2009 Vienna - European Society of Human Genetics

Genomics, Genomic technology and Epigenetics
pable sequences, presence of Ref Seq, and coverage across genes.
Libraries representing the transcriptome were constructed from as little
as 0.4 ug of rRNA-depleted RNA without appreciable loss of coverage.
The correlation among the sites for Ref Seq was > 0.90. We cloned
and sequenced rRNA depleted total RNA. Additionally, the same RNA
samples were cloned and sequenced using the same protocol by 6
independent field confirmation sites. In addition to technical reproducibility,
we will show the ability of the system to detect rare transcripts,
alternative splicing events as well as putative fusion transcripts.
P11.120
Expression analysis of immunorelevant genes in type 1 diabetes
pathogenesis
M. Hubackova 1 , Z. Halbhuber 2 , S. Kolouskova 1 , V. Stavikova 1 , T. Ulmannova 1 ,
D. Chudoba 1 , M. Krivjanska 2 , K. Stechova 1 ;
1 2nd Faculty of Medicine of Charles University and University Hospital Motol,
Prague 5, Czech Republic, 2 Central European Biosystems, Prague 4, Czech
Republic.
We analysed expression of genes relevant to immunoregulation in
peripheral blood mononuclear cells (PBMC). We compared basal expression
versus expression after stimulation with diabetes-associated
beta-cell autoantigens. For some parameters (some Th1, Th2, Th3
and Th17 cytokines) we correlated gene data with protein microarray
results.
Gene expression of 58 genes of immune regulation was analysed
using high density Phalanx gene microarray, containing total 30968
genomic probes. Cytokines were detected by ELISA and quantitative
protein microarray. PBMCs were stimulated by diabetogenic peptides
(3 GAD65 derived peptides, IA2-peptide and proinsulin peptide). Study
cohort consisted of 6 patients with T1D, 14 of their first-degree relatives
(5/14 positive for at least one autoantibody - DRLpos group) and
4 healthy controls.
The highest gene expression after specific stimulation was observed
mainly within the DRLpos group. These persons are autoantibody
positive but with normal intravenous glucose tolerance test. After specific
stimulation in DRLpos group, significant gene expression was observed
for: IFN-gamma, IL-1,-2,-6,-13,-22,-31, GATA-3, JUNB, IL-6R,
STAT-6, TGF-beta. The most important difference was observed for
IL-23R which was downregulated in DRLpos group (12 fold) and also
in T1D patients (23 fold). In T1D patients we observed an important
activation of IL-2,- IL-33, JUNB genes after specific stimulation and
strong downregulation of IL-4 and slightly downregulation of STAT-6
and GATA-3. Protein array data are in agreement with gene expression
analysis results.
Our results indicated that Th2/Th17 imbalance along with Th1 predominance
may be important in human T1D pathogenesis but further
study is necessary.
Supported by projects No.00064203, NPVII 2B06019.
P11.121
Ultraconserved elements are enriched among pathogenic copy
number variants causing mental delay and congenital anomalies
C. Orellana1 , S. Monfort1 , M. Roselló1 , I. Ferrer-Bolufer1 , D. Blesa2 , S. Oltra1 , F.
Martínez1 ;
1 2 Hospital Universitario La Fe, Valencia, Spain, Centro de Investigación Príncipe
Felipe, Valencia, Spain.
The ultraconserved elements (UCEs) are defined as stretches of at
least 200 base pairs of DNA that match identically with corresponding
regions in the mouse and rat genomes, albeit their real significance
remains an intriguing issue. These elements are most often located
either overlapping exons in genes involved in RNA processing or in
introns or nearby genes involved in the regulation of transcription and
development. Interestingly, human UCEs have been reported to be
strongly depleted among segmental duplications and benign copy
number variants. No comprehensive survey of a putative enrichment
of these elements among pathogenic dose variants has yet been reported.
A survey for UCEs was performed among the cryptic genomic rearrangements
detected in our series of patients with idiopathic neurodevelopmental
disorders associated to congenital anomalies. A total of 25
different elements, out the 481 described UCEs, were contained in 10
of the 21 pathogenic gains or losses detected in our series, what represents
a highly significant enrichment of ultraconserved elements.
