2009 Vienna - European Society of Human Genetics

Molecular basis of Mendelian disorders
known. While hemizygosity of the elastin gene has been associated
with supravalvular aortic stenosis, studies of patients with atypical deletions
and mouse models have suggested that LIMK1, CYLN2 and
GTF2IRD1 might play a role in some aspects of the phenotype.
To identify pathways and processes perturbed in WBS, we used
microarrays to profile the transcriptomes of skin fibroblast cell lines
from eight young WBS and nine age-matched control girls. Using an
iterative signature algorithm on our dataset combined with other skin
fibroblast transcriptomes publicly available, we identified modules of
coherently regulated transcripts. Among our findings, two GABA-receptor
genes (GABBR1 and GABRE), one glutamate receptor subunit
(GRIA3) as well as a few other genes possibly related to neurological
processes (SLIT3, GOLSYN and NAV1) were downregulated in WBS
patients. Other modules were significantly enriched in cell adhesion
and proliferation factors.
These dysregulations, combined with that of at least 10 extracellular
matrix proteins, constitute a potential cause for cognitive deficits and
other neurological features, as they may influence CNS development
by affecting cell and axonal migration. They could also modulate the
function of neuronal connections by accelerating or impairing neurotransmitter
release and recycling at the synaptic level.
P11.125
A comprehensive characterisation of human cD46, cD55 and
CD59 transgenic swine fibroblasts - a potential source of nuclei
for somatic cloning.
J. E. Zeyland 1 , R. Slomski 1,2 , A. Wozniak 2 , A. Nowak 1 , D. Lipinski 1,2 ;
1 Department of Biochemistry and Biotechnology, University of Life Sciences,
Poznan, Poland, 2 Institute of Human Genetics, Polish Academy of Sciences,
Poznan, Poland.
Transgenic swines, especially those expressing, not a single one, but
a combination of the complement system regulators are essential to
help overcoming a hyperacute rejection (HAR) and necessary to estimate
a potential ratio of a strategy collapse.
Single and triple transgenic swine foetal fibroblasts for human coding
sequences of CD46, CD55 and CD59 using a promoter of a human
elongation factor 1 alpha gene were generated by lipofection method
with 80% capacity. After blasticidine selection stable lines were molecularly
characterised and checked for transgene integration by PCR.
Forward primers were located in the EF-1α promoter region and reverse
primers in the region coding CD46, CD55 or CD59 respectively.
Lines with confirmed transgene integration were subjected for characterisation
of expression by RT-PCR. The transgene expression and
its impact on human complement system was assessed by human
complement-mediated cytolysis assay. Human serum (HS) contains
complement system components which are the main reason of HAR.
Each transgene expressed in single transgenic line had a protective
effect on the tested cells in HS cytotoxicity assay. Also in triple transgenic
lines the expression of the transgenes had a wide positive impact
on the protection of cells from human complement-mediated lysis,
however it was not additive. Cytogenetic analysis was performed to
evaluate the chromosomal stability in the transgenic cells. Several cytogenetic
staining procedures revealed, inter alia, anomalous number
of the chromosomes, structural aberration and dicentromeric chromosomes.
Only fully characterised cell lines can be used as nuclei donors
for a somatic cloning and producing healthy transgenic animals.
P11.126
sOLiDtm Sequencing of Whole Genome Bisulfite Converted
Libraries Prepared from Nanogram Quantities
C. Lee1 , T. Halama2 , S. Ranade2 , V. Boyd2 , B. Coleman1 , K. Pearlstein1 , K.
Pearlstein1 , Y. Sun2 , Z. Zhang2 , H. Peckham1 , G. Costa1 , M. Rhodes2 , K. McKernan1
, F. Raffaldi3 ;
1 2 Applied Biosystems, Beverly, MA, United States, Applied Biosystems, Foster
City, CA, United States, 3Applied Biosystems, Italy.
DNA methylation is the most characterized epigenetic mechanism,
playing an essential role in normal mammalian development and is
associated with gene expression and
carcinogenesis. In animals, DNA methylation normally involves the
modification of cytosine residues at the 5-carbon position. One popular
method to study DNA methylation is to treat the sample with bisulfite.
