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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 BarcelonaGenome Instability Group: http://gig.uab.catGenome Instability Group: http://gig.uab.cat
LA ANEMIA DE FANCONILA ANEMIA DE FANCONILA ANEMIA DE FANCONILA ANEMIA DE FANCONILA ANEMIA DE FANCONILA ANEMIA DE FANCONILA ANEMIA DE FANCONILA 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
Disease evolution
Kutler et al., Blood, 2003
Dr.Eunike Velleuer (Dusseldorf) in action in Barcelona, March 2010
Xeroderma pigmentosum
Chromosome fragility in Fanconi anemia
Control
Fanconi
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.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
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
15 complementation Groups in 15 complementation Groups in FanconiFanconi AnemiaAnemia
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
Common phenotype in all genetic subtypes
FA-A FA-C
100
80
60
FANC-CFANC-G
FANC-FFANC-A
Rel
ativ
e su
rviv
al %
EGFP
Retrovirus-meditated genetic subtyping
1 10 100 1000
40
20
0
MMC (nM)
Rel
ativ
e su
rviv
al %
Genetic subtyping of 111 Spanish FA patients
D1
D26%
E3%
G4%
J3%
Unknown
8%
Retroviral subtyping, mutational screening, western blot
A74%
D11%
C1%
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
whole exome sequencing
ERCC4/XPF mutations and XPF-deficiency in Fanconi anemia patients (FANCQ)
c.2065C>A c.689T>C
c.1484_1488delCTCAA c.2371_2398dup28
Am J Hum Genet 2003
XPF cDNA genetically complements MMC sensitivity of FA104 lymphoblasts
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
Interstrand crosslink repair (ICLR)
Fork stalling
Nucleotide excision repair (NER)
recognition
dual incision
UV
Unhooking
Homologous recombination
excision
DNA synthesis
ligation
NER
ICLR
NER
ICLR NER
ICLR
NER
ICLR NER ICLRNER ICLR
1 gene (XPF), 2 repair pathways, 3 syndromes
Xeroderma pigmentosumXeroderma pigmentosum Fanconi anemiaFanconi anemia ProgeriaProgeria
Bogliolo et al., Am J Hum Genet 2013
Skin cancer Leukemia and SCC Cancer?
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
16 complementation Groups in 16 complementation Groups in FanconiFanconi AnemiaAnemia
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
Spanish FANCA mutational spectrum
Point mutationPoint mutationSplicing mutationMicrodeletionMicroinsertionDeletion
130 mutations, 52 different, 20 novelCallén et al., 2005; Blood; Kalb et al, Am J Hum Genet 2007;
Castella et al., Blood 2011
HN-SCC Cancer
Bowel Ca.Colorectal Ca
Colorectal Ca.56 y.o
Lung Ca58 y.o.
Lung + Breast Ca, 74 y.o.
Cancer pedigree due to mutations in BRIP1/FANCJ found by exome sequencing
Cervical 33 y.o
Protate Ca. 60 y.o.
Prostate Ca58 y.o.
Kindey Ca
Brain Ca55 y.o
Dead at birth Down Synd.
Lymphoma70 y.o.
Fanconi anemiaIntrauterine growth retardation
Dead at birth Congenital malformations
Saviour babies: embryo selection for HLA matched
Molly Nash
Preimplantational genetic diagnosis with HLA-matching selection: savior babies
(38 cycles, 7 families)
524 oocytes
299 embryos299 embryos
75 healthy26 HLA compatible
16 tranferred to uterus
5 implanted (pregnancy)1 born
Barcelona, August 15th 2006
Bone marrow transplant increases cancer risck (SCC)
J Natl Cancer Institute, 2008
Blood CD34+ cells Selection
Gene therapy: Genetic Correction of Hematopoietic Stem Gene therapy: Genetic Correction of Hematopoietic Stem Cells from Patients with Fanconi anemiaCells from Patients with Fanconi anemia
Transduction with therapeutic lentiviral vectors
Infusion of transduced graft
“Natural” vs “medical” gene therapy
Mutation Back mutation (mosaics)Gene therapy
Mosaic FA patient
FA patientFA patient
Mosaicism
DEB test: ?DEB test: positive
DEB test: positive
Back mutation
Clonal expansion
Mosaicism often results in clinical improvement:
“natural” gene therapy
RE
LAT
IVE
VA
LUE
(%
)
Age (months)
Figure 3 Gene therapy of FANCD1 KO mice (BRCA2-/-)B
120
3 Gy
FA-D1Males
FA-D1Females
+ EGFP-LVs
+ BRCA2- LVs
3 Gy
0
20
40
60
80
100D
onor
Chi
mer
ism
(%) # 3
# 1# 4# 6# 5
# 7
# 9
# 8
1 2 3 4 5 6
Months after Bone Marrow Transplantation
0 10 30 MMC (nM)
CF
Cs
Sur
viva
l ( %
)# 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
Sur
viva
l ( %
)
B
6 months after BMTA
Gene therapy of FANCD1 KO mice (BRCA2-/-)
FA-D1+ EGFP-LV FA-D1+ BRCA2-LVWT
- MMC
+ MMC
B
- +
WT
MMC
% A
berr
ant
Cel
ls
0
20
40
60
80
100
- +
FA-D1 + EGFP-LV
# 9
FA-D1 + BRCA2-LV
- +
# 4
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 ActionCoordinated International Action
Coordinator: Juan Bueren (Madrid)
� Fanconi anemia diagnosis.
