2009 Vienna - European Society of Human Genetics

Genomics, Genomic technology and Epigenetics
and MCT8L. Transfection of long MCT8_M75L or MCT8_M75A cDNA
resulted only in the production of MCT8L. Transport studies showed
that uptake of thyroid hormone in cells expressing MCT8L was lower
than in cells expressing MCT8S. However, the G41_S42 duplication
in the MCT8 protein did not change thyroid hormone transport activity,
surprisingly in contrast to the studied human MCT8 mutations so far.
We found that both TLSs in MCT8 are used to produce two isoforms
which are both capable of transporting thyroid hormones in vitro. It is
yet unknown whether these isoforms are differentially expressed in
human tissues.
P11.070
Whole Genome Amplification of single cell: application in
forensic SNP profiling.
E. Giardina1 , I. Pietrangeli1 , M. De Felici2 , G. Arcudi3 , A. Spinella4 , G. Novelli1,5 ;
1 2 Biopathology, Rome, Italy, Department of Public Health, University of Rome
Tor Vergata, Rome, Italy, 3Department of Public Health-Institute of Forensic
Medicine, Faculty of Medicine, University of Rome Tor Vergata, Rome, Italy,
4Direzione Centrale Anticrimine, Servizio di Polizia Scientifica, Rome, Italy,
5Division of Cardiovascular Medicine, Department of Medicine, University of
Arkansas for Medical Sciences, Little Rock, AR, United States.
The scarcity of genomic DNA can be limiting factor in several fields of
genetic research such as forensic, prenatal of preimplantation diagnosis.
Multiple displacement amplification (MDA) is a whole genome amplification
technique developed to amplify a limited DNA sample nonspecifically
to generate a new sample that is indistinguishable from the
original but has a higher DNA concentration.
In this work we evaluated the applicability of MDA for forensic purposes.
We amplified DNA, extracted from 30 single amniocyte cells
and 10 single lymphocytes and typed a 36-SNP panel. Single cell
MDA DNA was typed twice for the same set of SNPs to evaluate the
reproducibility of results. We performed a total of 2160 typing reactions
observing a positive typing rate in amplified DNA (99,65%) and
97,87% of concordance rate between amplified vs control (genomic)
DNA. The absence of perfect concordance rate revealed the failure
of amplification of alleles in heterozygous samples (ADO). We also
performed traditional STR-based profiling on single cell amplified DNA
obtaining a higher ADO rate. These results suggest that MDA should
be considered as a suitable option for SNP typing in challenging forensic
casework.
Acknowledgements
This work was supported by financing from EU FP6 projects NACBO:
Novel and Improved Nanomaterials, Chemistries and Apparatus for
Nano-Biotechnology (contract no. NMP4-CT-2004-500804).
P11.071
Multiplexed DNA methylation testing for efficient tumor marker
definition and microarray validation
A. Weinhaeusel 1 , M. Hofner 1 , C. Fürhauser 2 , M. Wielscher 1 , S. Schönthaler 1 ,
R. Pichler 1 , C. Singer 2 , D. Kandioler 3 , R. Panzer 4 , C. Noehammer 1 ;
1 Austrian Research Centers GmbH – ARC, Health & Environment, Molecular
Medicine, Seibersdorf, Austria, 2 Department of Obstetrics and Gynaecology,
Medical University of Vienna, Vienna, Austria, 3 Department of Surgery, Medical
University of Vienna, Vienna, Austria, 4 CCRI - Children’s Cancer Reseacrh
Institute, St Anna Children’s Hospital, Vienna, Austria.
Here we present the design principle and performance of a combined
multiplex-PCR and microarray hybridization technique for multiplexed
methylation testing.
Targeting 323 published DNA regions hypermethylated in several neoplasias,
methylation analysis is performed via methylation dependent
restriction enzyme digestion of 500ng of starting DNA. DNA is amplified
within 16 multiplex PCRs covering 360 different amplicons and
detected via microarray hybridization. After PCR amplicons are pooled
and positives are detected using strepavidin-Cy3 via microarray hybridization.
