ETEROGENEITA’ GENETICA DELL’ANEMIA DI FANCONI Anna Savoia Università di Trieste XXXIII...
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Transcript of ETEROGENEITA’ GENETICA DELL’ANEMIA DI FANCONI Anna Savoia Università di Trieste XXXIII...
ETEROGENEITA’ GENETICA DELL’ANEMIA DI FANCONI
Anna Savoia
Università di Trieste
XXXIII CONGRESSO NAZIONALE AIEOPPadova e Abano Terme
22-24 ottobre 2006
FANCONI ANAEMIA
Clinical symptoms
• Progressive pancytopaenia
• Congenital malformations
• Predisposition to malignancies
• Spontaneous chromosomal instability
• Hypersensitivity to:crosslinking agents (MMC, DEB) oxygen radicalstumor necrosis factor (TNF)interferon-gamma
• G2 phase prolongation and/or arrest
Cellular phenotype
DEB test
Flow cytometry
Diagnosis
Complementation Groups
Genes Localization Proteins a.a. (kDa)
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)
Howlett et al. Science 297:606, 2002; Mathew. Oncogene 25:5875, 2006
• 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 tumorMedulloblastoma
Wilms tumorAML
T-ALL
FA Complementation Groups(241 European patients)
D2D1
C
B
A
LJIGFE
Levitus et al, Blood 103:2498, 2004
Taniguchi and D’Andrea. Blood 107:4223, 2006
Ubiquitin ligase activity
DNA-dependent ATPase and 5’-3’ helicase
Endonuclease and helicase activity
FA/BRCA pathway: a network of processes
Cytoplasm Nucleus
Nuclear foci(DNA replication
DNA recombinationDNA repair)
D2
ATM
Radiation
Crosslinking agentsS-phase
BLMC
AG
F
P
D2D2
RAD51
BRCA1
RAD50MRE11NBS1
D1-BRCA2
L
UbB
E
Ub
USP1
ATR
P
J-BRIP1
M
(3) Molecular diagnosis: genetic heterogeneityAt least 11 genes responsible for FALow correlation between genotype and phenotype
(1) Clinical diagnosis: phenotypic heterogeneityAbsence of malformation (25-40%)Late onset of aplastic anemiaSolid tumor as first clinical manifestation
(2) Cytogenetic diagnosi hematopoietic mosaicism
Fanconi anemia: diagnostic difficulties
Fanconi anemia: somatic mosaicism
FORWARD MOSAICISM de novo deleterious
mutation
Lymphocyte culturesDEB test and cell cycle analysis
Resistant EBV-immortalized lymphoblasts (20%)
REVERSE MOSAICIM in vivo reversion to normal
1 2
Hypothesis: Resistant cells derive from a subpopulation of B lymphocytes whose FA phenotype has reverted to wild type
Gene conversion
Intragenic mitotic recombination
Compensatory secondary mutation in cis
Mechanisms for reversion
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 CfoIFANCA-393r TGG AGG GAG ACA CTG CCA GAG CCC GCT GCG G CTG CCC CGG Trp Arg Glu Thr Leu Pro Glu Pro Ala Ala Leu Pro Arg
Ly Ly Ly Fi Pb1 Pb2
FANCA-393r complementationCfoI digestion
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 abnormalitiesRequirement for transfusion
Solid tumors
Faivre et al, Blood 96:4064, 2000
EUFA397 IVS3+3A>C IVS3+3A>C EUFA388 Q264X Q264X
FA-63 Q264X Q264X FA-73 Q264X IVS2-1G>T
EUFA330 IVS9+3delA IVS9+3delA EUFA262 IVS26+1T>C IVS10+1G>T
FA-25 IVS14+1G>C IVS14+1G>C EUFA265 IVS16-20A>G IVS16-20A>G
FA-38 Q669X n.i. EUFA268 Q772X Q772X EUFA232 S858R 3761insAG EUFA578 IVS28+83C>G (Homo) Deletion (?)
FA-19 2830ins19 n.i. FA-32 2830ins19 (Homo) Deletion (?)
EUFA223 S947X S947X EUFA341 S947X S947X EUFA393 3559insG 3559insG
FA-37 3559insG n.i. FA-53 3559insG n.i. FA-41 3638-3639delCT 3638-3639delCT FA-58 3638-3639delCT FA-35 D1359Y n.i.
EUFA337 120 kb (entire gene) 5 kb (exons 18 to 21)
FANCA screening: private mutations and intragenic deletions
Savino et al, Hum Mutat 22:338-339, 2003
Molecular Diagnosis
Positive DEB test
Screening for mutations
LinkageComplementation
Mutated gene
PROTEIN
FANCAFANCBFANCC
D1-BRCA2FANCD2FANCEFANCFFANCGFANCJFANCLFANCM
Phenotype
FA-?
FANCA
FANCG
K56
2
VU
337
VU
388
VU
223
VU
232
VU
262
VU
263
VU
268
VU
389
VU
338
WT
WT
WT
FA
NC
A D
elE
x18-
21 D
el
Q26
4XQ
264X
S94
7XS
947X
S85
8R37
61
ins
AG
IVS
10+
1G>
TIV
S26
+2T
>C
Q77
2XQ
772X
IVS
10+
1G>
TIV
S26
+2T
>C
Western blot analysis of FA lymphoblastoid cell lines
Savino et al, Hum Mutat 22:338-339, 2003
Integrity of FA complex: FANCD2-Ub
B
Ub
D2 D2
Ub
L
A
GE
FC M
EU
FA
232
EU
FA
262
EU
FA
338
EU
FA
389
K56
2
FANCD2FANCD2-Ub
FA-A FA-A wt wt wt
Savino et al, Hum Mutat 22:338-339, 2003
Anti-FANCA
Defective
Anti-FANCD2
FANCD2Ub
Defective
Normal
Defective
Normal
DefectiveFANCD2nonUb
Normal
Anti-FANCBFANCCFANCEFANCFFANCGFANCLFANCM
Anti-FANCD1FANCJ
FANCX FANCX
POSITIVE DEB TEST
FA protein analysis: prescreening strategy
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
DEB TEST
Borriello et al. Leukemia 2006, doi: 10.1038/sj.leu.240446
Defective FA/BRCA in cancers
Germ cells Somatic cells Cancers
FA-/- FA-/-n mutations
AML, SCC
BRCA2-/- BRCA2-/-n mutations AML, SCC
brain, Wilms
FA+/-non BRCA2
FA-/-n mutations
Pancreas (<1%) Breast cancers? (<1% for J)Leukemia?
1 FA mutation
FA+/+ FA-/-n mutations
Leukemiaovary, pancreas
Epigenetic silencing2 FA mutations?
Modified from Mathew. Oncogene 25:5875, 2006