires St-Luc, UCL, Brussels, Belgium
2
Mutations in the endothelial-cell tyrosine kinase receptor TIE2 cause inherited and sporadic
forms of venous malformation. The most frequent somatic mutation, L914F, and the
common germline mutation, R849W, have been shown to differ both in terms of
phosphorylation-level, as well as sub-cellular localization and trafficking of the receptor. We
now show that PI3K/AKT and STAT1 are chronically activated by both mutant forms, with
L914F exerting a much stronger effect. Gene expression profiling of HUVECs overexpressing
the mutant or wild-type forms of TIE2, indicates that L914F dysregulates multiple pathways,
with more than 80 differentially expressed genes. By contrast, R849W has very weak effects,
making it indistinguishable from wild-type cells in global analyses. The genes modulated by
L914F-cells are predominantly involved in vascular development and cell migration, and
provide us with insights into the mechanisms involved in lesion-formation.
58
Next Generation Sequencing and Recent Advances in Genetics
Belgian Society of Human Genetics
P24: IRF6 Screening of Syndromic and a priori Non-Syndromic Cleft Lip and Palate
Patients: Identification of a New Type of Minor VWS Sign
Laurence Desmyter1, Michella Ghassibe1, Nicole Revencu1, Odile Boute2, Melissa Lees3,
Geneviève François4, Christine Verellen-dumoulin5, Yves Sznajer6, Katrien Claes7, Koen
Devriendt8, Lionel VanMalderghem9, Geert Mortier10, Catherine Vilain11, Geneviève
Pierquin9, Catherine vincent-Delorme12, Yves Gillerot5, Romain Vanwijck4, Bénédicte Bayet4,
CLP group 13 & Miikka Vikkula1
1
Laboratory of Human Molecular Genetics, de Duve Institute,Brussels, Belgium
Service de génétique clinique, Hôpital Jeanne de Flandre, Lille , France
3
Clinical Genetics Unit, Great Ormond Street Hospital for Children, NHS Trust, London, UK
4
Centre Labiopalatin, Division of Plastic Surgery, Cliniques Saint-Luc, Brussels, Belgium
5
Center for Human Genetics, Cliniques Saint-Luc, Université catholique de Louvain, Brussels,
Belgium
6
Unité de génétique clinique pédiatrique, Hôpital des enfants Reine Fabiola, Brussels, Belgium
7
Centrum voor Medische Genetica, UZ-Gent, Gent, Belgium
8
Center for Human Genetics, KUL, Leuven , Belgium
9
Centre de Génétique Humaine, CHU, Université de Liège, Liège , Belgium
10
Centrum Medische Genetica, UZ-Antwerpen, Edegem , Belgium
11
Centre de Génétique, U.L.B.-Hôpital Erasme, Brussels , Belgium
12
Génétique médicale, Centre hospitalier d’Arras, Arras , France
13
European collaborators
2
Van der Woude syndrome (VWS), caused by dominant IRF6 mutation, is the most common
cleft syndrome. In 15% of the patients, lip pits are absent and the phenotype mimics
isolated clefts. Therefore, we hypothesized that some of the families classified as having
non-syndromic inherited cleft lip and palate could have an IRF6 mutation. We screened in
total 170 patients with cleft lip with or without cleft palate (CL/P): 75 were syndromic and
95 were a priori part of multiplex non-syndromic families. A mutation was identified in 62.7
and 3.3% of the patients, respectively. In one of the 95 a priori non-syndromic families with
an autosomal dominant inheritance (family B), new insights into the family history revealed
the presence, at birth, of lower lip pits in two members and the diagnosis was revised as
VWS. A novel lower lip sign was observed in one individual in this family. Interestingly, a
similar lower lip sign was also observed in one individual from a 2nd family (family A). This
consists of 2 nodules below the lower lip on the external side. In a 3rd multiplex family
(family C), a de novo mutation was identified in an a priori non-syndromic CL/P patient. Reexamination after mutation screening revealed the presence of a tiny pit-looking lesion on
the inner side of the lower lip leading to a revised diagnosis of VWS. On the basis of this
data, we conclude that IRF6 should be screened when any doubt rises about the normality
of the lower lip and also if a non-syndromic cleft lip patient (with or without cleft palate) has
a family history suggestive of autosomal dominant inheritance.
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P25: BMP2-Smad-Runx2 over-expression provides evidence for the involvement of a proosteogenic signalling pathway in pseudoxanthoma elasticum.
Mohammd J. Hosen1, Olivier Vanakker1, Paul Coucke1, Olivier Le Saux2 & Anne De Paepe1
1
Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium
Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of
Hawaii, US
2
Aims
Pseudoxanthoma elasticum (PXE) is characterized by oculocutaneous and cardiovascular
manifestations, due to mineralization and fragmentation of elastic fibers. The causal ABCC6
gene encodes a transmembrane transporter; however, the pathogenetic link with the elastic
fiber abnormalities remains unknown. Current pathophysiological hypotheses include a role
for oxidative stress and an unidentified PXE serum factor. We have previously shown that
PXE patients have a diminished vitamin K (VK) serum concentration, leading to inefficient
carboxylation or activation of VK dependent inhibitors of mineralization, such as matrix gla
protein (MGP). MGP is known to inhibit Bone Morphogenetic Protein 2 (BMP2), a master
regulator of osteogenesis. Several pathways are associated with BMP2, one of which is the
BMP2-Smad-RUNX2 pathway in which RUNX2 acts as a transcriptional regulator of proteins
involved in mineralization of osteogenesis. We aimed to study the role of this signaling
pathway in the PXE pathogenesis.
Methods & Results
Alizarin red calcium stains were performed to detect mineralization around the whiskers of
ABCC6 knockout mice and human PXE dermis. Immunohistochemical stains on calcium
positive adjacent slides for BMP2, Smad 1-4-5-8 and RUX2 showed positive labeling, colocalizing with mineralization, in all tissues compared to controls. These qualitative results
were quantitatively confirmed via qPCR on human PXE fibroblasts. Comparable qPCR results
were obtained on control fibroblasts inoculated with human PXE serum.
Discussion & Conclusion
Our results indicate the involvement of the pro-osteogenic cellular BMP2-Smad-RUNX2
pathway in PXE. Calcification-specific upregulation of RUNX2 provides a pathophysiological
mechanism for the up- or downregulation of proteins previously implicated in PXE (such as
osteocalcin, alkaline phosphatase, osteopontin, bone sialoprotein and VEGF-A), leading to
ectopic mineralization of neovascularisation. In addition, our results for the first time merge
three principal pathophysiological observations in PXE. Indeed, besides VK-deficiency also
oxidative stress has been shown to contribute to RUNX2 overexpression and we show a
similar effect of PXE serum on RUNX2 expression. Thus, our results provide evidence that
vitamin K deficiency, oxidative stress as well as the PXE serum factor co-operate in an
integrated pathophysiological process leading to ectopic mineralization in PXE.
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P26: A family with autosomal dominant CLP due to an IRF6 mutation
Mirta Basha1, Laurence Desmyter1, Mickael Quentric1, Herve Bénateau2, Benedicte Bayet3,
Nicole Revencu1 & Miika Vikkula1
1
Laboratory of Human Genetics, de Duve Institute, Université catholique de Louvain,
Brussels, Belgium
2
Centre Hospitalo-Universitaire de Caen, Département de Chirurgie Maxillo-Faciale,
Plastique et Rec
3
Centre Labiopalatin, Service de Chirurgie Plastique, Cliniques universitaires Saint-Luc,
Brussels, Belgium
In 2002, it was established that mutations in the gene encoding interferon regulatory factor
6 (IRF6) cause VWS in majority of cases. Van der Woude syndrome is an autosomal
dominant disorder accounting for 2% of all cleft lip and palate cases. In addition to CLP, it is
characterized by a high penetrance rate of lower lip pits (80%) and hypodontia (50%). Lip
pits have a variable expressivity ranging from clear bilateral fistulae with salivary leakage to
subtle microforms. During clinical examination, these microforms can be overlooked,
sometimes rendering the diagnosis of this syndrome difficult (see poster of L.D.).
Here we report on familial orofacial clefting with five affected members, all a priori without
lower lip pits. The pedigree illustrates an autosomal dominant pattern of inheritance. We
screened the IRF6 gene in four affected and two unaffected members of the family and
identified a mutation: c.390delG; p. Ser131Leufs*36 in all affected members. This mutation
has not been reported before.
Based on our findings, familial CLP with an autosomal dominant pattern of inheritance
should be screened for the IRF6 gene even if lip pits have not been documented. As a result,
this will improve genetic counseling in predicting the disorder.
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P27: Legislation on direct-to-consumer genetic testing in seven European countries
Pascal Borry1, Rachel E. van Hellemondt2, Dominique Sprumont3, Camilla F. Duarte Jales4,
Emmanuelle Rial-Sebbag5, Tade M. Spranger6, Liam Curren7, Jane Kaye7, Herman Nys1 &
Heidi Howard8
1
University of Leuven, Belgium
Leiden University Medical Centre, the Netherlands.
3
Institute of Health Law, University of Neuchâtel, Switzerland.
4
Institute of Bioethics, Portuguese Catholic University, Portugal.
5
UMR U 1027, Inserm, Université de Toulouse - Université Paul Sabatier -Toulouse III
6
Institute for Public Law, University of Bonn, Germany
7
HeLEX Centre for Health, Law and Emerging Technologies, University of Oxford, U.K.
8
University of Basel, Switserland
2
An increasing number of private companies are now offering direct-to-consumer (DTC)
genetic testing services. The tests offered range from tests for single gene, highly penetrant
disorders to susceptibility tests for genetic variants associated with common complex
diseases or with particular non-health-related traits. Although a lot of attention has been
devoted to the regulatory framework of DTC genetic testing services in the U.S.A., only
limited information about the regulatory framework in Europe is available. We will report
on the situation with regard to the national legislation on direct-to-consumer (DTC) genetic
testing in seven European countries (Belgium, the Netherlands, Switzerland, Portugal,
France, Germany, United Kingdom). The paper will address whether these countries have
legislation that specifically address the issue of DTC genetic testing or have relevant laws
that is pertinent to the regulatory control of these services in their countries. The findings
show that France, Germany, Portugal, Switzerland have specific legislation that defines that
genetic tests can only be carried out by a medical doctor after the provision of sufficient
information concerning the nature, meaning and consequences of the genetic test and after
the consent of the person concerned. In the Netherlands, some DTC genetic tests could fall
under legislation that provides the Minister the right to refuse to provide a license to
operate if a test is scientifically unsound, not in accordance with the professional medical
practice standards or if the expected benefit is not in balance with the (potential) health
risks. Belgium and the United Kingdom allow the provision of DTC genetic tests. Although
relevant legislation that bind DTC companies exists at the European level (E.g. the in vitro
medical diagnostic devices, consumer protection legislation, data protection legislation), the
lack of a harmonized (European) approach at all levels is problematic in a context where
services are offered through the internet.
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P28: Accelerated TH-MYCN driven neuroblastoma formation in 129x1/SvJ mice is
characterized by absence or very few cooperative genomic imbalances
Anneleen Beckers1, Candy Kumps1, Alexander Schramm2, Angelika
Vandesompele1, Katleen De Preter1, Johannes Schulte2 & Frank Speleman1
Eggert2,
Jo
1
Center for Medical Genetics, Ghent University, Belgium
Department of Pediatric Oncology and Haematology, University Children's Hospital Essen,
Germany
2
Neuroblastoma (NB) is a clinically and genetically heterogeneous pediatric tumor arising
from precursor cells of the sympathetic nervous system. Despite intensive treatment of
aggressive NB survival rates in these patients are still disappointingly low. Therefore,
amongst others, establishing mouse NB models that faithfully recapitulate human disease is
of utmost importance in order to understand the complex biology of the disease in more
detail and to offer modalities for in vivo testing of new therapeutic compounds. Several
years ago, the first mouse neuroblastoma model was established by Weiss and colleagues
[Weiss et al., EMBO 1997]. Using a rat tyrosine hydroxylase promotor driven human MYCN
gene, tumors developed in 25 to 35% in a mouse in C57Bl/6 background. We observed a
significant increase in penetrance of tumor formation to <70% in the 129x1/SvJ background.
Interestingly, few or little genomic imbalances were detected in these tumors as compared
to aberrations observed in F1 hybrids from a C57BL/6 x M. musculus castaneus cross (with
tumor incidence ~10%) [Weiss et al., Cancer Research 2000 and Hackett et al., Cancer
Research 2003]. We hypothesize that due to the accelerated tumor formation less
cooperative genetic defects are required. Of further interest, the only consistent observed
aberration, a trisomy 3, is also recurrently detected in the original TH-MYCN mouse model
tumors further supporting the importance of dosage increase of one or more genes on
mouse chromosome 3 in neuroblastoma tumor formation. We will present in depth mousehuman synteny analysis and comparison of mouse and human neuroblastoma array CGH
profiles and the resulting selected candidate genes contributing to tumor formation.
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P29: FAM20A mutations are responsible for a new syndromic form of amelogenesis
imperfecta
Mickaël Quentric1,2,6, Muriel Molla1,3,6, Arnaud Picard1,3,4, Alain Verloes5, Miikka Vikkula2,7 &
Ariane Berdal1,3,7
1
Laboratory of Oral Molecular Physiopathology, INSERM UMR 872, Cordeliers Research
Center, Team 5, Paris, France
2
Laboratory of Human Molecular Genetics, de Duve Institute, Université catholique de
Louvain, Brussels, Belgium
3
Oral and Facial Rare Malformations Reference Center - Rothschild and Trousseau
Hospitals, APHP, Paris, France
4
AP-HP, Hôpital d'enfants Armand-Trousseau, Service de Chirurgie Maxillo-Faciale et
Chirurgie Plastique, Paris, France
5
AP-HP, Hôpital Robert Debré, Unité fonctionnelle de génétique clinique, Paris, France.
6,7
Equal contribution
Amelogenesis imperfecta (AI) is a genetically and clinically heterogeneous group of inherited
dental enamel defects, affecting 1/700 to 1/14000 newborns, depending on the populations
studied. Fourteen different subtypes have been listed depending on their clinical description
and their mode of inheritance.
So far, 8 genes have been linked to AI. Six are for the isolated forms: AMELX (MIM300391),
ENAM (MIM 606585), MMP20 (MIM 604629), KLK4 (MIM 603767), FAM83H (MIM 611927),
WDR72 (MIM613214), and 2 for syndromes in which AI is a key feature: tricho-dentoosseous syndrome (MIM 190320) for DLX3 and Jalili syndrome (MIM 217080) for CNNM4.
Recently, a homozygous nonsense mutation (c.406C>T) in FAM20A was identified in a large
consanguineous family affected by AI with gingival hyperplasia.
We performed mutational analyses on 14 AI individuals recruited in the Oral and Facial Rare
Malformations Reference Center (Rothschild and Trousseau Hospitals, Paris). Four patients,
coming from 3 unrelated consanguineous families, presented the same phenotype i.e.
hypoplastic AI, gingival hyperplasia, delayed tooth eruption and intra-pulpal calcifications.
The other ten presented an AI without any of the other inclusion criteria.
All syndromic AI patients screened had mutations in FAM20A, whereas none of the 10
isolated AI patients had. Three homozygous mutations were identified altering the splicing
or leading to a premature stop codon.
This study confirms that FAM20A loss of function leads to a new syndromic form of
amelogenesis imperfecta.
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P30: Exome sequencing and data analysis of consanguineous families with assumed
autosomal recessive inheritance patterns: the (in)significance of linkage studies
Mala Isrie1, Alejandro Sifrim2, Masoud Z. Eskteki1, Luc Dehaspe1, Jeroen V. Houdt1, Hilde
Peeters1, Joris R. Vermeesch1 & Hilde V. Esch1
1
2
Center for Human Genetics, KU Leuven - UZ Leuven, Leuven, Belgium
Department of Electrical Engineering (ESAT/SCD), KU Leuven, Heverlee, Belgium
Introduction
Two consanguineous families with multiple affected children, suggesting autosomal
recessive inheritance of a homozygous identical by descent (IBD) mutation, were selected.
The phenotype in Family 1 consisted of hypotonia, intellectual disability (ID) and mild
dysmorphism. Affected children in Family 2 had non-syndromic ID combined with seizures.
Conventional karyotyping, FMR1 and array-CGH analyses were normal. Due to absence of a
specific genetic diagnosis, next-generation sequencing (NGS) was performed, aimed at
identifying the disease-causing mutations.
Methods
DNA libraries of the proband, parents and – in the case of Family 2 - one additional
unaffected brother, were prepared according to the TruSeq Illumina protocol. Targeted
capturing was performed for Family 1 using a custom-made Nimblegen array. Exome
capturing in solution was performed for both families according to the Nimblegen protocol.
Sequencing was carried out on the Illumina HiSeq 2000 platform. Data analyses for Family 1
and Family 2 were based on the CASAVA and GATK pipeline, respectively. Annotation was
performed using Annovar as well as our internally developed software Annotate-it.
Confirmation of candidate variants and segregation analyses were done by Sanger
sequencing. Linkage analysis and homozygosity mapping were performed using data from
the Affymetrix SNP array 6.0.
Results
Linkage analysis in Family 1 was performed prior to NGS. A 1.8 Mb candidate region was
found on chromosome 16p12, but array capturing of this region was ineffective. Exome
sequencing did reveal potentially causative variants, but not inside the linkage region.
Segregation studies of these variants are ongoing. Data analysis of the complete exome in
Family 2 revealed a dozen candidate variants. One of these segregated in the family. This
concerned a missense mutation in a gene not previously associated with ID and/or epilepsy.
It was predicted to be damaging by different software programs and subsequent family
studies including homozygosity mapping supported the possible causality. The variant was
present at low frequency in the general population, but combined with an ethnicity bias.
This candidate gene is currently being investigated further.
Conclusion
Exome capturing in solution has proven to be a successful tool to identify candidate
variants. Filtering the data for the linkage region in Family 1 did, however, not produce any
obvious causative variants. Based on non-IBD mutations reported in consanguineous
families in literature, we extended our data analysis outside the linkage region. In Family 2
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SNP segregation obtained from exome analysis confirmed the homozygosity mapping data
from SNP arrays and we identified one good candidate gene to cause the phenotype in this
family. We will present the advantages as well as pitfalls of using linkage in NGS assays. Also
the presence of candidate variants in the general population, ethnic variability and means to
determine causality will be discussed.
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P31: Array-CGH analysis in male infertility
Katrien Stouffs1, Deborah Vandermaelen1, Björn Menten2, Gwenda Vandyck1, Herman
Tournaye3 & Willy Lissens1
1
Center for Medical Genetics, UZ Brussel; Reproduction and Genetics, Vrije Universiteit
Brussel
2
Center for Medical Genetics, Ghent University Hospital
3
Center for Reproductive Medicine, UZ Brussel; Reproduction and Genetics, Vrije
Universiteit Brussel
In the past decades, many (mutation) studies have been performed aiming to identify
genetic causes of male infertility. However, these studies were rather disappointing. In the
current study, we looked for the presence of copy number variations (CNVs) in 9 infertile
men with a maturation arrest of spermatogenesis and 20 controls with normal sperm
parameters. After several elimination steps, 11 candidate regions remained. Where
necessary, the presence of mRNA was examined/confirmed in testis and control tissues.
One extra region could be eliminated as the single gene present in this region was not
expressed in testis. The remaining 10 regions were investigated by qPCR in order to
investigate more controls (>130). The analysis showed that two regions were also aberrant
in our control group, and are therefore likely not involved in male infertility. One more
alteration has been detected in a proven fertile father group. The remaining seven
rearrangements varied from 21kb to 263kb, and included four deletions containing the
genes SLC25A24, FAM82A1, C17ORF51 and SIRT4. Since all deletions were present in
heterozygous form, the remaining copy of these genes was sequenced for the patients
having these deletions. No mutations were detected; and therefore presumably at least one
functional copy is present. Three duplications involve the genes THRAP3+C1ORF113, SYT6
and PLSCR2. The genes SYT6, PLSCR2 and SIRT4 have a known function in the last stages of
spermatogenesis and/or fertilization. Despite a potential role in male infertility, we presume
that an alteration involving these genes might not cause an arrest at the spermatocyte
stage. The function of the remaining genes is largely unknown. We could prove that all of
these genes are expressed in spermatogenesis. SLC25A24 has two transcript variants of
which variant 2 is testis-specific. The start codon of this variant, however, is located in exon
2, in which multiple deletions were reported in the database of genomic variants. Besides
multiple heterozygous deletions, we detected one patient with a maturation arrest of
spermatogenesis who was having a homozygous deletion of this region. Therefore, larger
(and other) patient and control groups are currently investigated. Furthermore,
immunohistochemistry is performed to determine the localization of the SLC25A24 proteins
in testicular tissues. In the near future, experiments will be set up to get more insight into
the role of the genes FAM82A1 and C17ORF51. These studies will elucidate whether
deletions of these genes are linked to male infertility.
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P32: Exclusion of known lymphedema genes in two families with severe congenital
lymphedema
M Amyere1, S Greenberger2, D Chitayat 3, E Pras 4, K Chong 3, T Uster 3, H Reznik-Wolf 4, D
Marek-Yagel4, L Boon 5 & M Vikkula1
1
Laboratory of Human Molecular Genetics, de Duve Institute, Université catholique de Louvain
Vascular Biology Program and Department of Surgery, Children’s Hospital Boston and HMS
3
Prenatal Diagnosis and Medical Genetics Program, Mount Sinai Hospital; University of Toronto
4
Danek Gartner Institute of Human Genetics, Sheba Medical Center, Ramat-Gan, Israel
5
Centre for Vascular Anomalies, Division of Plastic Surgery, Cliniques Universitaires St-Luc,Brussels
2
Lymphedema is a soft tissue swelling resulting from abnormal accumulation of interstitial
fluid containing high molecular weight proteins due to abnormal drainage of lymph by the
lymphatic vasculature. Primary lymphedema is due to an abnormal lymphangiogenesis
which usually starts in utero. Some of the cases are inherited and in most cases have
autosomal dominant or recessive mode of inheritance. Some of the autosomal dominant
cases have incomplete penetrance and variable expression. Both syndromic and nonsyndromic cases have been caused by mutations in genes with major role in
lymphangiogenesis including FOXC2, VEGFR3, SOX18, CCBE1, PTPN14 and GCJ2.
