2008 Barcelona - European Society of Human Genetics

Therapy for genetic disease
Sciences, Tehran, Islamic Republic of Iran.
The K562 cell line is an erythroleukemia cell line that is widely used for
studies of globin gene regulation . In this study, we have tested several
transient transfection methods in K562 cells. The PSV-β-Galactosidase
vector was used as a control to monitor transfection efficiency. We
tested commercially available reagents such as Lipofectamin TM 2000
(Invitrogen), FuGENE ® HD transfection Reagent (Roche), X-treme-
GENE siRNA transfection Reagent (Roche), HiPerfect Transfection
Reagent (Qiagen) and DEAE-Dextran (Sigma) . Also K562 cells were
electroporated under different conditions . Our Result demonstrated
that Lipofectamin TM 2000 provide a reliable, effective and reproducible
method for transfecting K562 cells . We have also used Lipofectamin TM
2000 to successfully transfect positive and negative control siRNAs
into K562 cells . We are now in the process of using this system to
study the effect of siRNAs against potential γ-globin repressors (in the
hope of inducing the γ-globin gene).
P10.18
Loeys-Dietz syndrome: In- vitro restoration of fibrillin and elastin
production after treatment with losartan and dexamethasone
C. P. Barnett, A. Hinek, T. J. Bradley, D. Chitayat;
Hospital for Sick Children, Toronto, ON, Canada.
A 2-year-old boy born to non-consanguineous parents was seen by us
following his fourth inguinal hernia repair . He was noted to have facial
dysmorphism including brachycephaly, high forehead, hypoplastic supraorbital
ridges and malar areas, proptosis with ptosis, posteriorly rotated
ears, high arched palate with a normal uvula, arachnodactyly and
camptodactyly . Echocardiography demonstrated a dilated aortic root
and tortuous aortic arch and branches . X-rays revealed craniocervical
instability, craniosynostosis and generalized osteopenia . Loeys-Dietz
syndrome (LDS) was suspected clinically and DNA analysis of the
TGFBR1 gene revealed a missense mutation in codon 4 (c .722C>T)
leading to substitution p .S241L . No mutations in the TGFBR2 or FBN1
genes were detected .
In vitro studies of skin fibroblasts derived from this patient indicated
that they were significantly deficient in elastin and fibrillin gene expression
(RT-PCR technique) . Consistently, immunohistochemistry confirmed
a lack of adequate production of elastin and fibrillin. Production
of fibulins 1, 2 and 5, auxiliary components of elastic fibers shown to
be important in normal elastogenesis, was not affected . Treatment of
the cultured fibroblasts with losartan (angiotensin II receptor blocker)
or dexamethasone restored normal production of elastic and fibrillin
fibers. In vitro treatment of fibroblasts derived from other LDS patients
is underway to assess the reproducibility of these findings and may
provide more information on the potential for future clinical use .
Our findings further highlight the overall role of imbalanced signal
transduction through TGF receptors that lead to impaired elastogenesis
in LDS and similar diseases .
P10.19
towards a pharmacological therapy for mandibuloacral
Dysplasia syndrome
A. Vielle-Canonge 1 , F. Gullotta 2 , S. Salvatori 2 , P. Molinaro 2 , F. Lombardi 1 , S. Ciacci
1 , M. D’Adamo 3 , P. Sbraccia 3 , A. M. Nardone 2 , M. R. D’Apice 1 , G. Lattanzi 4 ,
N. M. Maraldi 4 , G. Novelli 1,5 ;
1 Departments of Biopathology and Diagnostic Imaging, Rome, Italy, 2 Departments
of medical genetics, A.O.U. Policlinico Tor Vergata, Rome, Italy, 3 Department
of Internal Medicine, University of Rome Tor Vergata, Rome, Italy, 4 IGM-
CNR, Unit of Bologna, c/o Istituti Ortopedici Rizzoli, Bologna, Italy, 5 University
of Arkansas for Medical Sciences, Little Rock, AR, United States.
