2008 Barcelona - European Society of Human Genetics

Concurrent Sessions
of all individual genes from a single human chromosome (HSA21) in
mouse ES cells to identify the effects of gene dosage imbalance on the
global transcriptome and on the ability of pluripotent ES cells to differentiate
into lineages relevant to human aneuploidy phenotypes .
c05.5
A large human miRNA library screen reveals a potential role of
miRNAs in the fine tuning of fibrinogen levels
A. Fort1 , C. Borel1 , R. J. Fish1 , S. E. Antonarakis1 , M. Neerman-Arbez1,2 ;
1 2 University Medical Center, Geneva, Switzerland, Division of Angiology and
Haemostasis, University Hospital, Geneva, Switzerland.
In humans, plasma fibrinogen levels are maintained between 2-4
g/L with a wide variability in the population . Here, we addressed the
role of microRNAs in the regulation of fibrinogen gene expression by
screening the regulatory potential of 471 human microRNAs (from the
~1’000 annotated miRNAs in the human genome) on all five fibrinogen
transcript 3’UTRs (FGA, FGAα-E, FGB, FGG and FGG’). For this
purpose, we cloned each fibrinogen 3’UTR behind a firefly luciferase
reporter gene . Co-transfections in HEK-293 cells included one of the
firefly luciferase reporter gene constructs, a microRNA precursor and
a transfection efficiency control (expressing renilla luciferase). The
regulatory effects of microRNAs on each fibrinogen 3’UTR was calculated
by dividing the ratio of luciferase intensities (firefly/renilla) of
each transfection with a microRNA by the ratio from a reaction without
microRNA . We also screened human liver RNA for expression of 362
microRNAs, by qPCR . 57% of the tested microRNAs are expressed
in the liver. With these experiments, we identified 8 microRNAs, expressed
in the liver, showing -13% to -40% down-regulating potential;
4 of these acting specifically on FGA, 2 on FGB, 1 acting on FGA
and FGG and 1 down-regulating all fibrinogen 3’UTRs. None of these
microRNAs showed an effect on the control firefly luciferase vector
lacking a 3’UTR, demonstrating fibrinogen 3’UTR-specific down-regulation
potential . We also detected 16 liver-expressed microRNAs with
up-regulating potential of + 16% to + 176% . Ongoing experiments are
assessing the effects of these candidate microRNAs on endogenous
fibrinogen synthesis in HepG2 cells.
c05.6
Visualization of molecular interactions in situ, with singlemolecule
resolution
O. Söderberg;
Uppsala University, Uppsala, Sweden.
New, powerful techniques are required to observe individual protein
molecules and their interactions in situ, in order to analyze, with high
precision, in which cells the interacting proteins are located, and in
what sub-cellular compartments .
We therefore developed a method called in situ proximity-ligation assay
(in situ PLA) that requires multiple recognition events, by a pair of
antibodies, for detection . By conjugating DNA-strands to the antibodies
we convert the binding of the antibodies to a protein or a protein
complex into an amplifiable DNA molecule, thus increasing both the
selectivity and sensitivity of the assay. Rolling-circle amplification generates
a concatemeric product for localized detection by fluorescently
labeled probes . Using the in situ PLA, we could detect interactions between
two (c-Myc/Max) and three (c-Myc/Max/RNA pol II) endogenous
proteins, visualizing the active fraction of c-Myc as it exert its function
in promoting gene transcription (Söderberg et al . Nature Methods,
2006), as well as post-translational modifications, i.e. phosphorylation
of PDGFRβ, in cultured cells and fresh frozen tissues sections in situ
(Jarvius et al . Molecular & Cellular Proteomics, 2007) . In situ PLA is
applicable for detection of all types of biomolecules (e .g . proteins, DNA
and RNA) and interactions thereof, allowing detection and enumeration
of biomolecules within cells and tissue, at a single-molecule resolution.