We report two consanguineous families with recurrence of an autosomal recessive form of
congenital lymphedema. One of the families is of Iranian-Jewish and Israeli descent and the
other of Iraqi descent. Direct sequencing of the known lymphedema genes including coding
sequences and intron–exon boundaries, did not identify any mutation. Whole genome scan
using Affymetrix SNP-Chip 250K was thus performed in both families and no copy number
change was identified in the affected members. Autozygosity mapping and parametric
linkage analysis also excluded the candidate lymphedema-genes. Three overlapping
autozygous regions were identified in the two families and were confirmed by linkage
analysis. Deep sequencing of whole exome of affected patients were performed and the
data analysis are on going.
Our findings provide evidence for the existence of new causative gene or genes associated
with the autosomal recessive form of congenital lymphedema.
(E-mail: [email protected]).
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P33: Impact of MDM2 SNP309 on the survival of neuroblastoma patients
Ali Rihani, Tom Van Maerken, Nurten Yigit, Jo Vandesompele & Frank Speleman
Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium
Neuroblastoma is the most common extracranial solid tumor in children. Less than 2% of
patients exhibit a p53 mutation at diagnosis. Intact p53 suggests that the p53 pathway may
be under negative control of upstream or downstream components. A particular
polymorphism located within the MDM2 promoter region, SNP309, has previously been
associated with increased cancer risk in some tumor entities or protection against certain
other cancers. Using TaqMan SNP genotyping, we evaluated the effect of MDM2 SNP309 on
the survival and clinicogenetic characteristics in a large cohort of neuroblastoma patients.
No significant difference in overall survival was observed among neuroblastoma patients
comprising all stages. However, stage 4 patients who are homozygous for the G allele (G/G)
or T allele (T/T) have significantly shorter overall survival than patients that are
heterozygous (G/T). The presence of the SNP was not associated with the timing of cancer
onset or MYCN status. We are currently investigating the functional impact of this SNP in
neuroblastoma and are further looking into the relationship between the different allele
situations, expression levels of MDM2 and survival of neuroblastoma patients.
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P34: Evaluation of a CGG Repeat Primed PCR system designed for detection of Fragile X
expanded alleles in clinical prenatal samples
Sara Seneca1, Sonia Van Dooren1, Willy Lissens1, Kathelijn Keymolen2, Marjan De
Rademaeker2, Urielle Ullmann3, Kim Van Berkel2 & Maryse Bonduelle1
1
CMG, UZ Brussel & VUB
CMG, UZ Brussel
3
CMG, UZ Brussel & IPG, Gosselies
2
Abnormal expansion of the (CGG)n repeat in the 5’untranslated region (UTR) of the FMR1
gene is known to generate Fragile X syndrome (FXS), the most common form of cognitive
impairment, and several other diseases in patients. The expansion reduces the expression of
the FMR1 by promoting DNA hypermethylation of this region when exceeding ≈ 200
repeats. Molecular diagnosis relies on determination of the number of allele repeats in the
DNA template, and assessment of the methylation state of the FMR1 locus. Current
conventional PCR amplification is only successful for normal and small permutation alleles,
and is not informative for homozygous repeat alleles. Expanded alleles too large to amplify
efficiently and female homozygous samples require additional Southern Blot (SB) analysis
for categorization and sizing. Although regarded as gold standard, this technique is
laborious, time consuming and involves (not always available) large amounts of DNA.
However, accurate and efficient quantification of the number of repeats in the 5’ UTR of
FMR1 gene is essential as premutation alleles are common in the general population.
We evaluated a commercially available assay (Asuragen) using a large set of blinded
archived prenatal samples that were previously analyzed for FXS with conventional PCR and
SB analysis in our Center. The PCR reagentia were able to identify accurately and quickly
FMR1 genotypes, ranging from normal over premutation to full mutation alleles, in patient
and control samples. Exact sizing was possible for a spectrum of permutation alleles. The
assay is also sensitive to size mosaicism and AGG interruptions.
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P35: Maternal 16p11.2 mosaic deletion transmitted to her affected child
Bernard Grisart, Christine Verellen-Dumoulin & Dominique Roland
Center for Human Genetics, Inst. Pathology and Genetics, 25 Av G. Lemaitre, 6041,
Gosselies, Belgium
A 18 years old patient was referred for genetic consultation because of morbid obesity,
manic depression and learning difficulty. Clinical history was characterized by language
delay from the age of 2 and obesity without hyperphagy from the age of 3. Later on, this girl
displayed learning difficulties at school. She follows now an adapted scholar program and
receives risperdone treatment for bipolar disorder. At the age of 15, she shows morbid
obesity (BMI=49), hypertension, auto-immune hypothyroidism, insulin resistance and
hyperandrogenism. Clinical examination reveals mild facial dysmorphism with large
palpebral fissures, acanthosis nigricans and adipomasty.
CGH analysis (Agilent microarray ; ISCA design 60K) showed a 517 kb deletion at position
16p11.2. This deletion was initially associated with autism (see review by Slavotinek et al.,
2008; Hum Mut 124 :1-17). Subsequently others studies enlarged the clinical spectrum
associated with the deletion 16p11.2 (Shiwani et al., 2009; J Med Genet 47(5): 332-341)
suggesting that this rearrangement is a susceptibility factor for language delay, intellectual
disability, motor delay and attention deficit, congenital malformation and epilepsy. This
rearrangement was also linked to progressive obesity (Walters et al., 2010; Nature 463: 671675). Therefore the deletion observed in our patient can clearly explain her symptoms.
Deletion 16p11.2 can occur de novo or be inherited from one parent (who can be
asymptomatic or less severely affected). At first, parental screening was performed by
quantitative PCR using primers developed within deletion 16p11.2. With this technique, we
could not clearly establish if the mother was carrier of the deletion; the father was clearly
negative. With CGH analysis we could show that the mother was carrier of the deletion. Log
ratio values in this interval (-0,356) suggested that the deletion was present as a mosaic at
an estimated frequency of 31%. FISH analysis confirmed that the deletion was present as
mosaic in 42% of blood lymphoid cells. Obesity is the only symptom expressed in the
mother.
The results obtained in our patient and her mother demonstrate that deletion 16p11.2 can
exist as a mosaic state in blood (probably as a result of a mitotic recombination event at the
beginning of embryogenesis) and can later be transmitted at homoplasmic state to children.
Recurrence risk in sibship is difficult to evaluate as maternal germinal mosaicism can be
different from the one measured in blood.
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P36: Mutations in SERPINF1 cause recessive Osteogenesis imperfecta
Helena Soenen, Sofie Symoens, Paul Coucke, Fransiska Malfait & Anne De Paepe
Centre for Medical Genetics, Ghent University Hospital, Ghent, Belgium
Background:The brittle bone disease, Osteogenesis imperfecta (OI) is a heritable connective
tissue disorder which is characterised by susceptibility to fractures and bone deformities.
In the majority of cases, OI is inherited in an autosomal dominant manner and is caused by
mutations in type I collagen genes, COL1A1 and COL1A2, thereby interfering with the type I
collagen production or structure. In the minority of cases, OI is inherited in an autosomal
recessive manner with homozygous or compound heterozygous mutations in genes
encoding proteins involved in collagen post-translational modifications, folding and
secretion (LEPRE1, CRTAP, PPIB, FKPB10, SP7, and SERPINH1).
Recently, a novel disease locus, SERPINF1, coding for pigment epithelium derived factor
(PEDF), has been found to be associated with severe forms of OI. PEDF is a 50 kDA secreted
glycoprotein of the serpin superfamily. PEDF is believed to play a role in bone homeostasis
as an inhibitor of bone resorption. Its balance with vascular endothelial growth factor
(VEGF) probably plays a crucial role in the process of bone formation. In the OI cases
described up to date, only nonsense mutations in SERPINF1 have been reported and the
biochemistry (the biosynthesis) of type I collagen was reported to be normal.
Methods: We selected 22 OI patients, whom were previously shown to be negative for
mutations in all known OI genes. These patients were sequenced for SERPINF1 using Sanger
sequencing. Next, the expression of the SERPINF1 gene was evaluated using reverse
transcription quantitative-PCR (RT-qPCR).
Results: We identified two novel homozygous loss-of-function mutations in the SERPINF1
gene for two non-related patients with a progressively worsening form of OI, characterised
by vertebral compression fractures, severe deformity of the long bones, normal dentition,
and normal sclera. A homozygous nonsense mutation was identified in exon 6 for patient 1
(p.Tyr253X) and a homozygous missense mutation was identified in exon 4 for patient 2
(p.Gly95Glu), which affects a highly conserved amino acid. Using RT-qPCR, we observed no
expression of SERPINF1 for patient 1 and significantly reduced expression of SERPINF1 for
patient 2. Biochemichal analysis of type I collagen was normal.
Conclusions: The homozygous nonsense mutation observed in patient 1 generates a
premature stop codon which is expected to result in deficiency of SERPINF1. This is
supported by the loss of SERPINF1 expression using RT-qPCR. The homozygous missense
mutation observed in patient 2 occurs in the first nucleotide of exon 4 making it prone to
alternative splicing. The mutation likely results in alternative splicing through the insertion
or deletion of a fragment of DNA during translation. This can result in an out-of-frame
transcript and will be targeted for degradation, rendering a null allele. The remaining
SERPINF1 transcripts will contain the missense mutation. This is supported by the
significantly reduced expression of SERPINF1 using RT-qPCR. These mutations have not been
described or reported. We are currently further characterising the missense mutation.
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P37: Somatic mutations in the isocitrate dehydrogenase (IDH) genes are associated with
Maffucci Syndrome and Ollier Disease
Nisha Limaye1, Twinkal C. Pansuriya2, Ronald van Eijk2, Pio d’Adamo3, Maayke A. J. H. van
Ruler2, Marieke L. Kuijjer2, Jan Oosting2, Anne-Marie Cleton-Jansen2, Jolieke G. van
Oosterwijk2, Sofie L. J. Verbeke2, Daniëlle Meijer2, Tom van Wezel2, Karolin H. Nord4, Luca
Sangiorgi5, Berkin Toker6, Bernadette Liegl-Atzwanger7, Mikel San-Julian8, Raf Sciot9, LarsGunnar Kindblom10, Soeren Daugaard11, Laurence M. Boon12, Kyle C. Kurek13, Karoly
Szuhai14, Pim J. French15, Judith V. M. G. Bovée2, Catherine Godfraind12 & Miikka Vikkula1
1
de Duve Institute, Université catholique de Louvain, Brussels, Belgium
Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands
3
University of Trieste, Trieste, Italy
4
Department of Clinical Genetics, Lund University Hospital, Lund, Sweden
5
Department of Medical Genetics, Rizzoli Orthopedic Institute, Bologna, Italy
6
Istanbul University Medical School, Istanbul, Turkey
7
Institute of Pathology, Medical University, Graz, Austria
8
Department of Orthopaedic Surgery and Traumatology, University Clinic of Navarra, Pamplona,
Spain
9
Department of Pathology, University of Leuven, Leuven, Belgium
10
Department of Musculoskeletal Pathology, Royal Orthopaedic Hospital, Birmingham, UK
11
Department of Pathology, University of Copenhagen, Copenhagen, Denmark
12
Cliniques universitaires Saint-Luc, Université catholique de Louvain, Brussels, Belgium
13
Children’s Hospital Boston, Harvard Medical School, Boston, Massachusetts, USA
14
Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands
15
Erasmus University Medical Center, Erasmus University, Rotterdam, The NetheRLANDS
2
Maffucci Syndrome and Ollier Disease are both non-hereditary enchondromatosis
syndromes, characterized by multiple benign central cartilaginous tumors. In the case of
Maffucci syndrome, these are accompanied by vascular tumors in the form of spindle cell
hemangiomas. Enchondromas can undergo malignant transformation, and patients show an
increased incidence of cancers such as glioma. Mutations in the isocitrate dehydrogenases
IDH1 and IDH2 had been descibed in glioma, as well as solitary enchondromas, making them
attractive candidates to be involved in the pathogenesis of Maffucci syndrome and Ollier
disease. Targeted sequencing of these genes identified mutations in cartilaginous and
vascular tumors, from 81% of patients with Ollier Disease, and 77% of patients with
Maffucci syndrome. The mutations were somatic and mosaic, affecting particular protein
residues: R132 (C or H) in IDH1, and R172 (G) in IDH2. Besides glioma, these mutations have
been identified in myelodysplastic syndromes including acute myeloid leukemia (AML), as
well as other cancers. As in the case of gliomas and AML, the presence of IDH-mutations
correlates with global DNA hypermethylation and differential gene expression in Maffucci
and Ollier tissues. We now investigate whether these changes are in fact necessary and/or
sufficient to cause the tumors, as they do not show an increased incidence in patients with
the inherited disease D-2-hydroxyglutaric aciduria, which can be caused by germline R172
IDH2 mutations. To this end, we will carry out deep exome sequencing on tumors from our
cohort of Maffucci patients, to assess for the presence of other somatic gene mutations.
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P38: Prader Willi syndrome with mosaic monosomy 15q11.2-q13.3 related to an unstable
translocation t(5;15)(p15.3;q13)
Benoît Parmentier, Bernard Grisart, Sébastien Boulanger, Christine Verellen-Dumoulin &
Isabelle Maystadt
Department of Human Genetics, Institut de Pathologie et de génétique, Charleroi, Belgium
Prader Willi syndrome (PWS) is a well documented neurobehavorial disorder caused by
various genetic mechanisms such as paternal microdeletions 15q11-q13 (76%), maternal
uniparental disomy (21%), and imprinting defects (3%). Here, we report the case of a
newborn male patient refered for genetic evaluation because of severe neonatal hypotonia,
cryptorchidism, mild dysmorphism and pes cavus.
Cytogenic analyses, established from peripheral blood lymphocytes using GTG banding and
fluorescent in situ hybridization, revealed a mosaic deletion of the 15q11.2 region resulting
from an autosomal translocation t(5;15) which was balanced in 30% of mitoses and
unbalanced in 70% with loss a of the derivative chromosome 15.
The
karyotype
was
defined
as
45,XY,der(5)t(5;15)(p15.3;q13),-15[32]/
46,XY,t(5;15)(p15.3;q13)[18].
Cytogenetic and molecular analyses were performed to delineate the deleted 15q region.
Multiplex ligation-dependent probe amplification confirmed the deletion and revealed an
abnormal methylation pattern, compatible with a mosaic PWS diagnosis. Microarray CGH
showed that the deletion spans a 8 Mb region, which is larger than that usually seen in PWS.
This region extends distally and partially includes the recently described 15q13.3
microdeletion syndrome associated with mental retardation and epilepsy.
Developmental prognosis of the patient is difficult to predict given the mosaicism and the
variable expressivity of the 15q13.3 microdeletion syndrome.
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P39: Dissecting ALK downstream signaling as a strategy towards multi-targeted therapy
Candy Kumps1, Irina Lambertz1, Marilena De Mariano2, Michael Porcu 3, Piotr Zabrocki3,
Johannes Schulte4, Angelika Eggert4, Jo Vandesompele 1, Alexander Schramm 4, Karin
Verstraeten5, An De Bondt 5, Jorge Vialard5, Jan Cools 3, Katleen De Preter 1 & Frank
Speleman1
1
Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium
Translational Oncopathology, National Cancer Research Institute (IST), Genoa, Italy
3
Center for Human Genetics, K.U.Leuven - VIB, Leuven, Belgium
4
Department of Pediatric Oncology and Haematology, University Children's Hospital Essen,
Germany
5
Oncology Discovery Research and Early Development, Johnson & Johnson, Beerse, Belgium
2
Background
ALK is a tyrosine kinase receptor expressed in the developing nervous system and activated
in several tumor entities, such as anaplastic large cell lymphoma (ALCL) and non-small cell
lung cancer (NSCLC). Recently, activating ALK mutations were discovered in the majority of
familial neuroblastomas (NB) and nearly 10% of sporadic NB. Two hotspot mutations were
identified affecting residues F1174 and R1275, respectively. Evidence is accumulating that
small molecule inhibitors such as crizotinib may be effective in treatment of malignancies
with activated ALK. Understanding the dynamics and complexity of ALK signaling is a
prerequisite in order to understand differential response and resistance to molecular
therapy and to identify new vulnerables nodes for combined pharmacological targeting of
such cancers. To achieve this goal we set out to investigate the direction and components of
signaling following oncogenic ALK activation.
Methods
Gene expression data (Affymetrix HG133Uplus2) were obtained in triplicate after inhibition
using the small molecule ALK inhibitor NVP-TAE684 in NB cell lines with activating ALK
mutations (F1174L (n=3), R1275Q (n=3)) and amplification (n=1) as well as wild type cell
lines (n=3). Cell growth upon RNAi treatment was measured using XCELLigence real-time
monitoring. ALKF1174L and ALKR1275Q transformed Ba/F3 cells were used for expression
analysis.
Results
Differential gene expression analysis was performed following pharmacological ALK
inhibition in ALK mutated and wild type NB cell lines. This allowed to establish a 150-gene
ALK driven mRNA expression signature. Gene Ontology analysis showed predominant
involvement of genes implicated in MAPK/ERK signaling pathway (e.g. SPRY4, EGR1, DUSP4,
DUSP6, ETV5). One of the most consistently regulated genes was ETV5, a member of the
PEA3 group of transcription factors, for which an oncogenic function has been described in
multiple malignancies (including prostate cancer). It is an important player in neuronal fate
decision of neural crest stem cells and plays a significant role in metastasis and invasion of
breast, ovarium and endometrial cancer. ETV5 mRNA levels are down regulated after
pharmacological ALK inhibition and shRNA mediated knockdown.
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Additionally, ALK transformed Ba/F3 cells show increased ETV5 expression as compared to
parental Ba/F3 cells. Furthermore, RNAi mediated ETV5 knockdown in NB cell lines, showed
drastic reduction in cell growth in cells with activated ALK, indicating that ETV5 expression
contributes to the NB oncogenic phenotype. Functional in vitro and in vivo assays are
ongoing to further elucidate the contribution of ETV5 to the oncogenic NB phenotype.
Conclusion
In a first step towards elucidating the activated ALK signaling cascade we have investigated
the transcriptional consequences of pharmacological inhibition of activated ALK. In this
study, we have identified the MAPK/ERK pathway as an important ALK signaling pathway
and propose ETV5 as a major transcriptional effector contributing to the NB malignant
phenotype, thus offering new therapeutic targets for molecular therapy.
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P40: Combined Identity-by-descent Mapping And Exome Sequencing Identifies A Novel
MERTK Mutation In Autosomal Recessive Retinitis Pigmentosa.
Frauke Coppieters1, Kristof Van Schil1, Steve Lefever1, Julie De Zaeytijd2, Bart P. Leroy3,
Ingele Casteels4, Thomy de Ravel5 & Elfride De Baere1
1
Center for Medical Genetics Ghent, Ghent University Hospital, Ghent, Belgium
Department of Ophthalmology, Ghent University Hospital, Ghent, Belgium
3
Department of Ophthalmology, Center for Medical Genetics Ghent, Ghent University
Hospital, Ghent, Be
4
Department of Ophthalmology, Leuven University Hospitals, Leuven, Belgium
5
Center for Human Genetics, Leuven University Hospitals, Leuven, Belgium
2
Purpose: Autosomal recessive retinitis pigmentosa (ARRP) is a genetically heterogeneous
disorder, with 35 disease genes and 3 loci associated thus far. The goal of this study was to
identify the molecular defect in a consanguineous ARRP family with one affected individual
and three unaffected siblings (parents: first cousins once removed).
Methods: Genomewide identity-by-descent (IBD) mapping was performed in the patient
and three unaffected siblings using GeneChip Human Mapping 250K Nsp arrays (Affymetrix).
Subsequently, the patient underwent exome enrichment (TruSeq Exome Enrichment Kit,
Illumina) and sequencing (2x100 cycles, HiSeq, Illumina). The CLC Genomics Workbench (CLC
bio) was employed for read mapping and variant calling. Variants were confirmed using
Sanger sequencing (ABI 3730xl, Applied Biosystems).
Results: IBD mapping revealed 5 homozygous regions larger than 10 Mb in the patient.
However, 4 of these were also homozygous in one or more healthy siblings, thus leaving a
37.6 Mb region on chromosome 2 as the best candidate.
In total, 80/89 million reads could be mapped against the human genome reference
sequence (NCBI, GRCh37.p5), resulting in an average coverage of 45x. The candidate region
on chromosome 2 contained 836 substitutions and 24 deletions/insertions with a coverage
and a variant allele frequency equal to or above 10x and 75%, respectively. After candidate
gene analysis of all RetNet genes, the following novel homozygous missense mutation was
identified in MERTK: c.2180G>A (p.Arg727Gln) (NM_006343.2). This mutation affects a
highly conserved nucleotide and amino acid, is located in the active site of the tyrosine
protein kinase domain, and is predicted to abolish protein function by both SIFT and
PolyPhen. Moreover, the carbonyl oxygen of the Arg727 residue is known to be involved in a
hydrogen-bond with the hydroxyl group of the ligand Compound-52 (Huang et al., 2009, J
Struct Biol). In addition, the mutation was heterozygous in the parents and heterozygous or
absent in the healthy siblings. Notably, the age-of-onset (12 years), photophobia, and
macular pigment alterations observed in the patient are in agreement with previously
reported MERTK-associated phenotypes.
Conclusions: In this study, we identified the causal genetic defect in a consanguineous
family with a single ARRP patient by combining IBD mapping with exome sequencing. This
approach is a powerful tool to establish a molecular diagnosis in genetic heterogeneous
conditions such as most retinal dystrophies.
