2008 Barcelona - European Society of Human Genetics

Genomics, technology, bioinformatics 0
P08.63
Prader-Willi syndrome: multiplex Ligation Dependent Probe
Amplification Diagnosis (MLPA) and Microsatellites
A. Rodriguez, M. Miramar, M. Calvo, F. Lorente, E. Barrio, S. Izquierdo, N.
Martinez;
Molecular Genetics, H.U.Miguel Servet. University of Zaragoza, Zaragoza,
Spain.
Introduction: PWS is a neuroendocrine genetic disease, with a global
frequency estimated between 1/10 .000 - 1/25 .000 livebirths . Clinical
symptoms are characterized by hipotonia, hyporreflexia, lack of movements
and deglutory alterations . Typical dismorphologic and behavioural
phenotype include hyperphagia-obesity, hypogonadism, hypogenitalism,
mental retardation and bone maturation retardation .
Lack of paternal contribution results in PWS either by paternal deletion
in 15q11-q13 region (70%) or from maternal disomy (25%) . A few
cases (2-5%) are due to imprinting center mutations .
Aims: We have performed a mutational screening of large deletions
in 15q11-q13 region and the metilation analysis of SNRPN gene in 37
patients with PWS clinical features. The aim of this study is to confirm
clinical diagnosis, to evaluate the main genetic alterations in our population
and provide supportive genetic counselling to the families .
Material and Methods: Patients DNA was obtained by robotic extraction
(EZ1, Qiagen) . The MLPA technique was performed by the commercially
available PWS Kit from MRC Holland . This kit allows large deletions
screening in 15q11-q13 region genes in two PCR reactions and
the methylation study of SNRPN by HhaI enzyme digestion . Analysis
was performed with an ABI PRISM 310 sequencing analyzer (Applied
Biosystems) and further data Excel normalization . In abnormal methylation
cases microsatellites D15S10, D15S11, D15S113, D15S128,
GABRA3, GABRB5 were studied .
Results and Conclusion: The MLPA genetic analysis has allowed PWS
diagnostic confirmation in 16.2% patients with clinical criteria. Two deletions
in 15q11-q13 region and 4 maternal uniparental disomy were
found . We conclude that PWS is well characterized by the MLPA technique
.
P08.64
ProSeeK: A web server for MLPA probe design
L. Pantano, L. Armengol, S. Villatoro, X. Estivill;
Center for Genomic Regulation, Barcelona, Spain.
The technological evolution of platforms for assessing genome-wide
copy number imbalances has allowed the discovery of an unexpected
amount of human sequence involved in duplications and deletions
(termed copy number variants or CNVs) . In terms of sequence coverage,
this is the most important type of human variation identified so
far and can make an important contribution to human diversity and
disease susceptibility . While different methods exist to asses genomewide
changes in gene dosage at high-throughput, methods for validating
these findings are tedious and expensive. Multiplex Ligation-dependent
Probe Amplification (MLPA) is one of the available technologies,
used even in diagnostic settings, to asses copy number variation
in the human genome . One of the dull and time-consuming steps of
MLPA is probe design step . Because of the multiplexing capacity and
the required specificity, the oligonucleotides targeting specific regions
have to meet a large number of requirements for an efficient design of
the experiment . ProSeeK is designed to perform all steps automatically
and to provide the best candidate probes for each individual assay
. ProSeeK is integrated as a user-friendly web server to ensure
portability .
P08.65
the OpenArray tm platform: enabling high-throughput sNP
genotyping and real-time PcR applications in nanoliter volumes
S. Liu-Cordero, D. G. W. Roberts, E. Ortenberg, J. Cho, J. Hurley, M. Kopczynski,
K. D. Munnelly;
BioTrove, Inc., Woburn, MA, United States.
