anemia de fanconi: el síndrome de los cromosomas de cristal · la anemia de fanconi-first...
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I Congreso Nacional de Anemias Raras, Barcelona, 20-21 Set 2013
Anemia de Fanconi: el síndrome de los
cromosomas de cristal
Dr. Jordi SurrallésCatedrático de Genética
Universitat Autònoma de Barcelona
Genome Instability Group: http://gig.uab.cat
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LA ANEMIA DE FANCONI
-First described by Guido Fanconi in 1927
-Autosomal recessive (one subtype is X linked)
-A very rare disease with a frequncy of ~1/400.000
-highly hetyerogenous (genetically and clinically)
Ghido Fanconi con Andrea Lee Kuritzky Children's Hospital, Los Angeles, 1959
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Kutler et al., Blood, 2003
Disease evolution
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Dr.Eunike Velleuer (Dusseldorf) in action in
Barcelona, March 2010
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Xeroderma pigmentosum
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Chromosome fragility in Fanconi anemia
Control
Fanconi
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OVERLAPPING SYNDROMES
Inherited bone marrow failure syndromes: Dyskeratosis congenita,
Diamond-Blackfan anemia,
Shwachman-Diamond syndrome,
severe congenital neutropenia,
thrombocytopenia absent radii (TAR) syndrome,
amegakaryocytic thrombocytopenia.
Other overlapping syndromes:Baller-Gerold syndrome,
Nijmegen breakage syndrome (MMC+)
Rothmund-Thomson syndrome,
Roberts syndrome (MMC+)
Warsaw Breakage syndrome (MMC+)
DK-phocomelia,
VACTERL hydrocephalus syndrome,
Wiskott-Aldrich syndrome
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C. Group Gene Reference
FA-A FANCA Lo Ten Foe et al. Nature Genetics 1996
FABC Consortium. Nature Genetics 1996
FA-B FANCB Meetei et al Nature Genetics 2004
FA-C FANCC Strathdee et al. Nature, 1992
FA-D1 FANCD1/BRCA2 Howlett et al. Science, 2002
FA-D2 FANCD2 Timmers et al. Mol Cell, 2001
FA-E FANCE De Winter et al. Am. J. Hum Genet 2000
FA-F FANCF De Winter et al. Nature Genet. 2000
FA-G FANCG De Winter et al. Nature Genet. 1998
FA-I FANCI Smogorzewska et al. Cell 2007
FA-J FANCJ/BRIP1 Levran et al. Nature Genet. 2005; Levitus et al. Nature Genet. 2005;
FA-L FANCL Ruhikanta et al. Nature Genet. 2003
FA-M FANCM Meetei et al Nature Genetics 2005
FA-N FANCN/PALB2 Reid et al Nature Genetics 2007
Xia et al Nature Genetics 2007
FA-O FANCO/RAD51C Vaz et al Nature Genetics 2010
FA-P FANCP/SLX4 Stoepker et al Nature Genetics 2011
Kim et al Nature Genetics 2011
15 complementation Groups in Fanconi Anemia
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Common phenotype in all genetic subtypes
FA-A FA-C
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1 10 100 1000
100
80
60
40
20
0
FANC-C
FANC-G
FANC-F
FANC-A
MMC (nM)
Re
lati
ve
su
rviv
al
%
EGFP
Retrovirus-meditated genetic subtyping
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Genetic subtyping of 111 Spanish FA patients
A74%
D11%
D26%
E3%
G4%
J3%
C1%
Unknown
8%
Retroviral subtyping, mutational screening, western blot
Callen et al., Blood 2005; Casado et al., J Med Genet 2007; Kalb et al Am J Hum Genet
2007; Castella et al Blood 2011
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whole exome sequencing
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ERCC4/XPF mutations and XPF-deficiency
in Fanconi anemia patients (FANCQ)
c.1484_1488delCTCAA
c.2065C>A c.689T>C
c.2371_2398dup28
Am J Hum Genet 2003
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XPF cDNA genetically complements MMC
sensitivity of FA104 lymphoblasts
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XPF/ERCC4 mutations lead to three rare
disorders: XP, XFE-progeria, and FA (FA-Q)
Xeroderma pigmentosum XFE-progeria Fanconi anemia
FANCQ alias for XPF
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Interstrand crosslink
repair (ICLR)
Fork stalling
Unhooking
Homologous recombination
Nucleotide excision
repair (NER)
recognition
dual incision
excision
DNA synthesis
ligation
UV
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NER
ICLR
Xeroderma pigmentosum
NER
ICLR
Xeroderma pigmentosum
NER
ICLR
Fanconi anemia
NER
ICLR
Fanconi anemia
NER ICLR
Progeria
NER ICLR
Progeria
1 gene (XPF), 2 repair pathways, 3 syndromes
Bogliolo et al., Am J Hum Genet 2013
Skin cancer Leukemia and SCC Cancer?