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P41: Identification of a novel, homozygous PCDH15 deletion in an USH1 patient using
identity-by-descent mapping with a high-density SNP microarray.
Miriam Bauwens1, Frauke Coppieters1, Hannah Verdin1, Françoise Meire2 & Elfride De
Baere1
1
Center for Medical Genetics Ghent, Ghent University Hospital, Ghent, Belgium
Department of Ophthalmology, University Hospital for Children Queen Fabiola, HUDERF,
Brussels, Be
2
Purpose
Usher syndrome is an autosomal recessive condition characterized by hearing loss and
retinitis pigmentosa. Three major subtypes can be distinguished. Mutations in the CDH23,
MYO7A, PCDH15, USH1C and USH1G genes can explain circa 80% of cases with Usher type I
(USH1), the most severe type. Here, we aimed to identify the underlying genetic defect in a
patient with USH1, characterized by congenital, bilateral, profound sensorineural hearing
loss, vestibular areflexia, and adolescent-onset retinitis pigmentosa, originating from a
consanguineous couple.
Methods
We performed genomewide homozygosity or identity-by-descent (IBD) mapping, with a
high-density SNP microarray (Affymetrix, 250K, NspI). Data analysis was performed using an
in-house Perl script. The largest homozygous regions were evaluated for the presence of
candidate genes that underwent Sanger sequencing. An identified deletion was delineated
using conventional PCR, followed by long-range PCR and Sanger sequencing of the junction
product.
Results
IBD mapping in this family revealed several large IBD regions. In two regions of 25.9 Mb and
13.2 Mb, respectively, two known USH1 genes (CDH23 and PCDH15) are located. PCR failure
of several amplicons of PCDH15 was suggestive for a homozygous partial PCDH15 deletion.
This was confirmed by a stretch of SNPs located in the PCDH15 gene that contained missed
calls. The deletion was delineated using conventional PCR amplicons and the deletion
junction was obtained using long-range PCR. Sequencing of the junction product led to the
delineation of the deletion at the nucleotide level. This corresponds with a deletion of the
first exons of PCDH15, presumably resulting in nonsense mediated decay and loss of
function of PCDH15. Breakpoint analysis suggested the deletion is mediated by
microhomology.
Conclusions
Previous studies demonstrated the importance of copy number screening such as MLPA of
PCDH15 in USH1 patients, revealing several deletions and duplications with non-recurrent
breakpoints. Here, we identified a novel partial PCDH15 deletion using IBD mapping with a
high-density SNP chip, in a single index case from a consanguineous pedigree. In conclusion,
we identified the underlying genetic defect in this patient, in agreement with the USH1
phenotype.
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P42: Identification of the cause of Blue Rubber Bleb Nevus Syndrome
Julie Soblet1, Nisha Limaye1, Maria Cordisco2, Anne Dompmartin3, Odile Enjolras4, Simon
Holden5, Alan D. Irvine6, Christine Labrèze7, Augustina Lanoel2, Paul N. Rieu8, Samira Syed9,
Carine J. van der Vleuten 10, Rosemarie Watson6, Steven J. Fishman11, John B. Mulliken11,
Laurence M. Boon12 & Miikka Vikkula1
1
Human Molecular Genetics, de Duve Institute, Université catholique de Louvain, Brussels,
Belgium
2
Department of Dermatology, Hospital Garrahan, Buenos Aires, Argentina
3
Department of Dermatology, Université de Caen Basse Normandie, CHU Caen, France
4
Consultation des Angiomes, Hôpital Lariboisière, Paris, France
5
Department of Clinical Genetics, Guy's Hospital, London, UK
6
Department of Paediatric Dermatology, Our Lady's Children's Hospital, Dublin, Ireland
7
Dermatology Department, Hôpital Pellegrin Enfants, Bordeaux, France
8
Department of Pediatric Surgery, Radboud University Nijmegen Medical Center, Nijmegen,
Holland
9
Department of Dermatology, Great Ormond Street Hospital for Children, London, UK
10
Department of Dermatology, Radboud University Nijmegen Medical Center, Nijmegen,
The Netherlands
11
Vascular Anomalies Center, Children's Hospital, Boston, MA, USA
12
Center for Vascular Anomalies, Cliniques universitaires Saint-Luc, UCL, Brussels, Belgium
Blue rubber bleb nevus syndrome (BRBN) is a rare congenial disorder (OMIM # 112200)
originally reported by Gascoyen in 1860, and described in detail by Bean in 1958. The
syndrome is characterized by widely distributed multiple cutaneous and internal venous
malformations. Patients with BRBN can present with a few to hundreds of lesions. In
addition to cutaneous lesions, the patients have pathognomonic gastrointestinal lesions,
most commonly in the small intestine, documented by endoscopy, colonoscopy, or
magnetic resonance imaging (MRI). Lesions may however be found anywhere from the
mouth to the anus. Significant complications of gastrointestinal involvement include
gastrointestinal hemorrhage, intussusception, volvulus, internal hemorrhage, and infarction.
Although several case reports have been published, the etiopathology of BRBN is still
unknown.
We hypothesized that BRBN may be due to somatic changes, within the lesions, of a gene
implicated in the regulation of angiogenesis.
To test this, we screened the coding region of our most important candidate gene, by direct
sequencing. The screen was done on cDNA derived from resected lesions in a series of 14
patients. In 16 tissues from 10 patients, we identified amino acid substitutions in highly
conserved positions. These changes are not known in dbSNP and not found in controls nor
in the blood of the patients.
Thus, BRBN is due to postzygotic changes often hitting a single gene.
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P43: New TUBB3 gene mutation in a woman with CFEOM3 and brain abnormalities.
Anaïs Drielsma1, Isabelle Pirson2, Patrice Jissendi3, Massimo Pandolfo4 & Marc Abramowicz5
1
Department of Medical Genetics and IRIBHM, ULB, Brussels, Belgium
IRIBHM, Université Libre de Bruxelles (ULB), Brussels, Belgium
3
Department of Neuroradiology, Hôpital Erasme-ULB, Brussels, Belgium
4
Department of Neurology, Hôpital Erasme-ULB, Brussels, Belgium
5
Department of Medical Genetics, Hôpital Erasme-ULB, Brussels, Belgium
2
Congenital fibrosis of extraocular muscles type 3 (CFEOM3) is a cranial dysinnervation
disorder characterized by ophthalmoplegia and ptosis, thought to result from defect in
axonal guidance. Isolated CFEOM3 results from mutations in microtubules-associated
KIF21A and TUBB3 genes. CFEOM3 caused by TUBB3 gene mutation may also be associated
with facial weakness, congenital contractures, intellectual deficiency, progressive
polyneuropathy and brain anomalies as dysgenesis of the corpus callosum, anterior
commissure, internal capsule, corticospinal tracts and basal ganglia. Other mutations of the
same gene are reported in patients without CFEOM who present a wide range of cortical
dysplasia including neuronal migration disorders associated with pontocerebellar
hypoplasia.
We report on a 26 years old woman who suffered from severe congenital strabismus,
ophthalmoplegia, unilateral ptosis and psychomotor retardation. CFEOM3 was diagnosed
during strabismus surgery. She also has minimal pyramidal syndrome with conserved
muscular strength but fatigability and slowness. Electroencephalogram, somatosensory
evoked potentials and intelligence were normal. Magnetic resonance imaging showed
dysplastic and hypoplastic vermis, dysmorphic and small corpus callosum, hypoplastic right
cerebral hemisphere and peduncle, asymmetric caudate nuclei, anterior commissure
agenesis and dilated ventricles.
Therefore, we sequenced TUBB3 gene in this patient and found a de novo heterozygous
missense mutation. This mutation affects a highly conserved amino acid, is predicted to be
damaging by Polyphen2 software and was not reported in patients with CFEOM3 (Tischfield,
2010) nor cortical dysplasia (Poirier, 2010) until now. Our finding will help to determine the
genotype-phenotype correlation of the TUBB3-related spectrum.
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P44: Co-occurrence of the SMMCI syndrome and a 5q21 deletion in young girl patient
Annette Uwineza, Geneviève Pierquin, Anne-Cécile Hellin, Mauricette Jamar, Jean-Hubert
Caberg, Karin Segers, Vinciane Diderberg & Vincent Bours
Center for Human Genetics, CHU Sart-Tilman, University of Liège, Belgium.
Solitary median maxillary central incisor (SMMCI) or single central incisor is a rare dental
anomaly. It is estimated to occur in 1:50,000 live births. SMMCI syndrome is a complex,
autosomal dominant developmental disorder in which an SMMCI is seen in association with
midline nasal cavity defects (choanal atresia, mid-nasal stenosis, nasal pyriform aperture
stenosis) and variably holoprosencephaly.
We report a 21month-old girl, who was the second child of unrelated parents. She was
referred to our genetic counseling service for evaluation of dysmorphic features suggesting
the SMMCI syndrome and psychomotor development delay. On physical examination, she
presented a single central incisor, choanal atresia, nasal pyriform aperture stenosis, ocular
hypotelorism, short stature and microcephaly. No brain anomalies were identified by
cerebral CT-scan.
A de novo heterozygote missense mutation 494C>T was identified within SHH gene, leading
to the replacement of Alanine at amino acid position 165 with Valine. Affymetrix WholeGenome 2.7M Array Chip revealed a deletion of 695kb of the 5q21.1-q21.2 region.
In conclusion, it difficult to establish genotype-phenotype correlations of the 5q21 deletion
because of the simultaneous presence of the SMMCI syndrome.
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P45: Further insights into the involvement of GLUT10 in the pathogenesis of Arterial
Tortusosity Syndrome
Andy Willaert1, Sandeep Khatri2, Bert Callewaert1, Paul Coucke1, Seth Crosby3, Joseph Lee4,
Elaine Davis4, Sruti Shiva5, Michael Tsang6, Anne De Paepe1 & Zsolt Urban2
1
Department of Medical Genetics, Ghent University Hospital, Ghent 9000, Belgium
Department of Human Genetics, University of Pittsburgh, Pittsburgh, PA 15261, USA
3
Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110,
USA
4
Department of Anatomy and Cell Biology, McGill University, Montreal, H3A 2B2, Canada
5
Vascular Medicine Institute, Department of Medicine, University of Pittsburgh, PA 15261,
USA
6
Department of Developmental Biology, University of Pittsburgh, PA 15213, USA
2
Growth factor signaling results in dramatic phenotypic changes in cells, which require
commensurate alterations in cellular metabolism. Mutations in SLC2A10/GLUT10, a member
of the facilitative glucose transporter family, are associated with altered transforming
growth factor-β (TGFβ) signaling in patients with arterial tortuosity syndrome (ATS). The
objective of this work was to test if SLC2A10/GLUT10 can serve as a link between TGFβrelated transcriptional regulation and metabolism during development. In zebrafish
embryos, knockdown of slc2a10 using antisense morpholino oligonucleotide injection
caused a wavy notochord and cardiovascular abnormalities with a reduced heart rate and
blood flow, that was coupled with incomplete and irregular vascular patterning. This was
phenocopied by treatment with a small-molecule inhibitor of TGFβ receptor (tgfbr1/alk5).
Array hybridization showed that the changes at the transcriptome level caused by the two
treatments were highly correlated, revealing that reduced tgfbr1 signaling is a key feature of
ATS in early zebrafish development. Interestingly, a large proportion of the genes, which
were specifically dysregulated after glut10 depletion and not by tgfbr1 inhibition play a
major role in mitochondrial function. Consistent with these results, slc2a10 morphants
showed decreased respiration, and reduced TGFβ reporter gene activity. Finally, co-injection
of antisense morpholinos targeting slc2a10/glut10 and smad7 (a TGFβ inhibitor) resulted in
a partial rescue of smad7 morphant phenotypes, suggesting slc2a10/glut10 functions
downstream of smads. Taken together, scl2a10/glut10 is essential for cardiovascular
development by facilitating both mitochondrial respiration and TGFβ signaling.
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P46: A 273-kb Duplication at 22q13.33 encompassing the SHANK3 gene in 2 sibs with
microcephaly, behavioral disorder and learning disabilities
Anne Destree, Pascale Hilbert, Sébastien Boulanger, Christine Verellen-Dumoulin & Isabelle
Maystadt
Centre de Génétique Humaine, IPG, avenue Georges Lemaître 25, 6041 Charleroi (Gosselies)
The 22q13 deletion syndrome is a not uncommon condition associated with global
developmental delay, absent or delayed speech and hypotonia. Pure distal trisomy of the
long arm of chromosome 22 are rare. Twenty patients, including our two cases, with
variable clinical phenotype extending from mild psychomotor delay to severe delay with
congenital malformations, have been shown to have a pure distal 22q trisomy. The size of
the duplicated segment is extremely variable. Here we report on a brother and a sister
presenting the smallest cryptic 22q13.33 duplication ever reported, detected by a salsa
MLPA P188 kit 22q13. The duplicated region spans about 273-kb which encompasses 11
genes, including SHANK3. Both patients live in an institution because of the desertion of the
parents and show developmental delay, especially in the language sphere, mild intellectual
disabilities, behavioral disturbance, microcephaly, growth retardation and mild dysmorphic
features. This phenotype was previously described in patients with larger duplication
22q13.3 and a recognizable phenotype was suggested. We confirm here this phenotype and
delineate a critical chromosomal region of 273-kb, including the SHANK3 gene, which
appears to be a strong candidate gene for the 22q13.33 duplication phenotype.
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P47: Surveying the Imprinted Genome in Normal and IUGR Human Placentas
Andreas I. Diplas1, Luca Lambertini2, James Wetmur3 & Jia Chen4
1
Department of Medical Genetics, Saint-Luc Hospital, Brussels, 1200, Belgium
Community and Preventive Medicine, Mount Sinai School of Medicine, New York, 10029,
USA
3
Microbiology, Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York,
10029, USA
4
Pediatrics and Oncological Science, Mount Sinai School of Medicine, New York, 10029, USA
2
Objectives
Dysregulation of imprinted genes and loss of genomic imprinting (LOI) have been
independently linked to intra-uterine growth restriction (IUGR). Herein, we examined
expression of imprinted genes, LOI and their correlation in normal and IUGR human
placentas.
Methods
We developed a panel of 74 genes that had been shown to be imprinted in humans. Using
placental tissue from 10 normal and 7 IUGR pregnancies, we conducted a systematic survey
of expression of these genes by quantitative RT-PCR. Quantitative allele-specific PCR was
used to determine the LOI levels in RNAs from 14 normal and 24 IUGR placentas.
Results
We found that 52/74 (~70%) of the genes were expressed in human placentas. Nine of the
52 (17%) expressed genes were significantly differentially expressed between normal and
IUGR placentas, among which 5 were upregulated and 4 were downregulated. We also
assessed LOI profile of 14 imprinted genes in 14 normal and 24 IUGR placentas. LOI was
found as a common phenomenon.
Conclusions
Differential expression of imprinted genes indicates that genomic imprinting might
contribute to the etiology of IUGR. We found no correlation between gene expression and
LOI profile and mechanisms other than LOI may contribute to the dysregulation of imprinted
genes. More studies are needed to explore the genetic/epigenetic mechanisms that link LOI
to changes in the expression of imprinted genes and possibly leading to IUGR development.
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P48: Case report: an aspecific chronic thrombocytopenic purpura may lead to a syndrome
Annelyse Bruwier1, Philippe Clapuyt2, Jean-Jacques De Bruycker1, Bénédicte Brichard1,
Catherine Heijmans3, Yanick Crow4 & Nicole Revencu5
1
Department of Paediatric Haematology Oncology, Cliniques universitaires Saint-Luc, UCL,
Brussels
2
Department of Radiology, Cliniques universitaires Saint-Luc, UCL, Brussels
3
Department of Paediatrics, Hopital de Jolimont, Haine-Saint-Paul, Belgium
4
Manchester Academic Health Sciences Centre (MAHSC), St. Mary's Hospital, Manchester,
UK
5
Centre for Human Genetics, Cliniques universitaires Saint-Luc, UCL, Brussels
Herein, we report on a 10-year-old girl with short stature and chronic thrombocytopenia,
who has been treated by intermittent corticosteroid for 4 years. She is the 5th child of
healthy, consanguineous parents of Turkish origin. Her four sisters are healthy. She has a
normal, general and psychomotor development. At 7 years of age, she was found to have
autoimmune hypothyroidism and positive antinuclear antibodies (ANAs). She has been
complaining of back pain for several months. The X-ray investigation showed platyspondyly
and lacunar lesions localized at the posterior third of the vertebral bodies. The
complementary X-ray workup revealed radiolucent lesions extending from the growth plate
into the metaphysis of the long bones: distal radius and ulna, distal femora, proximal fibula
and tibia. The constellation of radiological features led to the diagnosis of
spondyloenchondrodysplasia (SPENCD).
The correlation of chronic thrombocytopenia and autoimmune hypothyroidism with these
specific skeletal abnormalities suggested the diagnosis of SPENCD syndrome, an autosomal
recessive syndrome, firstly recognised as a distinct entity in 1976 (1). Neurological
involvement, absent in our patient, could also be observed, such as pyramidal signs, seizures
and cerebral calcifications.
Recently, mutations in the ACP5 gene were identified in patients with SPENCD syndrome (2,
3). This gene encodes TRAP protein expressed in osteoclasts and macrophages, which derive
from a common hematopoietic lineage. A homozygous mutation in the ACP5 gene
(c.155A>C; p.Lys52Thr) was identified in our patient, confirming the clinical diagnosis.
In conclusion, chronic thrombocytopenia associated with other clinical signs should be
further investigated, as it can be part of a more complex disorder.
1) Radiology. 1976 Jan;118(1):133-9
2) Nat Genet. 2011 Feb;43(2):127-31
3) Nat Genet. 2011 Feb;43(2):132-7
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P49: First steps towards the elucidation of the apoptotic mechanisms of DFNA5 based on
humanized yeast models.
Sofie M. L. Van Rossom1, Ken Op de Beeck1, V. Franssens2, J. Winderickx2 & Guy Van Camp1
1
2
Dept. of Medical Genetics, University of Antwerp, Belgium
Departement of Biology, Faculty of Science, 3001 Heverlee, Belgium
DFNA5 was originally discovered as a gene responsible for hereditary hearing impairment. In
several families with hereditary deafness, a mutation in DFNA5 causes skipping of exon 8.
Later research revealed that DFNA5 is involved in several forms of cancer. DFNA5 is
epigenetically inactivated in colorectal, gastric and breast tumours, indicating a possible role
as a tumour suppressor gene.
The exact function of DFNA5, however, remains to be elucidated. Recent experiments have
revealed that mutant DFNA5 induces a growth defect in yeast and mammalian cells. In order
to reveal the mechanisms underlying this growth defect, we monitored specific apoptotic
markers, like a DHE and an Annexin/PI staining. This confirmed that yeast transfected with
mutant DFNA5 induces apoptotic cell death. We also performed intracellular co-localisation
studies of DFNA5 and, interestingly, found that mutant DFNA5 co-localizes to the
mitochondria while wild-type protein remains cytoplasmatic. A preliminary experiment
revealed that only wild-type DFNA5 forms ‘aggresomes’ adjacent to the vacuoles. This could
be an indication of the involvement of IPODs, one of the cellular quality control networks
present in cells (Kaganovich et al., 2008).
In order to elucidate pathways involved in DFNA5 induced cell death we screened eighteen
specific deletion strains lacking various proteins involved in programmed cell death. The
results of our experiments indicated a role of Yca1, the yeast metacaspase, as well as of
Fis1, which is involved in mitochondrial fission. Our data led us to conclude that the clinical
mutation of DFNA5 induces mitochondrial defects in yeast. Further elucidation of the
phenotype triggered by the mutant DFNA5 can potentially contribute to new therapies for
deafness or cancer by interfering with the underlying pathways.
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P50: Establishment of a miRNA/mRNA regulatory network through integrated analysis of
neuroblast and neuroblastoma expression profiles
Sara De Brouwer1, Pieter Mestdagh1, Nicky D'Haene2, Irina Lambertz1, Fanny De Vloed1, Gert
Van Peer1, Johannes H. Schulte3, Angelika Eggert3, Alexander Schramm3, Rosa Noguera4,
Geneviève Laureys5, Jo Vandesompele1, Frank Speleman1 & Katleen De Preter1
1
Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium
Laboratoire d’Anatomie Pathologique, ULB, Brussels, Belgium
3
Department of Pediatric Oncology and Haematology, University Children's Hospital Essen,
Germany
4
Department of Pathology, Medical School, University of Valencia, Valencia, Spain
5
Department of Pediatric Hematology and Oncology, Ghent University Hospital, Ghent,
Belgium
2
MicroRNAs (miRNAs) play an essential regulatory role in many normal cellular functions and
development as well as diseases including cancer. For neuroblastoma, like for other
embryonic tumours, it has been assumed that oncogenesis results from the disruption of
normal developmental processes. The normal counterpart for the neuroblastoma cells are
the sympatho-adrenal progenitor cells located within the adrenal gland and sympathetic
ganglia. To investigate whether miRNAs, implicated in the development of the sympathetic
nervous system, are also implicated in neuroblastoma development, we compared miRNA
expression profiles of 101 primary untreated neuroblastoma tumours to those of human
neuroblasts, dissected from foetal adrenal glands. We identified 60 differentially expressed
miRNAs that were used for an integrative genomic analysis with mRNA expression profiles in
order to build a miRNA/mRNA transcriptional network controlling genes implicated in
neuronal development and neuroblastoma. MiR-204 emerged as a strong putative network
component and was shown to directly target PHOX2B, a gene known to act as master
regulator of sympathetic nervous system development and implicated in familial
neuroblastoma. The network was further expanded using target enrichment analysis, gene
ontology data, literature data, miRNA target prediction databases and miRNA-mRNA
correlation analyses. One of the components of this network was SOX11, experimentally
shown to be directly targeted by miR-204 and miR-542-3p and indirectly regulated by miR133b, all of which are higher expressed in the neuroblasts as compared to tumours. A
reduction in colony formation capacity was observed upon knockdown of SOX11 thus
underscoring the oncogenic potential of this gene. In summary, our data allow to describe a
putative miRNA/mRNA regulatory network implicated in neuroblastoma. Further functional
in vitro and in vivo studies will establish more firmly the contribution of the identified
network components and may provide a rationale for development of miRNA-targeted
therapies.
