2009 Vienna - European Society of Human Genetics

Genomics, Genomic technology and Epigenetics
P11.100
Expression Profiling of both miRNAs and mRNA targets using a
novel nanofluidic real-time qPCR technology
A. Bond, E. Grigorenko, E. Ortenberg, J. Hurley, J. White, K. Munnelly;
BioTrove, Inc., Woburn, MA, United States.
Previously, profiling both microRNAs and their mRNA targets was difficult,
time consuming, and involved extensive post-profiling validation.
Our system allows for easy, rapid and inexpensive quantitation of both
miRNA and mRNA targets with no need for validation. The reliability of
real-time qPCR, the flexibility of an array format and novel nanofluidics
provide the backbone of this elegant system. Using the OpenArray
system, we profiled RNA from a panel of human adult tissue types for a
set of well characterized miRNAs. In our second study, total RNA was
obtained from normal tissues and tumor tissues, reverse-transcribed
and loaded on plates preformatted with primer pairs against hundreds
of genes that have been found to be differentially expressed in cancer.
This pre-validated qPCR panel covers disparate, but linked pathways
such as DNA repair, angiogenesis, cell adhesion and cell cycle. Using
this nanofluidic system we were able to identify genes that consistently
changed expression across cancer types as well as genes differentially
expressed in specific cancers. The OpenArray system allowed us
to concurrently chart both the modulator and the modulated. An analysis
of the data provides us with a rich view of the complexities of gene
expression and further validated our technique. The simple power of
this technique is that the researcher does not need to further validate
their results with qPCR; they have simultaneously performed both the
discovery and validation phases.
P11.101
Reference materials for genetic diagnostics and HLA-typing
J. Boyle, M. Hawkins, W. Pickering, E. Byrne, E. Gray, P. Metcalfe, R. Hawkins;
National Institute for Biological Standards and Control, South Mimms, Hertfordshire,
United Kingdom.
The National Institute for Biological Standards and Control (NIBSC)
in collaboration with diagnostic laboratories have developed a programme
to generate genetic reference materials (GRMs) as WHO
International Standards and CE-marked In Vitro Diagnostic Controls.
The materials are used for genetic diagnostics, both for genetic disease
and tissue typing (HLA-typing), and are typically positive control
DNA samples of known genotype which serve to verify assay, operator
and data tracking performance. The genetic material is sourced from
cell lines established from primary lymphocytes to produce quality-assured
cell banks.
CE-marked HLA-typing panels are produced within the ISO13485
quality system. The HLA-A genotyping panel comprises 24 genomic
DNA samples and provides single-use positive controls in DNA-based
low resolution HLA-A typing for many common alleles in the UK Caucasian
population. The panel has been awarded the CE-mark and is
expected to be available in 2009. A HLA-DRB1 panel of 40 genomic
DNA samples is currently in production.
WHO International Standards of genomic DNAs have been produced
for Factor V Leiden, Prothrombin G20210A, Haemophilia A intron 22
inversion and Fragile X syndrome. A genomic DNA GRM for Prader
Willi and Angelman syndromes will be submitted to the WHO or another
international organisation in 2009. GRMs for other genetic disorders,
including a panel for hereditary non-polyposis colorectal cancer
with genomic DNA deletion samples are planned. NIBSC aims to address
the rapid increase in the numbers of tests performed and the
development of new technologies by the provision of GRMs as a key
tool in successful genetic diagnostics.
P11.102
the Locus Reference Genomic (LRG) DNA sequence format
R. Dalgleish, the GEN2PHEN Consortium, the European Bioinformatics Institute
(EBI), the National Center for Biotechnology Information(NCBI);
University of Leicester, Leicester, United Kingdom.
A crucial element of sequence variant nomenclature is the reference
DNA sequence used to describe the variant. NCBI has established
curated non-redundant reference sequences of genomes, transcripts,
proteins and genes (RefSeq & RefSeqGene). However, these are not
ideal for reporting variants in Locus-Specific Databases (LSDBs).
The primary limitation is that the reference DNA sequence might be
revised over time, reflecting current knowledge, but failing to provide a
stable reference for inter-generational genetic diagnoses. Additionally,
there may be several alternatively-spliced transcripts. Furthermore,
legacy exon- and amino-acid-numbering systems may have arisen for
particular genes and be in common use, even though they do not comply
with current variant reporting standards.
To address these limitations, we have developed a new sequence format
known as Locus Reference Genomic (LRG) that will have both
locked and updatable sections.
The locked section will primarily comprise:
• The DNA sequence
• Coordinates of all identified exons
• Details of coding transcripts and their conceptual translation
The updatable section will primarily comprise:
• Coordinates and other information to map a LRG onto the current
human genome build
• The exon-numbering scheme
• Details of legacy DNA reference sequences, RefSeq and RefSeq-
Gene sequences
• Details of legacy exon- & amino-acid-numbering systems
• Chromosome number
• Known sequence variants
• Audit data for each feature
Details of LRGs, which are implemented in XML, accompanied by
sample sequences can be found at http://www.lrg-sequence.org
Acknowledgements: GEN2PHEN is funded by the European Community’s
Seventh Framework Programme (FP7/2007-2013) under grant
agreement 200754.
P11.103
Dual luciferase reporter assay system: limitation of its
interpretation
L. Grodecka 1 , H. Grombirikova 1 , B. Ravcukova 1 , J. Litzman 2 , T. Freiberger 1 ;
1 Molecular Genetics Laboratory, CKTCH, Brno, Czech Republic, 2 Institute of
Clinical Immunology and Allergology, St. Anne’s University Hospital, Brno,
Czech Republic.
Genetic reporter systems are popular tools for studying many processes
that take place in eukaryotic cells, e.g. transciriptional regulation,
mRNA processing, etc. Usually, dual reporter enzymes, an experimantal
reporter and a normalizing standard, are used in one experiment to
minimize an interassay variation caused by differences in transfection
efficiency and cell viability. Consequently, the results are expressed
as the reporter enzyme activity normalized to the activity of internal
standard. Assuming that the same amount of reporter genes is always
used in one experiment, the ratio of its expression (and thus ratio of
enzyme activities) should be a constant. No correlation is supposed
between the ratio of reporter activities and the number of transfected
cells or the activity of the reporters. However, in our experiment, there
is a correlation between the ratio of reporter activities (luminescence
of different luciferases) and the experimental reporter activity. Using
one mixture of reporter and standard gene contructs for transfection of
different amounts of cells, we obtained ratios of luciferases activities
strongly correlated to the firefly luciferase activity (r = 0,84; p < 0,001).
This effect was specific for the particular construct since neither the
construct with shorter insert of the same type nor the reporter genecontaining
plasmid without any insert showed this effect. We speculate
that some specific expression activator could induce such non-standard
outcomes. These results clearly indicate that the reporter gene
assays data should always be evaluated with care.
This study was supported by grant of IGA MZ CR No. NR 9192-3.
P11.104
identifying Users and contributors on the Biomedical internet
G. A. Thorisson, P. Burton, A. J. Brookes;
University of Leicester, Leicester, United Kingdom.
A number of ostensibly separate initiatives have begun considering
the risks, benefits, and practicalities of unambiguously identifying researchers
as they use and contribute to biomedical data sources on
the Internet. The GEN2PHEN project (http://www.gen2phen.org) is
one such initiative, given its general aim of helping to unify human
and model organism genetic variation databases towards increasingly
holistic views into Genotype-To-Phenotype (G2P) information. More
specifically, the GEN2PHEN project considers researcher identification
to be an absolutely central part of how biomedical databasing, and
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