2008 Barcelona - European Society of Human Genetics

Genomics, technology, bioinformatics 0
Oligo-Array (Agilent) was created . Multiple probes on this array spanning
the amplified region of exon 2 of the dystrophin gene.
In a next step Array-CGH data will provide a basic for the design of
PCR systems for exact determination of the amplification limits.
P08.26
Role of the kinase DYRK1A in cerebral cortex development:
effects on the transcriptome
E. Balducci 1,2 , M. J. Barallobre 1,2 , M. C. Ruiz de Villa 3 , A. Sánchez 3 , M. L. Arbonés
1,2 ;
1 Center for Genomic Regulation (CRG), Barcelona, Spain, 2 Centre for Biomedical
Research on Rare Diseases (CIBERER), Barcelona, Spain, 3 Statistics Department,
University of Barcelona, Barcelona, Spain.
DYRK1A is a dual-specificity protein kinase involved in brain development
. Human DYRK1A is located in the Down Syndrome (DS) Critical
Region of chromosome 21 and its overexpression has been associated
to the neurological defects observed in DS . Nevertheless, DYRK1A
function at the molecular level remains poorly understood . As DYRK1A
substrates include several transcription factors, we have analyzed the
impact of Dyrk1a dose reduction on the transcriptome of mouse cerebral
cortex at postnatal day 0 (P0) and 7 (P7) . To this end, global gene
expression of Dyrk1a+/- and Dyrk1a+/+ cortices were compared using
Affymetrix chips .
Microarray results revealed deep changes in gene expression extending
into the set of 3169 genes analyzed . Among those, 22% and 5%
showed different expression levels between genotypes at P0 and P7
respectively (adj-p2 . Gene Ontology analysis revealed enrichment in transporters within
the down-regulated genes and in DNA binding molecules within the upregulated
genes both, at P0 and at P7 . Among the genes deregulated
between genotypes, 61 were common to both developmental stages .
Interestingly, their regulation showed a similar trend at P0 and P7 but
always opposite to the developmental trend (defined by the expression
of such genes in Dyrk1a+/+ mice at P7 vs P0) .
These results suggest that DYRK1A plays a pivotal role in cerebral
cortex development acting on the regulation of the transcriptome .
P08.27
Re-examining the human genome
R. J. Kinsella, I. Barnes, C. Snow, A. Frankish, J. Mudge, L. Wilming, J. Loveland,
D. Carvalho-Silva, J. Rajan, E. Hart, L. Gordon, J. Gilbert, S. Trevanion, T.
Hubbard, J. L. Harrow;
Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire, United Kingdom.
Recent improvements in sequencing technologies enable the routine
sequencing of large complex genomes . However the annotation of
such genomes is still complex and there is no higher eukaryote that
has all its coding sequences predicted to 100% accuracy . The HA-
VANA group (http://www .sanger .ac .uk/HGP/havana/) at the Wellcome
Trust Sanger Institute is involved in manually annotating all the coding
transcripts within the human genome through various international collaborations
. The team is involved in the scale up of the ENCODE pilot
gene annotation project (GENCODE) to the whole human genome .
The project includes seven other partner institutes and will integrate
computational approaches, expert manual annotation and targeted
experimental approaches to generate a reference gene set . This will
include analysis of pseudogenes, experimental validation of putative/
novel genes and examination of the protein-coding potential of genes
using comparative and structural analysis .
In addition, the Havana group collaborates with RefSeq at NCBI,
UCSC and Ensembl to produce a core set of human and mouse coding
genes for the Consensus CDS (CCDS) project . Any CCDS candidate
transcripts where there is disagreement between WTSI/EBI and NCBI
annotation are manually re inspected, discussed and, where possible,
an accord is reached on a structure (18293 human CCDS agreed to
date) . The end result is a combined, non-redundant gene set agreed
by several of the major genome centers .
All annotation is displayed on the Vertebrate Genome Annotation
(VEGA) database, a central repository for high quality manual annotation
of finished vertebrate genome sequence with a three monthly
release cycle (http://vega .sanger .ac .uk/index .html) .
