2008 Barcelona - European Society of Human Genetics

Genomics, technology, bioinformatics 0
brain . However, in human genome other brain-expressed genes having
SINE-associated PitX2/Nkx2.5 sites are identified (e.g. KIF17). It is
likely that SINEs contributed to regulatory evolution of brain-expressed
genes through their associated binding sites for PitX2 in a lineage specific
manner.
P08.44
KingFisher® Flex - Versatile automated nucleic acid, protein and
cell purification
S. Suomalainen, M. Partanen, V. Puro, A. Lamberg;
Thermo Fisher Scientific, Vantaa, Finland.
Sample preparation is often a limiting step for genomics and proteomics
studies. Rapid and efficient isolation of nucleic acids, proteins
and cells from complex biological matrixes is needed to get high quality
starting material for various experiments . KingFisher, the magnetic
bead based, automated purification system from Thermo Fisher Scientific,
provides a quick and easy solution to achieve high quality and
reproducible results in purification of nucleic acids, proteins and cells
with minimal hands-on time . The technology is based on magnetic
rods which move particles through the various purification phases -
binding, mixing, washing and elution . The KingFisher is an open and
flexible system, letting the user to choose any available magnetic particle
based purification kit suitable for the application.
Thermo Scientific KingFisher family consists of three instruments with
different throughput and working volume capacities . The latest member
of the family is KingFisher Flex, which is truly flexible solution for
different types of sample processing needs . There are four magnetic
heads available for the instrument depending of the needed processing
volumes. With the 24-rod configuration the processing volume can
be raised up to 5 ml and with the 96-rod configuration it is possible to
achieve the highest throughput in working volumes 20-1000 µl . The instrument
is compatible with robotic platforms providing a high throughput
automated solution for all biotech and pharmaceutical laboratories
. The data from different applications shows that KingFisher Flex
provides a rapid method for purification of high quality and quantity
biological molecules .
P08.45
Diagnosis optimization by using mLPA in the investigation
protocol in mentally retarded children - Romanian experience
A. Sireteanu 1 , E. Neagu 2 , G. Schobers 3 , V. Bica 4 , C. Skrypnyk 5 , M. Puiu 6 , V.
Cret 7 , L. Barbarii 2 , C. Rusu 1 ;
1 Department of Human Genetics, University of Medicine and Pharmacy, Iasi,
Romania, 2 ”Mina Minovici” Forensic Medicine Institute, Bucuresti, Romania,
3 Department of Human Genetics, Radboud University Nijmegen Medical
Centre, Nijmegen, The Netherlands, 4 Institute for Mother and Child, Bucuresti,
Romania, 5 Faculty of Medicine and Pharmacy, University of Oradea, Oradea,
Romania, 6 University of Medicine and Pharmacy, Timisoara, Romania, 7 Children’s
Hospital, Cluj, Romania.
Mental retardation (MR) is a heterogeneous entity, genetic causes
being involved mainly in moderate/severe forms . Subtelomeric rearrangements
(SR) play an important role in idiopatic MR determinism .
Recently introduced MLPA (Multiplex Ligation Probe Amplification)
technique seems to provide good results in SR’s identification. We
have used MLPA to identify SR in children with idiopatic MR, the protocol
consisting of the following sequence: clinical selection based on de
Vries score; karyotype; antiFMRP test for Fragile X syndrome screening;
PCR for Fragile X syndrome diagnosis; MLPA testing using 2 independent
kits in order to separate polymorphisms . Parents were tested
if an abnormality was detected in their child (by karyotype/MLPA) .
The study group was formed of 223 children that had a de Vries score
of 3/more . All patient data were recorded in a database specially designed
. In 28 of them (12 .5%) the karyotype revealed different abnormalities
. 17 cases (7 .6%) presented speech delay/ autism, but anti-
FMRP test and PCR were normal . Out of the 195 MLPA tests done:
168 cases (86 .1% were normal, 13 (6,7%) abnormal and 14 (7 .1%)
had polymorphisms . The most frequently involved SR were 1p del and
7q del. Clinical features of the cases identified with different SR will be
illustrated with photos and discussed .
