ETEROGENEITA’ GENETICA DELL’ANEMIA DI FANCONI Anna Savoia Università di Trieste XXXIII CONGRESSO NAZIONALE AIEOP Padova e Abano Terme 22-24 ottobre 2006.

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

Genetics

Autosomal and X-linked recessive

Incidence <1:100,000 live births

Genetic heterogeneity

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

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-/- 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

Defective FA/BRCA pathway

“Classical” Fanconi anemia

“Atypical” Fanconi anemia (Germline mutations in both alleles)

Chemotherapy sensitivitySolid tumours

Sporadic tumors (Germline and somatic mutations)

Dosage of radiation and chemotherapeutic agents