European Human Genetics Conference 2007 June 16 – 19, 2007 ...

Genomics, technology, bioinformatics
P1249. Impact of Histone Modifications on Gene Expression and
Differentiation
J. F. Fischer1 , J. Toedling2 , T. Krueger1 , M. Schueler1 , W. Huber2 , S. Sperling1 ;
1 2 Max-Planck-Institut für Molekulare Genetik, Berlin, Germany, European Bioinformatics
Institute, Cambridge, United Kingdom.
Histones together with DNA form the nucleosome that provides the
scaffold for the compaction of DNA. Histone tails are susceptible to
a wide variety of modifications and the type of histone modification
contributes to the degree of DNA accessibility and gene transcription.
However, it is not clear whether histone modification marks are primary
or secondary to transcription, whether histone modifications act synergistically
to form a histone code and to what extent they function as
signaling marks for the recruitment of effectors.
We investigated the relationship between transcript levels and four histone
modifications (H4ac, H3ac, H3K4me2, H3K4me3). We performed
ChIP-chip and expression array analysis in two independent mouse
cell lines (C2C12, HL-1), and C2C12 in two differentiation stages.
Our results revise previous findings, based on univariate data, which
had led to the belief that all of these modification marks are associated
with elevated expression levels. The apparent positive effect of histone-3-lysin-4-dimethylation
is caused by its correlated co-occurrence,
and an effect that disappears when it is present by itself. Our results
suggest that histone modifications form a code, as their combinatorial
composition is associated with distinct read-outs. We show that
modifications are highly dynamic during differentiation. Upregulation of
transcripts is associated with a gain of histone-4-acetylation, but many
modification conversions occur without a change of transcript levels.
This indicates that histone modifications precede transcription, rather
than being a consequence of it, and primarily function as signalling
marks for specific effectors, but are not by themselves a sufficient driving
force for transcription.
P1250. DS5: Biological storage and retrieval: Bringing e-Science
to embryo bioinformatics resources.
M. K. Scott 1 , J. van Hemert 2 , Y. Chen 2 , X. Wang 1 , S. Lisgo 1 , S. Lindsay 1 , M.
Atkinson 2 , R. A. Baldock 3 ;
1 Institute of Human Genetics, Newcastle upon Tyne, United Kingdom, 2 National
e-Science Centre, Edinburgh, United Kingdom, 3 MRC Human Genetics Unit,
Edinburgh, United Kingdom.
The current technologies for storage, accession and manipulation of
biological data have focused on efficient management, curation and
analysis of data in terms of a centralised resource, by enabling largescale
data-download for analysis. Bio-resources that require a more
distributed approach (e.g. for shared material, inter-institute collaboration
or large volume data-analysis) are currently unacceptably low in
power for the tasks required, or suffer limitations affecting usability and
usefulness. These types of requirements are a central concern of development
in e-Science and therefore, as part of the FP6 funded DGE-
Map (Developmental Gene Expression Map) project we are assessing
the current architectures to improve on restrictions experienced. This
is a collaborative effort between the e-Science Institute, the Institute of
Human Genetics and the MRC-Wellcome Trust Human Developmental
Biology Resource. The three main areas of focus have been: a) a
database for the storage and access of internal experimental details,
specimen records and users details, with an emphasis on upgrading
the current technology to utilise web portal access, b) 3D mapping
and visualisation software to increase accuracy and automation for
data mapping, including inter-stage and inter-species analysis, and c)
establishing a web presence equipped with search, analysis and datamining
tools, for the dissemination of data to the wider community.
P1251. Development and validation of the “chromosome X
exon-specific array” that enables identification of copy number
changes in genes of the X chromosome
L. K. Kousoulidou 1 , S. Bashiardes 1 , H. van Bokhoven 2 , H. Ropers 3 , J. Chelly 4 ,
C. Moraine 5 , A. de Brouwer 2 , H. Van Esch 6 , G. Froyen 7 , P. C. Patsalis 1 ;
1 Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus, 2 Department of
human genetics, Radboud University Nijmegen Medical Centre., Nijmegen, The
Netherlands, 3 Max Planck Institute for Molecular Genetics, Human Molecular
Genetics Department, Berlin, Germany, 4 INSERM U129-ICGM, Faculte de
Medecine Cochin., Paris, France, 5 Centre Hospitalier Universitaire de Tours,
Service de Genetique, Hospital Bretoneau., Tours, France, 6 Centre for Human
Genetics, University Hospital Gasthuisberg., Leuven, Belgium, 7 Human Ge-
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nome Laboratory, VIB, Dept. Human Genetics, K.U.Leuven., Leuven, Belgium.