� Diagnosis of the pathogenic mutations.
� Early diagnosis of myelodysplastic syndromes orleukemia .
WP1: To determine the genetic and hematopoietic characteristics of FA patients
leukemia .
� Diagnosis of mosaic patients with revertant mutationsaccounting for spontaneous hematological recovery.
� Subtyping of Fanconi anemia patients.
� Prediction of the hematopoietic reserve of thepatients.
AIM: To collect 4x10 6 CD34+ cells / kg of weight projected to 5 years.
WP2: To assess the safety and efficacy of an improv ed mobilization and HSC collection method based on a new mobilizatio n regimen
for FA patients with plerixafor and 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
1 2 3 4 5 6 7 8 Days:
Apheresis
Mozobil
Filgrastim
WP3: To validate the safety and efficacy of the the rapeutic clinical-grade lentiviral vector
Antecedents:
cPPT Wpre*
GA
∆∆∆∆U3 R U5
PGK
SD SA
RRE
ψψψψFANCA
CMV R U5
PGK-FANCA.Wpre* LV
Orphan Medicinal Product Designation: EU 3/10/822
Lentiviral vector containing the Fanconi anemia A (FANCA) gene
WP3: To validate the safety and efficacy of the the rapeutic clinical-grade lentiviral vector
AIMs:
-Production of the therapeutic vector under GMP conditions.
- Manufacturing of the medicinal product, genetically
- Validation of the safety of the medicinal product.
- Manufacturing of the medicinal product, genetically modified FA-A CD34 + cells, under GMP conditions.
WP4: To assess the safety and efficacy of the infus ion of CD34 +
cells in FA patients, after transduction with the t herapeutic lentiviral vector
AIMTo demonstrate the safety and obtain the first evid ences of clinical efficacy associated to the infusion of the medicinal
product: Genetically modified autologous CD34 + cells.
� Patients complementation group: FA -A
Inclusion Criteria
Exclusion Criteria
� Patients with a HLA-identical related donor� Nº of cryopreserved or fresh CD34 + cells: <10 5 CD34+/kg weight
� Evidence of CD34 + cells transformation� Evidence of somatic mosaicism in HSCs associated wi th
hematological improvement
� Patients complementation group: FA -A� Moderate to severe aplasia
FutureFuture
IPScytokines Blood
3-4 transcription
FA gene (lenti)
Disease-corrected haematopoietic progenitors from Fanconi anemia induced pluripotent stem (iPS) cells
FA fibro Haematopoietic progenitors
3-4 transcription factors* (retro)
*KLF4, cMYC, OCT4, SOX2
Raya et al. Nature, 2009
DSB
mutation repair
DSB
save integration (save harbour)
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)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)
Surrallés’ lab, Barcelona, SpainDr. M. Bogliolo
Dr. L. Mina
Dr. J. Minguillón
Dr. R. Pujol
Dr. MJ Ramírez
Dr. J. Surrallés
Dr. G. HernándezDr. G. Hernández
Dr. M. Aza-Carmona
A. Molina
J.P. Trujillo
M. Marin
H. Montanuy
S. Sánchez
Genome Instability Group: www.gig.uab.cat