2008 Barcelona - European Society of Human Genetics

Genomics, technology, bioinformatics 0
P08.35
complete sequencing of the cFtR gene using the new
generation Gs-FLX sequencing technology
H. Cuppens, L. Vliegen, J. Cassiman;
Center for Human Genetics, Leuven, Belgium.
In most genes involved in genetic diseases, a broad spectrum of mutations
is found. Even for diseases such as cystic fibrosis, genetic testing
can be very challenging . Indeed, routine CFTR genetic tests only
screen for the most common mutations (88-92% sensitivity in most
European countries) .
New generation sequencing technology, such as picotiter pyrosequencing
on a GS-FLX system, has been recently introduced . However,
this technology was initially developed for whole genome sequencing
purposes .
We adopted this technology for complete sequence analysis of the
CFTR coding region, and its exon/intron junctions .
To this aim we have developed a robust multiplex amplification assay
in which biotinylated amplicon-specific primers are locally restricted
through streptavidin/biotin crosslinking . Indeed, 30 amplicons should
be analyzed for the CFTR gene, which can be only economically feasible
if amplified in one, or a limited number, PCR multiplex reaction(s).
For a 50x coverage, only half a million nucleotides are needed for
CFTR sequence analysis, i .e . 0 .5% of the full capacity of the GS-FLX
system . Therefore, 100-200 samples should be pooled in order to use
the full capacity of the GS-FLX system . We therefore also developed
an universal sample tagging approach allowing the pooling of more
than 100 samples with one set of 260 primers (60 amplicon-specific
and 200 tagging primers) . This compares to 6000 primers if ampliconspecific
PCR primers are tagged as such.
This technique is readily transferable to any gene, allowing sequencing
of more than 100 samples for the same gene, or even different
genes, in an economically feasible way .
P08.36
Nuclear localization of sm22 alpha during heart development
E. Bregant 1 , R. Lonigro 1 , N. Passon 1 , A. Scaloni 2 , G. Renzone 3 , M. Pandolfi 4 ,
C. Di Loreto 4 , G. Damante 5 ;
1 1.Department of Biomedical Sciences and Technologies, Udine, Italy, 2 2.Proteomics
and Mass Spectrometry Laboratory, ISPAAM , Napoli, Italy, 3 2.Proteomics
and Mass Spectrometry Laboratory, ISPAAM, , Napoli, Italy, 4 3.Department
of Medical Morphological Research , Udine, Italy, 5 1. Department of
Biomedical Sciences and Technologies, Udine, Italy.
The molecular mechanisms that control heart development have been
the subject of intense investigation . The transition of embryonic to
adult cardiomyocytes is associated to changes in the expression patterns
of different proteins .
Aim of this study is the identification of nuclear proteins whose expression
is modified during cardiac differentiation. A proteomic approach,
based on two-dimensional electrophoresis was utilized . The experimental
model is the H9C2, a myoblast cell line derived from embryonic
rat ventricle . These cells proliferate in medium with 10% serum,
instead low serum and stimulation with retinoic acid induce differentiation
versus cardiomyocytes .
We have analyzed the nuclear extracts of H9C2 that are grown in
proliferation medium and in differentiation medium by proteomic approach
.
Seven different proteins have been identified as differentially expressed
after MALDI-TOFF, LC-ESI-MS/MS mass spectrometry . An interesting
protein is SM22 alpha (transgelin), a 22-KD cytoskeletal protein that is
a marker of smooth muscle cells .
The level of the SM22 alpha is reduced at 20 th day of differentiation
condition. These data are confirmed by Western-Blot analysis and
quantitative RT-PCR .
By immunochemistry, we confirmed the nuclear localization of the protein
in H9C2 cell line . Furthermore, in histological section of human
embryonic heart we show that SM22 alpha is located at nuclear level
in heart vessels and in myocytes of the cardiac outflow.
Thus, our data indicate that SM22 alpha can be localized in the nucleus
and suggest that this localization is regulated during development
P08.37
methylation-sensitive High Resolution melting Analysis as a
diagnostic tool for Beckwith-Wiedemann and silver-Russell
syndromes.