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P51: Comprehensive clinical and molecular analysis of 13 families with type 1 recessive
cutis laxa
Bert Callewaert1, Chi-Ting Su2, Tim Van Damme1, Philip Vlummens1, Fransiska Malfait1,
Olivier Vanakker1, Zsolt Urban2 & Anne De Paepe1
1
2
Ghent University Hospital, Belgium
Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA, USA
Autosomal recessive cutis laxa type I (ARCL type I) typically associates generalized cutis laxa
with severe pulmonary emphysema and/or vascular complications. In a handful of cases
mutations in the FBLN4 or FBLN5 genes have been identified. Recently, mutations in the
LTBP4 gene have been implicated in a similar phenotype with, in addition, marked gastrointestinal and genito-urinary involvement. This constellation was denoted the Urban-RifkinDavies syndrome (URDS). We studied FBLN4, FBLN5 and LTBP4 in 12 families with type I
recessive cutis laxa. We found homozygous FBLN5 mutations in 2 probands, whereas 9
probands harbored biallelic loss-of-function mutations in LTBP4. In one proband, no
mutations were found. Our results showed that LTBP4 and FBLN5 mutations cause a very
similar cutis laxa phenotype associated with severe pulmonary emphysema, in the absence
of vascular tortuosity or aneurysm formation. We observed more severe gastro-intestinal
and genitourinary tract involvement and a higher prevalence of diaphragmatic hernia in
patients with LTBP4 mutations. Supravalvular aortic stenosis remains thus far only
associated with FBLN5-mutations. Functional studies showed that most premature
termination mutations in LTBP4 result in severely reduced mRNA and protein levels. This
correlated with increased transforming growth factor beta (TGFβ) signaling. However, we
found a mutation, c.4128dupC, which escaped nonsense-mediated decay. Production of the
corresponding mutant protein (p.Pro1376fsX27) caused abnormal morphology of fibrillin-1
microfibrils in skin fibroblast cultures, but normal TGFβ signaling. We conclude that LTBP4
mutations cause disease through several distinct molecular mechanisms.
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P52: Study of the role of microRNAs in the development of Colitis Associated Cancer.
Nassim Bouznad*, Claire Josse*, Vincent Bours & Cécile Oury
University of Liège, GIGA-R Unit of Human Genetics, Liège, Belgium
Objectives
MicroRNAs are small non-coding RNAs (18-25 nucleotides) that regulate gene expression
post-transcriptionally by targeting messenger RNAs for translational repression or mRNA
degradation. The diversity of miRNAs and of their mRNA targets indicates that they play a
key role in the regulation of different cellular processes. MicroRNAs can act as tumor
suppressors or oncogenes and have also been involved in inflammatory responses.
The aim of our work is to study the role of microRNAs in the route from chronic
inflammation to colorectal cancer.
Methods and results
We used a well known mouse model of colitis associated carcinogenesis in which mice were
treated with an unique dose of the carcinogen azoxymethane (AOM) followed by 3 cycles of
dextran sulfate sodium (DSS) administered in the drinking water for 5 days. MiRNA
expression profiles were analyzed in mouse whole colons at different time intervals using
the miRXplore microarray (Miltenyi Biotech). We identified 81 miRNAs that were modulated
in our model. Some of these miRNAs (miR- 146a; miR-34a, miR- 26b, miR-29c, miR-18a, miR223) have already been implicated in tumorigenesis or in inflammatory diseases. We further
validated our microarray results by quantitative Real-Time PCR. We then decided to focus
on miR-223 that was up-regulated in mouse colon tumors when compared to adjacent
normal tissues. Notably, changes in miR-223 expression have previously been associated
with some classes of human colorectal cancers.
Using the same mouse colon samples, we then showed down-regulation of several miR-223
targets that have been validated and/or predicted by 5 different algorithms (miRanda,
miRDB, miRWalk, RNA22, TargetScan).
We generated lentiviral vectors to either over-express or antagonize miR-223 effects in
human epithelial and immune cell lines. We confirmed that the expression of the studied
miR-223 targets was accordingly modified both at the mRNA level by RT-qPCR and at the
protein level by western blotting experiments.
Conclusions
Our study suggests that microRNAs play a crucial role in the physiopathology of colitis
associated cancers. Dysregulation of miRNAs can constitute an early step in the progression
of chronic inflammation to cancer. We have developed tools that will enable us to further
analyze the role of miR-223 in this process.
* these authors contributed equally to this work.
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P53: Biochemical and clinical outcome of mutations in the C-propeptide encoding domains
of the COL1A1 and COL1A2 gene
Sofie Symoens, Fransiska Malfait, Delfien Syx, Paul Coucke & Anne De Paepe
Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium
Biochemical and clinical outcome of mutations in the C-propeptide encoding domains of the
COL1A1 and COL1A2 gene
Sofie Symoens, Fransiska Malfait, Delfien Syx, Paul Coucke and Anne De Paepe
Center for Medical Genetics Ghent, Ghent University Hospital, 9000 Ghent, Belgium
Mutations in COL1A1 and COL1A2 affecting the type I procollagen C-propeptide domain are
a rare cause of autosomal dominant Osteogenesis imperfecta (OI), accounting for only 4%
(74/1824) of reported mutations.
We reviewed clinical, biochemical and molecular data on 20 OI patients in whom we
identified a α1(I)- or α2(I)-C-propeptide mutation.
In nine OI type I patients we identified a COL1A1-mutation generating a premature
termination codon (PTC), leading to diminished production of type I (pro)collagen, as
reflected by a decreased intensity of type I (pro)collagen.
Five patients with OI type III or IV harbour a COL1A1-missense mutation, which most likely
alters the C-propeptide structure causing them to interfere with chain selection, association
and nucleation. This results in delayed incorporation of this mutant α1-chain as reflected by
overhydroxylation of full-length type I (pro)collagen. Interestingly, these missense
mutations are located near a cysteine-residu, involved in intermolecular disulfide bonds, or
near the most C-terminal part of the proα1-chain.
In a foetus with OI type II, aborted at 22-weeks of gestational age, we observed decreased
intensity, but no overmodification of type I (pro)collagen. Remarkably, COL1A1-mutation
screening revealed a 1-bp deletion resulting in elongation of the mutant transcript, escape
of NMD, translation into protein and probably interference with chain selection and
incorporation.
Five patients with a COL1A2-C-propeptide mutation display an OI type I phenotype.
Biochemical analysis showed near-normal migration patterns.
Conclusion: Our findings expand this rare class of OI mutations and underscore the data
presented in literature. Structural alterations of the C-propeptide impede chain selection,
association and nucleation, possibly due to interference with the formation of
intermolecular disulphide bridges, thereby allowing overmodification of the helical domains.
If assembly occurs, triple helix formation proceeds without interruption and without
structural alterations, but with posttranslational overmodification. In the α(I)-procollagen
chain, these structural defects mostly lead to severe-to-lethal OI. In contrast, PTC mutations
cause NMD and a diminished amount of proα1(I)-collagen and result in OI type I, as
expected. COL1A2-mutations usually result in milder phenotypes that their COL1A1counterparts. Interestingly, mutations affecting the α1(I)- and α2(I)-C-proteinase cleavage
site have been described and result in mild OI, associated with fractures and bone areas
with increased mineralization. However, exceptions occur and one should be careful when
interpreting the biochemical analyses, as illustrated by our findings on the presented
aborted fetus.
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Belgian Society of Human Genetics
P54: Functional evaluation of the recurrent MLH1 variant c.*35_*37del: no evidence for
posttranscriptional regulation by hsa-miR186-2 and hsa-miR1276 binding
Peter Schietecatte, Jens Vandenhaute, Martine De Bleeckere, Fanny De Vloed, Gert Van
Peer, Anne De Paepe, Bruce Poppe & Kathleen Claes
Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium
Introduction
MicroRNAs (miRNAs) are important negative regulators of gene expression which mainly
function through base-pairing with the 3’ UTR of target genes. In the MutL homolog1
(MLH1) DNA mismatch repair gene, a 3’ UTR recurrent variant (c.*35_*37del) has been
described to lead to decreased MLH1 expression levels. Although this variant was shown not
to be a highly penetrant mutation, modifier effects can not be excluded. In silico algorithms
predict that this variant creates novel miRNA binding sites for hsa-miR186-2 and hsamiR1276.
Objectives
We aim to investigate whether the MLH1 c.*35_*37del variant results in a decreased MLH1
expression level and if this reduction results from binding of hsa-miR186-2 and hsamiR1276.
Methods
A dual luciferase reporter system was used to study the interactions between the miRNAs of
interest and both the wildtype MLH1 3’ UTR and 3’UTR with the c.*35_*37del variant. In
addition, a quantitative PCR (qPCR) experiment will be performed to compare MLH1
expression levels between normal individuals and individuals carrying the MLH1
c.*35_*37del variant.
Results
Our results demonstrate that hsa-miR186-2 binds to neither the wildtype or variant MLH1 3’
UTR. In contrast, hsa-miR1276 binds to both the wildtype and variant MLH1 3’ UTR, and
results in minor reductions in luciferase activity (30% and 40%, respectively).
Conclusions
We could not demonstrate that the MLH1 c.*35_*37del variant creates a novel binding site
for hsa-miR186-2 and hsa-miR1276. Therefore, it is not likely that this variant affects MLH1
expression through these miRNAs.
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P55: Facilitating the Fragile X post- and prenatal genetic diagnostic testing workflow by
use of the Abbott FMR1 TP-PCR and FMR1 sizing PCR products.
Sonia Van Dooren, Sara Seneca, Kristof Endels, Kathelijn Keymolen, Marjan De Rademaeker,
Kim Van Berkel, Willy Lissens & Maryse Bonduelle
UZ Brussel -VUB, Centre for Medical Genetics, Brussels
Molecular testing of FMR1 (CGG)n expanded repeats remains hard to tackle given the
limitations of sizing PCR in detecting large and uninformative alleles, resulting in a significant
number of samples that require confirmation through laborious southern blot analysis.
Abbott Molecular recently developed a TP-PCR to be used in combination with their sizing
PCR to facilitate the Fragile X diagnostic testing workflow.
This study aimed at comparing Abbott Molecular FMR1 Sizing PCR and TP-PCR versus inhouse sizing PCR and southern blot in order to evaluate the sizing accuracy and detection
sensitivity of normal, intermediate, premutation and full mutation alleles. Over 100 samples
of different sources (approximately 50% whole blood, 40% chorion villi and 10%
amniocytes) and a panel of artificially mimicked mosaic samples were evaluated.
Signal intensity and sizing accuracy met expectations in the normal to small premutation
range. The sizing ‘long run’ greatly improved the detection capacity and sizing accuracy of
longer range (large premutation to full mutation) fragments, although inspection of raw
data is recommended. TP-PCR allowed discrimination between normal/intermediate and
premutation/full mutation alleles. However, premutation and full mutation TP-PCR signal
patterns were very similar, therefore requiring the Abbott FMR1 Sizing PCR to distinguish
them.
Mosaic detection in Sizing- or TP-PCR ranged from 20% down to 10%, depending on the
repeat range mixture used.
Our results corroborate the workflow proposed by Abbott Molecular of using FMR1 TP-PCR
as a first line screening platform in combination with the FMR1 Sizing PCR as second line
test.
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Belgian Society of Human Genetics
P56: Evidence of association between interferon regulatory factor 5 gene polymorphisms
and asthma
Vinciane Dideberg1, Chuan Wang2, Johanna Sandling2, Gerard Koppelman3, Matthew RoseZerilli 4, John Holloway4, Dirkje Postma5, Stephen Holgate4, Vincent Bours1 & Ann-Christine
Syvänen2
1
Department of Human Genetics, CHU of Liège, University of Liège, Liège, Belgium.
Department of Medical Sciences, Uppsala University, Uppsala, Sweden.
3
Department of Pediatric Pulmonology, University Medical Center Groningen, The
Netherlands.
4
Division of Infection and Immunity, Southampton General Hospital, Southampton, United
Kingdom.
5
Department of Pulmonology, University Medical Center Groningen, Groningen, The
Netherlands.
2
Objectives
Asthma pathogenesis is complex and is the outcome of the combination of several factors as
genetic predisposition, viral infection and immune response. The aim of this study was to
investigate a potential implication of the interferon regulatory factor 5 (IRF5) gene in the
genetic susceptibility to asthma, considering the implication of IRF5 in adaptive as in innate
immunity.
Methods
Ten polymorphic loci in the IRF5 gene, which encodes an important transcription factor in
the type I IFN system, were genotyped in two European family-based asthmatic cohorts.
The first cohort included 1467 individuals from 340 families, ascertained though 2 affected
sibs, which were from the Southampton area of the United Kingdom and the second cohort,
composed of 200 families which were from the northern part of the Netherlands.
Results
The most common haplotype displayed association signals with asthma in the UK cohort. In
attempt to replicate these findings, similar analyses were conducted in the Dutch cohort. In
this cohort a suggestive association was found between this haplotype with asthma, which
was also under-transmitted to the affected offspring. In the combined analysis
encompassing both the UK and the Dutch cohort, the significant association signal was
retained (p = 0.00066).
Conclusions
Although the influence of the IRF5 gene in immune/inflammatory diseases has been widely
confirmed by genetic association and functional studies, our findings that the IRF5 gene
polymorphisms might also be implicated in asthma provide the first evidence which links
IRF5 to this disease.
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P57: The 6q24.3q25.2 Deletion Syndrome.
Catheline Vilain1, Guillaume Smits1, Claudine Heinrichs2, Marc Abramowicz3, Anne De
Leener3, Montse Urbina3 & Bruno Pichon3
1
ULB Center of Human Genetics, and Clinical Genetics, HUDERF-ULB, Brussels, Belgium
Department of Endocrinology, HUDERF-ULB Queen Fabiola Children Hospital, Brussels,
Belgium
3
ULB Center of Human Genetics, Brussels, Belgium
2
By cytochip oligo 44K CGH array analysis we found a 7.9 Mb 6q24.3q25.2 deletion in a 2 year
old boy with the association of a prenatal onset of growth retardation, dysmorphism, and
mild motor delay.
Short long bones were observed during the third trimester of pregnancy (normal standard
caryotype, absence of p.Gly380Arg FGFR3 achondroplasia mutation). The patient was born
at term, small for gestational age (2330g-46cm-HC: 33cm) with club feet and a large atrial
septal defect.
Evolution is in accordance with patients with 6q24.3q25.3 deletion previously described in
literature, and consists in growth delay (at 2 years 3months, height at - 4 SD with short limbs
(upper/lower ratio : 0.91, N :1,4), and head circumference at - 1 SD), motor delay (sitting
position acquired at the age of 10 months, independent gait at the age of 26 months), joint
hyperlaxity, redundant skin, and facial dysmorphism (high and prominent forehead,
upslanting palpebral fissure, epicanthus, depressed nasal bridge, long filtrum, low set ears).
This patient, as well as another 3 year old patient reported in the literature, has normal
intellectual development so far (appropriate verbal and communication skills at age 3
years).
This independent observation confirms the clinical phenotype of patients with 6q24.3q25.2
deletion syndrome.
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P58: Regulatory networks governed by microRNAs in T-ALL oncogenesis and normal T-cell
development
Joni Van der Meulen1, Evelien Mets1, Pieter Mestdagh1, Peter Pipelers2, Gert Van Peer1, Tom
Taghon3, David Camacho Trujillo1, David Avran4, Nadine Van Roy1, Olivier Thas2, Yves
Benoit5, Jo Vandesompele1, Bruce Poppe1, Pieter Van Vlierberghe1, Pieter Rondou1, Jean
Soulier4 & Frank Speleman1
1
Center for Medical Genetics, Ghent University, Belgium
Department of Mathematical Modelling, Statistics and Bioinformatics, Ghent University,
Belgium
3
Department of Clinical Chemistry, Microbiology and Immunology, Ghent University
Hospital, Belgium
4
Hôpital Saint Louis, Institut Universitaire d'Hématologie, Paris, France
5
Department of Pediatric Hemato-Oncology, Ghent University Hospital, Belgium
2
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes
affecting children, adolescents and adults. T-ALL is characterized by a differentiation arrest
at specific stages of T-cell development, primarily originating from the ectopic expression of
T-cell oncogenes. Based on the mutation status of these genes and mRNA profiles, at least 5
different molecular-cytogenetic subgroups have been delineated. In addition, genetic
abnormalities affecting cell cycle, proliferation, survival and self-renewal are present in all TALL subgroups. In a recent study, we discovered five oncogenic microRNAs (miRNAs) coregulating the expression of key tumor suppressor genes implicated in the pathogenesis of
T-ALL (Mavrakis et al., Nature Genetics, 2011). In this study, we established the extended
miRNAome (756 miRNAs) in a genetically well characterized T-ALL patient cohort (n=65), 20
T-ALL cell lines as well as 9 different subsets of sorted T-cell populations from 3 human
donors in parallel to mRNA and gene copy number profiles thus allowing integrative data
mining. Cross-comparison between the different datasets resulted in the identification of
miRNAs with presumed function in normal T-cell development as well as novel candidate
miRNAs in T-ALL pathogenesis. The candidates were validated in an independent patient
series (n=50). To better understand our previously established miRNA-mRNA regulatory Tcell/T-ALL network, we performed miRNA-mRNA correlation analysis followed by gene set
enrichment analysis in order to assign putative functions to the selected miRNAs
(http://www.mirnabodymap.org/; Mestdagh et al., Nucleic Acids Research, 2011). In
addition, the regulatory network was consolidated and extended through identification of
miRNAs targeting selected key T-ALL oncogenes and tumor suppressor genes using a robust
high throughput 3’UTR screening assay. In conclusion, this study further details the
regulatory networks controlling normal T-cell development and T-ALL oncogenesis.
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P59: The combined experience of three European centres with preimplantation genetic
diagnosis for Huntington disease
Marjan De Rademaeker1, Maartje Van Rij2, Céline Moutou3, Jos Dreesen2, Stéphane Viville3,
Joep Geraedts4, Martine De Rycke5, Willem Verpoest6, Christine De Die-Smulders4 & Inge
Liebaers5
1
Centre Medical Genetics, Universitair Ziekenhuis Brussel
Dept.Clinical Genetics, Maastricht University Medical Centre
3
Service de Biologie de la Reproduction, Université de Strasbourg
4
Dept.Clinical Genetics, Maastricht University Medical Centre
5
Centre Medical Genetics, Universitair Ziekenhuis Brussel
6
Centre of Reproductive Medicine, Universitair Ziekenhuis Brussel
2
Objective
To study experience of PGD Huntington disease (HD) in three European PGD centres.
Materials and methods
Data on intakes, couples' reproductive history and outcome of PGD treatment cycles
between 1995 and 2008 were collected. Three hundred thirty one couples had an intake for
PGD for HD. PGD workup was based on two approaches:(1) direct testing of the CAG-triplet
repeat and (2) linkage analysis using intragenic or flanking microsattelite markers of the HTT
gene.
Results
68% (225/331) of the couples requested direct testing and 32% (106/331) exclusion PGD. At
the time of PGD intake, 39% of women had experienced one or more pregnancies. A history
of pregnancy termination after prenatal diagnosis was observed more frequently in the
direct testing group (25%) than in the exclusion group
174 (53%) of the 331 couples requesting PGD had at least one PGD cycle with oocyte
retrieval(OR). In 310 (79.9%) of the 389 cycles with oocyte retrieval at least one embryo
could be transferred to the uterus, leading to 84 clinical pregnancies, resulting in 77
deliveries and the birth of 90 children.
The delivery rates per OR were 19.8% and 24.8% per embryotransfer.
Conclusions
The results obtained are similar to usual PGD success rates. PGD seems a valuable and safe
reproductive option for both cariers and at risk persons for HD
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P60: Characterization of dysmorphic features in 1q21.1 microduplication syndrome:
description of three patients and review of the literature.
Stephanie Moortgat, Anne Destree, Bernard Grisart, Christine Verellen-Dumoulin & Isabelle
Maystadt
Centre de Génétique Humaine, Institut de Pathologie et de Génétique, Charleroi (Gosselies),
Belgium
Array-CGH screening in large patient cohorts with unexplained mental retardation,
congenital anomalies or autism has recently lead to the characterisation of novel
microdeletion/microduplication syndromes. The low-copy-repeats spanning the region
1q21.1 mediate nonallelic homologous recombinations that result in rearrangements of
1q21.1.
Recent data suggest that a recurrent 1,35 Mb microduplication on chromosome 1q21.1
predisposes to autism spectrum disorder, developmental delay and mental retardation.
Other clinical features include macrocephaly, hypertelorism and a wide range of congenital
anomalies. Variable expressivity, incomplete penetrance and non-specific phenotypic
features associated with 1q21.1 microduplication complicate genetic diagnosis and
counselling.
We report on three unrelated girls presenting with developmental delay and similar facial
dysmorphism such as hypertelorism, flat nasal bridge, short nose with anteverted nostrils,
prominent philtrum and thin upper lip. Head circonferences were in normal ranges (≤ 2SD).
One was two years old and the two others were three. At the age of three, one of them
developed behavioral disorder with hallucinations and agressivity and the other was
impulsive and presented bruxism.
Array-CGH detected an identical 2.5Mb microduplication on 1q21.1, encompassing the
minimal critical region of 1.35Mb. Parental origin is undetermined (paternal DNA was not
available for analysis).
These three additional patients confirm that identification of 1q21.1 microduplication by
array-CGH should be considered as a predisposition factor for developmental delay and
behavioral disorders. We refine the 1q21.1 microduplication syndrome by suggesting a
distinctive common phenotype, comparing facial dysmorphic features of our patients with
previously reported cases.
