European Human Genetics Conference 2007 June 16 – 19, 2007 ...

Genomics, technology, bioinformatics
Leiden, The Netherlands, 5 Erasmus MC, Rotterdam, The Netherlands.
The panoply of human globin gene mutation detection methods has become
significantly enriched with the advent of microarray-based genotyping
platforms. So far, a handful of microarray-based methods have
been described for mutation screening in the human globin genes region,
namely a microelectronic array, arrayed primer extension-based
(APEX) systems and microarrays involving tagged single-base extension
(SBE) with hybridization to universal glass microarrays. We report
here a new flow-through microarray for human β-globin gene mutation
detection. This microarray has a porous surface and uses Al 2 O 3 as
a solid support, which allows not only diffusion-independent binding
kinetics, but also monitoring of hybridization of the extended primers
in real-time by repeated cycling of the sample through the array. The
hybridization process is therefore fully reaction-rate limited and completed
within a few minutes. The microarray is read by a dedicated
instrument (PamStation TM ), while data are analyzed using specialized
software. The microarray is being developed to screen for both causative
β-globin gene mutations leading to β-thalassemia and associated
SNPs for haplotype analysis. For this reason, 20 different β-globin
gene mutations and 15 SNPs located in the human β-globin locus can
be currently addressed using this platform (Phase I) with the ultimate
goal to expand the microarray’s capacity to screen for a total of 108
causative mutations and 30 SNPs (Phase II). Taken together the low
analysis costs (i.e. 50-70€ per patient), this platform might develop into
an option for numerous potential applications from human molecular
diagnostics to pre-implantation genetic diagnosis and non-invasive
prenatal diagnosis of hemoglobinopathies.
P1274. Microarray analysis of bone metabolism genes in
patients with osteoporosis
D. N. Ghorab 1 , M. V. Moskalenko 1 , A. S. Glotov 2 , E. S. Vashukova 2 , L. B. Polushkina
2 , T. E. Ivaschenko 2 , M. V. Assev 2 , V. G. Pinelis 3 , V. S. Baranov 2 ;
1 Saint-Petersburg State University, Saint-Petersburg, Russian Federation, 2 Ott’s
Institute of Obstetrics & Gynecology RAMS, Saint-Petersburg, Russian Federation,
3 Scientific Center of Children Health RAMS, Moscow, Russian Federation.
Osteoporosis is a complex desease with a strong genetic component.
Various genes encoding bone associated proteins, cytokines and receptors,
have been shown to contribute to genetic basis of the disease.
For analysis following loci were selected: Col1a1 (-1997 G/T, 1245 G/
T), BGP (-198 C/T), IL-6 (-174 C/T), TNFα (-308 G/A), VDR (-3731
A/G, 61968 C/T), ER (36627 C/T, 36673 A/G) and GR (N363S A/G).
These loci feature significant genetic polymorphism, and are thought
to be associated with genetic predisposition to osteoporosis. A genetic
study of predisposition to the osteoporosis is believed to extend our
knowledge about the disease and to determine the optimal terms for
antiosteoporotic therapies.
We have started to introduce in practice a new molecular approach,
developed with using of multiplex PCR followed by allele-specific hybridization
on biochip for SNPs detection. Amplified DNA with fluorescent
labels was hybridized with oligonucleotide DNA probes immobilized
in gel pads on a biochip. The efficiency of the protocol was tested
in our laboratory. The results showed 100% concordance between the
biochip-based approach and the established PCR protocol.
The genotyping procedure, which is faster, reliable and can be used for
rapid screening on the biochip is suggested for the analysis of genetic
predisposition to multifactorial diseases, including osteoporosis.
P1275. A new method for separation and characterization of
small RNA by On-Chip Electrophoresis
R. Salowsky1 , C. Tissot1 , M. Valer2 , Y. Filaudeau3 ;
1 2 Agilent Technologies, Waldbronn, Germany, Agilent Technologies, Inc., Santa
Clara, CA, United States, 3Agilent Technologies, Nice, France.
MicroRNAs have just been recognized to play important roles in regulation
in the genomes of animals and plants. To understand and describe
their functional roles many questions still have to be answered.