M. Alders, J. Bliek, K. vd Lip, R. vd Boogaard, M. Mannens;
Academic Medical Center, Amsterdam, The Netherlands.
The Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome
(SRS) are caused by disturbed imprinting at 11p15 . This region
harbors two independently regulated clusters of imprinted genes . The
first cluster is under control of the IC1 upstream of the H19 promoter,
which is methylated only at the paternal allele . The second cluster is
controlled by IC2 upstream of the KCNQ1OT1 promoter and is methylation
on the maternal allele only .
BWS is an overgrowth syndrome . The majority of the patients display
hypermethylation of IC1, hypomethylation of IC2 or both . SRS is a
growth retardation syndrome and in a subset of patients a hypomethylation
of IC1 is found, opposite to the aberration found in BWS patients
.
Molecular confirmation of BWS and SRS is done by methylation analysis
of IC1 and IC2 . Since the methylation defects in BWS and SRS are
mosaic the test must be quantitative .
We set out to validate High Resolution Melting Analysis (HRMA) for
methylation analysis in BWS/SRS diagnostics . Advantages of this
method are that it is fast, cost effective and requires no post PCR
handling . We tested a group of 17 BWS/SRS patients with different
levels of hyper- and hypomethylation at IC1 and/or IC2 and 45 normal
controls. All patients showed a melting profile different from the normal
controls and the degree of deviation was consistent with the degree
of hypo- or hypermethylation as determined by southern blotting . In
conclusion, HRMA analysis proofs to be a fast, reliable and sensitive
diagnostic tool for BWS and SRS .
P08.38
calibraton improves methylation-sensitive high resolution
melting results
C. N. Gundry1 , M. Wall1 , J. McKinney1 , J. D. Phillips2 , M. K. Yu3 , D. H. F. Teng1 ;
1 2 Idaho Technology, SLC, UT, United States, University of Utah, SLC, UT,
United States, 3Myriad Genetics, SLC, UT, United States.
High resolution melting has been shown to be a sensitive method for
methylation detection of CpG sites . In high resolution melting, multiple
CpG sites within the same PCR fragment can be detected homogeneously.
Unlike the common real-time methylation-specific PCR technique
(MethylLight Taqman probe detection) hi-res melting provides
more information with fewer PCRs . As 5-methylcytosines are resistant
to conversion to uracils, there can be substantial sample-to-sample
melting differences depending on the number of CpG dinucleotide
sites and the sequence context within the fragment . However, depending
upon fragment and the exact number of 5-methylcytosines actually
converted during the bisulfite treatment, there can be both variation in
replicate conversions of the same sample and very subtle melting differences
between samples . Both of these problems are resolved with
calibrated melting . Calibration has especially been helpful to increase
accuracy of genotyping when melting differences are subtle . This techniques
improves hi-res melting reproducibility and genotyping calls via
synthetic oligonucleotide probes . We used melt calibration in conjunction
with amplicon methylation analysis to improve our detection in a
highly methylated region . We obtained excellent resolution of amplicon
fragments within a hypermethylated region of the miRNA-195 genomic
sequence. Sequence verification showed that our homogeneous technique
is comparable to this gold standard in accuracy .
P08.39
interlaboratory validation of High Resolution melting (HRm) for
BRcA1 and BRcA2 on the Lightcycler ® 480
T. Janssens 1 , N. van der Stoep 2 , R. Buser 3 , G. Michils 1 , A. Corveleyn 1 , E. Dequeker
1 , P. Maillet 3 , E. Bakker 2 , G. Matthijs 1 ;
1 Center for Human Genetics, Leuven, Belgium, 2 Center for Human and Clinical
Genetics, Leiden, The Netherlands, 3 Laboratory of Oncogenetics, Geneva,
Switzerland.
High Resolution Melting (HRM) was selected as a technology for which
a thorough validation would be very timely . In a collaborative EuroGentest
study, we extensively tested it on the LightCycler® 480 .
HRM is a fast, simple and cost-effective high-throughput scanning