ETEROGENEITA GENETICA DELLANEMIA DI FANCONI

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XXXIII CONGRESSO NAZIONALE AIEOP Padova e Abano
Terme 22-24 ottobre 2006
ETEROGENEITA GENETICA DELLANEMIA DI
FANCONI Anna Savoia Università di Trieste
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FANCONI ANAEMIA

• Clinical symptoms
• Progressive pancytopaenia
• Congenital malformations
• Predisposition to malignancies

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Cellular phenotype
Diagnosis

• Spontaneous chromosomal instability
• Hypersensitivity to
• crosslinking agents (MMC, DEB) oxygen radicals
• tumor necrosis factor (TNF)
• interferon-gamma
• G2 phase prolongation and/or arrest

DEB test
Flow cytometry
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Genetics Autosomal and X-linked recessive
Incidence lt1100,000 live births Genetic
heterogeneity
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(No Transcript)
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Biallelic BRCA2/FANCD1 mutations (16 kindreds
from literature)

• Early onset leukemia (2.2 ys vs 13.4 ys for all
  other FA)
• Mainly AML but also T-ALL and B-ALL
• Medulloblastomas and Wilms tumors

Medulloblastoma
Wilms tumor Medulloblastoma
Wilms tumor AML
T-ALL
Howlett et al. Science 297606, 2002 Mathew.
Oncogene 255875, 2006
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FA Complementation Groups (241 European patients)
A
B
C
L
D1
J
I
D2
E
G
F
Levitus et al, Blood 1032498, 2004
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DNA-dependent ATPase and 5-3 helicase
Ubiquitin ligase activity
Endonuclease and helicase activity
Taniguchi and DAndrea. Blood 1074223, 2006
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FA/BRCA pathway a network of processes
RAD50 MRE11 NBS1
ATM
ATR
BRCA1
Radiation
USP1
RAD51
D2
D2
Nuclear foci (DNA replication DNA
recombination DNA repair)
Crosslinking agents S-phase
L
M
BLM
Nucleus
Cytoplasm
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Fanconi anemia diagnostic difficulties
(1) Clinical diagnosis phenotypic
heterogeneity Absence of malformation
(25-40) Late onset of aplastic anemia Solid
tumor as first clinical manifestation
(2) Cytogenetic diagnosi hematopoietic mosaicism
(3) Molecular diagnosis genetic heterogeneity At
least 11 genes responsible for FA Low correlation
between genotype and phenotype
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Fanconi anemia somatic mosaicism
1
2
FORWARD MOSAICISM de novo deleterious mutation
REVERSE MOSAICIM in vivo reversion to normal
Lymphocyte cultures DEB test and cell cycle
analysis Resistant EBV-immortalized lymphoblasts
(20)
Hypothesis Resistant cells derive from a
subpopulation of B lymphocytes whose FA
phenotype has reverted to wild type
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Mechanisms for reversion
Gene conversion Intragenic mitotic
recombination Compensatory secondary mutation in
cis
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FANCA TGG AGG AGA CAC TGC CAG AGC CCG CTG
CCC CGG Trp Arg Arg His Cys Gln Ser
Pro Leu Pro Arg FANCA-393m TGG AGG GAG ACA CTG
CCA GAG CCC GCT GCC CCG G Trp Arg
Glu Thr Leu Pro Glu Pro Ala Ala Pro 18/stop

CfoI FANCA-393r TGG AGG GAG ACA CTG CCA GAG CCC
GCT GCG CTG CCC CGG Trp Arg Glu Thr
Leu Pro Glu Pro Ala Ala Leu Pro Arg
CfoI digestion
FANCA-393r complementation
Ly
Ly
Ly
Fi
Pb1
Pb2
Waisfisz et al. Nat Genet 22 379-383, 1999
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FA mosaicism of hematopoietic system Reversion
of the FA phenotype can occur spontaneously in
hematopoietic stem or progenitor cells A single
reverted stem cell may have the capacity to
gradually replace affected progenitor cells Risk
of malignancy? Bone marrow transplantation?
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Correlation between genotype-phenotype
Significant differences Pancytopenia FA-G gt
FA-C AML FA-G gt FA-A and
FA-C Malformations FA-A gt FA-G gt FA-C
No significant difference Onset of hemathologic
abnormalities Requirement for transfusion Solid
tumors
Faivre et al, Blood 964064, 2000
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FANCA screening private mutations and intragenic
deletions
Savino et al, Hum Mutat 22338-339, 2003
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Molecular Diagnosis
FA-?
Positive DEB test
FANCA FANCB FANCC D1-BRCA2 FANCD2 FANCE FANCF FANC
G FANCJ FANCL FANCM
Linkage
PROTEIN
Complementation
Phenotype
Mutated gene
Screening for mutations
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Western blot analysis of FA lymphoblastoid cell
lines
S858R 3761 ins AG
IVS101GgtT IVS262TgtC
IVS101GgtT IVS262TgtC
FANCA Del Ex18-21 Del
Q264X Q264X
Q772X Q772X
S947X S947X
WT
WT
WT
K562 VU337 VU388 VU223 VU232 VU262 VU263 VU268 VU3
89 VU338
FANCA
FANCG
Savino et al, Hum Mutat 22338-339, 2003
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Integrity of FA complex FANCD2-Ub
M
FA-A
FA-A
wt
wt
wt
FANCD2-Ub
FANCD2
K562
EUFA262
EUFA338
EUFA389
EUFA232
Savino et al, Hum Mutat 22338-339, 2003
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FA protein analysis prescreening strategy
POSITIVE DEB TEST
Anti- FANCA
Anti- FANCD2
FANCD2 Ub
FANCD2 nonUb
Defective
Defective
Normal
Anti- FANCB FANCC FANCE FANCF FANCG FANCL FANCM
Anti- FANCD1 FANCJ
Normal
Normal
Defective
Defective
FANCX
FANCX
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DEB TEST
T-acute lymphoblastic leukemia (T-ALL) Severe
chemotherapy toxicity
No Fanconi anemia clinical features No
congenital malformation No aplastic anemia
antecedent to the onset of T-ALL
Borriello et al. Leukemia 2006, doi
10.1038/sj.leu.240446
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Low FANCD2 expression level
Borriello et al. Leukemia 2006, doi
10.1038/sj.leu.240446
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Identification of the Leu153Ser mutation
Borriello et al. Leukemia 2006, doi
10.1038/sj.leu.240446
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Defective FA/BRCA in cancers
Germ cells
Somatic cells
Cancers
n mutations
AML, SCC
n mutations
AML, SCC brain, Wilms
Pancreas (lt1) Breast cancers? (lt1 for
J) Leukemia?
1 FA mutation
n mutations
Epigenetic silencing 2 FA mutations?
Leukemia ovary, pancreas
n mutations
Modified from Mathew. Oncogene 255875, 2006
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Defective FA/BRCA pathway
Classical Fanconi anemia Atypical Fanconi
anemia (Germline mutations in both
alleles) Chemotherapy sensitivity Solid
tumours Sporadic tumors (Germline and somatic
mutations) Dosage of radiation and
chemotherapeutic agents