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

Concurrent Sessions
C57. RNAi-based allele specific silencing of the ataxin-7 gene in
South African patients with SCA7
J. Scholefield 1 , M. Weinberg 2 , P. Arbuthnot 2 , M. Wood 3 , J. Greenberg 1 ;
1 Institute of Infectious Disease and Molecular Medicine, UCT, Cape Town,
South Africa, 2 Department of Molecular Medicine and Haematology, Wits, Johannesburg,
South Africa, 3 Department of Physiology, Anatomy and Genetics,
Oxford, Oxford, United Kingdom.
RNA interference (RNAi) is a mechanism that occurs naturally in eukaryotes
and mediates post-transcriptional gene silencing and has
been shown to have application to treatment of many debilitating genetic
disorders. One such disease is the polyglutamine disorder, spinocerebellar
ataxia 7 (SCA7). SCA7 has a higher frequency in South
Africa and has only been diagnosed here in black African patients to
date. A recent study demonstrated that an exonic single nucleotide
polymorphism (SNP) is tightly linked to the disease-causing (CAG) n
expansion within ataxin 7. The aim of this project is to exploit the RNAi
pathway to achieve specific knockdown of the mutant mRNA transcript
of ataxin 7. A panel of 14 different Pol III U6 promoter-encoded short
hairpin RNAs (shRNAs) that target the ataxin 7 SNP were designed.
Knockdown effects against mutant and wild-type targets linked to a
luciferase reporter gene were measured in triplicate. Knockdown of the
targets was measured in triplicate. Luciferase emissions were reduced
in cell cultures containing the mutant target compared with those of
the wild-type, although the differences were for the most part, small.
However, one shRNA showed nearly 30% discrimination between the
wild-type and the mutant target cell cultures. Unexpectedly, shRNAs
with secondary mismatches abrogated discrimination and knockdown
with one exception. These preliminary results indicate that discriminatory
knockdown of ataxin 7 can be achieved using a weak mismatch.
Further shRNAs have been designed to maximise this initial discrimination.
This investigation shows promise for development of a gene
therapy-based approach to treating a disease of South African importance
as well as other similar conditions.
C58. Nonsense-mediated mRNA decay regulates response of
cystic fibrosis patients to gentamicin
L. Linde 1 , S. Boelz 2,3 , M. Nissim-Rafinia 1 , Y. S. Oren 1 , M. Wilschanski 4 , Y. Yaakov
4 , G. Neu-Yilik 2,3 , A. E. Kulozik 2,3 , E. Kerem 4 , B. Kerem 1 ;
1 Department of Genetics, Life Sciences Institute, The Hebrew University, Jerusalem,
Israel, 2 Molecular Medicine Partnership Unit, University of Heidelberg
and European Molecular Biology Laboratory, Heidelberg, Germany, 3 Department
for Pediatric Oncology, Hematology and Immunology, University Hospital
Heidelberg, Heidelberg, Germany, 4 CF Center, Hadassah University Hospital,
Mount Scopus, Jerusalem, Israel.
Aminoglycosides can readthrough premature termination codons
(PTCs), permitting translation of full-length proteins. Previously we
have found variable efficiency of readthrough in response to the aminoglycoside
gentamicin among cystic fibrosis (CF) patients all carrying
the W1282X nonsense mutation. Here we demonstrate that there are
patients in whom the level of CFTR nonsense transcripts is markedly
reduced while in others it is significantly higher. Response to gentamicin
was found only in patients with the higher level. We further investigated
the possibility that the nonsense-mediated mRNA decay (NMD)
might vary among cells and hence governs the level of nonsense transcripts
available for readthrough. Our results demonstrate differences
in NMD efficiency of CFTR transcripts carrying the W1282X mutation
among different epithelial cell lines, even derived from the same tissue.
Variability was also found for β-globin transcripts carrying a diseasecausing
PTC as well as for five physiologic NMD substrates, RPL3,
SC35 1.6 kb, SC35 1.7 kb, ASNS and CARS. Importantly, our results
demonstrate existence of cells in which NMD of all transcripts was efficient,
while others in which the NMD was less efficient. Downregulation
of NMD in cells carrying the W1282X mutation increased the level
of CFTR nonsense transcripts and enhanced the CFTR chloride-channel
activity in response to gentamicin. Together our results show that
the efficiency of NMD might vary and hence regulate the response to
treatments aiming to promote readthrough of PTCs in many human
genetic diseases.