Recently, different groups have demonstrated that the farnesyl transerase
inhibitors (FTIs) were able to reverse in-vitro and in-vivo some
phenotypic manifestations of progeroid syndromes secondary to LMNA
and/or ZMPSTE24 mutations (Hutchinson-Gilford progeria syndrome
and restrictive dermopathy) . The rationale of this treatment is based
on blockage of the toxic effect of the farnesylated forms of prelamin
A which in turn is responsible of cellular morphology alterations and
genomic instability . In order to verify if this treatment is reproducible
also in the mandibuloacral dysplasia (MADA), we studied the cellular
effects of FTIs treatment on primary fibroblasts cell lines after 72 hrs
at different concentrations (10 - 500 nM) of the drug . We observed that
this treatment induces in MADA’s cells, an increase of abnormal nuclei
in a dose-manner dependent effect . On the basis of these results, we
decided to test the effect of two different drugs (bisphosphonates and
statins) known to act on the same biochemical pathway at different
levels. We treated MAD fibroblasts in a two steps model (24 hrs statins
treatment and then 12hrs bisphosphonate in a single dose) . This treatment,
showed an improvement of the cellular phenotype (reduction
of the number of misshapen nuclei and a partial rescue of the heterochromatin
organization) . Singularly, these drugs were ineffective . All
together these results, suggest that FTI treatment is ineffectiveness
versus MADA patients, while inhibitors of prenylation pathways could
be considered as potential .
This work was supported by the AIFA (Italy) and EURO-Laminopathies
(Contract LSHM-CT-2005-018690) .
P10.20
A tendency in the treatment of mitochondrial complex i
deficiency
B. Radeva1 , M. Stancheva1 , E. Naumova2 ;
1 2 University Children Hospital, Sofia, Bulgaria, Central laboratory of Clinical
Immunology, UMBAL”Alexandrovska”’, Sofia, Bulgaria.
The treatment of the mitochodrial disorders is difficult and not well
known .With an integral clinical-laboratory methode were diagnosed 5
children with mitochondrial complex I deficiency: 2 patients with mutation
of ND5 gene and 3 - with ND3 , ND2, ND1 .Their mutations were
confirmed by PCR SBT methode of mitochodrial regions in peripheral
intravenous blood .The clinical symptoms were various .The child with
ND5 mutation- T 12880 C (with aminoacid change phenylalanine with
leucine) had hemiparesis, weakness , muscle hypotonia, decreased
sensitivity, the other boy (synonim basal changes), T 11311 C and polymorphisms-
convulsions, ophthalmoplegia, ataxia ,decreased hearing
and vision, weakness and fatiguity . The patient with ND3 mutation had
the same symptoms, but more severe: atrophy of n . opticus , deafness
due to neuritis nervi acustici and severe ataxia . The child with ND2 mutation
C 5472 G and multiple polymorphysms had muscle hypotonia,
hyporeflexia, myoclonic convulsions.After treatment with high doses
vitamins B1, B2, B6, B12, Q10, L-carnitine the general condition and
neurologic status improved . The children with ND5 mutation recovered
at all , as the patients with ND 3 and other mutations who showed an
improval too . The treatment of the children proceeds .
P10.21
indicating FtA Elute: for the collection, processing, and elution
of DNA from biologically clear samples for use in downstream
amplification technologies
J. Dinan 1 , S. Judice 2 , N. Nelson 3 , L. Battalagine 2 , M. Green 1 , B. Moran 4 , M.
Harvey 3 , B. Parker 3 ;
1 Whatman International, Maidstone, Kent, United Kingdom, 2 Whatman Inc,
sanford, ME, United States, 3 Whatman Inc, Sanford, ME, United States, 4 Whatman
Inc, New Jersey, NJ, United States.
Collecting samples using an oral swab and Indicating FTA Elute is a
non-invasive procedure that can be carried out easily and safely by the
layperson, thus providing an ideal format for collecting human genetic
material from virtually anywhere in the world .
To visualize sample collection and placement, Indicating FTA Elute has
been developed for use with clear biological samples by the incorporation
of a colour indicating dye that distinguishes the clear sample once
it is applied to the matrix . It is functionally equivalent to traditional FTA
Elute in that it protects DNA samples from degradation and provides a
source of amplifiable DNA which is eluted from its matrix with a simple
heat and water elution step . We ran a number of tests on various biological
samples to show that DNA eluted from Indicating FTA Elute is
functionally identical to that eluted from traditional FTA Elute . The aim
was to demonstrate that Indicating FTA Elute provides a template for
amplification dependent assays such as STR and allelic discrimination
analysis but greatly simplified and optimized sample extraction from
the matrix .
In summary, Indicating FTA Elute in combination with an oral swab
represents a revolutionary, non-invasive method for simplified genetic
sample collection, sample transport in the mail, simplified DNA extraction
requiring nothing more than water and heat, plus the added benefit
of storing the samples at room temperature for literally years if this is
a requirement . Indicating FTA Elute is therefore perfectly positioned to
support the emerging discipline of pharmacogenomics or personalized
medicine .