As the read-out of the method is based upon DNA amplifications,
in situ PLA can easily be multiplexed for simultaneously detection of
multiple analytes . In situ PLA provides a unique opportunity to monitor
interaction patterns for diagnostic purposes .
c06.1
mutations in the cyclin family member FAm58A cause a novel
X-linked dominant disorder characterized by syndactyly,
telecanthus, anogenital and renal malformations (stAR
syndrome)
J. Kohlhase 1 , D. Boehm 1 , F. J. Kaiser 2 , S. Kaulfuss 3 , W. Borozdin 1 , K. Buiting 4 ,
P. Burfeind 3 , J. Boehm 5 , F. Barrionuevo 5 , A. Craig 5 , K. Borowski 6 , K. Keppler-
Noreuil 6 , T. Schmitt-Mechelke 7 , B. Steiner 8 , D. Bartholdi 8 , G. Mortier 9 , R. Sandford
10 , B. Zabel 11 , A. Superti-Furga 11 , S. Unger 5,11 ;
1 Center for Human Genetics Freiburg, Freiburg, Germany, 2 University Clinic
Schleswig-Holstein, Campus Luebeck, Luebeck, Germany, 3 Human Genetics,
University of Goettingen, Goettingen, Germany, 4 University Clinic Essen, Essen,
Germany, 5 Human Genetics, University of Freiburg, Freiburg, Germany,
6 Medical Genetics, University of Iowa, Iowa City, IA, United States, 7 Neuropediatrics,
Kinderspital Luzern, Luzern, Switzerland, 8 Medical Genetics, University
of Zurich, Schwerzenbach, Switzerland, 9 Medical Genetics, University of Ghent,
Ghent, Belgium, 10 Medical Genetics, University of Cambridge, Cambridge,
United Kingdom, 11 Pediatrics, University Clinic Freiburg, Freiburg, Germany.
We recently identified four girls with a consistent constellation of toe
Syndactyly, Telecanthus, Anogenital and Renal malformations, and we
propose the name STAR syndrome for this condition . A single mother-daughter
pair had previously been reported by Green et al . with
a similar combination of malformations . The authors noted that this
condition was autosomal dominantly inherited and overlapping with
but distinct from Townes-Brocks syndrome (OMIM #601446) . Array
CGH performed with DNA of one of our patients revealed a de novo
heterozygous deletion of 37 .9-50 .7 kb including exons 1 and 2 of the
gene FAM58A on Xq28, and qPCR detected a de novo deletion of
FAM58A exon 5 in a second case . Point mutation analysis revealed
one truncating and two splice mutations in FAM58A in three further
cases including the family reported by Green thus confirming this disorder
as a distinct recognizable X-linked dominant condition . FAM58A
encodes a Cyclin box fold domain, and in accordance with that siRNA
mediated knockdown in cultured cells revealed a proliferation defect .
FAM58A interacts with SALL1 but not SALL4 as determined by co-immunoprecipitation,
corresponding to the close phenotypic overlap with
Townes-Brocks syndrome .
c06.2
KCNQ2 mutations and implications for counselling and perinatal
care in Benign Familial Neonatal convulsions
N. Verbeek 1 , M. Poot 1 , W. Arts 2 , K. Flipsen-ten Berg 1 , B. Gunning 3 , D. Lindhout
1 , M. van Kempen 1 ;
1 Division of Biomedical Genetics, Utrecht, The Netherlands, 2 Erasmus MC,
Rotterdam, The Netherlands, 3 Epilepsy Centre Kempenhaeghe, Heeze, The
Netherlands.
Background: Benign familial neonatal convulsions (BFNC) is an autosomal
dominantly inherited form of epilepsy characterized by seizure
onset around the third day of life, spontaneously resolving within a few
months . In general, psychomotor development is normal, but 10-15%
of patients develop epilepsy later in life . BFNC is caused by mutations
in the voltage-gated potassium channel subunit gene KCNQ2
(20q13 .3) or, less frequently, KCNQ3 (8q24) . Purpose of this study is
to determine the frequency of KCNQ2-mutations in BFNC families and
sporadic cases .
Methods: MLPA- and sequence analysis of the KCNQ2 gene was performed
in 28 probands/families with neonatal seizures . Larger deletions
were analysed with Infinium humanhap300 SNP-array. Phenotypic
details were provided by the referring physician .
Results: In eleven families and three sporadic cases, three missense,
eight frameshift or nonsense mutations, and three large deletions were
detected . All large deletions (ranging form 49 to 479 kbp) contained
one to fourteen annotated genes, including the frontal lobe epilepsy
gene, CHRNA4, in two families . Associated mental retardation (without
active epilepsy), autism or (febrile) seizures later in life, occurred
in multiple families . Perinatal mutation analysis was performed in two
families .
Conclusions: Mutations were found in 50% of probands with (benign)
neonatal convulsions . Large deletions comprise a substantial portion
(21%) of KCNQ2-mutations . Mutation detection in neonates with benign
convulsions can prevent superfluous diagnostic procedures. Furthermore,
perinatal mutation analysis in sibs with 50% risk to BFNC
could significantly improve perinatal management.