Bisulfite converts cytosine to uracil residues, but methylated cytosines
are not affected. Hence, the DNA is subjected to a very specific modifi-
cation dependent on methylation and sequencing of bisulfite converted
DNA can yield detailed information about modified segments within a
genome. Here we present a novel bisulfite conversion technique that
requires only nanogram quantities of genomic DNA. The methodology
was applied to two different genomes: Dh10b and Yoruba. Fragment
libraries were constructed with methyl C protected adaptors and libraries
were sized on Polyacrylamide gel. The DNA was bisulfite converted
while still embedded in the gel piece and the library was amplified
using an in-gel PCR method. These libraries were then sequenced on
the SOLiD TM System, generating 50 bp reads. The high throughput of
the SOLiD TM System and the unique advantages of two base encoding
allow the researcher to study the methylation of an entire complex genome
in unprecedented detail. The results from SOLiD TM sequencing
of the converted genomes and the analysis will also be discussed.
P12. molecular basis of mendelian disorders
P12.001
the novel iVsii-i (G>A) splice donor site mutation on the alpha 1
globin gene
M. Taghavi*, F. Bayat, A. Amirian, A. Valaei, N. Saeidi, Z. Kaini Moghaddam, A.
Kordafshari, S. Fathiazar, M. Mossayebzadeh, M. Karimipour, S. Zeinali**;
Pasteur Inistitute of Iran, Tehran, Islamic Republic of Iran.
More than 30 different point mutations and small deletions and insertions
have been reported in alpha globin gene. Point mutations are
less common, but they may occur at high frequencies in certain areas
under selective pressure by malaria. Here we report the combination
of IVSII-I (G>A) mutation at α1 -globin gene an β:HbD(CD121).
After obtaining informed consent, blood samples (10mL) were collected
in tubes containing EDTA and extracted by salting out method.
Multiplex Gap PCR and direct α-globin gene sequencing techniques
were used to analyze alpha globin gene mutations. Exon 3 of β-globin
gene was amplified for determination of HbD variant and digested by
EcoRI.
Here we report a novel mutations causing α-thalassemia that has
not been reported previously. The IVSII-I(G>A) mutation in α1-globin
gene detected in the family with thalassemia phenotype.This mutation
disrupts donor splice site of second intron of α1-globin gene.
Direct sequencing revealed that the proband (7 years old child) was
homozygous and the parents were heterozygous carrier. The other
nucleotide change for this proband and her mother, was β:CD121
GAA>GCC (known as HbD Punjab). This mutation has not been reported
in globin gene server. Because it disrupts the splice site of
second exon, so it could be a pathologic mutation. It seems that the
homozygous form of this mutation does not make HbH disease (in our
case Heinz bodies were negative) and heterozygous from make a mild
α-thalassemia trait.
P12.002
Molecular analysis of γ-globin promoters, HS-111 and 3`HS1 in
beta thalassemia intermedia patients associated with high levels
of HbF
M. Hamid 1,2 , F. Mahjoubi 3 , A. Arab 2 , S. Zeinali 2 , M. Akbari 4 , M. Karimipoor 2 ;
1 Clinical Genetics Department, National Institute of Genetic Engineering and
Biotechnology (NIGEB), Tehran, Iran., Tehran, Islamic Republic of Iran, 2 Department
of Molecular Medicine, Biotechnology Research Center,Pasteur Institute
of Iran, Tehran, Islamic Republic of Iran, 3 Clinical Genetics Department,
National Institute of Genetic Engineering and Biotechnology, Tehran, Islamic
Republic of Iran, 4 Department of Medical Genetics, School of Medical Sciences,
Tarbiat Modaras University,, Tehran, Islamic Republic of Iran.
We have studied the nucleotide variations in promoter region of
gamma globin genes, HS-111 and 3`HS1 regions in β-thalassaemia
intermedia, thalassemia major patients and normal individuals from
Iranian origin. The five nucleotide variations in the 5’ sequences of the
Aγ globin gene including -369 (C > G), -611 deletion T, -603 GA>AG
in all samples, -588(A>G) in heterozygous and homozygous form and
-AAGC at - 222 to -225 were found with different frequencies. In our
study the -369(C>G), -611(-T),
-603/604(GA>AG) mutations might as common polymorphism in our
population, didn’t show any effect on expression of HbF and not correlated
with β-globin mutations, Whereas most of the -588 (A) variation is
related to β-thalassemia intermedia pateints especially in IVSII-I/IVSII-
0