C59. Harmless selection of genetically manipulated human stem
keratinocytes
T. Magnaldo, V. Bergoglio, E. Warrick, F. Larcher, O. Chevallier-Lagente, M.
Del Rio;
CNRS, Villejuif, France.
Ex-vivo gene therapy of monogenic and recessively inherited genodermatoses
prone to cancer require the selection of transduced epidermal
keratinocytes in a manner compatible with skin graft perspectives. We
have set up a selection system which aims at : i-preserving growth
and differentiation potentials of transduced keratinocytes, ii-reduce the
risk of immune response in grafted patients, iii-maintain sustained expression
of the corrective gene. In this system, selection is based on
ectopic expression of the small cell surface marker CD24 in proliferative
keratinocytes. In human epidermis, CD24 is normally expressed
in post mitotic, differentiated keratinocytes. Several primary strains of
normal keratinocytes could be successfully transduced using a CD24-
IRES-GFP MoMLV retroviral viral vector. CD24-selected cells could
be passaged serially over more than one year, attesting the conservation
of stem cell growth potential. Reconstruction of organotypic skin
cultures using transduced cells, indicated normal differentiation and
proliferation capacity. Transduced cells were grafted onto the nu/nu
athymic mouse and regenerated a full thickness, normally differentiated
epidermis, over a period of 20 weeks. Expression of the GFP
reporter gene was maintained without attenuation. The encouraging
results strongly stimulate our prospects of genetic correction of
epidermal keratinocytes from patients suffering from the DNA repair
deficient / cancer prone disease, xeroderma pigmentosum or for any
other genodermatose candidate for ex vivo cutaneous gene therapy.
In addition, our system now allows any application of long term and
harmless gene transfer such as gene extinction or mutation expression
in human primary cells.
C60. Development of liver disease despite mannose treatment in
two patients with CDG-Ib
K. Mention, F. Lacaille, V. Valayannopoulos, S. Romano, F. Jaubert, Y. De Keyser,
N. Seta, P. De Lonlay;
Necker Enfants Malades, Paris, France.
Congenital disorders of glycosylation (CDG) result from a defect in
N-glycosylation. Phosphomannose isomerase (PMI) deficiency is the
only treatable CDG (CDG-Ib). The first clinical description of CDG-Ib
was made by Pelletier et al. in 1986, as a lethal disease in 4 patients
from the “Saguenay-Lac St-Jean” area in Quebec. Since this description,
mannose therapy improved the general condition and the digestive
symptoms in all reported patients as well as in ours.
We report here the 6 year follow-up of two patients with CDG-Ib treated
with oral mannose, who were diagnosed at 2 months of age with
digestive symptoms, liver involvement and hyperinsulinemic hypoglycemia.
Both developed portal hypertension while general condition
improved and other symptoms disappeared. #The development of the
characteristic histological lesions of congenital hepatic fibrosis was
observed despite an early onset of treatment and several years on
mannose. In both patients, improvement of the transferrin profile was
noted, although complete normalization was never observed. We can
speculate that persistently abnormally glycosylated proteins interfere
with the normal development of the liver, either by accumulation of an
abnormal product, or more probably by a loss of function.
In conclusion, the efficacy of mannose might transform this lethal disease
into a treatable one. However, in some patients this treatment
does not seem to protect against the liver disease.
C61. Acetylcholinesterase deficiency with neuromuscular
junction remodeling in a mouse model of Schwartz-Jampel
syndrome
M. Stum 1 , E. Girard 2 , V. Bernard 3 , M. Bangratz 1 , C. Davoine 1 , N. Tabti 1 , J.
Willer 4 , B. Fontaine 1,5 , J. Molgó 6 , E. Krejci 3 , S. Nicole 1,5 ;
1 U546, INSERM; UMRS546 UPMC-Paris6, Paris, France, 2 UPR9040, CNRS,
laboratoire de Neurobiologie Cellulaire et Moléculaire, Institut de Neurobiologie
Alfred Fessard, Gif-sur-Yvette, France, 3 U686, INSERM, Paris, France, 4 Assitance
Publique-Hôpitaux de Paris, Fédération de neurophysiologie clinique,
Groupe Hospitalier de la Pitié-Salpêtrière, Paris, France, 5 Assistance Publique-
Hôpitaux de Paris, Fédération des maladies du système nerveux & centre de
référence “canalopathies musculaires”, Groupe Hospitalier de la Pitié-Salpêtrière,
Paris, France, 6 UPR9040, CNRS, laboratoire de Neurobiologie Cellulaire
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