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C. Group Gene Reference
FA-A FANCA Lo Ten Foe et al. Nature Genetics 1996
FABC Consortium. Nature Genetics 1996
FA-B FANCB Meetei et al Nature Genetics 2004
FA-C FANCC Strathdee et al. Nature, 1992
FA-D1 FANCD1/BRCA2 Howlett et al. Science, 2002
FA-D2 FANCD2 Timmers et al. Mol Cell, 2001
FA-E FANCE De Winter et al. Am. J. Hum Genet 2000
FA-F FANCF De Winter et al. Nature Genet. 2000
FA-G FANCG De Winter et al. Nature Genet. 1998
FA-I FANCI Smogorzewska et al. Cell 2007
FA-J FANCJ/BRIP1 Levran et al. Nature Genet. 2005; Levitus et al. Nature Genet. 2005;
FA-L FANCL Ruhikanta et al. Nature Genet. 2003
FA-M FANCM Meetei et al Nature Genetics 2005
FA-N FANCN/PALB2 Reid et al Nature Genetics 2007
Xia et al Nature Genetics 2007
FA-O FANCO/RAD51C Vaz et al Nature Genetics 2010
FA-P FANCP/SLX4 Stoepker et al Nature Genetics 2011
Kim et al Nature Genetics 2011
FA-Q FANCQ/ERCC4 Bogliolo et al Am J Hum Genet 2013
16 complementation Groups in Fanconi Anemia
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Spanish FANCA mutational spectrum
Point mutationSplicing mutation
Microdeletion
MicroinsertionDeletion
130 mutations, 52 different, 20 novel
Callén et al., 2005; Blood; Kalb et al, Am J Hum Genet 2007;
Castella et al., Blood 2011
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HN-SCC
Cancer
Bowel Ca.
Colorectal Ca
Colorectal Ca.
56 y.o
Cervical
33 y.o
Protate Ca.
60 y.o.
Lung Ca
58 y.o.
Lung + Breast Ca, 74 y.o.
Prostate Ca
58 y.o.
Kindey Ca
Brain Ca
55 y.o
Dead at
birth Down
Synd.
Lymphoma
70 y.o.
Fanconi
anemia
Intrauterine
growth
retardation
Dead at birth
Congenital
malformations
Cancer pedigree due to mutations in
BRIP1/FANCJ found by exome sequencing
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Saviour babies: embryo selection for HLA matched
Molly Nash
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Preimplantational genetic diagnosis with
HLA-matching selection: savior babies (38 cycles, 7 families)
524 oocytes
299 embryos
75 healthy
26 HLA compatible
16 tranferred to uterus
5 implanted (pregnancy)
1 born
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Barcelona, August 15th 2006
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J Natl Cancer Institute, 2008
Bone marrow transplant increases cancer risck (SCC)
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Transduction with therapeutic
lentiviral vectors
Blood CD34+ cells
Selection
Infusion of transduced graft
Gene therapy: Genetic Correction of Hematopoietic Stem
Cells from Patients with Fanconi anemia
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“Natural” vs “medical” gene therapy
Mutation Back mutation (mosaics)Gene therapy
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Mosaic FA
patient
DEB test: ?
FA patient
DEB test:
positive
FA patient
DEB test:
positive
Back
mutation
Clonal
expansion
Mosaicism
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REL
ATI
VE
VA
LUE
(%)
Age (months)
Mosaicism often results in clinical improvement: “natural” gene therapy
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Figure 3 Gene therapy of FANCD1 KO mice (BRCA2-/-)
B
0
20
40
60
80
100
120D
on
or
Ch
ime
ris
m (
%) # 3
# 1
# 4
# 6
# 5
# 7
# 9
# 8
1 2 3 4 5 6
Months after Bone Marrow Transplantation
3 Gy
FA-D1
Males
FA-D1
Females
+ EGFP-LVs
+ BRCA2- LVs
3 Gy
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FA-D1+ EGFP-LV FA-D1+ BRCA2-LVWT
- MMC
+ MMC
0 10 30 MMC (nM)
CF
Cs
Su
rviv
al
( %
)
# 1
# 3
# 4
# 5# 6
# 8# 7
# 90
20
40
60
80
100
120
MMC (nM)0 10 30
# 1
# 3
# 9
# 7
# 8
# 6
# 4
# 5
2 months after BMT
0
20
40
60
80
100
120
CF
Cs
Su
rviv
al
( %
)
B
6 months after BMT
- +
WT
MMC
% A
berr
an
t C
ells
0
20
40
60
80
100
A
- +
FA-D1 +
EGFP-LV
# 9
FA-D1 +
BRCA2-LV
- +
# 4
Gene therapy of FANCD1 KO mice (BRCA2-/-)
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Phase I/II Gene therapy trial of Fanconi anemia
patients with a new Orphan Drug consisting of
a lentiviral vector carrying the FANCA gene: A
Coordinated International Action
Coordinator: Juan Bueren (Madrid)
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Fanconi anemia diagnosis.