In conclusion, we appreciate that the diagnostic score is useful in case
selection for further testing, MLPA proves to be efficient in diagnosing
SR and the frequency of SR as a possible cause of idiopatic MR is
similar to other studies in the literature .
P08.46
confocal morphometry of the transfected human mesenchymal
stem cells
A. V. Lavrov 1 , T. V. Karamysheva 2 , N. B. Rubtsov 2 ;
1 Research Center of Medical Genetics RAMS, Moscow, Russian Federation,
2 Institute of Cytology and Genetics SB RAS, Novosibirsk, Russian Federation.
Transfected human stem cells (SC) demonstrate grate potential for
clinical applications . They are extensively investigated to elucidate
their efficiency and safety. We focused on studying the reaction of SC
on the introducing the genetic construction . Morphometry of nuclei of
the transfected mesenchimal stem cells (MSC) was performed .
Materials and methods: The culture of MSC at the “late” passage (12)
was used . Lipid-mediated transfection was performed with a non-viral
vector based on ps415 containing VEGF121 using UF-56 as the lipid
media . After 90 minutes media was changed . Cells were washed and
cultivated for two days, then seeded on cover-glasses and cultivated
24 hours more. Cells were fixated with paraformaldehyde. Nuclei were
contrasted with DAPI . Scanning was performed using confocal microscope
Axioimager A1 .
Results: average nucleus dimensions of the transfected human
mesenchimal stem cells: 14,6x8,7x8,3mkm and of control MSC -
14,8x8,9x8,3mkm .
Conclusions: transfected MSC have the same nucleus dimensions
as the control MSC . Additional studies should be performed to check
chromatin structure and the form of the nuclei which appeared slightly
different in our study . Objective formal criteria should be formulated to
perform such analysis . The integrity of the nucleus may prove the stability
of MSC after transfection forming the basis for further application
of the transfected MSC in clinical trials .
P08.47
RmetaWeb: meta-analysis online tool
A. Kastrin;
Division Of Medical Genetics, Ljubljana, Slovenia.
Meta-analysis is a statistical procedure that integrates the results of
several independent studies . The number of papers published on
meta-analyses in biomedical domain has increased sharply in the past
15 years . The merits and perils of the somewhat mysterious procedure
of meta-analysis, however, continue to be debated in the medical
community . Given the increasing pace of published meta-analysis
studies, their methodological quality, however, still leaves much to be
desired . We have developed RMetaWeb, an online system for comprehensive,
fast, and reliable research synthesis analysis . The web
interface is based on a Perl CGI script that communicates with R using
the CGIwithR library . An interactive viewer provides straightforward
navigation through various procedures . Output is exported to an HTML
and PDF, which offers convenient views on the results in both tabular
and graphical formats. RMetaWeb is intended to serve to the scientific
community and it is hoped that it will become a useful tool on conducting
reliable meta-analysis in biomedical research . The RMetaWeb is
freely available from http://genepark .mf .uni-lj .si .
P08.48
Optimizing bisulfite DNA conversion method for methylated CpG
island discovery and screening
E. Currie-Fraser, B. Finkelnburg, S. Jankowski, L. Xu;
Applied Biosystems, Foster City, CA, United States.
DNA methylation at the 5’ position of cytosine in promoter CpG islands
plays a critical role in regulating gene expression . Typically methylation
is inversely correlated with the transcription status of the gene .
Bisulfite DNA conversion is one of the most used techniques for methylation
studies because of its relative simplicity, whereas other methods
are frequently cumbersome and require significant optimization.
The bisulfite conversion method allows precise analysis of methylation
in a target region by converting all nonmethylated cytosines into
uracils, while methylated cytosines remain unchanged. The workflow
described here provides an effective solution for methylation analysis
with straightforward protocols. We recognize that bisulfite conversion
rate and primer design are the most critical steps in this approach . In
this presentation, we demonstrate that a promoter region’s methylation
status can be identified with confidence using an optimized approach,
which requires small amounts of genomic DNA and generates high
quality data. This workflow, therefore, is very useful for the analysis of