We therefore propose that these elements may be interpreted as hallmarks
for dose-sensitive genes, particularly for those genes whose
gain or loss may be directly implied in neurodevelopmental disorders.
P11.122
Effect of semax and PGP treatment on expression of Vegf in
ischemic rat brain
V. V. Stavchansky1 , L. V. Dergunova1,2 , I. M. Maksimov1 , A. B. Botsina2 , T. V.
Tvorogova2 , V. I. Skvortsova2 , S. A. Limborska1,2 ;
1 2 Institute of Molecular Genetics RAS, Moscow, Russian Federation, Institute of
Stroke RSMU, Moscow, Russian Federation.
Vascular endothelial growth factor (Vegf) is a hypoxia-inducible angiogenic
peptide with recently identified neurotrophic effects. On the other
hand early post-ischemic delivery of Vegf increased blood-brain barrier
leakage and tissue damage. We analyzed the effect of synthetic
polypeptide Semax (Met-Glu-His-Phe-Pro-Gly-Pro) and its C-terminal
fragment Pro-Gly-Pro upon expression of Vegf in rat brain after global
cerebral ischemia. The study was carried out on 2-3-month-old male
Wistar rats (n=85). After 15 minutes of irreversible bilateral common
carotid artery occlusion the animals were exposed to intraperitoneal
injection of either Semax, PGP or saline 1, 4 and 8 hours after the occlusion.
Ischemic rats injected with saline were used as control groups.
The mRNA expression of Vegf was assessed by relative quantification
using real-time RT-PCR. Gapdh was used as the reference gene. The
level of Vegf mRNA was decreased compare with control animals: in
cortex of sham-operated and rats treated with Semax at 4h, 8h and
24h after occlusion; in hippocampus of rats treated with PGP - at 12h,
24h and treated with Semax - at 4h. The level of Vegf mRNA was
increased: in cerebellum of rats treated with PGP - at 12h, and in hippocampus
of rats treated with Semax - at 24h.
P11.123
GPGraphics: A Universal Graphical Backend for sNP microarray
Analysis
S. Uebe, F. Pasutto, M. Krumbiegel, D. Schanze, A. B. Ekici, A. Reis;
Institute of Human Genetics, Erlangen, Germany.
Whole genome association (WGA) studies using microarray platforms
are currently one of the most popular methods to search for diseaseassociated
genes throughout the human genome. Several analysis
programs are available, most of them, however, lack both a graphical
user interface as well as graphical output. Most analysis software
packages are designed to run in high power computing (HPC) environments,
where GNU/Linux and a variety of Unix flavors are the predominant
operating systems. These systems are by their very nature rather
text- than graphics-oriented, thus making it difficult for a researcher
to get an actual overview of the huge amount of data these programs
produce. We now present GPGraphics, which can graphically evaluate
data from pooling as well as single sample based WGA software, such
as GenePool or PLINK. GPGraphics provides a series of mathematical
filters to visualize even faint signals in noisy data. Due to the modular
nature of the software, the more processing intensive analyses may
still be performed on a non-graphical HPC system, while the evaluation
of the data generated by those systems can be performed in the
graphical environment of a Microsoft Windows computer. Since the
software is written for the Microsoft .NET framework, it will even run on
non-Windows systems, provided they have a .NET runtime environment
fully compatible to the .NET 2.0 specification. The presented software
has been successfully used at our institute to analyze whole genome
microarray data for both qualitative and quantitative traits, such
as pseudoexfoliation syndrome and cornea thickness, respectively.
P11.124
Transcriptome profiling of Williams-Beuren syndrome
C. N. Henrichsen 1 , G. Csardi 1 , M. Zabot 2 , S. Bergmann 1 , G. Merla 3 , A. Reymond
1 ;
1 University of Lausanne, Lausanne, Switzerland, 2 Hospices civils de Lyon,
Hôpital Debrousse, Lyon, France, 3 IRCCS “Casa Sollievo della Sofferenza”,
San Giovanni Rotondo, Italy.
Williams-Beuren syndrome (WBS), a neurodevelopmental disorder
characterized by mental retardation with unique cognitive and personality
profile, is caused by an interstitial deletion on chromosome
7q11.23 encompassing 28 genes. Although the primary cause of WBS
is well-understood, the molecular basis of the phenotype is largely un-
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