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P61: To Tell or Not to Tell? A Review of Ethical Reflections on Incidental Findings Arising in
Genetics Contexts
Gabrielle M. Christenhusz1, Koenraad Devriendt2 & Kris Dierickx1
1
2
Centre for Biomedical Ethics and Law, K. U. Leuven
Centre for Human Genetics, K. U. Leuven
Incidental findings, a common issue in neurology and oncology, are a pressing ethical issue
for new genetic technologies because of the potential of these technologies to deliver an
enormous amount of often unsought for information of widely varying significance. This
presentation is based on two systematic reviews. One review looks at the ethical reasons
presented in the literature for and against the disclosure of incidental findings arising from
clinical and research genetics. 21 articles were selected for review as presenting ethical
reasons either supporting or opposing the disclosure of incidental findings arising in general
contexts or in clinical and research genetic contexts. A third group of reasons offer caution
when making decisions about disclosure. A second review looked at the empirical research
that has been done on the ethical issues involved in the disclosure of incidental findings. 16
articles were selected for review as presenting the attitudes and opinions of professionals,
research participants and lay people.
In the present presentation, we consider how the recommendations from the articles in the
first review, supporting a qualified form of disclosure or caution in disclosure, compare with
the findings from the articles in the second review, in which research participants and lay
people are shown to have high expectations regarding the disclosure of incidental findings.
We recommend limiting the number of possible incidental findings even before genetic
testing is carried out, while recognising that the conflict this may generate with the
expectations of research participants and lay people needs to be addressed.
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P62: Genomic analysis reveals enrichment for MYCN pathway genes in chromosomal
regions affected by targeted DNA copy number alterations in neuroblastoma
Annelies Fieuw1, Candy Kumps1, Pieter Mestdagh1, Björn Menten1, Filip Pattyn1, Steve
Lefever1, Sara De Brouwer1, Alexander Schramm2, Johannes H. Schulte2, Nadine Van Roy1,
Rosa Noguera3, Valerie Combaret4, Angelika Eggert2, Jo Vandesompele1, Frank
Westermann5, Katleen De Preter1 & Frank Speleman1
1
Center for Medical Genetics, Ghent University, Ghent, Belgium
Department of Pediatric Oncology and Haematology, University Children's Hospital, Essen,
Germany
3
Department of Pathology, Medical School, University of Valencia, Valencia, Spain
4
Centre Léon Bérard, FNCLCC, Laboratoire de Recerche Translationnelle, Lyon, France
5
Department of Tumor Genetics, German Cancer Research Center, Heidelberg, Germany
2
Neuroblastoma is an embryonic tumor arising from immature sympathetic nervous system
cells. DNA copy number alterations have been extensively studied in this tumor, revealing
mainly recurrent large segmental DNA copy number losses and gains as well as frequent
MYCN amplification. The finding of a focal 5 kb gain containing exclusively the MYCN
activated oncogenic miR-17-92 cluster in a neuroblastoma cell line suggested that clinically
relevant small focal genomic gains and losses are implicated in neuroblastoma and that such
DNA copy number changes may specifically target MYCN regulated genes.
To further test this hypothesis, we investigated unpublished high resolution DNA copy
number data of 190 neuroblastoma tumor samples and 33 cell lines for which for the
majority also accompanying mRNA and miRNA data were available. We detected significant
enrichment for (in)direct up regulated MYCN target genes in focally gained and amplified
regions thus indicating that rare and/or recurrent copy number variants can further
reinforce particular MYCN downstream effects in tumor cells. In the deleted regions, we
observed enrichment for predicted target genes of MYCN up regulated miRNAs. Using an
integrated data mining approach, we searched for new putative MYCN regulated genes
located within these regions of loss and identified RGS5, a gene encoding a regulator of G
protein signaling implicated in vascular normalization. RGS5 expression levels were strongly
inversely correlated to MYCN expression in the primary neuroblastoma tumor cohort.
Combined miRNA expression data and 3’UTR seed sequence information allowed us to
identify and subsequently validate several MYCN regulated miRNAs including miR-9
targeting RGS5 thus further expanding the previously published network of miRNA
mediated down regulation of MYCN effectors. Given the emerging role of RGS5 in tumor
angiogenesis, this gene may represent an important new target for anti-angiogenic therapy.
In conclusion, this study identifies biologically relevant genes that are targeted by focal gains
or deletions and that may represent important targets for future neuroblastoma therapy.
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P63: ASCAT: a versatile framework for allele-specific copy number profiling of tumors
from SNP array or next-generation sequencing data
Peter Van Loo1, David C. Wedge1, Gro Nilsen2, Hans Kristian Moen Vollan3, Jiqiu Chen4, Bina
P. Pandey4, Peter Marynen5, Andrew Futreal1, Anne-Lise Børresen-Dale3, Vessela N.
Kristensen3, Peter J. Campbell1, Ole Christian Lingjærde2 & Michael R. Stratton1
1
Wellcome Trust Sanger Institute
Department of Informatics, University of Oslo
3
Department of Genetics, Oslo Univeristy Hospital Radiumhospitalet
4
Department of Electrical Engineering, Univeristy of Leuven
5
Department of Human Genetics, University of Leuven
2
Cancer biopsies contain multiple populations of cells, including normal cells and possibly
multiple subclones of cancer cells. We recently developed ASCAT (Allele-Specific Copy
number Analysis of Tumors), a novel bioinformatics approach to accurately dissect the
allele-specific copy number of solid tumors, and applied ASCAT to Illumina 109k SNP array
data of 112 breast cancers with matched normal data (Van Loo et al., 2010, PNAS
107:16910-16915). Here, we extend this approach to unmatched tumor data, to multiple
SNP arrays platforms and to next-generation sequencing data.
We developed a novel method to infer germline genotypes from tumor SNP array data and
obtain 99% concordance with genotypes obtained from matched germline data. Hence, this
method allows ASCAT to perform comparably whether matched normal samples are
available or not. We extended the ASCAT framework to multiple SNP array platforms from
both Affymetrix and Illumina. In a series of samples where both Affymetrix and Illumina SNP
array data is available, we obtain similar results. In addition, we show that ASCAT can be
used to gain insight into intra-tumor heterogeneity.
We further extend the ASCAT framework to next-generation sequencing data. This allows
accurate estimates of tumor cellularity from both exome sequencing and whole-genome
sequencing data. In addition, allele-specific copy number profiles are obtained from which
gains, losses, loss-of-heterozygosity and copy-number-neutral events can be inferred.
ASCAT-derived cellularity estimates can be used to direct required sequencing depth and
ASCAT-derived copy numbers can be used to increase sensitivity of variant detection
methods. In addition, copy number states can be assigned to predicted mutations, allowing
inference of mutation zygosity and subclonality.
In conclusion, ASCAT is a versatile method to infer tumor cellularity and allele-specific copy
number from both matched and unmatched SNP array or next-generation sequencing data.
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P64: Prenatal Array CGH Identifies Genomic Imbalances Associated With Congenital
Diaphragmatic Hernia (CDH)
Paul D. Brady1, Jean-Pierre Fryns1, Koenraad Devriendt1, Jan Deprest2 & Joris Vermeesch1
1
2
Centre for Human Genetics, KU Leuven
Fetal Medicine Unit, Department Women & Child, UZ Leuven
Congenital Diaphragmatic Hernia (CDH) is caused by a defect in the formation or closure of
the diaphragm, with an incidence of 1.7 – 5.7 per 10,000 live-births (Kotecha, et al, 2011).
Genetic factors are considered to play an important role in the pathogenesis of congenital
diaphragmatic hernia (CDH) (Brady, et al, 2010). UZ Leuven is a specialist referral centre for
foetuses undergoing foetal therapy by fetoscopic tracheal occlusion (FETO). We have
previously presented our findings from a retrospective study using a targeted custom design
array in foetuses presenting with isolated CDH (Srisupundit & Brady, et al, 2010). Here we
present our findings from an on-going prospective study into the use of a high-resolution
genome-wide oligonucleotide array for prenatal diagnosis, in which we have now screened
over 60 cases of CDH, which has revealed both recurrent and novel genomic imbalances
associated with development of CDH.
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P65: Experience and future plans for the molecular diagnosis of the Adenomatous
Polyposis Coli (APC) gene at UCL
Xavier Pepermans, Jean-Luc Guarin & Hélène Antoine-Poirel
Human Genetics Center, Université catholique de Louvain - Cliniques Universitaires SaintLuc
The global incidence of Familial Adenomatous Polyposis (FAP) is 1/8000 (0.5% of all
Colorectal Cancers). The genetic cause of FAP lies in autosomal dominantly inherited
mutation of the APC gene (OMIM175100). APC gene (NM_000038) consists of 15 coding
exons and encodes a 2843-aa protein acting as a tumor suppressor by controlling B-catenin
turnover.
Till 2009, the screening of APC gene was performed with Sanger sequencing (14 first exons)
and PTT (last exon). Since 2009, we combine sequencing of the whole ORF (28 amplicons)
with MLPA.
Since 2009, we have tested 43 new unrelated propositus patients with distinct FAP
phenotype. Different types of mutations were found in 10/43 patients:
- 6 deleterious mutations (3 del-ins, 2 nonsense, 1 splice defect), two are unpublished
(c.4242insT, c.1312+2T>A); 1/6 exhibit an attenuated form (AFAP);
- 4 missense mutations considered as variants with unknown significance, two are
unpublished (p.Ala1670Val, p.Ser2640Thr); the 3 patients with available clinical data exhibit
an AFAP; genotype-phenotype is under way to assess the pathogenicity of these
substitutions.
We acquired an experience with FAP screening (FAP-EMQN-2010: our laboratory ranked
within the top three). We recently added the detection of deletions within a new alternative
transcript (Rohlin et al. 2011). Moreover, we plan on introducing the technology of NGS into
the screening of the APC gene, which is a nice candidate gene (great variety of mutations on
a wide ORF size).
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P66: Presymptomatic Iodotyrosine deiodinase-deficient hypothyroidism identified via
genome-wide approach
Catheline Vilain1, Isabelle Pirson2, Willem Kulik3, Gijs Afink4, Rodrigo Moreno-Reyes5,
Bernard Corvilain6, Marc Abramowicz1 & Agnes Burniat6
1
ULB Center of Human Genetics, Brussels, Belgium
I.R.I.B.H.M., ULB, Brussels, Belgium
3
Laboratory for Genetic Metabolism Diseases, AMC, Amsterdam, The Netherlands
4
Reproductive Biology Laboratory, AMC, Amsterdam, The Netherlands
5
Nuclear Medecine, ULB Erasme, Brussels, Belgium
6
Department of Endocrinology, ULB Erasme, Brussels, Belgium
2
By genome-wide homozygosity mapping, we identified a c.658G>A homozygous mutation of
the iodotyrosine deiodinase (IYD) gene in a 15.9 year old male with hypothyroidism, large
goiter, stunted growth, and normal Iodine perchlorate discharge test. The patient was the
eldest child of seven siblings born to healthy first cousins. The youngest sibling, a 4.5 year
old girl, was also found homozygous for the mutation. Although asymptomatic, she had
elevated MIT and DIT urinary excretion. While the mutation had been reported previously
with a possible dominant effect, in the present family it behaved as a strictly autosomal
recessive trait. We delineated the phenotype of the defect in more detail, and showed that
the urinary MIT and DIT excretion was limited in the patients. This finding probably points to
the lack of local iodine in the thyroid gland as a main mechanism responsible for
hypothyroidism in IYD deficiency. The cause for the wide inter- and intra-familial variability
of the disease severity remains unclear. Besides refining the phenotype of the IYD defect,
this is the first report of a genome-wide approach allowing presymptomatic diagnosis of an
inborn thyroid defect that escaped neonatal screening.
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P67: Complex genetics of radial ray deficiencies: Screening of a cohort of 54 patients
Sarah Vergult1, Jeannette Hoogeboom2, Emilia Bijlsma2, Tom Sante1, Eva Klopocki3, Bram De
Wilde1, Marjolijn Jongmans4, Christian Thiel5, Joke Verheij6, Antonio Perez-Aytez7, Hilde Van
Esch8, Alma Küchler9, Diane Barge-Schaapveld10, Yves Sznajer11, Geert Mortier12 & Björn
Menten1
1
Center for Medical Genetics, Ghent University, Belgium
Department of Plastic, Reconstructive and Hand Surgery, Erasmus MC, Rotterdam
3
Institut für Medizinische Genetik, Charité Universitätsmedizin Berlin, Berlin, Germany
4
Department of Human Genetics, Radboud University Nijmegen Medical Center, Nijmegen, The
Netherlands
5
Institute of Human Genetics, University Hospital Erlangen, Erlangen, Germany
6
Department of Genetics, University Medical Center Groningen, Groningen, The Netherlands
7
Grupo de Investigación en Perinatología, Instituto de Investigación Sanitaria, Valencia, Spain
8
Center for Human Genetics, Leuven, Belgium
9
Institut für Humangenetik, Universitätsklinikum Essen, Essen, Germany
10
Department of Clinical Genetics, Academic Medical Centre, Amsterdam, The Netherlands
11
Pediatric Clinical Genetics, Center for Human Genetics, ULB, Brussels, Belgium
12
Department for Medical Genetics, Antwerp, Belgium
2
Radial ray deficiencies (RRDs) are characterized by unilateral or bilateral absence of varying
portions of the radius and thumb. Both isolated and syndromic forms have been described,
and although for some of the syndromes the causal genes have been identified e.g. TBX5
(Holt-Oram Syndrome), the genetics of RRDs remain complex. Given this complexity we
searched for genomic aberrations in 54 patients with RRDs by means of molecular
karyotyping. In eight patients, a genomic aberration was detected. We identified known
microdeletion/-duplication syndromes such as the 1q21.1 microduplication and the
16p13.11 microduplication syndrome in three patients. Beside these known CNV
syndromes, some rare copy number variations were detected. Of these, two were of high
interest: a deletion at chromosome band 7p22.1 and a deletion at chromosome band
10q24.3.
The 7p22.1 deletion contains the RAC1 gene. Conditional knockout mice of Rac1 exhibit
truncated fore- and hindlimbs (Wu et al., 2008), making it an interesting candidate gene for
RRDs. However, sequence analysis of our cohort did not reveal any other patient with a
causal mutation in this gene. To investigate the possibility of a recessive allele being
unmasked by the 7p22.1 deletion, target capture followed by next generation sequencing
was performed for the other allele in the patient with the 7p22.1 deletion. No mutation was
detected in the RAC1 gene nor in any other gene within the deletion interval. So most likely
the deletion itself is causal for the RRDs observed in this patient.
The 10q24.3 deletion contains only one gene, FBXW4. This gene is of high interest since in
mice insertions in the orthologue dactylin gene give rise to dactylaplasia. In humans,
duplications of this region are associated with Split Hand Foot Malformation (SHFM3). How
these duplications cause SHFM3 is still not known.
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Since several genes in the vicinity have a role in limb development, it has been suggested
that these duplications could disrupt the interaction between cis-regulatory elements and
their target genes, resulting in the SHFM phenotype. In our patient it could also be possible
that the 10q24.3 deletion gives rise to RRDs by deletion of one or more these specific
regulatory sequences. To test this hypothesis, six CNEs (conserved non-coding elements) in
the deletion interval were selected for sequencing. The analysis of these CNEs revealed one
substitution (g.103380009A>G), which was absent in 96 control samples. Unfortunately
parental DNA was not available to check the inheritance of this substitution. Sequence
analysis of the exons of FBXW4 in our entire patient cohort, did not reveal any causal
mutations.
This is the first microarray study in a larger cohort of patients with RRDs. Some interesting
aberrations were detected that need further investigations.
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P68: Identification of IDH1&2 mutations and SNP rs11554137 in AML using HRM.
Magali Dodemont, Frederic Lambert & Vincent Bours
Laboratory of Molecular Haematology-Center of Human Genetics- CHU Liège-Ulg
Background
Recently, mutations of either IDH1 (IDH1R132) and IDH2 (IDH2R140 and IDH2R172) have
been described in CN-AML. These mutations might correlate with poorer prognostic,
especially in FLT3-ITD negative/NPM1+ patients.
Moreover, a SNP in IDH1 (rs11554137) has been reported in 12% of CN-AML, potentially
associated with a worse survival.
The aim of our study was to develop a screening strategy for fast and sensitive detection of
those mutations and SNP based on High Resolution Melting analysis followed by sequencing
confirmation.
Method
DNA was extracted from 64 de novo AML.
Screening for mutations and SNP was performed using a new HRM based LightCycler® 480
assay adapted from the publication of Noordermeer et al.
Abnormal profiles were confirmed by direct sequencing.
Results
Four specific groups were detected after HRM analysis of IDH1: one corresponded to the
rs11554137 SNP, the second one to the IDH1R132 mutation, the third profile characterized
samples with both IDH1R132 mutation and the SNP whereas the last one corresponded to
wild-type samples.
Two different clusters characterized IDH2: one corresponding to wild-type samples and the
second one to samples presumably mutated.
After analysis, fifteen abnormal HRM profiles were obtained reflecting the presence of
either IDH1&2 mutations and SNP whereas the others seven were supposed to reflect the
presence of an isolated rs11554137 SNP. These results were subsequently confirmed by
sequencing. Two ‘abnormal profiles’ were not confirmed by sequencing giving a method
specificity of 95.5 %.
Conclusion
This pre-screening method reduces the number of samples to be sequenced to the
abnormal HRM profiles saving time and reducing cost.
Rs11554137 SNP is associated with a characteristic profile and doesn’t require to be
sequenced.
We have developed a HRM assay to detect IDH mutations and SNP and provide evidence of
its applicability as a molecular diagnostic assay for clinical purposes.
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P69: Profiling intra tumour heterogeneity with SNP-CGH array based on mono-allelic
deletions.
Christophe Poulet1, James Swingland2, Manuel Deprez3, Pierre Robe4, Christian Herens5,
Federico Roncaroli2, Vincent Bours1 & Federico Turkheimer2
1
Ulg, GIGA-R, Human Genetics, Liège, Belgium
MRC, Imperial College, Clinical Neuroscience, London, UK
3
Ulg, CHU, Neuropathology, Liège, Belgium
4
CHU, Neurosurgery, Utrecht, Netherland
5
Ulg, CHU, Human Genetics, Liège, Belgium
2
Diagnosis in cancer molecular cytogenetics relies on the ability of Comparative Genomic
Hybridisation (CGH) analysis to detect chromosomal alterations that frequently occur in
tumours. However, these tumours are often heterogeneous and most current CGH
algorithms do not take this heterogeneity into account, leading to frequent failures in
detection. Besides, many CGH data algorithms have numerous tuneable parameters urging
for the development of a simple analysis tool with a good visualisation display of candidate
alterations. This tool should also evaluate the intra-tumour heterogeneity (ITH) in non
paired samples.
We developed a new analysis method to estimate the ITH with SNP-CGH array based on
allelic deletions. Basically, the commonest alteration is hypothesised to represent the
earliest stable genetic alteration in the tumour and has therefore affected the majority of
the altered cells in the sample. Conversely, the latest stable genetic alteration has affected
the minority of the altered cells in the sample. These early and late events represent
therefore different percentages of altered cells. Our method transforms each deletion event
into its percentage of altered cells. Combining all these percentages, a heterogeneity profile
is drawn to reveal the different sub-populations of altered cells constituting the tumour.
We applied this ITH method on two available GEO datasets, one is composed with chronic
lymphocytic leukaemia samples and the other is composed with non-small cell lung cancer
samples. Our method obtained a good correlation with the results originally published in
these studies using cell counting methods such as FISH, flow cytometry or light microscopy.
Based on our method, the identification of cell sub-clones in heterogeneous tumours was
greatly facilitated. Moreover, our method is based on the algorithm “CHROMOWAVE” used
to denoise mRNA expression levels and therefore our method makes possible the direct
correlation of SNP-CGH array data with gene expression levels. The current method does
not provide ITH estimation based on allelic gains and further work in this direction is
needed. However, our method is an important step for future profiling of ITH and for
drawing a phylogenetic tree revealing the tumour development.
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P70: MYT1L, a new candidate gene for non-syndromic intellectual disability
Nina De Rocker1, Sarah Vergult1, Elke Van Oudenhove1, Carla Rosenberg2, Paula Frassinetti2,
Zeynep Tümer3, Frédérique Bena4, Armand Bottani4, Nele Bockaert5, Filip Roelens6, Orsetta
Zuffardi7, David A. Koolen8, Tjitske Kleefstra8, Ernie M. Bongers8, Thomy de Ravel9, JeanPierre Frijns9, Koenraad Devriendt9, Joris Vermeesch9, Geert Mortier10, Jill R. Mokry11, Lisa G.
Shaffer11 & Björn Menten1
1
Center for Medical Genetics, Ghent University, Belgium
Bioscience Institute, University of São Paulo, Brazil
3
Center for Applied Human Molecular Genetics, Glostrup, Denmark
4
Service of Genetic Medicine, University Hospitals of Geneva, Switzerland
5
COS, Ghent University Hospital, Ghent, Belgium
6
Heilig Hart Ziekenhuis Roeselare, Belgium
7
Medical Genetics, University of Pavia, Italy
8
Centre for Molecular Life Sciences, Radboud University Nijmegen, The Netherlands
9
Centre for Human Genetics, University Hospitals Leuven, Katholieke Universiteit Leuven, Belgium
10
Department for Medical Genetics, Antwerp, Belgium
11
Signature Genomic Laboratories, Perkin Elmer, Inc., Spokane, Washington, USA
2
Intellectual disability is a highly heterogeneous neurodevelopmental disorder with more
than 100 causal genes already identified, but many more await discovery. The introduction
of genomic microarray in clinical diagnostics has however greatly improved the detection of
small genetic aberrations in patients, with subsequent identification of candidate disease
causing genes.
In this study we defined a new microdeletion syndrome comprising chromosome band
2p25.3 in 11 patients with mild to moderate non-specific intellectual disability. The shortest
region of overlap contains one single gene, the myelin transcription factor 1-like (MYT1L),
pointing to this gene as a possible cause for the intellectual disability. MYT1L is highly
homologous to family member MYT1, which regulates neural differentiation in the brain.