BioTrove has developed the OpenArray platform based on throughhole
technology, a broadly applicable nanoliter fluidics technology for
parallel and low-volume solution phase reactions . OpenArray plates
are coated with hydrophilic coatings on the interior of each through hole
and hydrophobic coatings on the exterior of the through-holes . This
enables OpenArray plates to hold solutions in the open through-holes
via capillary action . The OpenArray plate consists of 3072 throughholes
that can be loaded with reagents to perform individual 33 nL reactions
for use in both real time PCR applications as well as endpoint
genotyping applications. The unique configuration of the through-holes
enables the researcher to interrogate a large number of nucleic acid
samples against a large number of assays in a flexible, configurable
format . By altering the number of assays or the number of samples the
researcher can easily customize the OpenArray to meet their changing
needs. Researchers using this technology benefit from the parallelism
of microarrays and the data quality of solution phase reactions .
P08.66
External cell control quantitative Rt-PcR (eccPcR): A new
technique for reliable detection of subtle changes in mRNA
expression
A. Bors 1 , P. Ribiczey 1 , G. Köblös 2 , A. Brózik 1 , Z. Ujfaludi 3 , M. Magócsi 1 , A.
Váradi 2 , A. Tordai 1 , T. Kovács 1 , T. Arányi 2 ;
1 Hungarian National Blood Transfusion Service, Budapest, Hungary, 2 Institute
of Enzymology, Hungarian Academy of Sciences, Budapest, Hungary, 3 University
of Szeged, Department of Biochemistry and Molecular Biology, Szeged,
Hungary.
Quantitative RT-PCR (qRT-PCR) is a widely used method to determine
relative gene expression levels. Quantification of the observed expression
levels becomes reliable after normalization to the expression of
an internal standard gene . If the mRNA level of the standard gene is
altered during the experiment, small changes of target mRNA levels
can be especially difficult to detect. However, the expression of commonly
used internal standard genes is often unstable, which considerably
bias quantification.
To overcome the drawback of unstable internal standards, we developed
a new method, called external cell control PCR (eccPCR) . This
method is based on the addition of control cells to the studied cells
before RNA extraction and qRT-PCR . Only the control cells express
the reference gene, while only the studied cells express the gene of interest
. This techique controls all steps of sample preparation and overcomes
the incertitude of normalization to internal standard genes .
We present the validation of the new method by detection the changes
of hSERCA3 mRNA expression in response to Na+-butyrate treatment
of KATO-III gastric cancer cells using F4-6 mouse erythroleukemia
cells as control and mPu .1 mRNA as the reference gene . In addition,
we demonstrate the instability of the expression of a commonly used
internal standard gene GAPDH by the eccPCR technique . The sensitivity
analysis of the new method showed that a 1 .5-fold gene expression
level difference can be systematically detected with the eccPCR
assay .
We conclude that eccPCR allows accurate quantification of small expression
level differences of a gene of interest .
P08.67
An integrated custom design tool for PcR resequencing
B. Turner, A. Sartori, S. Jankowski, L. Xu;
Applied Biosystems, Foster City, CA, United States.
Increasing attention has been devoted to SNP discovery and genotyping
in an effort to associate disease/phenotypes with gene variations
and mutations and to determine evolutionary relationships, but reliable
primer design and data analysis remain two of the major challenges to
overcome. Development of a flexible design tool that allows researchers
to select PCR-sequencing primers for different genomic targets
with user-defined parameters would greatly facilitate resequencing.
In order to help the scientific community to better use PCR and CEsequencing
for SNP discovery, we released the VariantSEQr primer
designs of 15K+ human genes to the public via NCBI’s ProbeDB .
To improve upon this one-size-fits-all approach, we have developed
an integrated web-based tool which incorporates target sequence selection/submission,
primer design, and data analysis into a connected
workflow. Users can choose genes, transcripts and other identifiers,
select any region in the genome, or upload their own sequence as
well as specify design parameters, e .g . amplicon length, primer Tm
etc . The web interface then submits the job to a backend pipeline,
which takes advantage of proprietary primer picking and predictive
quality assurance processes that generated Applied Biosystem’s VariantSEQr
primers . All currently known SNP/MNP sites in the genomic