2009 Vienna - European Society of Human Genetics

Therapy for genetic disorders
in the muscle,91% in epithelial cells and 97% in lymphocytes. Family
investigation revealed that the grandmother,mother,aunt and uncle of
the proband were all carriers with a mean mutant load of 10%,40%,5%
and 20% respectively. The heteroplasmy was constant across tissues.
These four relatives are asymptomatic and despite relatively high mutant
load (i.e.:40% in the mother),biochemical analysis revealed normal
complex I activity in the muscle biopsy performed in the mother
and her two siblings.
These results provide important insight on genotype-phenotype correlation.
Positive mutant load observed in an older(70y.) asymptomatic
individual as well as normal OXPHOS assays in the other unaffected
adults demonstrate that high mutant loads are probably necessary for
symptoms to develop. These findings as well as heteroplasmy consistency
through tissue type could represent valuable information for family
members seeking accurate genetic counseling or prenatal testing.
P13.49
spectrum of Wilson Disease Gene (AtP7B) and AtOX1
mutations in an isolated Romanian Population
F. Raicu1,2 , A. Şendroiu2 , C. Glavce2 , R. Cocoş1 , L. Bohîlţea1 , I. Şendroiu2 ;
1Carol Davila University of Medicine and Pharmacy, Bucharest, Romania,
2Francisc I Rainer Institute of Anthropology Romanian Academy, Bucharest,
Romania.
Wilson disease is an autosomal recessive disorder characterized by
dramatic build-up of intracellular hepatic and brain copper. WD is
caused by mutations in the gene ATP7B. We report the further results
of an ongoing project concerning the spectrum of mutations on the
ATP7B gene patients from an isolated Romanian population with high
prevalence of WD. Direct sequencing of all 21 exons within ATP7B
gene revealed that four WD patients are heterozygotes or compound
heterozygotes bearing three previously reported mutations: P767Pfs,
H1069G and K832R (considered by some researches a polymorphism).
We found that one WD patient has only K832R polymorphism
in homozygosis. His parents are homozygote for K832R and heterozygote
for P767P-fs. The boy has all WD manifestation associated with
a mild disruption of copper metabolism based only on the presence of
hepatic disturbance and we supposed the presence of two diseasecausing
mutations in his DNA. Proteins interacting with the ATP7B
copper transporter such as ATOX1 are important in explaining this
phenomenon. Human ATOX1 protein regulate the catalytic activity of
ATP7B protein by binding and transporting cytosolic copper to ATPase
proteins in the trans-Golgi network for later incorporation to the ceruloplasmin.
Mutation analysis of the four exons of the ATOX1 gene was
performed in all five WD patients diagnosed by DNA analysis.Direct
sequencing of the ATOX1 gene within the 5’-UTR region revealed one
known heterozygous polymorphism (T/C at 5’UTR -99) in one Wilson
patients. The genetic base of Wilson Disease in our K832R WD patient
remains undentified.
P14.01
Induction of γ-globin by knockdown of MBD and C C in K562
cells by siRNA
J. Gharesouran 1,2 , Z. Deilami 3 , A. Asgharian 4 , M. Banan 2 ;
1 Department of Medical Genetics, Faculty of Medicine, University of Medical
Sciences, Tabriz, Islamic Republic of Iran, 2 Genetics Research Center, University
of Social Welfare & Rehabilitation Sciences, Tehran, Islamic Republic of
Iran, 3 Department of Microbiology, Islamic Azad University, Science and Research
Unit, Zanjan, Islamic Republic of Iran, 4 Department of cell and molecular
biology, Islamic Azad University, Science and Research Unit, Tehran, Islamic
Republic of Iran.
At present a number of chemicals that induce expression of the fetal
γ-globin gene are used as treatment for β-thalassemia. One of these
chemicals is a DNA methyl transferase inhibitor called 5-Azacytadine
(5-Aza). A potential downstream effector (γ-globin repressor) of 5-
Aza is a protein called Methyl Binding Domain protein 2 (MBD2). We
sought to determine whether knockdown of MBD2 by siRNAs in human
erythroid cells would result in induction of γ-globin mRNA. This
information could used to develop more targeted β-thalassemia drugs.
To this end, an MBD2-specific validated siRNA was transfected into
K562 erythroleukemia cells and the levels of MBD2 and γ-globin compared
to cells transfected with a scrambled siRNA control by using
qRT- PCR. MBD2 levels were knocked down to 0.65±0.23 and γ-globin
levels were increased to 1.70±0.46. In contrast, the largest γ-globin
mRNA increase obtained after exposure of K562 cells to 5-Azacytadine
was 7.36 fold--seen after a 3 day exposure to 15 μM of 5-Aza.