Myt1l shows high expression in the developing rat brain from embryonic day 15, with
maximal expression at parturition and subsequent decline to low but detectable levels in
adult brain. Myt1l expression is restricted to neurons, with highest expression during fetal
development. Common SNPs in MYT1L have been described to be associated with major
depressive disorder and schizophrenia, and a duplication disrupting MYT1L has been
reported in a patient with schizophrenia. Our patients with intellectual disability may thus
represent the extreme spectrum of phenotypic effects of deregulated MYT1L expression.
Recently, the direct conversion of fibroblasts into functional neurons has been described by
making use of only three transcription factors: Ascl1, Brn2, and Myt1l. The authors assumed
that high expression levels of strong neural cell-fate determining transcription factors can
activate salient features of the neuronal transcriptional program, indicating that MYT1L may
play a pivotal role in neuronal development. Taken together, these data strongly pinpoint
loss of MYT1L as a candidate gene for the intellectual disability observed in our patients.
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P71: The 15q11.2 deletion and Autism Spectrum Disorders
Veerle De Wolf, Hilde Peeters & Koen Devriendt
Center for Human genetics, University of Leuven, Leuven, Belgium
Introduction
Autism Spectrum Disorders (ASDs) are frequent neurodevelopmental disorders with a
predominant genetic cause. Several studies have shown the association of CNVs (Copy
Number Variations), both de novo and rare inherited, with ASD. Deletions of chromosome
15q11.2 (BP1-BP2 region) including CYFIP1, NIPA1, NIPA2 and TUBGCP5, are associated with
intellectual disability, epilepsy, schizophrenia and autism. These four genes are expressed in
brain or neuronal tissue. Of interest, CYFIP1, located in the BP1-BP2 deleted region, is an
important binding partner of FMRP and FMR1 mutations cause the Fragile X syndrome,
often associated with autism (30%). Also, patients with the Prader-Willi phenotype caused
by paternal deletions of the 15q11-13 BP1-BP3 region present a more severe behavioural
phenotype (ADHD, autism and OCD) compared to those having the BP2-BP3 deletion.
Together, this pinpoints the 15q11.2 deleted region as a possibly causal CNV in ASD.
Methods
We present an association study using quantitative Real-Time Polymerase Chain Reaction
(q-RT-PCR), testing for deletions of CYFIP1 in ASD patients with intellectual disability
(IQ<70), ASD patients with normal intelligence (IQ>70) and controls.
Results
We will present the results of the association study of CYFIP1 deletions in ASD patients.
Interestingly, the phenotype of del15q11.2 is very variable and this variant is often inherited
from a normal parent. As a consequence, we hypothesize that additional variants in the
other allele or in other genes of the FMRP network can contribute to the phenotype of
these patients. This multi-hit model has already been suggested by several others and
second hits were also found in patients carrying a del15q11.2.
Conclusion
We performed an association study of the 15q11.2 deletion with ASD. Investigation of the
inheritance pattern of this deletion shows frequent inheritance from a normal parent. A
follow-up study by performing exome sequencing of these patients can reveal a second hit
variant that could aid to explain the variability of the phenotype.
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P72: Fetal death: a case of Toriello-Carey syndrome
Kim van Berkel, Mina Leyder, Tim Verhasselt, Annieta Goossens & Kathelijn Keymolen
Universitair Ziekenhuis Brussel, Belgium
Objective
We describe a fetus in which we diagnosed a Toriello-Carey syndrome during thorough
investigation of fetal demise at 19 weeks of gestation. This is the first report of a TorielloCarey syndrome with fetal death.
Materials and Methods
After accidental diagnosis of fetal demise, thorough examination of both mother and fetus
was carried out, following the available guidelines. Personal and familial history, clinical,
radiological and pathological examination, laboratory testing and prenatal genetic testing
(including CGH-array) were carried out.
Results
Examination of the fetus demonstrated a particular facial gestalt including telecanthus and
Pierre Robin sequence. These findings in association with a partial corpus callosum agenesis
and an agenesis of the right kidney led to the clinical diagnosis of a Toriello-Carey syndrome.
Prenatal karyotyping and CGH-array were normal. Other causes of fetal death, such as
maternal factors were ruled out.
Conclusion
We believe that the diagnosis of Toriello-Carey syndrome, based on the clinical findings and
the normal genetic test results, is the reason for the fetal demise in this case.
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P73: 22q11.2 microduplication in a patient with bladder exstrophy and delayed
psychomotor development
Annette Uwineza, Geneviève Pierquin, Anne-Cécile Hellin, Mauricette Jamar, Jean-Hubert
Caberg & Vincent Bours
Center for Human Genetics, CHU Sart-Tilman, University of Liège, Belgium.
Bladder exstrophy (BE) is a complex congenital anomaly characterized by a defect in the
closure of the lower abdominal wall and bladder. This condition is part of a clinical spectrum
of the bladder exstrophy-epispadias complex (BEEC). The BEEC represents a spectrum of
urological abnormalities in which part or all of the distal urinary tract fails to close and is
exposed on the outer abdominal wall. Previously, nine cases of classic exstrophy of the
bladder with underlying microduplication 22q11.2 have been reported (Lundin et al., 2010;
Draaken et al., 2010; Ludwig et al., 2011).
A 10-year-old boy was referred for genetic evaluation of his psychomotor retardation. The
past medical history revealed the existence of bladder exstrophy at birth. Familial history
was negative because he was adopted. The clinical evaluation of the proposita showed short
stature, scar of repair of bladder exstrophy, micropenis and a normal intelligence. Multiplex
ligation-dependent probe amplification (MLPA) analysis performed using the SALSA MLPA
KIT P250 DiGeorge (MRC-Holland, Amsterdam, Netherlands) detected a microduplication
22q11.2. The duplication was confirmed in FISH and array–CGH. The array-CGH (Affymetrix
Cytogenetics Whole-Genome 2,7 M Array) identified a duplication of 2419 kb in the 22q11.2
region.
In conclusion, this report extends the phenotypic spectrum of bladder exstrophy in
microduplication 22q11.2 and may point to possible gene (s) located on 22q11.2 playing a
putative role in urogenital development. It provides further evidence of genotypephenotype correlation.
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P74: Cost-effective and robust genotyping using double-mismatch allele-specific
quantitative PCR
Steve Lefever1, Ali Rihani1, Filip Pattyn1, Tom Van Maerken1, Jan Hellemans2 & Jo
Vandesompele3
1
Center for Medical Genetics Ghent, Ghent University, Ghent, Belgium
Biogazelle, Zwijnaarde, Belgium
3
Center for Medical Genetics Ghent, Ghent University, Ghent, Belgium; Biogazelle,
Zwijnaarde, Belgium
2
Various methods are available for single nucleotide polymorphism (SNP) genotyping, each
with its benefits and limitations. While SNP genotyping on a genome scale is currently
dominated by microarrays, assessment of a targeted selection of SNPs (or single base
mutations) is mainly done through PCR. PCR based methods are faster and less laborious,
and include high-resolution melting (HRM) and real-time PCR using hydrolysis probes.
Although these PCR based methods can be performed in high-throughput, are amenable for
automation, and generate data that is relatively easy analyzed, a major drawback is the
dependence on probes - either labeled or not - adding to their complexity and cost,
especially when the number of samples to be genotyped is low. As an alternative, we
present here a cost-effective and robust qPCR based genotyping method, called double
mismatch allele-specific qPCR (DMAS-qPCR), consisting of two parallel quantitative PCR
reactions, each including a modified allele-specific forward and wild-type common reverse
primer (or vice versa). The modification consists of an artificial mismatch on position 4
(starting from the 3’ end). The combined information of Cq values from the two allele
specific reactions enables robust and straightforward genotyping. Results obtained using
this simple setup, precluding the necessity for probes and specialized equipment (and
accompanying software) such as a high-resolution melting instrument, were compared with
corresponding TaqMan results (considered the gold standard in this experiment) for 12 SNPs
from the TP53 pathway on 48 cancer cell lines. DMAS-PCR showed a hundred percent call
rate with 574 correct calls out of 576 genotypes (99.6% concordance). TaqMan assays
resulted in approximately 4% undetermined calls, indicating a higher sensitivity and
robustness for DMAS-qPCR. In conclusion, our new method enables accurate genotyping
results with all types of SNPs across a wide range of DNA input concentrations and will
prove to be a powerful alternative to the commonly used genotyping methods. To increase
adoption in the community, DMAS-qPCR assay design is available for limited use on
www.primerxl.org.
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P75: primerXL – PCR primer design for targeted resequencing
Steve Lefever, Filip Pattyn, Bram De Wilde, Jan Hellemans & Jo Vandesompele
Center for Medical Genetics Ghent, Ghent University, Ghent, Belgium
Targeted resequencing has come of age in a clinical diagnostic setting. Different target
enrichment strategies have emerged, each with their own benefits and limitations. While
enrichment by PCR outperforms hybridization based enrichment strategies when looking at
specificity and sensitivity, coverage uniformity, and flexibility, PCR is currently less
frequently-used due to the absence of available assays to cover complete genes (including
splice variants and intron boundaries) and the requirement of large numbers of parallel PCR
reactions. The latter issue is being addressed through novel high-throughput PCR platforms,
such as droplet PCR (Raindance) and nanoliter reactions (Wafergen and Fluidigm), which are
bringing PCR enrichment into the mainstream. The large number of simultaneous smallvolume reactions reduces turnaround time, cost and the input amount of DNA significantly.
The remaining bottleneck for PCR based resequencing is the availability of PCR assays to
cover entire genes. Various primer design software packages are available but most are not
suited for amplicon generation in the context of targeted resequencing. To address these
shortcomings, we have developed primerXL, a state-of-the-art high throughput primer
design pipeline for massively parallel targeted resequencing. It employs an optimized design
parameter relaxation cascade and multiple primer pair quality control analyses, generating
high quality and robust assays resulting in extremely uniform sequencing coverage. In
addition, an accompanying user-friendly webtool has been developed that enables full
customization in assay design, starting with a gene symbol as input. Performance data for 4
projects (X genes, Y samples) will be shown. PrimerXL is available for limited use at
www.primerxl.org.
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P76: Single cell genomics from the cell cycle perspective
Niels Van der Aa1, Jiqiu Cheng2, Masoud Zamani Esteki1, Parveen Kumar1, Evelyne
Vanneste1, Yves Moreau2, Joris Vermeesch1 & Thierry Voet1
1
2
Center for Human Genetics, KULeuven
ESAT, KULeuven
Methods to profile a genome of a single cell for DNA-copy number aberrations and single
nucleotide polymorphisms using DNA-microarrays have been developed and are applied in
basic research as well as clinical practice. However, current methodology does not
discriminate between the analysis of a cell in G0/1-, S- or G2/M-phase of the cell cycle
although the cell’s DNA-content is different for those phases and in addition dynamic during
S-phase. Here, we profile DNA extracted from populations of cells enriched for S-, G0/1- or
G2/M-phase, as well as whole-genome amplified DNA from single S-, G0/1- or G2/M-phase
cells by array comparative genomic hybridization. Individual DNA replication domains were
detected in single S-phase cells. Hence, the accuracy of single-cell copy number profiling is
dependent on the cell cycle stage of the cell being analyzed. We provide a new tool to
investigate DNA-replication and a work-flow to detect S-phase cells. We are currently
increasing the resolution of this method by massively parallel genome sequencing of cells in
G1/0- and S-phase.
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P77: The Contribution of CLIP2 Haploinsufficiency to the Clinical Manifestations of the
Williams-Beuren Syndrome
Geert Vandeweyer, Nathalie Van der Aa, Edwin Reyniers & R F. Kooy
Department of Medical Genetics, University and University Hospital of Antwerp
The Williams-Beuren syndrome is a rare contiguous gene syndrome, characterised by
intellectual disability, facial dysmorphisms, connective tissue abnormalities, cardiac defects,
structural brain abnormalities and transient infantile hypercalcemia. It is currently thought
that genes lying telomeric to RFC2, including CLIP2, GTF2I and GTF2IRD1, are most likely
responsible for the typical Williams Syndrome Cognitive Profile, characterised by a better
than expected auditory rote memory ability, a relative sparing of language capabilities and a
severe visual-spatial constructive impairment. Atypical deletions in the region helped to
establish genotype-phenotype correlations. So far however, hardly any deletions affecting
only a single gene in the disease region have been described.
We present here two healthy siblings with a pure, hemizygous deletion of the CLIP2 gene.
Based on a knock-out mouse model, a putative role in the cognitive and behavioural
abnormalities seen in Williams-Beuren patients has been suggested for this gene. The
siblings did not show any of the clinical features associated to the syndrome. Cognitive
testing showed an average IQ for both with no indication of the Williams Syndrome
Cognitive Profile. This shows that CLIP2 haploinsufficiency by itself does not contribute to
the physical or cognitive characteristics of the Williams-Beuren syndrome, nor does it
contribute to the Williams Syndrome Cognitive Profile. Our results therefore support the
hypothesis that GTF2IRD1 and GTF2I are the main genes causing the cognitive defects
associated to the Williams-Beuren Syndrome.
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P78: Interindividual variations in DNA methylation
Emilie Théâtre1, Benoît Charloteaux1, Denis Baurain1, Valérie Deffontaine1, Yukihide
Momozawa1, François Crins1, Myriam Mni1, Naima Ahariz1, Catherine Reenaers2, Pierrette
Gast2, Catherine Van Kemseke2, Philippe Leclercq2, Edouard Louis2 & Michel Georges1
1
2
Animal Genomics Unit, GIGA-Research, University of Liège, Belgium
Hepato-Gastroenterology Unit, CHU Liège, Belgium
Objectives: DNA methylation is an important epigenetic mark involved in differentiation and
development. In addition to mutations, it is becoming evident that changes in DNA
methylation can significantly influence the pathogenesis of complex diseases, notably
cancer (1). However, the genetic and environmental factors governing these variations
remain poorly understood.
In this work, we investigate the impact of genetic variations on the control of DNA
methylation and the resulting effect on gene expression.
Methods: To investigate the relationships between genome, epigenome and transcriptome,
we collected blood and intestinal biopsies (ileum, transverse colon and rectum) from ~ 200
healthy Caucasians. Leukocytes were fractionated in five cell populations. Genomic DNA and
total RNA were extracted from all tissues. Each individual was genotyped genome-wide.
Genome-wide methylome analysis was performed on transverse biopsies and the
transcriptome of all tissues was analyzed.
Results: After a stringent quality control of the genomic and the epigenomic data, we
searched for variants affecting methylation, i.e. methylation Quantitative Trait Loci (mQTL),
using standard procedures. We identified cis and trans mQTL effects consistent with the
haplotype structure of the corresponding loci. Interestingly, some variants seem to act as
master regulators of DNA methylation, influencing the methylation state of many CpG sites.
Conclusions: We thus identified several cis and trans mQTL across the genome in transverse
biopsies of healthy Caucasians. Further characterization of the corresponding loci is in
progress. Latest results will be presented.
(1) Rakyan, V.K., Down, T.A., Balding, D.J., and Beck S. (2011) Epigenome-wide association
studies for common human diseases. Nature Reviews Genetics, 12: 529-541.
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P79: Whole exome sequencing of Nicolaides-Baraitser syndrome patients reveals the de
novo nature of the underling defects
Jeroen K. Van Houdt, Beata A. Nowakowska, Koenraad Devriendt & Joris R. Vermeesch
Center for Human Genetics, KU Leuven, UZ Gasthuisberg, Leuven, Belgium
Nicolaides–Baraitser syndrome (NBS) was first described in 1993, but only recently well
delineated in 25 patients. Main characteristics are short stature, sparse hair, typical facial
morphology, brachydactyly, interphalangeal joint swellings, and intellectual disability with
marked language impairment. The syndrome occurs in persons with different ethnic
backgrounds with no significant difference in occurrence in males and females, no familial
cases are known and parental consanguinity has not been reported. This suggests that NBS
is caused by a dominant de novo mutation in the affected individuals. We performed whole
exome sequencing on four unrelated NBS patients by targeted exome enrichment and
sequencing on an Illumina GAIIx. We obtained between 5.1 to 6.7 Gb of sequence data per
individual. On average, 60% of the bases originated from the targeted exome, with 80% of
the targeted exons covered at least ten times. The mean exome coverage was 40 fold.
About 8500 variants per sample were nonsynonymous, frame shift or splice variant changes.
Because of the probable dominant nature of this disease, we filtered out SNPs described
previously in the control populations, using the NCBI dbSNP build 131 as well as 1000
Genomes Project database. The comparison showed that only 1% of all initially found
variants were included for further investigation. We focused our analysis on genes for which
at least 3 out of 4 individuals carried a novel, non-synonymous variant at different genomic
positions. We identified 5 genes that were affected by distinct missense variants in 3
unrelated cases. Validation by Sanger sequencing confirmed that all detected variants were
heterozygous in the affected individuals. For the three patients, for which the parents could
be tested, de novo mutations were found in a single candidate gene.
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P80: A visualization platform for interpretation of structural genomic data
Tom Sante, Sarah Vergult & Björn Menten
Center for Medical Genetics, Ghent University, Belgium
Working with next generation sequencing technology such as mate-pair sequencing, is a
challenge. These new high-throughput sequencing techniques allow the detection of large
and small CNVs, inversions, translocations and complex rearrangements at unprecedented
resolutions. The growth in data size is accelerating greatly as the sequencing technology is
becoming the tool of the future in genetic diagnostics and research, generating an ever
increasing amount of sequence reads. The ability to visualize this data, to aid analysis, is a
challenging but essential problem. We present a visualization platform which tackles that
problem by developing a graphic web interface that allows the researcher to visually explore
genomic microarray data and mate-paired data, and turn abstract data files into useful
information. The platform provides a genome browser to inspect the copy number profile,
supporting both CNV information from genomic microarrays, sequencing depth of coverage
data and MatePair/PairedEnd-data. The browser uses a zoomable sliding window to explore
the data at different resolution levels, and allows easy comparison of multiple samples. A
circular view is available to facilitate comprehension of intra- and interchromosomal
translocations. Experimental data can be complimented with a multitude of annotation
tracks, to guide the interpretation. Segmental duplications and human chained selfalignments provide information on genomic architecture, RefSeq genes track place the data
in its local genetic context, OMIM morbid/gene-map and database of genomic variants track
provide a biological/clinical context. The custom tracks can be used to compare individual
results with existing data collections of experiments to find correlations in your internal
datasets. The platform employs the latest web technologies so it can be used in any modern
web browser. The visualizations use the scalable vector graphics format for optimal image
quality. Selected regions of interest can be downloaded as SVG or PNG files. The platform
supports genomic microarrays (Agilent, Affymetrix, Illumina, NimbleGen) and
MatePair/PairedEnd sequencing experiment data (BAM/SAM, read clusters) input. Our
experiences as a medical genetics center, enabled us to deliver a comprehensive
visualization platform for genomic microarray experiments complemented with
MatePair/PairedEnd sequencing data, and can be of great value in the toolbox of any
investigator, research or clinical diagnostics.
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P81: Whole Genome Sequencing for gene hunting in autosomal dominant epilepsy
families
Katia Hardies1, Arvid Suls1, Sarah Weckhuysen1, Peter De Rijk1 & Peter De Jonghe2
1
VIB-Department of Molecular Genetics, University of Antwerp, Belgium
VIB-Department of Molecular Genetics, University of Antwerp, Univerity Hospital of
Antwerp, Belgium
2
In the past genome wide searches could only be conducted in large families with multiple
affected family members. This by means of genome wide scans followed by genotyping of
several hundred microsatellite markers or genome high-density (several thousands) single
polymorphisms nucleotides (SNPs) and DNA microarrays. Subsequently linkage analysis and
haplotyping made it possible to determine regions cosegregation with the disease.
Febrile seizures (FS) are the most common form of childhood seizures but also occur in
familial forms of epilepsy. The latter are often associated with a heterozygous spectrum of
phenotypes and a reduced penetrance. Between 1996 and 2007 nine loci responsible for
familial epilepsy syndromes, with FS as its most prominent seizure type, were identified with
the classic genetic research methods. Furthermore, mutations were found in several genes.
However the causative genes in most patients is still not identified and for six out of nine
loci most positional candidate genes were excluded for pathogenic mutations by Sanger
sequencing.
Nowadays next generation sequencing techniques (NGS) are becoming less and less
expensive causing the rise of new kind of genetic research. It is not only becoming possible
to do extensive research in isolated cases (trio analysis) but also to resequence whole
genomes of family members for whom a locus but no causal gene mutation was identified.
In our lab we resequenced 2 family members of 4 epilepsy families with FS. For the moment
the expected gene discovery is however holding off. Also to date only a few cases are
reported in the literature were causal mutations were found by using the latest
technologies. More so, when reported, it mainly concerns exom data and patients with
Mendelian disorders or families with an autosomal recessive inheritance pattern. The
problem lies in the premature assumption that when you have the whole genome, the
mutation has to be in the data provided and you “just” need to find it. But a new era of
genetic research comes with a new set of issues concerning data analysis. With information
about roughly every base pair in the human genome and variations in almost all genes
analytical services are needed to interpreted this data correctly. Next validation of the data
seems to become another large problem. Therefore it might be helpful to provide some
strategic points and problems we encountered in our data analysis.
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P82: Mapping the neuroblastoma epigenome: perspectives for improved biomarkers
Maté Ongenaert, Anneleen Decock, Jo Vandesompele & Frank Speleman
Center for Medical Genetics, Ghent University
Neuroblastoma (NB) is a childhood tumor originating from sympathetic nervous system
cells. Although recently new insights into genes involved in NB have emerged, the molecular
basis of neuroblastoma development and progression still remains poorly understood.
Current risk assessment schemes unfortunately result in a significant proportion of patient
misclassifications, leading to undertreatment or overtreatment. Clearly, a more objective
and accurate classifier is needed for improved outcome prediction. Next to genomic
changes, epigenetic alterations have been described as well: in total, there are about 75
genes described as epigenetically affected in NB cell lines and/or NB primary samples. Most
of these methylation markers are found using ‘candidate gene’ approaches and the
methylation frequencies are usually very low. In order to find novel methylation markers
that can be used for improved prognosis, we applied a whole-genome methylation screen.
