2008 Barcelona - European Society of Human Genetics

Genomics, technology, bioinformatics
P08.77
Genomic regulation of gene-expression levels in the brain
during human evolution
M. Cáceres 1,2 , L. Armengol 1 , J. W. Thomas 2 , T. M. Preuss 3 , X. Estivill 1 ;
1 Genes and Disease Program, Center for Genomic Regulation (CRG-UPF) and
CIBERESP, Barcelona, Spain, 2 Department of Human Genetics, Emory University
School of Medicine, Atlanta, GA, United States, 3 Division of Neuroscience
and Center for Behavioral Neuroscience, Yerkes National Primate Research
Center, Atlanta, GA, United States.
For the last few years, several groups have used microarrays to compare
transcript levels in humans and non-human primates and have
identified hundreds of candidate genes with expression changes in the
human brain . Here, a multidisciplinary approach, including experimental
techniques and bioinformatic analysis of genome sequence and
gene-expression data, is proposed to obtain a more profound understanding
of the potential causes and effects of these gene expression
changes . Preliminary sequence comparisons have yielded examples
of diverse types of mechanisms that could account for the observed
differences in mRNA levels, such as multiple gene duplications, insertion
of transposable elements in 3’ UTR or upstream regions, and a
single nucleotide mutation in the polyA signal . In addition, as an example
of the proposed strategy, a global analysis of the regulatory sequences
of the thrombospondin 4 (THBS4) gene has been carried out .
This gene is involved in synapse formation and shows a six-fold upregulation
in humans that is specific of the forebrain. Interestingly, an
excess of nucleotide changes and an Alu insertion were found in the
THBS4 promoter region in the human lineage . However, in vitro gene
reporter assays indicated that the human and chimpanzee promoter
fragments have similar transcriptional activity, and computational prediction
of transcription factor binding sites suggests that THBS4 upregulation
could be due to changes in trans-acting regulators . Overall,
these results provide insights into the regulatory mechanisms acting at
various levels and the evolutionary role of gene-expression regulation,
and could help us to determine the molecular basis of human-specific
traits .
P08.78
Using genome-wide pathway analysis to unravel the etiology of
a complex disease like type 2 diabetes
C. C. Elbers 1,2 , K. R. van Eijk 1 , L. Franke 1,3 , F. Mulder 1 , Y. T. van der Schouw 2 ,
C. Wijmenga 1,3 , N. C. Onland-Moret 1,2 ;
1 Complex Genetics Section, Dept. of Biomedical Genetics, University Medical
Center Utrecht, Utrecht, The Netherlands, 2 Julius Center for Health Sciences
and Primary Care, University Medical Center Utrecht, Utrecht, The Netherlands,
3 Department of Genetics, University Medical Center Groningen and
University of Groningen, Groningen, The Netherlands.
Several genome-wide association studies (GWAS) have recently
been published on a variety of complex diseases . However, 99 .9% of
GWAS data is currently discarded and not analyzed to its full potential
. In this study, we took a different approach and aimed to evaluate
alternative methods of obtaining valuable information on disease etiology
from GWAS data . We assessed whether we could detect overrepresented
biological pathways in the GWAS datasets, by combining
a network-based tool, ‘Prioritizer’, with a pathway-classification tool,
‘PANTHER’ .
As an example, we used publicly available data from two type 2 diabetes
(T2D) GWAS; the Diabetes Genetics Initiative (DGI) and the Wellcome
Trust Case Control Consortium (WTCCC) . Of the 1,179 SNPs
in the DGI dataset and the 1,712 SNPs in the WTCCC dataset that
showed association with T2D with a p-value lower than 0 .003, we were
able to map 559 and 797 SNPs, respectively, to genomic loci that contained
one or more genes . Prioritizer then selected the most promising
gene(s) from each locus based on their functional interactions with
genes on the other loci . We used PANTHER to assign each of the
selected genes to a specific pathway and to test whether we saw more
genes in each pathway than expected .
Our results showed that the ‘inflammation mediated by chemokine and
cytokine signaling’ and ‘Wnt signaling’ pathways were consistently the
most strongly overrepresented in the T2D data . By taking an alternative
approach, we have shown it is possible to detect biological mechanisms
in GWAS data .
