2009 Vienna - European Society of Human Genetics

Genomics, Genomic technology and Epigenetics
sequence variation relative to a control sample; however, HRM does
not impart the identity of the variation, and sequencing is required to
exactly identify the nucleotide changes. Since HRM is an end-point
PCR analysis technique, we further developed a workflow enabling
the product of HRM PCR to be used directly in a Sanger sequencing
reaction after a simple 100 to 200 fold dilution. The direct sequencing
approach conserves sample by eliminating another round of PCR to
generate product for sequencing. And the high ratio dilution eliminates
the need for a purification step after PCR. The fluorescent dye used
for HRM does not interfere with the sequencing reaction. To further
simplify the workflow, universal M13 tags were added to the 5’ end
of the gene-specific primers allowing the HRM amplicons to be easily
sequenced using a universal protocol in any high throughput sequencing
setting.
P11.061
intra-intronic human minisatellite UPs29 associated with
neurological diseases regulates reporter gene activity
depending on its copy number in cell line F9
L. Sasina 1 , I. Suchkova 1 , K. Solovyev 1 , N. Slominska 1 , V. S. Baranov 2 , E. Patkin
1 ;
1 Institute of Experimental Medicine, St.Petersburg, Russian Federation, 2 Ott’s
Institute of Obstetrics & Gynecology, St.Petersburg, Russian Federation.
Earlier we found the association of increased rate of short alleles of
minisatellite UPS29 localized in intron of CENTB5 gene with some
forms of Parkinson disease and epilepsy. The molecular mechanism
of such correlation remains mainly unclear. To elucidate this problem
we generated PCR product corresponding to “healthy” (900bp) and
to “disease” (400bp) UPS29 alleles. These PCR products were introduced
into plasmid pEGFP with ROSA-betagco26 promoter and fused
to reporter gene GFP. These plasmids were transfected into cells of
embryonal carcinoma line F9. Number of GFP-positive cells, their
morphology and fluorescence intensity were analyzed with the help of
confocal microscopy after cultivation for 24 and 48h. Plasmids without
UPS29 served as controls. There were no signal-positive cells in case
of the same but minisatellite-free plasmids, though molecular analysis
shower the presence of plasmids in F9 cells. Thus an GFP expression
lacked in control, but appeared due to presence of UPS29 in construct.
The number of GFP expressing cells depended on repeated
units number in UPS29. The most high fluorescence was observed
in neuron-like derivatives of F9 cells induced to differentiate. We suppose
that such regularity could be explained by minisatellite-specific
TF binding, and as a result UPS29 served as enhancer relatively to
ROSA26 promoter. Obtained results point to possible mechanism of
regulatory function of intra-intronic and other non-protein coding repeats
as additional enhancers. The shortening of one of minisatellite
allele will lead to gene expression decrease and further to pathology.
Acknowledgement. The work was supported by RFBR grant 08-04-
12167
P11.062
Epigenetic study on regulation of normal and mutant kit gene in
mice
G. Cardos1 , A. Arghir1 , S. M. Chirieac1 , G. P. Savi1 , M. E. Hinescu1,2 ;
1 2 ”Victor Babes” National Institute of Pathology, Bucharest, Romania, „Carol
Davila” University of Medicine and Pharmacy, Bucharest, Romania.
The Kit protein is a tyrosine-kinase receptor with a key-role in normal
development of Interstitial Cajal Cells (ICC), the kit gene having different
expression levels in various tissues and developmental stages.
We report preliminary results of an epigenetic study on the kit gene
regulation in order to find correlation (if any) between gene expression
level and methylation status of its regulatory elements in mice, in different
tissues and organs in which ICCs were identified.
Different segments of gastrointestinal tract and extra-digestive organs
were sampled from mutant B6Cg-KitW-sh /HnihrJaeBsmJ and control
B6129PF2/J mice. RNA and DNA were extracted by DNA/RNA Mini Kit
(Qiagen). Gene expression analysis was performed by Reverse Transcription-PCR
methodology. The methylation status of the kit regulatory
elements was studied by both enzymatic restrictions with methylation-sensitive
BstUI endonuclease followed by PCR and Methylation
Specific-PCR after bisulfitic treatment of DNA by EpiTect ® Bisulfit Kit
(Qiagen).