This technique relies on capturing with the MBD2 protein, containing a methyl-binding
domain (MBD), with a very high affinity towards methylated genomic regions. In an initial
phase, MBD2-seq was performed on 8 NB cell lines (where we also had micro-array data of,
before and after treatment with the demethylating agent DAC to see re-expression after
demethylation).
An integrated analysis (MBD2-sequencing, re-expression analysis, analysis of public
expression data) led to the selection of 43 candidate biomarkers, which were validated
using real-time MSP (Methylation-Specific PCR) in 89 primary neuroblastoma patients in
three prognostic groups. More than ten novel biomarkers in neuroblastoma were found to
be related with risk factors (such as age at diagnosis, MYCN, stage) and with survival.
Currently, we are performing MBD-sequencing on 45 primary neuroblastoma tumors of the
same three prognostic groups, which will allow us to make a genome-wide map of the
epigenome of neuroblastoma. Of these samples, exon-arrays and aCGH profiles are
available for an extensive integrated data-analysis across the genomic and epigenomic
information. This data should lead to the identification of highly sensitive and specific
biomarkers for improved risk classification and treatment.
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P83: The NeXT generation Variant annotation Tracker: a one stop cloud solution to exome
sequencing data analysis
Bram De Wilde, Jasper Anckaert, Tom Sante, Jan Hellemans, Björn Menten, Frank Speleman,
Jo Vandesompele & Filip Pattyn
Center for Medical Genetics, Gent University
Background
As genetic variation data is being generated at an unprecedented scale, assessment of
functional consequences of the variants in a given patient or cohort is a challenging task,
both from a computational as well as from a data management perspective. It is expected
that in this era of personalised genomics, a clinical sample may need to be re-annotated
repeatedly as new annotation information on the genome becomes available or new
insights on variant interpretation accumulate. While various initiatives emerge to collect the
overwhelming amount of genomic variants currently generated, a central system to manage
and store the annotation of genomic variants as well as determining the functional effects is
still missing.
Results
Here we present a one stop solution to next generation sequencing data analysis. The ‘NeXT
generation Variant Annotation Tracker’ or ‘NXTVAT’ is a highly scalable cloud based
sequence analysis platform. The web based frontend interfaces with an object oriented
shardable database and fully distributed analysis pipelines and enables scaling of the
application to virtually any required size. A plug-in style organisation of the variant
annotation pipelines makes updating and extending variant annotation straightforward.
Currently, variant annotation and effect prediction is done using Ensembl API (1), polyphen2
(2) and genesplicer (3).
Submission of variants is supported for various formats, including the emerging standard
format VCF version 4.0 from the 1000 genomes consortium (4,5). A full next generation
sequencing data analysis can be triggered by uploading any kind of next generation
sequencing data. Users without bio-informatic skills can easily compare huge numbers of
variants across selected samples. As such, NXTVAT tremendously helps in making biological
sense out of next generation sequencing data. While NXTVAT is made publically available for
variant annotation, full next generation sequencing analysis is only available upon request.
1. Mclaren W, Pritchard B, Rios D, Chen Y, Flicek P, Cunningham F. Deriving the consequences of genomic
variants with the Ensembl API and SNP Effect Predictor. Bioinformatics. 2010 Aug. 15;26(16):2069–2070.
2. Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, et al. A method and server for
predicting damaging missense mutations. Nat Meth. 2010 Apr. 1;7(4):248–249.
3. Pertea M, Lin X, Salzberg SL. GeneSplicer: a new computational method for splice site prediction. Nucleic
Acids Res. 2001 Mar. 1;29(5):1185–1190.
4. Danecek P, Auton A, Abecasis G, Albers CA, Banks E, DePristo MA, et al. The variant call format and VCFtools.
Bioinformatics. 2011 Jul. 15;27(15):2156–2158.
5. Durbin RM, Altshuler DL, Durbin RM, Abecasis GR, Bentley DR, Chakravarti A, et al. A map of human genome
variation from population-scale sequencing. Nature. 2010 Oct. 28;467(7319):1061–1073.
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P84: Is CCDC54 involved in spermatogenesis?
Annelien Massart1, Willy Lissens1, Herman Tournaye1, Karine Breckpot2 & Katrien Stouffs1
1
Department of Embryology and Genetics, Vrije Universiteit Brussel, Brussels, Belgium
Laboratory of Molecular and Cellular Therapy, Department of Physiology-Immunology,
VUB
2
The coiled-coil domain containing 54 (CCDC54) protein, a protein with unknown function,
has been investigated. RNA of CCDC54 is mainly expressed in testis and in silico analysis of
the CCDC54 protein suggested it is a conserved protein.
In Genbank a c.316G>T SNP of CCDC54 was reported, introducing a stop codon after 105
amino acids and most likely causing loss of function of the protein. First of all, 150
normozoospermic controls were screened for the presence of this SNP using the restriction
enzyme BccI, but this SNP was not found. In a following step, the CCDC54 gene was
sequenced in 70 patients in whom spermatogenesis is abnormal, comprising 27 patients
with a maturation arrest of spermatogenesis (MA) and 43 patients with Sertoli cell-only
syndrome (SCOS), and in 50 normozoospermic controls. One change (c.638T>C or
p.Ile213Thr) was present only in one patient with MA. This variant was looked for in 45
more controls with the restriction enzyme Tsp509I and was not detected. The homozygous
variants c.113G>A and c.912C>A were both found in respectively four SCOS patients and
one patient with SCOS and one patient with MA, but were absent in the control group. Since
only c.113G>A causes a change on amino acid level (p.Arg38Gln), it was looked for in 50
more controls by restriction digestion with BanI. The change was present in a homozygous
form in 2 controls.
At the protein level, a yeast two hybrid screening was performed, which identified ZBTB32,
a transcriptional repressor, as a binding partner of CCDC54. Luciferase reporter assays were
then performed to investigate the effect of the binding of CCDC54 and ZBTB32. These
experiments suggested that CCDC54 is a negative co-regulator of ZBTB32. CCDC54 variants
were created with site-directed mutagenesis and used in the luciferase reporter
experiments to get an idea of the functional domains. Also a CCDC54 vector containing the
c.638T>C was used in the luciferase assays and gave, like the other variants, the same
results as wild type CCDC54. To see if CCDC54 binds directly to DNA or the androgen
receptor, electrophoretic mobility shift assay and co-immunoprecipitation are being
performed.
These preliminary results suggest that CCDC54 could be an important protein for
spermatogenesis.
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P85: Genetic causes of intellectual disability in D.R.Congo: An etiological diagnostic survey
in 46 males
Aimé Lumaka Zola, Prosper Tshilobo Lukusa & Koen Devriendt
Center for Human Genetics, KU Leuven
Genetic causes account for an estimated 17.4 to 41.1% of cases of Intellectual disability (ID)
in Western countries (Bernardini, Alesi et al. 2010). Currently, no data exist on the genetics
of ID in Central Africa. We have initiated a project to study the etiology of I.D. in Kinshasa,
Democratic Republic of Congo. The aims were (1) to determine the incidence of fragile-X
syndrome in clinics and special schools, (2) to evaluate the usefulness of caryotyping, MLPA
and array-CGH in this population.
46 male children and young adults were recruited from 2 schools for special education and
the University hospitals in Kinshasa, D.R.Congo. All individuals were clinically examined on
site, scored according 3 existing screening checklists for Fragile X syndrome (Hagerman,
Maes and Guruju), and a blood sample was obtained for genetic testing in Belgium (Center
for human genetics, KUL).
Results
A clinical diagnosis of Down syndrome was clinically made in 6 persons, and confirmed by
means of standard caryotyping. Caryotype analysis of the remaining 40 patients was normal.
Fragile-X was excluded in all these 40 cases although 12 were likely Fragile X positive
according to the Hagerman’s checklist. Among these 40 individuals, 9 were considered to be
dysmorphic, given the presence of at least 3 minor dysmorphic features. Thus, Array-CGH
was performed in all of them, and three causal imbalances were identified: one 3.473 Mb
deletion of 17p11.2 (Smith-Magenis syndrome), one 6.2 Mb terminal deletion of
22q13.31q13.33, including the SHANK3 gene, and one 1.6 Mb interstitial deletion of
chromosome 20q11.22. MLPA screening for recurrent microdeletions (SALSA MPLA P245
Microdeletion, MRC Holland) of the other cases is ongoing. Preliminary results of this
project already provide some evidence on the underlying genetic etiology of ID in Central
Africa, the necessity for a better clinical characterization of known syndromes and relevance
of genetic testing for screening in low income countries.
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List of Participants
Prof Marc Abramowicz
Genetics, ULB
Route de Lennik, 808, 1070 Brussels, Belgium
[email protected]
Miss Machteld Baetens
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Miss Evi Aerts
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Miss Riet Bammens
KULeuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Dr Robert Akkers
BIOKÉ
Plesmanlaan 1d, 2333BZ Leiden, Netherlands
[email protected]
Mrs Geneviève Ameye
Centre de Génétique UCL
Avenue E. Mounier , 1200 Brussels, Belgium
[email protected]
Dr Mustapha Amyere
Laboratory of Human Molecular Genetics
de Duve Institute, UCL
Avenue Hippocrate, 74, 1200 Brussels, Belgium
[email protected]
Prof Hélène Antoine-Poirel
Centre de Génétique UCL
Avenue E. Mounier , 1200 Brussels, Belgium
[email protected]
Mr Amin Ardeshirdavani
KU Leuven, ESAT-SCD
Kasteelpark Arenberg 10, 3001 Heverlee, Belgium
[email protected]
Mr Claude Bandelier
Centre de Génétique UCL
Avenue E. Mounier , 1200 Brussels, Belgium
[email protected]
Mrs Isabelle Bar
Institut de Pathologie et de Génétique
Avenue Georges Lemaître, 25, 6041 Gosselies, Belgium
[email protected]
Dr Mirta Basha
Laboratory of Human Molecular Genetics
de Duve Institute, UCL
Avenue Hippocrate, 74, 1200 Brussels, Belgium
[email protected]
Mr Siavash Bashiri
Agilent Technologies
Dag Hammarskjoldsvag 54a, 75183 Uppsala, Sweden
[email protected]
Miss Miriam Bauwens
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Dr Ingrid Arijs
Department of Gastroenterology, KU Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Dr Sigri Beckers
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Miss Maria Artesi
Department of Human Genetics-CHU-University of Liège
GIGA - Sart Tilman, 4000 Liège, Belgium
[email protected]
Miss Anneleen Beckers
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Miss Cindy Badoer
Hôpital Erasme, Laboratoire de génétique moléculaire
Route de Lennik, 808, 1070 Brussels, Belgium
[email protected]
Dr Bram Bekaert
Dienst forensische Geneeskunde - UZ Leuven
Kapucijnenvoer 33, 3000 Leuven, Belgium
[email protected]
Mrs Anne-Mie Bael
UZ Brussel, Centre for Medical Genetics
Laarbeeklaan 101, 1090 Brussels, Belgium
[email protected]
Mrs Valérie Benoit
Institut de Pathologie et de Génétique
Avenue Georges Lemaître, 25, 6041 Gosselies, Belgium
[email protected]
12th ANNUAL MEETING 2012
125
Belgian Society of Human Genetics
Mrs Clémence Beslin
Roche Diagnostics Belgium
Schaarbeeklei 198, 1800 Vilvoorde, Belgium
[email protected]
Prof Vincent Bours
Department of Human Genetics-CHU-University of Liège
GIGA - Sart Tilman, 4000 Liège, Belgium
[email protected]
Prof Bettina Blaumeiser
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Mr Nassim Bouznad
Department of Human Genetics-CHU-University of Liège
GIGA - Sart Tilman, 4000 Liège, Belgium
[email protected]
Mr François Boemer
Department of Human Genetics-CHU-University of Liège
GIGA - Sart Tilman, 4000 Liège, Belgium
[email protected]
Miss Nikhita Bolar
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Prof Maryse Bonduelle
UZ Brussel, Centre for Medical Genetics
Laarbeeklaan 101, 1090 Brussels, Belgium
[email protected]
Miss Sarah Bonneux
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Miss Vere Borra
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Prof Pascal Borry
K.U.Leuven
Kapucijnenvoer 35, 3000 Leuven, Belgium
[email protected]
Miss Eveline Boudin
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Mrs Meriem Boukerroucha
Department of Human Genetics-CHU-University of Liège
GIGA - Sart Tilman, 4000 Liège, Belgium
[email protected]
Mr Sebastien Boulanger
Institut de Pathologie et de Génétique
Avenue Georges Lemaître, 25, 6041 Gosselies, Belgium
[email protected]
126
Miss Sien Braat
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Mr Paul Brady
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Dr Pascal Brouillard
Laboratory of Human Molecular Genetics
de Duve Institute, UCL
Avenue Hippocrate, 74, 1200 Brussels, Belgium
[email protected]
Miss Vanessa Brys
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Mrs Vicky Buyse
AROS Applied Biotechnology A/S
Brendstrupgaardsvej 102, 8200 Aarhus N, Denmark
[email protected]
Mr Jean Hubert Caberg
Department of Human Genetics-CHU-University of Liège
GIGA - Sart Tilman, 4000 Liège, Belgium
[email protected]
Mr Ben Caljon
UZ Brussel, Centre for Medical Genetics
Laarbeeklaan 101, 1090 Brussels, Belgium
[email protected]
Dr Bert Callewaert
Center for Medical Genetics, Ghent University Hospital
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Miss Julie Cannuyer
UCL, De Duve Institute (GEPI
Rue de l'Arbre de Mai, 6, 7800 Ath, Belgium
[email protected]
Mrs Emilie Castermans
Department of Human Genetics-CHU-University of Liège
GIGA - Sart Tilman, 4000 Liège, Belgium
[email protected]
Next Generation Sequencing and Recent Advances in Genetics
Belgian Society of Human Genetics
Dr Tom Cattaert
Université de Liège - GIGA-R/Montefiore Institute
Grande Traverse 10, 4000 Liege, Belgium
[email protected]
Dr Anniek Corveleyn
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Dr Carole Charlier
University of Liège
Avenue de l'Hôpital, Sart Tilman, 4000 Liège, Belgium
[email protected]
Prof Paul Coucke
Center for Medical Genetics, Ghent University Hospital
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Dr Benoit Charloteaux
Animal Genomics Unit, GIGA-Research
University of Liège
Avenue de l'Hôpital, Sart Tilman, 4000 Liège, Belgium
[email protected]
Dr Winnie Courtens
Department of Human Genetics-CHU-University of Liège
GIGA - Sart Tilman, 4000 Liège, Belgium
[email protected]
Ms Huijun Cheng
Animal Genomics Unit, GIGA-Research
University of Liège
Avenue de l'Hôpital, Sart Tilman, 4000 Liege, Belgium
[email protected]
Mrs Jiqiu Cheng
KULeuven
Kasteelpark Arenberg 10 - bus 2446 , 3001 Heverlee,
Belgium
[email protected]
Ms Gabrielle Christenhusz
Centre for Biomedical Ethics and Law, K. U. Leuven
Kapucijnenvoer 35, 3000 Leuven, Belgium
[email protected]
Prof Kathleen Claes
Center for Medical Genetics, Ghent University Hospital
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Miss Charlotte Claes
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43 , 2650 Edegem, Belgium
[email protected]
Miss Isabelle Cleynen
KU Leuven, Department of Pathophysiology
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Mrs Katrijn Cobbaut
UZ Brussel, Centre for Medical Genetics
Laarbeeklaan 101, 1090 Brussels, Belgium
[email protected]
Mrs Pascale Cochaux
Hospital Erasme - Genetic Department
Route de Lennik, 808, 1070 Brussels, Belgium
[email protected]
Ms Marianne Crespin
Institut de Pathologie et de Génétique
Avenue Georges Lemaître, 25, 6041 Gosselies, Belgium
[email protected]
Miss Francesca Cristofoli
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Miss Sanne D'hondt
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Prof Karin Dahan
Institut de Pathologie et de Génétique
Avenue Georges Lemaître, 25, 6041 Gosselies, Belgium
[email protected]
Miss Dorien Daneels
UZ Brussel, Centre for Medical Genetics
Laarbeeklaan 101 , 1090 Brussels, Belgium
[email protected]
Prof Elfride De Baere
Center for Medical Genetics, Ghent University Hospital
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Miss Sylvia De Brakeleer
VUB, Lab Molecular Oncology
Laarbeeklaan 103, 1090 Brussels, Belgium
[email protected]
Miss Sara De Brouwer
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Ms Ilse De Clercq
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Dr Frauke Coppieters
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
12th ANNUAL MEETING 2012
127
Belgian Society of Human Genetics
Miss Annelies De Jaegher
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Miss Bieke Decaesteker
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Miss Kim De Leeneer
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Miss Anneleen Decock
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Dr Anne De Leener
ULB Human Genetic Center
Route de Lennik, 808, 1070 Brussels, Belgium
[email protected]
Miss Valerie Deffontaine
Animal Genomics Unit, GIGA-Research
University of Liège
Avenue de l'Hôpital, Sart Tilman, 4000 Liège, Belgium
[email protected]
Prof Anne De Paepe
Center for Medical Genetics, Ghent University Hospital
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Dr Katleen De Preter
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Dr Marjan De Rademaeker
UZ Brussel, Centre for Medical Genetics
Laarbeeklaan 101, 1090 Brussels, Belgium
[email protected]
Prof Thomy de Ravel
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Miss Nina De Rocker
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Mrs Martine De Rycke
UZ Brussel, Centre for Medical Genetics
Laarbeeklaan 101 , 1090 Brussels, Belgium
[email protected]
Mr Bram De Wilde
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Miss Veerle de Wolf
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Dr François Guillaume Debray
Department of Human Genetics-CHU-University of Liège
GIGA - Sart Tilman, 4000 Liège, Belgium
[email protected]
128
Prof Jurgen Del-Favero
Multiplicom N.V.
