2008 Barcelona - European Society of Human Genetics

Concurrent Sessions
reporting, and literature mining .
Array-CGH technology is currently on its way to replace classical
karyotyping as primary diagnostic tool in copy number screening . Its
vast importance in clinical diagnosis and research are underlined by
large number of genes in human development and disease still unknown.
A data storage and mining tool aimed specifically at leveraging
the power of Array-CGH in a clinical context will aid aetiology of
rare and complex genetic diseases by characterising genomic rearrangements,
annotating and analysing clinical features, and providing
advanced data mining and integration . For example, through an
automated analysis of PUBMED abstracts, Bench allows to prioritize
candidate genes in genomic deletions and duplications by phenotype
characteristics annotated to the patient . Also, known Copy Number
Variations, genes and their functions, and other genome annotations,
when integrated with patient related data, aid in diagnosis and aetiology
of new submicroscopic chromosomal imbalance syndromes .
Bench provides a collaborative Array-CGH LIMS system that allows
to maintain records of phenotype and chromosome rearrangements
in patients, augmented with data mining, reporting and visualisation
facilitating research, diagnostics, research and genetic counseling by
involving relevant information from a variety of sources . Bench can
be used free of charge in a research collaboration . http://www .esat .
kuleuven .be/cghgate/ .
c05.1
Functional interactions of conserved non-coding (cNc)
sequences with other cNc using circular chromosome
conformation capture (4c)
D. Robyr, G. Duriaux-Sail, S. E. Antonarakis;
University of Geneva Medical School, Geneva, Switzerland.
The comparison of human chromosome 21 (Hsa21) sequences with
the mouse syntenic regions led to the identification of roughly 3500
regions displaying an identity of >70% over a length of a least 100
nucleotides of ungaped alignment . About 65% (~ 2300) of these are
conserved non-coding sequences (CNCs) . Very little is known about
the function of most CNCs . We speculated that a functional CNC may
interact with its genomic target (i .e . an enhancer would bind to it’s cognate
gene promoter). Thus, the identification of any part of the genome
that interacts directly with a CNC could provide clues on the function of
the latter . We have generated libraries of CNC-interacting DpnII fragments
by chromosome conformation capture (4C) whose identity is
determined by subsequent high-throughput sequencing .
We are currently screening for the interactions of 18 CNCs located in
the two ENCODE regions of HSA21 in different cell lines . Preliminary
results for two CNCs in K562 cells indicate that these may interact
near regions of the genome that show conservation among vertebrates
. Indeed, the median distances from the sequenced DpnII fragments
to the nearest conserved region are 381 .5 bp (P = 0 .0583) and
764bp (P = 0 .023) respectively for the 2 CNCs analysed . These results
provide initial evidence that the function of CNCs is mediated by their
interactions with other conserved regions . Interestingly, these CNCs
are capable of interactions with loci not only in cis and over several Mb,
but also in trans with loci located on other chromosomes .
c05.2
A high-resolution structural variation map of a human genome
by next-generation, high-throughput paired-end sequencing
F. M. De La Vega 1 , H. E. Peckham 2 , S. S. Ranade 2 , S. F. McLaughlin 2 , C. C.
Lee 2 , Y. Fu 2 , Z. Zhang 1 , F. C. L. Hyland 1 , C. L. Clouser 2 , A. A. Antipova 2 , J. M.
Manning 2 , C. L. Hendrickson 2 , L. Zhang 2 , E. T. Dimalanta 2 , T. D. Sokolsky 2 ,
M. W. Laptewicz 2 , B. E. Coleman 2 , J. K. Ichikawa 2 , J. B. Warner 2 , B. Li 1 , J. M.
Kidd 3 , J. A. Malek 4 , G. L. Costa 2 , E. E. Eichler 3 , K. J. McKernan 2 ;
1 Applied Biosystems, Foster City, CA, United States, 2 Applied Biosystems, Beverly,
MA, United States, 3 HHMI, University of Washington, Seattle, WA, United
States, 4 Weill Cornell Medical College in Qatar, Doha, Qatar.
The human genome and HapMap projects have considerably increased
our understanding of the role of sequence variation in evolution
and disease . Hybridization microarrays and fosmid-end sequencing
reveal that structural variants (SVs) including insertions, deletions,
duplications, inversions and translocations are common and extensive .