The aim of this study was to design and produce a specialized and novel
chromosome X exon-specific oligonucleotide microarray for screening
of all X-chromosomal genes and detection of copy number changes.
Identification of genetic alterations is extremely important for research
and clinical purposes. Recent studies have indicated that microdeletions
and microduplications occur at a high frequency in the human
genome, while advances in high-density oligonucleotide arrays have
paved the way with unparalleled resolution. The new “chromosome
X exon-specific array” contains about 22,000 60mer oligonucleotides
covering more than 92% of all chromosome X exons and miRNA regions,
as well as control regions from other chromosomes. Two known
abnormal control samples, one with a well-characterized isochromosome
X and another with a Xp22.2 duplication were interrogated on
the array to test its efficiency and identify probes that do not perform
efficiently. Modifications were carried out and the final “chromosome
X exon-specific array” was used for screening of 20 XLMR families
from the EURO-MRX Consortium. One out of the twenty families was
identified by array-CGH as having a 1.78-kb deletion involving all exons
of one gene and a second family was found to carry a 27-kb deletion
involving all exons of two contiguous genes. The above genetic
changes were confirmed with PCR. The “chromosome X exon-specific
array” could be utilized to detect genetic changes in X-linked disorders
and other chromosome X abnormalities. The “chromosome X exonspecific
array” will become available from “Oxford Gene Technology”
(OGT) biotechnology company within the next months.
P1252. Validation of a commercially available PCR kit for
diagnostic testing for Fragile X CGG repeat
P. A. van Bunderen, M. M. Weiss, E. Bakker;
Laboratory for Diagnostic Genome Analysis, Leiden, The Netherlands.
Almost all cases of Fragile X syndrome are caused by an expansion
of a CGG repeat in the 5’ UTR of the FMR1 gene to more than 200 repeats.
Molecular testing is usually performed by both PCR of the CGG
repeat (up until 100 repeats) and Southern blot analysis.
We are currently validating a new kit from Abbott. With this kit it should
be possible to detect large expanded CGG repeats. By calculating a
peakheight ratio of the CGG repeat and of a control X fragment, information
is gained about the status “heterozygous or homozygous”
in female samples. We tested 39 known samples (13 female and 26
male) including full expanded alleles, homozygous normal females,
and premutations.
Of the 13 females analysed, two homozygous and two full mutations
were confirmed. Of the 26 males, one showed a full mutation and two
had premutations. Of the 3 full mutations, 2 were visible in raw data.
Using the Abbott kit in Fragile X diagnostic testing is fast and only a
small amount of DNA is necessary. There are no discrepancies in all
39 samples. Two of three full mutations were visible in the raw data.
However, interpretation of peak ratios is sometimes difficult because
the reliability of the ratios is dependent on the size of the normal allel.
At this moment the size standard is not suitable for full mutation sizing,
so checking the raw data is necessary.
P1253. A PCR-based assay detecting pre- and full mutations of
the FMR1 gene
M. Pomponi, R. Pietrobono, E. Tabolacci, G. Neri;
Università Cattolica del S. Cuore, Roma, Italy.
We evaluated the Fragile X PCR Assay (Abbott Molecular, Inc.) effectiveness
in distinguishing the size of FMR1 alleles. The assay is
based on fluorescent multiplex PCR where the CGG repeat sequence
is co-amplified with undisclosed gender-specific targets. We tested
122 samples, whose FMR1 status had been ascertained by Southern
blotting (HindIII/EagI digestion , hybridization with probe Ox1.9).
The samples included 93 females (31 wt homozygotes, 24 wt heterozygotes,
20 premutations, 18 full mutations) and 29 males (10 wt, 7
premutations, 3 full mutations, 2 mosaics wt/full mutation, 8 mosaics
premutation/full mutation). Results obtained with the PCR assay coincided
with those obtained by Southern blotting. The determination of
female zygosity was done through the calculation of the TR/X ratio,
comparing the height of the peak corresponding to the CGG sequence
with that of the gender-specific target. A ratio>1 indicates wt homozygosity.
Of 31 homozygous females, 23 (74%) had TR/X>1, 7 (23%)
TR/X