Galileilaan 18, 2845 Niel, Belgium
[email protected]
Dr Julie Desir
Hospital Erasme- ULB
Route de Lennik, 808, 1070 Brussels, Belgium
[email protected]
Miss Laurence Desmyter
Laboratory of Human Molecular Genetics
de Duve Institute, UCL
Avenue Hippocrate, 74, 1200 Brussels, Belgium
[email protected]
Dr Barbara Dessars
Genetics'Department - Erasme Hospital
Route de Lennik, 808, 1070 Brussels, Belgium
[email protected]
Dr Anne Destree
Institut de Pathologie et de Génétique
Avenue Georges Lemaître, 25, 6041 Gosselies, Belgium
[email protected]
Dr Helena Devos
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Prof Koen devriendt
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Annelies Dheedene
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Dr Vinciane Dideberg
Department of Human Genetics-CHU-University of Liège
GIGA - Sart Tilman, 4000 Liège, Belgium
[email protected]
Next Generation Sequencing and Recent Advances in Genetics
Belgian Society of Human Genetics
Prof Kris Dierickx
Centre for Biomedical Ethics and Law, K. U. Leuven
Kapucijnenvoer 35, 3000 Leuven, Belgium
[email protected]
Miss Marylène Focant
ANALIS
Rue de Néverlée 11, 5020 Suarlee, Belgium
[email protected]
Dr Andreas Diplas
Centre de Génétique UCL
Avenue E. Mounier , 1200 Brussels, Belgium
[email protected]
Miss Katrien Francois
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Miss Magali Dodemont
Laboratory of Molecular Haematology
Center of Human genetics-CHU Liège
rue de l'enclos, 16, 4680 Hermée, Belgium
[email protected]
Mr Wim Dorlijn
Agilent Technologies
Groenelaan 5, 1186 XR Amstelveen, Netherlands
[email protected]
Dr Anais Drielsma
Université Libre Bruxelles-IRIBHM
Rue des recollectines, 19, 6940 Durbuy, Belgium
[email protected]
Miss Kaat Durinck
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Mr Hakim El Housni
Medical Genetic Dpt Erasme Hospital ULB
Route de Lennik, 808, 1070 Brussels, Belgium
[email protected]
Mrs Corinne Fasquelle
Department of Human Genetics-CHU-University of Liège
GIGA - Sart Tilman, 4000 Liège, Belgium
[email protected]
Ms Nathalie Fieremans
KU Leuven Department of Human Genetics
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Miss Annelies Fieuw
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Mr Igor Fijałkowski
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Mr Sebastien Floor
ULB - IRIBHM
Route de Lennik, 808, 1070 Brussels, Belgium
[email protected]
Mrs Sabine Franke
Department of Human Genetics-CHU-University of Liège
GIGA - Sart Tilman, 4000 Liège, Belgium
[email protected]
Dr Erik Fransen
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Dr Suzanna Frints
MUMC
postbox 5800, 6202 AZ Maastricht, Netherlands
[email protected]
Mrs Farzaneh Ghazavi
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Dr Lidia Ghisdal
Hôpital erasme-ULB
département de Néphrologie-Transplantation rénale
Route de Lennik, 808, 1070 Brussels, Belgium
[email protected]
Mrs Stéphanie Gofflot
Department of Human Genetics-CHU-University of Liège
GIGA - Sart Tilman, 4000 Liège, Belgium
[email protected]
Dr Bernard Grisart
Institut de Pathologie et de Génétique
Avenue Georges Lemaître, 25, 6041 Gosselies, Belgium
[email protected]
Miss Anneke Grunewald
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Mr Lionel Habran
Department of Human Genetics-CHU-University of Liège
GIGA - Sart Tilman, 4000 Liège, Belgium
[email protected]
Miss Hannelore Hamerlinck
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
12th ANNUAL MEETING 2012
129
Belgian Society of Human Genetics
Miss Katia Hardies
VIB-UA
Universiteitsplein 1, 2610 Wilrijk, Belgium
[email protected]
Mrs Mala Isrie
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Dr Raphael Helaers
Laboratory of Human Molecular Genetics
de Duve Institute, UCL
Avenue Hippocrate, 74, 1200 Brussels, Belgium
[email protected]
Miss Sigrun Jackmaert
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Mrs Anne-Cécile Hellin
Department of Human Genetics-CHU-University of Liège
GIGA - Sart Tilman, 4000 Liège, Belgium
[email protected]
Miss Céline Helsmoortel
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Miss Hetty Helsmoortel
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Miss Gretl Hendrickx
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Mr Christian Herens
Department of Human Genetics-CHU-University of Liège
GIGA - Sart Tilman, 4000 Liège, Belgium
[email protected]
Ms Inge Heulens
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Dr Pascale Hilbert
Institut de Pathologie et de Génétique
Avenue Georges Lemaître, 25, 6041 Gosselies, Belgium
[email protected]
Mrs Kerstin Hillmayer
Roche Diagnostics Belgium
Schaarbeeklei 198, 1800 Vilvoorde, Belgium
[email protected]
Mr Mohammad Jakir Hosen
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Mr Sajid Iqbal
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
130
Mr Kurt Jacobs
Research Group Reproduction and Genetics
Vrije Universiteit Brussel
Laarbeeklaan 103, 1090 Brussels, Belgium
[email protected]
Dr Mauricette Jamar
Department of Human Genetics-CHU-University of Liège
GIGA - Sart Tilman, 4000 Liège, Belgium
[email protected]
Dr Nicolas Janin
Service de Génétique Cliniques Universitaires Saint Luc
Rue des Vingt-Deux, 10, 4000 Liège, Belgium
[email protected]
Mrs Yaojuan Jia
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Dr Claire Josse
Department of Human Genetics-CHU-University of Liège
GIGA - Sart Tilman, 4000 Liège, Belgium
[email protected]
Mrs Storm Katrien
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Ms Hannah Kiss
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Dr Frank Kooy
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Dr Jerome Kroonen
Department of Human Genetics-CHU-University of Liège
GIGA - Sart Tilman, 4000 Liège, Belgium
[email protected]
Mr Bas Kulderij
PerkinElmer, Diagnostics, BeNeLux
Imperiastraat 8, 1930 Zaventem, Belgium
[email protected]
Next Generation Sequencing and Recent Advances in Genetics
Belgian Society of Human Genetics
Mr Parveen Kumar
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Mr Pierre-Emmanuel Leonard
Institut de Pathologie et de Génétique
Avenue Georges Lemaître, 25, 6041 Gosselies, Belgium
[email protected]
Miss Candy Kumps
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Miss Marie Léonard
ULB
Av des Commandants Borlée, 43, 1370 Jodoigne,
Belgium
[email protected]
Mr Maxime Labee
ANALIS
Rue de Neverlée 11, 5020 Suarlee, Belgium
[email protected]
Dr Frédéric Lambert
Department of Human Genetics-CHU-University of Liège
GIGA - Sart Tilman, 4000 Liège, Belgium
[email protected]
Dr Nelle Lambert
ULB, medical genetics departement, IRIBHM
Route de Lennik, 808, 1070 brussels, Belgium
[email protected]
Dr Irina Lambertz
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Mrs Nathalie Lannoy
Centre de Génétique UCL
Avenue E. Mounier , 1200 Brussels, Belgium
[email protected]
Dr Soazig Le Pennec
IRIBHM, Brussels
Route de Lennik, 808, 1070 Brussels, Belgium
[email protected]
Mr Wanbo Li
Animal Genomics Unit, GIGA-Research
University of Liège, Belgium
Avenue de l'Hôpital, Sart Tilman, 4000 Liege, Belgium
[email protected]
Mrs Cécile Libioulle
Department of Human Genetics-CHU-University of Liège
GIGA - Sart Tilman, 4000 Liège, Belgium
[email protected]
Miss Croes Lieselot
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Dr Nisha Limaye
Laboratory of Human Molecular Genetics
de Duve Institute, UCL
Avenue Hippocrate, 74, 1200 Brussels, Belgium
[email protected]
Prof Willy Lissens
UZ Brussel, Centre for Medical Genetics
Laarbeeklaan 101, 1090 Brussels, Belgium
[email protected]
Miss Christelle Lecut
Department of Human Genetics-CHU-University of Liège
GIGA - Sart Tilman, 4000 Liège, Belgium
[email protected]
Prof Bart Loeys
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Dr Damien Lederer
Institut de Pathologie et de Génétique
Avenue Georges Lemaître, 25, 6041 Gosselies, Belgium
[email protected]
Ms Axelle Loriaux
BIOKÉ
Plesmanlaan 1d, 2333BZ Leiden, Netherlands
[email protected]
Mr Steve Lefever
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Dr Jacoba Louw
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Prof Eric Legius
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Dr Aimé Lumaka Zola
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
12th ANNUAL MEETING 2012
131
Belgian Society of Human Genetics
Mrs Ann Löfgren
VIB-DMG-IBB
Universiteitsplein 1, 2610 Wilrijk, Belgium
[email protected]
Ms Camilla Mackenzie
Eurogentec
Rue du Bois St. Jean 5, 4102 Seraing, Belgium
[email protected]
Miss Annelien Massart
UZ Brussel, Centre for Medical Genetics
Laarbeeklaan 101, 1090 Brussels, Belgium
[email protected]
Dr Ligia Mateiu
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Prof Gert Matthijs
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Dr Bärbel Maus
University of Liège
Grande Traverse 10, 4000 Liège, Belgium
[email protected]
Prof Isabelle Maystadt
Institut de Pathologie et de Génétique
Avenue Georges Lemaître, 25, 6041 Gosselies, Belgium
[email protected]
Miss Cindy Melotte
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Miss Antonella Mendola
Laboratory of Human Molecular Genetics
de Duve Institute, UCL
Avenue Hippocrate, 74, 1200 Brussels, Belgium
[email protected]
Prof Björn Menten
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Miss Elke Mersy
Department of Clinical Genetics
Prenatal Diagnosis and Therapy
Maastricht University Medical Center
Noordgebouw, Joseph Bechlaan 113 , 6229 GR
Maastricht, Netherlands
[email protected]
Ms Afroditi Mertzanidou
Research Group Reproduction and Genetics
Vrije Universiteit Brussel
Laarbeeklaan 103, 1090 Brussels, Belgium
[email protected]
Dr Pieter Mestdagh
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Miss Evelien Mets
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Miss Sofie Metsu
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Prof Lucienne Michaux
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Dr Isabelle Migeotte
Hopital Erasme center of medical genetics
Route de Lennik, 808, 1070 Brussels, Belgium
[email protected]
Mrs Sarah Miolet
Roche Diagnostics Belgium
Schaarbeeklei 198, 1800 Vilvoorde, Belgium
[email protected]
Dr Colin Molter
InSilico - ULB
50, av. F. Roosevelt, 144, Belgium
[email protected]
Dr Stéphanie Moortgat
Institut de Pathologie et de Génétique
Avenue Georges Lemaître, 25, 6041 Gosselies, Belgium
[email protected]
Dr Deepti Narasimhaiah
Centre de Génétique UCL
Avenue E. Mounier, 1200 Brussels, Belgium
[email protected]
Mrs Ha Nguyen Thi
Research Group Reproduction and Genetics
Vrije Universiteit Brussel
Laarbeeklaan 103, 1090 Brussels, Belgium
[email protected]
Dr Beata Nowakowska
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
132
Next Generation Sequencing and Recent Advances in Genetics
Belgian Society of Human Genetics
Dr Maté Ongenaert
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Dr Jacques Poncin
Department of Human Genetics-CHU-University of Liège
GIGA - Sart Tilman, 4000 BELGIUM, Belgium
[email protected]
Dr Ken Op de Beeck
Center for Medical Genetics
Laboratory of Cancer Research and Clinical Oncology
Universiteitsplein 1, 2610 Wilrijk, Belgium
[email protected]
Prof Bruce Poppe
Center for Medical Genetics, Ghent University Hospital
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Dr Cécile Oury
Department of Human Genetics-CHU-University of Liège
GIGA - Sart Tilman, 4000 Liège, Belgium
[email protected]
Dr Stéphanie Paquay
Institut de Pathologie et de Génétique
Avenue Georges Lemaître, 25, 6041 Gosselies, Belgium
[email protected]
Miss Nancy Paredis
UZ Brussel, Centre for Medical Genetics
Laarbeeklaan 101, 1090 Brussels, Belgium
[email protected]
Mr Christophe Poulet
Department of Human Genetics-CHU-University of Liège
GIGA - Sart Tilman, 4000 Liège, Belgium
[email protected]
Miss Dorien Proost
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Mr Mickaël Quentric
Laboratory of Human Molecular Genetics
de Duve Institute, UCL
Avenue Hippocrate, 74, 1200 Brussels, Belgium
[email protected]
Jasmine Parma
Laboratoire de Génétique Moléculaire ULB
Hôpital Erasme
Route de Lennik, 808, 1070 Brussels, Belgium
[email protected]
Dr Valérie Race
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Mr Benoît Parmentier
Institut de Pathologie et de Génétique
Avenue Georges Lemaître, 25, 6041 Gosselies, Belgium
[email protected]
Dr Marilou Ramos-Pamplona
University of Liège
Avenue de l'Hôpital, Sart Tilman, 4000 Liège, Belgium
[email protected]
Prof Hilde Peeters
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Dr Marie Ravoet
Centre de Génétique UCL
Avenue E. Mounier , 1200 Brussels, Belgium
[email protected]
Ms Uschi Peeters
UZ Brussel, Centre for Medical Genetics
Laarbeeklaan 101, 1090 Brussels, Belgium
[email protected]
Marjolijn Renard
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Greet Peeters
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Dr Nicole Revencu
Centre de Génétique UCL
Avenue E. Mounier , 1200 Brussels, Belgium
[email protected]
Mr Xavier Pepermans
Centre de Génétique UCL
Avenue E. Mounier , 1200 Brussels, Belgium
[email protected]
Mr Edwin Reyniers
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Dr Geneviève Pierquin
Department of Human Genetics-CHU-University of Liège
GIGA - Sart Tilman, 4000 Liège, Belgium
[email protected]
Mr Ali Rihani
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
12th ANNUAL MEETING 2012
133
Belgian Society of Human Genetics
Mr Simon Robberts
Solis BioDyne OÜ
Riia 185a, 51014 Tartu, Estonia
[email protected]
Dr Peter Schols
Orbicule
Middelweg 129, 3001 Heverlee, Belgium
[email protected]
Mr Raf Roelands
Eurogentec
Rue du Bois St. Jean 5, 4102 Seraing, Belgium
[email protected]
Mr Stephan Schrooten
BIOKÉ
Plesmanlaan 1d, 2333BZ Leiden, Netherlands
[email protected]
Dr Dominique Roland
Institut de Pathologie et de Génétique
Avenue Georges Lemaître, 25, 6041 Gosselies, Belgium
[email protected]
Mrs Karin Segers
Department of Human Genetics-CHU-University of Liège
GIGA - Sart Tilman, 4000 Liège, Belgium
[email protected]
Mrs Sonia Rombaut
Institut de Pathologie et de Génétique
Avenue Georges Lemaître, 25, 6041 Gosselies, Belgium
[email protected]
Prof Sara Seneca
UZ Brussel, Centre for Medical Genetics
Laarbeeklaan 101, 1090 Brussels, Belgium
[email protected]
Dr Pieter Rondou
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Prof Karen Sermon
Research Group Reproduction and Genetics
Vrije Universiteit Brussel
Laarbeeklaan 103, 1090 Brussels, Belgium
[email protected]
Mrs Kristel Ruelle
Roche Diagnostics Belgium
Schaarbeeklei 198, 1800 Vilvoorde, Belgium
[email protected]
Dr Eva Sammels
Orbicule
Middelweg 129, 3001 Heverlee, Belgium
[email protected]
Mr Tom Sante
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Mr Rajendra Bahadur Shahi
UZ Brussel, Centre for Medical Genetics
Laarbeeklaan 101 , 1090 Brussels, Belgium
[email protected]
Mr Lode Sibbens
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Mr Alejandro Sifrim
University of Leuven
Kasteelpark Arenberg 10, 3001 Heverlee, Belgium
[email protected]
Miss Dorien Schepers
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Dr Nicolas Simonis
ULB, Bigre
91 avenue Edmond Parmentier, 1150 Brussels, Belgium
[email protected]
Mr Peter Schietecatte
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Dr Eric Smeets
Clinical Genetics
Weggevoerdenstraat 43, 3500 Hasselt, Belgium
[email protected]
Mr Matthieu Schlögel
Laboratory of Human Molecular Genetics
de Duve Institute, UCL
Avenue Hippocrate, 74, 1200 Brussels, Belgium
[email protected]
Dr Guillaume Smits
HUDERF-ULB
Av JJ Crocq 15, 1020 Brussels, Belgium
[email protected]
Dr Els Schollen
Roche Diagnostics Belgium
Schaarbeeklei 198, 1800 Vilvoorde, Belgium
[email protected]
134
Miss Julie Soblet
Laboratory of Human Molecular Genetics
de Duve Institute, UCL
Avenue Hippocrate, 74, 1200 Brussels, Belgium
[email protected]
Next Generation Sequencing and Recent Advances in Genetics
Belgian Society of Human Genetics
Mrs Helena Soenen
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Mrs Emilie Theatre
Animal Genomics Unit, GIGA-Research
University of Liège
Avenue de l'Hôpital, Sart Tilman, 4000 Liège, Belgium
[email protected]
Miss Manou Sommen
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Koen Theunis
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Prof Frank Speleman
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 ghent, Belgium
[email protected]
Dr Sébastien Toffoli
Institut de Pathologie et de Génétique
Avenue Georges Lemaître, 25, 6041 Gosselies, Belgium
[email protected]
Prof Claudia Spits
Research Group Reproduction and Genetics
Vrije Universiteit Brussel
Laarbeeklaan 103, 1090 Brussels, Belgium
[email protected]
Miss deborah trajman
ULB
Route de Lennik, 808, 1650 Beersel, Belgium
[email protected]
Dr Catherine Staessen
UZ Brussel, Centre for Medical Genetics
Laarbeeklaan 101, 1090 Brussels, Belgium
[email protected]
Mr Wouter Steyaert
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Mr Tiberio Sticca
Department of Human Genetics-CHU-University of Liège
GIGA - Sart Tilman, 4000 Liège, Belgium
[email protected]
Prof Katrien Stouffs
UZ Brussel, Centre for Medical Genetics
Laarbeeklaan 101, 1090 Brussels, Belgium
[email protected]
Mrs Sofie Symoens
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Miss Delfien Syx
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Prof Yves Sznajer
Centre de Génétique UCL
Avenue E. Mounier , 1200 Brussels, Belgium
[email protected]
Erik Teugels
VUB, Lab Molecular Oncology, Belgium
Laarbeeklaan 103, 1090 Brussels, Belgium
[email protected]
Mr Léon-Charles Tranchevent
Department of Electrical Engineering ESAT-SCD, IBBT
Future Health Department, KUL
Kasteelpark Arenberg 10, 3001 Heverlee, Belgium
[email protected]
Ms Melanie Uebelhoer
Laboratory of Human Molecular Genetics
de Duve Institute, UCL
Avenue Hippocrate, 74, 1200 Brussels, Belgium
[email protected]
Dr Urielle Ullmann
Institut de Pathologie et de Génétique
Avenue Georges Lemaître, 25, 6041 Gosselies, Belgium
[email protected]
Miss Montse Urbina
Laboratoire de Cytogénétique ULB Erasme
Route de Lennik, 808, 1070 Brussels, Belgium
[email protected]
Dr Annette Uwineza
Department of Human Genetics-CHU-University of Liège
GIGA - Sart Tilman, 4000 Liège, Belgium
[email protected]
Mrs Elvire Van Assche
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Dr Kim van Berkel
UZ Brussel, Centre for Medical Genetics
Laarbeeklaan 101, 1090 Brussels, Belgium
[email protected]
12th ANNUAL MEETING 2012
135
Belgian Society of Human Genetics
Prof Griet Van Buggenhout
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Mrs Lindsey Van Haute
Research Group Reproduction and Genetics, Vrije
Universiteit Brussel
Laarbeeklaan 103, 1090 Brussels, Belgium
[email protected]
Ms Jasmijn Van Camp
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Miss Clarissa Van Hecke
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Miss Caroline Van Cauwenbergh
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Dr Jeroen Van Houdt
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Kris Van Den Bogaert
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Mr Steven Van Hove
Illumina
Freddy van Riemsdijkweg 15, 5657 EE Eindhoven,
Netherlands
[email protected]
Dr Ann Van Den Bogaert
UZ Brussel, Centre for Medical Genetics
Laarbeeklaan 101, 1090 Brussels, Belgium
[email protected]
Dr Jenneke van den Ende
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Mr Niels Van der Aa
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Miss Morgane Van der Eecken
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Mrs Jo-Anne van der Krogt
KULeuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Dr Sonia Van Dooren
UZ Brussel, Centre for Medical Genetics
Laarbeeklaan 101, 1090 Brussels, Belgium
[email protected]
Prof Hilde Van Esch
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Dr Winfried van Eyndhoven
Agilent Technologies
Groenelaan 5, 1186 AA Amstelveen, Netherlands
[email protected]
136
Prof Wim Van Hul
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Ms Els Van Hul
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Dr Lut Van Laer
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Dr Peter Van Loo
Wellcome Trust Sanger Institute
Wellcome Trust Genome Campus, CB10 1SA Hinxton
Cambridge, United Kingdom
[email protected]
Mrs Conny Van Loon
INNOGENETICS
Technologiepark 6, 9052 Zwijnaarde, Belgium
[email protected]
Dr Tom Van Maerken
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Mr Kurt Van Mol
INNOGENETICS
Technologiepark 6, 9052 Zwijnaarde, Belgium
[email protected]
Next Generation Sequencing and Recent Advances in Genetics
Belgian Society of Human Genetics
Mr Gert Van Peer
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Dr Geert Vandeweyer
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Ms Sofie Van Rossom
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Miss Suzanne Vanhauwaert
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Prof Nadine Van Roy
Center for Medical Genetics, Ghent University Hospital
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Mr Bruno Vankeirsbilck
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Mr Kristof Van Schil
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Mr Pascal Vannuffel
Institut de Pathologie et de Génétique
Avenue Georges Lemaître, 25, 6041 Gosselies, Belgium
[email protected]
Prof Kristel Van Steen
Université de Liège - GIGA-R/Montefiore Institute
GIGA - Sart Tilman, 4000 Liège, Belgium
[email protected]
Miss Elise Vantroys
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Dr Olivier Vanakker
Center for Medical Genetics, Ghent University Hospital
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Tine Venken
UZ Brussel, Centre for Medical Genetics
Laarbeeklaan 101, 1090 Brussels, Belgium
[email protected]
Mr Jean-François Vanbellighen
Institut de Pathologie et de Génétique
Avenue Georges Lemaître, 25, 6041 Gosselies, Belgium
[email protected]
Dr Celine Vens
KU Leuven, Dept. Computer Science, Belgium
Celestijnenlaan 200A, 3001 Leuven, Belgium
[email protected]
Miss Kim Vancampenhout
UZ Brussel, Centre for Medical Genetics
Laarbeeklaan 101, 1090 Brussels, Belgium
[email protected]
Miss Sherly Verbauwhede
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Ms Ann Vandenbroucke
Diagnostic Service Facility
University of Antwerp, Belgium
Universiteitsplein 1, 2610 Wilrijk, Belgium
[email protected]
Miss Hannah Verdin
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Mr Jens Vandenhaute
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Mr Johan Vanderhoeven
Integrated DNA Technologies
Interleuvenlaan 12A, 3000 Leuven, Belgium
[email protected]
Joke Vandewalle
KU Leuven Department of Human Genetics Belgium
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Dr Pieter Verdyck
UZ Brussel, Centre for Medical Genetics
Laarbeeklaan 101, 1090 Brussels, Belgium
[email protected]
Prof Christine Verellen-Dumoulin
Institut de Pathologie et de Génétique
Avenue Georges Lemaître, 25, 6041 Gosselies, Belgium
[email protected]
Miss Sarah Vergult
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
12th ANNUAL MEETING 2012
137
Belgian Society of Human Genetics
Prof Joris Vermeesch
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Dr Andy Willaert
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Dr Ilse Verstraete
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Miss Marie Willems
Department of Human Genetics-CHU-University of Liège
GIGA - Sart Tilman, 4000 Liège, Belgium
[email protected]
Miss Aurélie Verstraete
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Prof Miikka Vikkula
Laboratory of Human Molecular Genetics
de Duve Institute, UCL
Avenue Hippocrate, 74, 1200 Brussels, Belgium
[email protected]
Dr Catheline Vilain
ULB Center of Human Genetics
Route de Lennik, 808, 1070 Brussels, Belgium
[email protected]
Prof Thierry Voet
Laboratory of Reproductive Genomics
Department of Human Genetics
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Mr Pieter-Jan Volders
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Mr Raf Winand
KU Leuven, ESAT-SCD, Belgium
Kasteelpark Arenberg 10, 3001 Heverlee, Belgium
[email protected]
Dr Mira Wouters
KULeuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Prof Wim Wuyts
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Dr Xuewen Xu
Animal Genomics Unit, GIGA-Research
University of Liège
Avenue de l'Hôpital, Sart Tilman, 4000 Liège, Belgium
[email protected]
Mr Masoud Zamani Esteki
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Miss Annelynn Wallaert
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
Ms Doreen Zegers
Center for Medical Genetics
University and University Hospital of Antwerp
Prins Boudewijnlaan 43, 2650 Edegem, Belgium
[email protected]
Ms Qian Wang
Center for Human Genetics Leuven
Herestraat 49, 3000 Leuven, Belgium
[email protected]
Ms Fjoralba Zeka
Center for Medical Genetics, Ghent University
De Pintelaan 185, 9000 Ghent, Belgium
[email protected]
138
Next Generation Sequencing and Recent Advances in Genetics