These results suggest that MBD2 siRNAs can indeed induce γ-globin
at significant levels. The DNA binding site of MBD2 is not clear. We
sought to determine whether MBD2 was mediating its repressive activity
via LARC. To this end siRNAs against C1C2 shRNP, the DNA binding
protein of LARC, were transfected into K562 cells and C1C2 and
γ-globin levels measured as discussed above. C1C2 levels were decreased
to 0.54±0.16, whereas levels of γ-globin remained unchanged
(1.07±0.25). These results suggest that the repressive effect of MBD2
is not mediated through its binding to LARC.
P14. therapy for genetic disorders
P14.02
Breast cancer prevention by letrozole in post menopausal
BRcA1/2 mutations carriers : the onco-03/LiBER trial
P. Pujol1 , S. Mijonnet2 , K. Samb3 , A. Martin2 ;
1oncogenetique CHU, INSERM-CRCM Val d’Aurelle, Montpellier, France,
2 3 FNCLCC, paris, France, oncogenetique CHU, INSERM- CRCM Val d’Aurelle,
Montpellier, France.
Women carrying germline BRCA1/2 deleterious mutations represent
an extreme risk population for developing breast cancer, with a cumulative
life-time risk of 56-80%. Although it greatly affects the quality
of life, breast prophylactic surgery in BRCA1/2 mutation carriers have
increased in Europe and in US over the last decade. Medical prevention
of breast cancer could thus provide a precious alternative to prophylactic
mastectomy.
The major breast cancer prevention trials using tamoxifen and raloxifen
showed an approximately 50% risk reduction in high risk women.
The contralateral risk reduction in current adjuvant trials comparing
aromatase inhibitors (AI) to tamoxifen reveals a higher preventive efficacy
of AI after menopause. Two ongoing randomized studies using
exemestane (MAP3) or anastrozole (IBIS2) are assessing the risk reduction
of breast cancer but none are designed for BRCA1/2 carriers.
The French federation of cancer centres, “FNCLCC” and the French
national cancer institute (INCa) have developed a randomized phase
III study to determine the efficacy of an aromatase inhibitor (letrozole)
to reduce the incidence of invasive breast cancer in post menopausal
BRCA1/2 carriers. The ONCO-03 (LIBER) study is a double-blind, letrozole
versus placebo, controlled study involving 30 centres in France.
The study opened for recruitment in february 2008. The study design,
procedures and first analysis of patients enrolment are presented.
For women bearing a BRCA1/2 genetic predisposition, evaluation of
medical prevention of breast cancer risk is needed to offer an additional
option to surveillance or bilateral mastectomy.
P14.03
Endothelial function and plasma thiols in patient with cADAsiL
(cerebral autosomal dominant arteriopathy with subcortical
infarct and leukoencephalopathy)
S. Romano 1 , J. Campolo 2 , E. Puca 1 , M. Frontali 1 , F. Pescini 3 , L. Pantoni 3 , C.
Tomasello 4 , M. Stromillo 5 , M. Dotti 5 , C. Mariotti 4 , C. Pelucchi 6 , R. De Maria 2 , D.
Inzitari 3 , F. Taroni 4 , A. Federico 5 , O. Parodi 2 ;
1 Istituto di Neurobiologia e Medicina Molecolare CNR, Roma, Italy, 2 Istituto
Fisiologia Clinica CNR, Milano, Italy, 3 Dipartimento di Scienze Neurologiche e
Psichiatriche Univ. di Firenze, Firenze, Italy, 4 Fondazione IRCCS Istituto Neurologico
Carlo Besta, Milano, Italy, 5 Dipartimento di Scienze Neurologiche e
del Comportamento Univ. di Siena, Siena, Italy, 6 Laboratorio di Epidemiologia
Malattie Croniche Dipartmento di Epidemiologia Istituto di Ricerche Farmacologiche
“Mario Negri”, Milano, Italy.
CADASIL, a rare disorder due to point mutations of Notch3 gene, is
characterized by recurrent strokes, serious motor disability, pseudobulbar
paralysis and subcortical dementia. The mutation modifies a
transmembrane receptor involved in the arterial maturation inducing
structural anomalies of endothelial cells and smooth muscle of small
vessels. Endothelial dysfunction (ED), an important prognostic marker
in cardiovascular disorders, is associated to defective endothelial production
of nitric oxide (NO); the essential cofactor of NO-synthase,
BH4, increases NO bioavailability. We designed a study to assess
whether endothelial function may be improved by BH4 administration
in 60 CADASIL patients enrolled at 5 Italian centres. We report on