A major drawback for current experiments is the lack of adequate analytical
methods for the analysis of small RNA samples and understanding
on how RNA integrity and different purification protocols affect its
qualitative and quantitative analysis.
Here we describe a novel Microfluidic assay that is able to perform
very sensitive high resolution analyses of small RNA samples on a
commercial lab-on-a-chip platform commonly used for RNA QC analysis.
The assay delivers information about size and concentration of
small RNA species like miRNA, siRNA, t-RNA etc, in the range from
10 to 150nt. Purified or enriched small RNA fractions, as well as total
RNA samples with miRNA concentrations down to 50 pg/µl can be
analyzed.
Verification of quality and quantity of miRNA after extraction protocols
is a major application for this Assay. Capability for separation and efficient
detection of both single- and double strand nucleic acids widens
the range for sensitive analysis with this On-Chip assay.
P1276. Functional assessment of DNA loop repair in human
nuclear extracts
S. Jaroudi, J. C. Harper, S. B. SenGupta;
University College London, London, United Kingdom.
Introduction: Mismatch repair (MMR) corrects small base mismatches
and insertion/deletion loops that arise within microsatellite repeats.
Current repair assays predominantly rely on cloning techniques. We
have designed an alternative functional assay for MMR.
Methods: Heteroduplex DNA molecules containing an insertion loop
were created by hybridisation of purified complementary single strands
of DNA from PCR products of the DMPK1 trinucleotide repeat locus
from separate homozygous samples. A nick was introduced 5’ to the
heterology to direct the repair to a specific strand. Heteroduplex constructs
were exposed to nuclear extracts from HeLa (MMR efficient)
and LoVo (MSH2 deficient) cells to repair the insertion loop. Using
semi-quantitative fluorescence analysis, the ratio of fluorescence from
the nicked strand and the complementary un-nicked strand could be
determined (R-value). Repair efficiency was assessed by comparing
R-values before and after exposure to nuclear extracts.
Results: Exposure of heteroduplexes to HeLa nuclear extracts resulted
in change in the R-value, indicative of repair. Repair was independent
of loop size (2, 21 and 24-bases) and nick directed. Little or no
repair was detected for 2-base loops in absence of a 5’-nick, however,
for larger loops repair was nick independent. Change in the R-value
was proportional to the duration of exposure to nuclear extracts. LoVo
nuclear extracts showed little repair for large loops and no repair was
detected for the 2-base loops.
Conclusion: This construct is easily generated and modified to vary the
size of the insertion loop. The same approach can be used to assess
repair of base-base mismatches.
P1277. The mitochondrial DNA content per cell in human cord
blood leukocytes gradually decreases during gestation
M. Pejznochova, M. Tesarova, T. Honzik, H. Hansikova, M. Magner, J. Zeman;
Dept. of Pediatrics, Faculty of Medicine, Charles University, Prague, Czech
Republic.
Most diseases in premature neonates are secondary to immaturity of
various organ systems. In addition, the inadequate capacity of mitochondrial
energy production may play an important role in the neonatal
morbidity. Isolated human cord blood leukocytes (HCBL) contribute
very little to the overall metabolic turnover, but they may serve as easily
available marker cells for studying the changes of mtDNA amount
during fetal development. Therefore the aim of our study was to analyze
the amount of mitochondrial DNA in HCBL during the gestation.
HCBL were isolated from blood samples of 107 neonates born between
25 th and 41 st week of gestation. Blood samples were obtained
after the delivery from placental part of umbilical cord. The mtDNA
amount was analyzed by the real-time PCR method on the instrument
Chromo4 (Bio-Rad) using SybrGreen I.
The significant negative correlations were found between the relative
mtDNA amount in HCBL and gestational age (r = - 0,54; p < 0,01) and
birth weight (r = - 0,43; p < 0,01), respectively.
The results revealed that the mtDNA content per cell decreases in
HCBL with onward fetal development. This may be explained by gradual
shift of the hematopoiesis from fetal liver to bone marrow during the
second half of pregnancy presumably accompanied by decreasing cell
volume of HCBL as it was shown similarly in red blood cells.
This work was supported by grant GACR 303/03/H065.
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