2008 Barcelona - European Society of Human Genetics

Therapy for genetic disease
found to be strong HbF inducers . This group includes compounds like
cisplantin analoqs, Chromomycin-Mithramycin, Tallimustine and Angelicin
. Some Ribonucleotide reductase inhibitors like Hydroxyurea,
Didox and Trimidox were also found to be strong HbF inducers . Ribonucleotide
reductase is an enzyme that plays an important role to DNA
replication and repair . These properties therefore amplify our speculation
that Anthracyclines might be inducers of HbF . We examined the
following compounds: Doxorubicin, Aclarubicin, Idarubicin, Bleomycin
and Daunorubicin on γ- and β- globin gene promoter activity in a dual
luciferase assay in the GM979 cells. All of them increased the γ-globin
promoter activity over 2 fold and were then tested in erythroid liquid
cultures derived from normal donors . Doxorubicin, Idarubicin and Daunorubicin
showed increasing levels of HbF and were then tested to
thalassemic patients (preliminary results). The findings so far from all
the measurements clearly show that the family of Anthracyclines could
be a possible target for finding new HbF inducers.
P10.03
medical rehabilitation for life quality improvement in a case with
arthrogryposis
M. Cevei1 , D. Stoicanescu2 , C. Avram1 ;
1 2 Faculty of Medicine and Pharmacy, Oradea, Romania, University of Medicine
and Pharmacy, Timisoara, Romania.
Arthrogryposis is a congenital disorder characterized by multiple joint
contractures found throughout the body at birth . We present the case
of a girl of 3 .5 year-old with multiple congenital defects: arthrogryposis
involving bilateral hip, knee and ankle joints, together with sacral agenesis
with lombar dysmorphism, anorectal agenesis, hydronephrosis of
the left kidney as the result from reflux, right kidney hypoplasia, renal
fusion and heart anomalies: tetralogy of Fallot . Immediately after birth,
in several steps, colostomy, left ureterocystoneostomy and suprapubic
cystostomy were performed . Later in infancy corrective surgery for the
heart defect was required . At the age of 2 years, surgery for the equinovarus
deformities and for left genu flexum was performed. Medical
rehabilitation tries to maximize independent function . The main goals
were increasing the muscle tonus of upper-limbs, increase the rate of
motion of the joints, establishment of stability for ambulation, learning
different schemes of movement according to her needs, obtaining of a
functional independence . The patient followed a complex rehabilitation
program with hydrokinetotherapy, electrotherapy, massage, occupational
therapy, psychological counseling . The therapies were successful,
after 10 weeks an improvement of the moving capacity and of the
transfer in orthostatism with minimal external assistance, with the obvious
increasing of the patient’s satisfaction, were noticed . In order to
fulfill our objectives the child will be hospitalized every two months for
functional evaluation and for continuing the physical therapies .
P10.04
Delineation of the 5-Azacytidine pathway by use of siRNAs
J. Gharesouran1 , A. Asgharian2 , Z. Deilami Khiabani2 , A. Aghajani1 , H. Khorram
Khorshid1 , H. Najmabadi1 , M. Banan1 ;
1 2 Genetics Research Center, Tehran, Islamic Republic of Iran, Cell and molecular
biology,Islamic Azad University, Tehran, Islamic Republic of Iran.
Induction of γ-globin may become a useful means for treatment of βthalassemia
. 5-Azacytidine (5-Aza) is a chemical compound that leads
to the induction of γ-globin, at least in part through blocking the function
of methyl transferases . In addition, it is known that the Methyl Domain
Binding Protein 2 (MBD2) binds to methylated DNA and may be
involved in repressing γ-globin expression. Therefore the function of 5-
Aza may in part be mediated through blocking MBD2 binding to DNA .
Here we have looked at whether the K562 erythroleukemia cell line is
an appropriate model system for studying this pathway . We have used
non-radioactive northern blot analysis and real time PCR to show that
γ-globin can be induced in K562 cells. In addition, we have validated
the function of a published MBD2 siRNA sequence by showing that it
can knock down MBD2-specific mRNAs in HeLa cells. We are in the
process of testing these siRNAs in K562 cells for 1) MBD2 knockdown
and 2) γ-globin induction. Upon validation, K562 cells will be used further
to delineate this pathway .