P08.28
An in vivo unbiased screen for enhancer activity using
lentivector-mediated transgenesis
M. Friedli1 , I. Barde2 , C. Attanasio1 , M. Arcangeli1 , A. Quazzola2 , S. Verp2 , F.
Spitz3 , J. Zakany4 , D. Duboule4,2 , D. Trono2 , S. E. Antonarakis1 ;
1University of Geneva, Department of Genetic Medicine and Development,
Geneva, Switzerland, 2EPFL, School of Life Sciences, Lausanne, Switzerland,
3 4 EMBL, Heidelberg, Germany, University of Geneva, Department of Zoology
and Animal Biology, Geneva, Switzerland.
Finding sequences that control spatial and temporal expression of
genes is important to understand genome function . Here, we present
an in vivo screen for enhancers in a contiguous 200-kilobase DNA
fragment using lentivector-mediated transgenesis . Previous studies
have used evolutionary conservation as an indicator of regulatory
potential, but increasing evidence suggests that this criterion systematically
overlooks functional sequences . We thus designed our study
without any bias towards a particular sequence feature . We chose a
mouse BAC corresponding to a region of Hsa21 because it contains
the olig1 and olig2 genes that are expressed specifically in the CNS.
In order to screen this fragment systematically for enhancer activity,
we generated a library of 121 overlapping clones (sizes: 2-4kb) in a
LacZ reporter lentiviral construct containing a minimal promoter . We
generated lentivectors individually for each segment and injected them
in pools of 10 or 20 in mouse oocytes . LacZ staining was performed
on E11 embryos to identify expression patterns. The first six pools
tested yielded 60 of 242 LacZ positive embryos with ~2 .5 transgenes
per embryo. To date, 7 fragments of 52 assessed were identified that
potentially contain gene expression regulators. To confirm regulatory
activity, 4 sequences were re-injected individually, 2 (one evolutionary
conserved) of which were confirmed as tissue specific enhancers with
stainings in the spinal chord and trigeminal ganglion compatible with
olig expression . The method could be scaled up to cover large chromosomal
regions, and determine what fraction of the constrained and
non-constrained genome has regulatory potential .
P08.29
Enhanced workflow for sequencing PCR products by capillary
electrophoresis
P. Kotturi, E. Currie-Fraser, S. Pickrell, M. Johnson, P. McNamara;
Applied Biosystems, Foster City, CA, United States.
Since the introduction of Sanger dideoxy sequencing, significant efforts
have been directed toward increasing throughput by streamlining
workflow. We describe here further enhancements for a PCR product
resequencing workflow capable of reducing the total time, from beginning
PCR reactions through completion of basecalling, to 6 hours or
less. The workflow shown employs a new AmpliTaq Gold ® Fast PCR
Master Mix in conjunction with modified thermal cycler conditions to
substantially reduce the time required for PCR amplification. Process
time is further reduced through optimization of cycle sequencing (BDT
v1 .1) conditions . We have coupled these improvements with an efficient
sequencing reaction cleanup protocol and decreased CE run
time using the fast plates on a 3130xL . Overall data quality compares
favorably with data obtained using previously documented methods .
The increased efficiency and generation of high quality results is vital
to both clinical and research applications in reducing time to discovery
.
P08.30
Analysis of copy number variation using formalin-fixed paraffinembedded
(FFPE) samples on BAc microarrays
D. Postma, S. Snoeijers, M. Lacombe, S. Schoenmakers;
Kreatech Diagnostics BV, Amsterdam, The Netherlands.
Array-based comparative genomic hybridization (aCGH) has become
a powerful tool to analyze DNA copy number changes . Especially in
combination with archival tissue samples with well-documented follow-up
data, it enables the analysis of genomic changes underlying
tumour development . Unfortunately, DNA isolated from archival, formalin-fixed,
paraffin-embedded (FFPE) material, is often degraded
and therefore difficult to label. In theory, labelling techniques that skip
the use of enzymes would be the best option . Therefore, we compared
the non-enzymatic Universal Linkage System (ULS) and conventional
random priming to label DNA isolated from FFPE material . Testing
of FFPE samples that had been stored for up to 17 years showed