Although the melting temperature of CpG rich DNA is very
high, primer and probe-design as well hybridization conditions have
been optimized, thus this assay enables multiplexed methylation testing
of human samples. The assay has been designed that 24 samples
x 360 reactions can be run in parallel on a single 384 well PCR plate.
Several tumor entities have been tested using the assay. Chip-data
derived from the multiplexed assay enabled classification and class
prediction and defined candidate-markers with potential diagnostic application.
P11.072
Expression profiles of small RNAs from various tissues
generated by sOLiD sequencing
R. Tanzi 1 , B. Nutter 1 , S. Kuersten 2 , J. Gu 2 , C. Barbacioru 1 , D. Wang 1 , B. Gardiner
3 , K. Lea 4 , S. Heater 2 , M. Barker 1 , L. Chapman 2 , T. Gulham 1 , L. Wong 1 , S.
Grimmond 3 ;
1 Life Technologies, Foster city, CA, United States, 2 Life Technologies, Austin,
TX, United States, 3 The university of Queensland, St Lucia QLD, Australia, 4 Life
Technologies, austin, TX, United States.
The combination of the SOLiD Small RNA Expression Kit (SREK)
with the SOLiD Sequencing System presents a unique opportunity to
study miRNA expression in a way not previously possible. We barcoded
and sequenced small RNA libraries from ten different human tissues
to saturating levels of detection; generating up to 200 million tags.
Comparing both independent sequencing runs and libraries indicates
the system is highly reproducible and has up to 6 logs dynamic range.
To analyze the quantitative ability of this approach we compared tag
count data to real-time PCR assays generated using TaqMan miRNA
low density arrays. Fold-change comparisons between platforms show
Pearson correlation values of 0.95. Detailed analysis indicates a far
greater repertoire of miRNA variants, or ‘isomirs’, than previously observed
suggesting a much broader range of mRNA targets for miRNAmediated
regulation. Using this approach we have identified hundreds
of potentially novel sequence tags. We chose a subset of these novel
transcripts and designed custom TaqMan miRNA assays to validate
them by real-time PCR analysis. We are able to demonstrate both the
presence and expression profile of >50% of the novel sequences, most
of which are present at relatively low levels in the ten tissues. Interestingly,
we failed to detect nearly all of these novel targets using conventional
northern blotting highlighting the need for qPCR sensitivity for
validation purposes. This human miRNA expression atlas provides a
unique opportunity to understand the sequence complexity and identity
of small noncoding RNAs present in a variety of human tissues.
P11.073
microRNA target prediction by expression analysis of host
genes
V. Gennarino, M. Sardiello, R. Avellino, N. Meola, V. Maselli, A. Ballabio, S.
Banfi;
TIGEM, Naples, Italy.
MicroRNAs (miRNAs) are small noncoding RNAs that control gene
expression by inducing RNA cleavage or translational inhibition. Most
human miRNAs are intragenic and are transcribed as part of their hosting
transcription units.
We hypothesized that the expression profiles of miRNA host genes
and of their targets are inversely correlated and devised a novel procedure,
HOCTAR (host gene oppositely correlated targets), which ranks
predicted miRNA target genes based on their anti-correlated expression
behavior relative to their respective miRNA host genes. HOCTAR
is the first tool for systematic miRNA target prediction that utilizes the
same set of microarray experiments to monitor the expression of both
miRNAs (through their host genes) and candidate targets. We applied
the procedure to 178 human intragenic miRNAs and found that it performs
better than currently available prediction softwares in pinpointing
previously validated miRNA targets. The high-scoring HOCTAR predicted
targets were enriched in gene ontology categories, which were
consistent with previously published data, as in the case of miR-106b
and miR-93. By means of overexpression and loss-of-function assays,
we also demonstrated that HOCTAR is efficient in predicting novel
miRNA targets and we identified, by microarray and qRT-PCR procedures,
34 and 28 novel targets for miR-26b and miR-98, respectively.
Overall, we believe that the use of HOCTAR significantly reduces the
number of candidate miRNA targets to be tested compared to the procedures
based solely on target sequence recognition. Finally, our data
further confirm that miRNAs have a significant impact on the mRNA
levels of most of their targets.