P10.05
Studying the γ-globin induction pathway by hydroxyurea in K562
cell lines
Z. Deilami Khiabani 1 , J. Gharesouran 2 , A. Asgharian 1 , H. Najmabadi 2 , M.
Banan 2 ;
1 Cell and molecular biology,Islamic Azad University, Tehran, Islamic Republic of
Iran, 2 Genetics Research Center,University of Social Welfare & Rehabilitation
Sciences, Tehran, Islamic Republic of Iran.
β-thalassemia is a genetic disorder which may be ameliorated by induction
of the fetal γ-globin gene. Several chemical agents can lead
to the induction of γ-globin. One such chemical is hydroxyurea. This
chemical is the only drug approved by the Federal Drug Administration
(FDA) for treatment of β-thalassemia. Despite its use in β-thalessemia
treatment, the mode of action hydroxyurea is poorly understood . Here,
we have examined the effect of different hydroxyurea concentrations
(50, 100, 200 µM) on γ-globin induction in the K562 erythroleukemia
cell line . We have used non-radioactive Northern blot analysis and RT-
Real time PCR to measure γ-globin levels. We have determined that
there is a 3-4 fold induction of γ-globin using 100 µM and 200 µM
hydroxyurea concentrations . We have also optimized transfection conditions
of K562 cells to show the effect of positive and negative control
siRNAs . We are currently in the process of testing siRNAs against candidate
gene(s) involved in this pathway .
P10.06
congenital Disorder of Glycosylation type ia: antisense
therapeutics for an intronic variation causing exonization of an
intronic sequence
A. I. Vega 1,2 , C. Pérez-Cerdá 1,2 , G. Matthijs 3 , M. Adamowicz 4 , M. Ugarte 1,2 , B.
Pérez 1,2 ;
1 Centro de Biología Molecular Severo Ochoa. Dpto Biología Molecular. UAM,
Madrid, Spain, 2 CIBERER, Madrid, Spain, 3 Center of Human Genetics, University
of Leuven, Leuven, Belgium, 4 The Children´s Memorial Health Institute,
Warsaw, Poland.
Congenital disorders of glycosylation (CDG) are a group of genetic
diseases resulting from defects in N-glycosylation of glycoconjugates .
The most common form of the disease is the CDG-Ia caused by a deficiency
in the cytosolic enzyme phosphomannomutase (PMM). PMM
converts mannose-6-phosphate to mannose 1-phosphate and is a key
enzyme in the generation of N-linked glycans . The clinical presentation
of PMM deficiency ranges from very severe to milder phenotype. In
this work we report the functional analysis of a nucleotide change identified
in the deep intronic region of intron 7 (c.639-15479C>T) of PMM2
gene and the use of antisense morpholino oligonucleotides (AMOs)
to restore normal splicing. First, we have confirmed using splicing assay
that this change provokes the exonization of 123bp between exon
7 and 8 in patient’s cDNA. Using modified morpholinos to block access
of the spliceosoma to 3´ and/or 5´ cryptic splice site we have
demonstrated that this insertion is a disease-causing mutation . After
transfection of a fibroblast cell line from the patient, the analysis of the
effect of the AMO was done by conventional RT-PCR, determination of
PMM activity and western blot using polyclonal antibodies . The results
obtained shown that a correctly spliced mRNA was rescued and efficiently
translated in a functional protein detected by western blot . PMM
activity was rescued close to the value of control cell line . Our results
offer a novel promise mutation-specific therapeutic approach in this
genetic disease where is not possible other effective treatment .
P10.07
treating congenital defects: a purposal of algorithm for
nutritional intervention in individuals with cleft lip/palate
R. J. Nogueira1 , A. E. S. Lima1 , L. G. Almstaden-Mendes2 , V. L. Gil-da-Silva-
Lopes2 ;
1Multiprofissional Nutritional Team-Clinical Hospital/UNICAMP, Campinas, Brazil,
2Department of Medical Genetics/UNICAMP, Campinas, Brazil.
Cleft lip and palate (CL/P) is one of the most common birth congenital
defects, occurring in approximately 1/600- 1000 newborns babies
worldwide. In view of the feeding difficulties presented by children with
a cleft lip and/or palate and the importance of food in their growth and
development, they should receive early and systematic healthcare, by a
specialized team besides of regular pediatric care . Therefore, the search
for strategies of low-cost and good effectiveness and improve of health
care are recommended by the World Health Organization (WHO) .