Diagnosis of the pathogenic mutations.
Early diagnosis of myelodysplastic syndromes or
leukemia.
Diagnosis of mosaic patients with revertant mutations
accounting for spontaneous hematological recovery.
Subtyping of Fanconi anemia patients.
Prediction of the hematopoietic reserve of the
patients.
WP1: To determine the genetic and hematopoietic
characteristics of FA patients
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AIM: To collect 4x106 CD34+ cells / kg of weight projected to 5 years.
WP2: To assess the safety and efficacy of an improved mobilization
and HSC collection method based on a new mobilization regimen
for FA patients with plerixafor and filgrastim.
1 2 3 4 5 6 7 8 Days:
Apheresis
Mozobil
Filgrastim
• HSC mobilization: Filgrastim (10-12 μg / kg every 12
hours) for up to 7 days and plerifaxor (240 μg / kg) up to 4
days, 6 to 11 hours before starting apheresis
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WP3: To validate the safety and efficacy of the therapeutic
clinical-grade lentiviral vector
Orphan Medicinal Product Designation: EU 3/10/822
Lentiviral vector containing the Fanconi anemia A (FANCA) gene
Antecedents:
cPPT Wpre*
GA
U3 R U5
PGK
SD SA
RRE
yFANCA
CMV R U5
PGK-FANCA.Wpre* LV
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WP3: To validate the safety and efficacy of the therapeutic clinical-
grade lentiviral vector
AIMs:
-Production of the therapeutic vector under GMP
conditions.
- Validation of the safety of the medicinal product.
- Manufacturing of the medicinal product, genetically
modified FA-A CD34+ cells, under GMP conditions.
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WP4: To assess the safety and efficacy of the infusion of CD34+
cells in FA patients, after transduction with the therapeutic
lentiviral vector
AIM
To demonstrate the safety and obtain the first evidences of
clinical efficacy associated to the infusion of the medicinal
product: Genetically modified autologous CD34+ cells.
Exclusion Criteria
Patients with a HLA-identical related donor Nº of cryopreserved or fresh CD34+ cells: <105 CD34+/kg weight
Evidence of CD34+ cells transformation
Evidence of somatic mosaicism in HSCs associated with
hematological improvement
Patients complementation group: FA-A
Moderate to severe aplasia
Inclusion Criteria
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Future
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FA fibro
IPS
cytokines Blood
Haematopoietic
progenitors
3-4 transcription
factors* (retro)
*KLF4, cMYC, OCT4, SOX2
FA gene (lenti)
Raya et al. Nature, 2009
Disease-corrected haematopoietic progenitors from
Fanconi anemia induced pluripotent stem (iPS) cells
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DSB
DSB
mutation repair
save integration (save harbour)
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acknowledgements:
Juan Bueren (CIEMAT-Madrid)
Red Española de Anemia de Fanconi
Juan Carlos Izpisua-Belmonte (CMRB-Barcelona)
Javier Benítez (CNIO-Madrid)
Sheila Zúñiga (Sistemas Genómicos, Valencia)
Arleen Auerbach (RU-New York)
Ruud Brakenhoff (VUMC-Amsterdam)
Detlev Schindler (Uni. Wursburg)
Johan de Winter (VUMC-Amsterdam)
Orlando Scharer (SBU-New York)
Koos Jaspers (EU-Rotterdam)
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Surrallés’ lab, Barcelona, SpainDr. M. Bogliolo
Dr. L. Mina
Dr. J. Minguillón
Dr. R. Pujol
Dr. MJ Ramírez
Dr. J. Surrallés
Dr. G. Hernández
Dr. M. Aza-Carmona
A. Molina
J.P. Trujillo
M. Marin
H. Montanuy
S. Sánchez
Genome Instability Group: www.gig.uab.cat
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