2008 Barcelona - European Society of Human Genetics

Genomics, technology, bioinformatics 0
method to detect sequence variations . PCR is performed in the presence
of a saturating dsDNA binding dye . Single-base variations in the
amplicon result in altered melting behaviour after heteroduplex formation,
visualised by HRM plots . The LightCycler® 480 performs both
PCR and HRM in one run .
BRCA1 and BRCA2 were chosen as target genes, because their size
and mutation spectrum represent a challenge in molecular diagnostics
. Current methods, like dHPLC and DGGE, despite their good performance,
are limited by throughput or labour-intensity .
HRM is potentially useful for solving these problems . However, it needs
to be shown that the sensitivity and the user-friendliness are at least as
good as the current state of the art . We set up an extensive validation
in 3 laboratories .
The first objective was to design a complete primer set for BRCA1
and BRCA2 . Indeed, the performance of HRM is largely depending on
PCR quality. Critical criteria were specific banding patterns after gel
electrophoresis, sigmoid curves on real-time PCR plots and less than
3 melting domains per amplicon .
About 300 known variations are being tested in a blinded way . This will
allow us to determine whether HRM reaches a sensitivity close to 99%,
which would make it a suitable new method for diagnostic use .
P08.40
interlaboratory validation study of High Resolution melting
curve Analysis for mutation scanning of BRcA1 using the
Lightscanner (it)
N. van der Stoep 1 , C. D. M. van Paridon 1 , A. Stambergova 2 , P. Norambuena 2 ,
M. Macek 2 , T. Janssens 3 , G. Matthijs 3 , E. Bakker 1 ;
1 Center for Human and Clinical Genetics, Leiden, The Netherlands, 2 Institute
of Biology and Medical Genetics, Prague, Czech Republic, 3 Center for Human
Genetics, Leuven, Belgium.
The current set up for mutation scanning of BRCA1 occurs through
sequence analysis, DGGE, PTT and DHPLC . All these techniques
are time consuming and expensive . Therefore we have evaluated the
High-Resolution Melting Curve Analysis (HR-MCA) as a high-throughput
mutation-scanning tool for the BRCA1 gene using LightScanner
from Idaho Technology (IT) and the LightScanner-MasterMix . This
study was implemented in the EuroGentest evaluation program for
new techniques in genome diagnostics . Investigations were carried out
using a panel of >189 variants and 327 wt controls . We optimized and
evaluated 58 primer-sets that cover BRCA1 . All heterozygous variants
could be detected using the Call-IT-1 .5 software resulting in a 100%
mutation-detection sensitivity . These variants also include small DNA
deletions and insertions . Out of 327 wts we observed 3,7% false positive
curves (FP) resulting in a specificity of 96%. Re-evaluation of ten
BRCA1-amplicons in the two other diagnostic laboratories gave rise
to identical results . Moreover using unlabeled probes we were able to
identify many frequent occurring polymorphisms, omitting unnecessary
sequence analysis . Finally we selected a set of 41 best-performing
primers that encompass the entire BRCA1 gene using stringent criteria
and performed 2 blind tests on 27 patient DNA samples that were sequenced
in parallel. Optimal HR-MC analyses setting were fixed based
on the results obtained with our large set of known control samples . In
both series we detected all heterozygous variants and observed a FPscore
of 1,8% . In conclusion, HR-MCA with the LightScanner appears
to be a rapid and sensitive method for mutation scanning .
P08.41
Isolation and transcriptional profiling of embryonic human
neural crest cells
S. Thomas 1 , M. Thomas 1 , P. Wincker 2 , P. Xu 3 , M. C. Speer 3 , A. Munnich 1 , S.
Lyonnet 1 , M. Vekemans 1 , H. C. Etchevers 1 ;
1 INSERM U781, PARIS, France, 2 GENOSCOPE (CEA) and UMR 8030 CNRS-
GENOSCOPE-Université d’Evry, Evry, France, 3 Center for Human Genetics,
Department of Medicine, Duke University Medical Center, Durham, NC, United
States.
