Transcript Document

XXXIII CONGRESSO NAZIONALE AIEOP
Padova e Abano Terme
22-24 ottobre 2006
ETEROGENEITA’ GENETICA DELL’ANEMIA DI FANCONI
Anna Savoia
Università di Trieste
FANCONI ANAEMIA
Clinical symptoms
• Progressive pancytopaenia
• Congenital malformations
• Predisposition to malignancies
Cellular phenotype
Diagnosis
• Spontaneous chromosomal instability
• Hypersensitivity to:
crosslinking agents (MMC, DEB)
DEB test
oxygen radicals
tumor necrosis factor (TNF)
interferon-gamma
• G2 phase prolongation and/or arrest
Flow cytometry
Genetics
Autosomal and X-linked recessive
Incidence <1:100,000 live births
Genetic heterogeneity
Complementation
Groups
Genes
Localization
Proteins
a.a. (k Da)
FA-A
FANCA
16q24.3
1455
FA-B
FANCB
Xp22.31
859
FA-C
FANCC
9q22.3
558
FA-D1
FANCD1
BRCA2
13q13.1
3418
FA-D2
FANCD2
3p26
1451
1451-Ub
FA-E
FANCE
6p21-22
536
FA-F
FANCF
11p15
374
FA-G
FANCG
XRCC9
9p13
622
FA-I
unknown
FA-J
BACH1
(BRIP1)
17q23.2
1249
FA-L
FANCL
2p16.1
375
FA-M
FANCM
14q21.2
2048
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 297:606, 2002; Mathew. Oncogene 25:5875, 2006
FA Complementation Groups
(241 European patients)
A
B
C
D1
D2
E F
G
L
I J
Levitus et al, Blood 103:2498, 2004
DNA-dependent ATPase and 5’-3’ helicase
Ubiquitin ligase activity
Endonuclease and helicase activity
Taniguchi and D’Andrea. Blood 107:4223, 2006
FA/BRCA pathway: a network of processes
J-BRIP1
BRCA1
RAD50
MRE11
NBS1
P
Nuclear foci
(DNA replication
DNA recombination
DNA repair)
Radiation
USP1
RAD51
D1-BRCA2
ATM
ATR
D2
D2
Ub
P
Ub
B
Nucleus
BLM
Crosslinking agents
S-phase
L
M
Cytoplasm
D2
E
C
F
G
A
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
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
Mechanisms for reversion
Gene conversion
Intragenic mitotic recombination
Compensatory secondary mutation in cis
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
Trp Arg Glu Thr Leu Pro Glu Pro Ala Ala Pro
CfoI
TGG AGG GAG ACA CTG CCA GAG CCC GCT GCG CTG
Trp Arg Glu Thr Leu Pro Glu Pro Ala Ala Leu
FANCA-393r
CfoI digestion
Ly Ly Ly Fi
G
+ 18/stop
CCC CGG
Pro Arg
FANCA-393r complementation
Pb1 Pb2
Waisfisz et al. Nat Genet 22: 379-383, 1999
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?
Correlation between genotype-phenotype
Significant differences
Pancytopenia
FA-G > FA-C
AML
FA-G > FA-A and FA-C
Malformations
FA-A > FA-G > FA-C
No significant difference
Onset of hemathologic abnormalities
Requirement for transfusion
Solid tumors
Faivre et al, Blood 96:4064, 2000
FANCA screening: private mutations and intragenic deletions
EUFA397
EUFA388
FA-63
FA-73
EUFA330
EUFA262
FA-25
EUFA265
FA-38
EUFA268
EUFA232
EUFA578
FA-19
FA-32
EUFA223
EUFA341
EUFA393
FA-37
FA-53
FA-41
FA-58
FA-35
EUFA337
IVS3+3A>C
Q264X
Q264X
Q264X
IVS9+3delA
IVS26+1T>C
IVS14+1G>C
IVS16-20A>G
Q669X
Q772X
S858R
IVS28+83C>G (Homo)
2830ins19
2830ins19 (Homo)
S947X
S947X
3559insG
3559insG
3559insG
3638-3639delCT
3638-3639delCT
D1359Y
120 kb (entire gene)
IVS3+3A>C
Q264X
Q264X
IVS2-1G>T
IVS9+3delA
IVS10+1G>T
IVS14+1G>C
IVS16-20A>G
n.i.
Q772X
3761insAG
Deletion (?)
n.i.
Deletion (?)
S947X
S947X
3559insG
n.i.
n.i.
3638-3639delCT
n.i.
5 kb (exons 18 to 21)
Savino et al, Hum Mutat 22:338-339, 2003
Molecular Diagnosis
FA-?
FANCA
FANCB
FANCC
D1-BRCA2
FANCD2
FANCE
FANCF
FANCG
FANCJ
FANCL
FANCM
Positive DEB test
Complementation
PROTEIN
Linkage
Phenotype
Mutated gene
Screening for mutations
Q772X
Q772X
VU268
VU338
WT
WT
IVS10+1G>T
IVS26+2T>C
VU263
VU389
IVS10+1G>T
IVS26+2T>C
3761 ins AG
S858R
S947X
S947X
VU262
VU232
VU223
VU388
WT
Q264X
Q264X
FANCG
FANCA Del
Ex18-21 Del
FANCA
VU337
K562
Western blot analysis of FA lymphoblastoid cell lines
Savino et al, Hum Mutat 22:338-339, 2003
Integrity of FA complex: FANCD2-Ub
F
B
C
G
A
FA-A
FA-A
wt
wt
M
E
wt
L
FANCD2-Ub
FANCD2
K562
EUFA389
EUFA338
EUFA262
EUFA232
Ub
D2
D2
Ub
Savino et al, Hum Mutat 22:338-339, 2003
FA protein analysis: prescreening strategy
POSITIVE DEB TEST
AntiFANCA
AntiFANCD2
FANCD2
nonUb
Defective
FANCD2
Ub
Normal
Defective
Normal
AntiFANCB
FANCC
FANCE
FANCF
FANCG
FANCL
FANCM
AntiFANCD1
FANCJ
Normal
FANCX
Defective
Defective
FANCX
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
Low FANCD2 expression level
Borriello et al. Leukemia 2006, doi: 10.1038/sj.leu.240446
Identification of the Leu153Ser mutation
Borriello et al. Leukemia 2006, doi: 10.1038/sj.leu.240446
Defective FA/BRCA in cancers
Germ cells
Somatic cells
Cancers
FA-/-
AML, SCC
n mutations
FA-/-
n mutations
BRCA2-/-
BRCA2-/-
FA+/-
1 FA mutation
non BRCA2
n mutations
FA-/-
Pancreas (<1%)
Breast cancers? (<1% for J)
Leukemia?
FA-/-
Leukemia
ovary, pancreas
Epigenetic silencing
2 FA mutations?
FA+/+
n mutations
AML, SCC
brain, Wilms
Modified from Mathew. Oncogene 25:5875, 2006
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