Microarray methods, however, lack resolution and are blind to unbalanced
events, while clone-based end-sequencing is time consuming
and expensive . Here, we present a high-resolution survey of SVs of a
human genome, a HapMap Yoruba sample (NA18507), by ultra-high
throughput sequencing of paired-end libraries with the AB SOLiD(TM)
System . We sequenced a variety of 2x25-bp paired-end libraries
(>15Gb) with insert sizes ranging from 600bp to 6kb (SD 10-23%) .
Each library provides over 10x physical (clone) coverage, with a total
combined physical coverage >60x for 90% of the genome . The high
physical coverage and diverse insert sizes allowed detecting small indels
within tags (1-10 bp), and approximately 70,000 indels of length
20 bp to >100 kb . Additionally, we sequenced 7Gb of 50-bp fragment
libraries, which combined with the paired libraries provided over 12x
sequence coverage, allowing us to discover millions of SNPs of which
75% are found in dbSNP . Inferred SVs were compared to a database
of end-sequence pairs of 10x physical coverage obtained by di-deoxy
sequencing of 40kb fosmid ends . A subset of novel SVs were validated
by PCR and Sanger sequencing . Our results serves as a model for
further high-resolution exploration of genetic variation in human populations
and cancer with next-generation sequencing .
c05.3
Expression analysis using deep solexa sequencing shows
major advances in robustness, resolution and inter-lab
portability over microarray platforms
J. T. den Dunnen 1,2 , Y. Ariyurek 2 , H. H. Thygesen 1 , E. Vreugdenhil 3 , J. M.
Boer 1 , G. B. van Ommen 1 , P. A. C. ‘t Hoen 1 ;
1 Human and Clinical Gentics, Leiden, The Netherlands, 2 Leiden Genome
Technology Center, Leiden, The Netherlands, 3 Medical Pharmacology, Leiden /
Amsterdam Center for Drug Research, Leiden, The Netherlands.
The hippocampal transcriptomes of wild-type mice and mice transgenic
for δC-doublecortin-like kinase were analyzed with the Solexa deep
sequencing technology . We determined around 2 million sequence
tags per sample and compared these data with results of the same
samples analysed using five different microarray platforms. Seventy
percent of the sequence tags were mapped to approximately 30,000
unique, high-confidence transcripts, their abundance spanning four
orders of magnitude . Antisense transcription, undetectable by microarrays,
was found in 51% of all genes, and alternative poly-adenylation
in 47% . With a dedicated Bayesian model and false discovery rate
of 8.5%, we measured statistically significant differential expression
for 3179 transcripts; many more and with higher fold-changes than
observed using microarrays . The deep sequencing technology demonstrates
superb reproducibility, not only between biological replicates
but even across laboratories . The described major advance in robustness,
comparability and richness of sequence -based transcriptomics
data is expected to boost in-depth collaborative, comparative and integrative
genomics studies .
c05.4
studying gene dosage imbalance in embryonic stem cells
G. Cobellis 1,2 , A. Romito 1 , R. De Cegli 1 , S. Iacobacci 1 , A. Fedele 1 , D. di Bernardo
1 , A. Ballabio 1 ;
1 TIGEM, Napoli, Italy, 2 Second University of Naples, Naples, Italy.
To gain insight into the alterations of the transcriptional pathways underlying
the pathogenesis of Down syndrome, we decided to use an
integrated strategy combining the systematic overexpression of chromosome
21 genes in ES cells, transcriptome analysis and systems
biology approaches .
We generated an ES cell clone bearing an inducible/exchangeable
cassette in the ROSA26 locus to be used to insert, one by one, every
murine orthologs of human chromosome 21 genes. Using this flexible
system, we developed a library of ES cells over-expressing, in
an inducible manner, murine orthologs of human transcription factors,
kinases and miRNAs mapping on HSA21 in order to perturb the physiological
genetic network at the cellular level . The biological pathways
affected by the over-expression of each coding and non-coding gene
and their regulators and regulated genes have been inferred using
system biology approach . We already mapped the regulatory gene
networks specifically altered by each transcription factors and kinases,
opening new hypothesis toward the understanding of pathogenesis of
Down syndrome .
Finally, the above-described ES cell lines have been used to study the
gene dosage imbalance effects on ES cell differentiation to cardiomyocytes,
myeloid and neuronal lineages, tissues affected by the DS .
This project represent the first to involve a systematic overexpression