Our study revealed comparable levels of the kit gene expression in
both mouse strains, high expression level being detected in bone marrow,
spleen, liver and the lowest level in lung. Particular methylation
pattern in a regulatory region (between -990 bp and -822 bp up-stream
to the coding region) of the kit gene was identified in spleen, which
may be correlated with high gene expression level. Our results suggest
that DNA methylation may play an important role in tissue and
organ-specific regulation of the kit gene, therefore our epigenetic study
will be extended to other kit gene regulatory elements.
Financial support: PN 06.26-02.18/2008 National Research Program.
P11.063
colocalisation of predicted exonic splicing enhancers in LAMA
gene with reported sequence variants
O. Siala, F. Fakhfakh;
Laboratory of Human Molecular Genetics, SFAX, Tunisia.
Translationally silent mutations were classified as polymorphisms.
This assumption is now being challenged through the analysis of the
mRNAs produced from mutant alleles, leading to the realization that
a higher proportion of polymorphisms affect splicing. In our study, we
analysed the colocalisation of exonic SNPs in LAMA2 gene related to
the MDC1A form of congenital muscular dystrophy with exonic splicing
enhancers (ESEs). Then, we searched the effect of allelic change on
ESEs efficacy. The LAMA2 sequence was searched for ESE motifs
using the web-based tool ESEfinder. Exons were screened for sequence
motifs likely to be recognised by the SR proteins SF2/ASF,
SC35, SRp40 and SRp55. Matching sequences are scored and only
scores above thresholds are predicted to act as ESEs. Results showed
the presence of 2709 ESEs in the 65 coding exons of LAMA2 gene;
this number was reduced to 480 significant ESEs after applying the
thresholds values filter. Secondly, the analysis of published sequence
variations in LAMA2 gene showed the presence of 41 exonic SNPs, 18
of them colocalize with the identified significant ESEs, representing a
fraction of about 44%. In addition, the allelic changes in 5 synonymous
or non synonymous SNPs can abolish or create an ESE suggesting
their potentially functional role in LAMA2 gene expression. These results
indicate the utility to consider the functional role of exonic SNPs
in splicing and can also answer to many questions about the disease
susceptibility and about the phenotypic variability observed in patients
sharing with the same mutation in LAMA2 gene.
P11.064
the Utility of capillary Electrophoresis and Next Generation
Sequencing platforms for scientific discovery
B. Finkelnburg1 , F. Raffaldi2 , A. Ferlinz1 , J. Walker3 , P. Vatta3 , C. Cummings3 , A.
Pradhan3 , M. Bozzini3 , A. Shah3 , A. Tam3 ;
1 2 Applied Biosystems Deutschland GmbH, Darmstadt, Germany, Applera Italia,
Monza, Italy, 3Applied Biosystems, Foster City, CA, United States.
With its long read lengths and high accuracy, capillary electrophoresisbased
sequencing is the gold standard technology for de novo projects.
Typically in de novo projects for genetic analysis of any organism
CE is considered ideal for creating high quality scaffolds. Now with the
availability of sequencing by short-read next generation sequencing
technologies system, this process is complemented through finishing
with high coverage. Alternatively, short-read sequencing technologies
offer the throughput requirement for assaying large numbers of candidate
regions or when resequencing pooled or heterogeneous samples.
Next-generation sequencing is suited for large-scale discovery experiments,
while CE with its high accuracy and unmatched data quality
can be used to validate structural genetic variations
We looked at de novo and targeted sequencing as two applications
where each technology could be applied. In our analysis we consider
sample preparation, and reagents, as well as key criteria that
researchers consistently demand including accuracy, coverage, read
length, quality values and ease of use.
P11.065
the Gen2Phen project: collecting gene sequence variants
and their phenotypic consequences in web-based LsDBs for
mendelian disorders
I. F. A. C. Fokkema, P. E. M. Taschner, G. B. van Ommen, J. T. den Dunnen;
Center for Human and Clinical Genetics, Leiden, The Netherlands.
The EU-funded Gen2Phen project aims to provide a holistic view of
genotype-phenotype information. Gen2Phen facilitates the collection