1. science
2. medicine
3. genetics

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