P08.79
Ultra-High-throughput sequencing in heterogeneous
inherited heart disorders: a first experience in Hypertrophic
cardiomyopathy
S. Fokstuen 1 , C. Iseli 2,3 , D. Robyr 4 , A. Munoz 4 , S. E. Antonarakis 4,1 , J. L. Blouin
1 ;
1 Genetic Medicine, University Hospitals of Geneva, Geneva, Switzerland, 2 Ludwig
Institute for Cancer Research, Lausanne, Switzerland, 3 Swiss Institute of
Bioinformatics, Lausanne, Switzerland, 4 Genetic Medicine, University of Geneva
School of Medicine, Geneva, Switzerland.
During last decade, molecular genetics has provided new insights into
the pathogenesis of inherited heart disorders and Hypertrophic Cardiomyopathy
(HCM) represents the most common one (1/500) . Genetic
testing has a growing impact on the management of patients and their
families . However, the extensive genetic heterogeneity of these disorders
requires the use of high-throughput mutation detection strategies
for clinical screening . We had developed a 30-Kb HCM-Custom-DNAresequencing-array
(HCM-RA) comprising all exons (n=160), splicesites
and 5’-UTR of 12 HCM genes. Although very efficient in detecting
single nucleotide variants, this approach did not identify small indels,
accounting for 14% of HCM mutations . Moreover, resequencing-arrays
lack flexibility since gene additions requires a new design.
In order to overcome the shortcomings of microarray-resequencing
we assessed the performance of the recently developed Ultra-High-
Throughput-Sequencing (UHTS) . We reanalysed these 12 genes in
a total of 19 patients, hybridized previously on HCM-RA (8 positivecontrols
as a composite-pool and 11 DNA without mutation), in a single
channel of a SOLEXA . Every single base of the sequence (570 fold
coverage accounting for 24 alleles) was analyzed using a newly developed
data analysis pipeline .
All the 8 control mutations and 18 SNPs previously identified by
HCM-RA were also observed in UHTS . Furthermore sequence alterations
and particularly indels were detected such as c .1028delC/
p .Thr343fsX349, [c .2146-9C>A+c .2146-2delA], c .506-12delC in gene
MYBPC3, c .53-15_-11delTTCTC in TNNT2 . Although evolution and
improvement in DNA target enrichment, specificity and data analysis
are needed, UHTS holds considerable promises in mutation/variant
analysis underlying highly heterogeneous or multigenic pathologic
conditions in clinical practice as well .
P08.80
Advantages of universal primers use in biochip analysis of
trombosis genes polymorphism
A. S. Glotov 1,2 , E. S. Vashukova 1,2 , L. B. Polushkina 1 , O. N. Mityaeva 3 , T. V.
Nasedkina 3 , I. V. Goldenkova-Pavlova 4 , T. E. Ivaschenko 1 , M. S. Zainulina 1 , V.
S. Baranov 1 ;
1 Ott’s Institute of Obstetrics&Gynecology, St.-Petersburg, Russian Federation,
2 Saint-Petersburg State University, Saint-Petersburg, Russian Federation, 3 Engelgardt
Institute of Molecular Biology, Moscow, Russian Federation, 4 Vavilov
Institute of General Genetics, Moscow, Russian Federation.
Genetic testing of inherited predisposition to frequent multifactor diseases
needs new screening methods of DNA-polymorphism analysis .
The most advanced approaches in genetic testing of multifactorial
diseases usually include multi-locus DNA amplification. The multiplex
PCR method could be significantly improved by means of the universal
sequences incorporated into locus-specific primers. We have selected
such DNA sequences and used them for two-step PCR genotyping of
F2, MTHFR, F5, PAI1, GPIIIa, FGB polymorphism with the subsequent
hybridization on oligonucleotide biochips . Use of this technique opens
an ample opportunity for the fast and reliable detection of inherited
thrombosis . The method could be easily adopted in any molecular diagnostic
laboratory .
The work was partly supported by BRHE Fellowship competition 2006
(Y4-B-12-02), Saint-Petersburg Personal grant for PhD (PD 07-1 .4-
129), and grant of Russian Foundation for Basic Research (N 07-04-
12271-ofi)
e-mail: anglotov@mail.ru