Developmental and stem cell biology ask the common question of how
functionally distinct cell types arise from one self-renewing founder
population . Human neural crest cells (hNCC) form in the embryo at the
end of the first month of pregnancy, although the precise dates have
remained elusive . Most NCC differentiate completely into melanocytes,
all components of the peripheral nervous system, certain endocrine
cells and connective and structural tissues in the head, although par-
tially competent NCC progenitors continue to reside in many tissues
in animal models . Due to their inaccessibility and despite their developmental
importance, the original properties and restriction of hNCC
potential over time have never been examined . Here, we demonstrate
when and how to isolate self-renewing hNCC and report the resultant
study of their transcriptome by serial analysis of gene expression
(SAGE) . In addition to highlighting candidate disease genes, we have
identified novel markers that distinguish hNCC from other human cell
types and distinguished between evolutionarily conserved and species-specific
NCC properties. Genome-wide analysis, using multiple
statistical criteria, demonstrates that the global molecular signature of
early migratory hNCC is remarkably similar to that of pluripotent human
embryonic stem cells . We show that most cell types of the human
pharyngula express a number of genes in situ that, individually, do not
autonomously confer pluripotency but taken within the context of the
transcriptome, may be permissive for the unique plasticity of hNCC
over time. Our findings pave the way for further studies of hNCC therapeutic
potential in neurocristopathies such as peripheral demyelinating
disorders .
P08.42
Human Leukocyte Antigen (HLA) typing and sequence-Based
typing (sBt) for Blood stem cell transplants
N. Cereb1 , L. L. Xu2 , E. Vennemeyer2 , S. Jankowski2 , S. Y. Yang1 ;
1 2 HistoGenetics, Ossining, NJ, United States, Applied Biosystems, Foster City,
CA, United States.
Human leukocyte antigens (HLA) are protein markers that are found
on most of the body’s cells . The immune system uses HLA to recognize
which cells belong in your body and which do not . These protein
markers help identify a person’s tissue type . The HLA proteins are important
in matching patients and donors for a bone marrow, peripheral
blood cell (PBSC)or cord blood transplant . When a transplant center
looks at the match level, it is looking at how alike the HLA tissue types
of the patient and the donor are to each other .
We will demonstrate that directed sequencing of over 40 PCR-amplicons
(including forward/reverse) in the HLA-Class I region provides a
complete test result for each patient . Even one amino acid variation in
each HLA gene between the donor and recipient increases transplant
mortality risk by 10% . Using an exact sequencing match will greatly
improve the patient’s chances of transplant success .
Direct sequencing for HLA typing gives the accurate and ultimate answer,
without a timely screening step prior to sequencing . Using stateof-art
automated capillary electrophoresis instrumentation and bioinformatics
tools to deliver these results saves lives by reducing time
before a donor is located, increasing successful transplant outcome
and lowering subsequent healthcare cost .
P08.43
conserved and non-conserved transcription regulatory elements
associated with siNEs in mammalian promoters
N. V. Tomilin;
Russian Academy of Sciences, St.Petersburg, Russian Federation.
About half of promoters of protein-coding genes in the mouse and
human genomes contain sequences of interspersed repeats which
can effect gene expression . Recent estimations suggest that some of
these sequences are evolutionary conserved and may represent >5%
of all constrained nonexonic elements which are under strong purifying
selection in mammalian genomes . However, major fraction of important
regulatory elements is not constrained in evolution . These elements
can be recruited from a large pool of lineage-specific dispersed
repeats . For example, human Alu and mouse B1 repeats contain short
conserved segments with potential binding site for transcription factor
PitX2 (binding motif YTGGGATTANW) which is important for establishement
of left-right asymmetry in development of multiple organs .
Alu-associated binding sites for PitX2 regulate expression of human
PLOD1 and ANF genes but most of downstream target genes for PitX2
is not established . Our results indicate that >1000 of mouse or human
genes contain Alu/B1 associated PitX2 binding sites and these genes
are expressed in almost all tissues . >100 of these promoters also have
potential binding site for transcription factor Nkx2 .5 (motif TYAAGTG)
which frequently cooperates with PitX2 in gene regulation . Among
PitX2 and Nkx2 .5 targets in the mouse genome we found promoters
of SMARCD3, PRKCZ and NIPA1 genes expressed at high level in