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ETEROGENEITA’ GENETICA DELL’ANEMIA DI FANCONI Anna Savoia Università di Trieste - PowerPoint PPT Presentation


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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.

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slide1

XXXIII CONGRESSO NAZIONALE AIEOP

Padova e Abano Terme

22-24 ottobre 2006

ETEROGENEITA’ GENETICA DELL’ANEMIA DI FANCONI

Anna Savoia

Università di Trieste

slide2

FANCONI ANAEMIA

  • Clinical symptoms
  • Progressive pancytopaenia
  • Congenital malformations
  • Predisposition to malignancies
slide3

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

slide4

Genetics

Autosomal and X-linked recessive

Incidence <1:100,000 live births

Genetic heterogeneity

slide6

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

slide7

FA Complementation Groups

(241 European patients)

A

B

C

L

D1

J

I

D2

E

G

F

Levitus et al, Blood 103:2498, 2004

slide8

DNA-dependent ATPase and 5’-3’ helicase

Ubiquitin ligase activity

Endonuclease and helicase activity

Taniguchi and D’Andrea. Blood 107:4223, 2006

slide9

D1-BRCA2

J-BRIP1

D2

B

P

A

F

G

Ub

Ub

P

C

E

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

slide10

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
slide11

Fanconi anemia: somatic mosaicism

1

2

REVERSE MOSAICIM

in vivo reversion to normal

FORWARD MOSAICISM

de novo deleterious mutation

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

slide12

Mechanisms for reversion

Gene conversion

Intragenic mitotic recombination

Compensatory secondary mutation in cis

slide13

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 AGGGAGACA CTG CCA GAG CCC GCT GCC CCG G

Trp ArgGlu Thr Leu Pro Glu Pro Ala Ala Pro + 18/stop

CfoI

FANCA-393r TGG AGGGAG ACA CTG CCA GAG CCC GCTGCG CTGCCCCGG

Trp ArgGlu Thr Leu Pro Glu Pro Ala AlaLeu Pro Arg

CfoI digestion

FANCA-393r complementation

Ly

Ly

Ly

Fi

Pb1

Pb2

Waisfisz et al. Nat Genet 22: 379-383, 1999

slide14

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?

slide15

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

slide16

FANCA screening: private mutations and intragenic deletions

Savino et al, Hum Mutat 22:338-339, 2003

slide17

Molecular Diagnosis

FA-?

Positive DEB test

FANCA

FANCB

FANCC

D1-BRCA2

FANCD2

FANCE

FANCF

FANCG

FANCJ

FANCL

FANCM

Linkage

PROTEIN

Complementation

Phenotype

Mutated gene

Screening for mutations

slide18

Western blot analysis of FA lymphoblastoid cell lines

S858R

3761 ins AG

IVS10+1G>T

IVS26+2T>C

IVS10+1G>T

IVS26+2T>C

FANCA Del

Ex18-21 Del

Q264X

Q264X

Q772X

Q772X

S947X

S947X

WT

WT

WT

K562

VU337

VU388

VU223

VU232

VU262

VU263

VU268

VU389

VU338

FANCA

FANCG

Savino et al, Hum Mutat 22:338-339, 2003

slide19

F

B

C

G

E

A

L

Ub

Ub

D2

D2

Integrity of FA complex: FANCD2-Ub

M

FA-A

FA-A

wt

wt

wt

FANCD2-Ub

FANCD2

K562

EUFA389

EUFA262

EUFA338

EUFA232

Savino et al, Hum Mutat 22:338-339, 2003

slide20

FA protein analysis: prescreening strategy

POSITIVE DEB TEST

Anti-

FANCA

Anti-

FANCD2

FANCD2

nonUb

FANCD2

Ub

Defective

Normal

Defective

Anti-

FANCB

FANCC

FANCE

FANCF

FANCG

FANCL

FANCM

Anti-

FANCD1

FANCJ

Normal

Normal

Defective

Defective

FANCX

FANCX

slide21

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

slide22

Low FANCD2 expression level

Borriello et al. Leukemia 2006, doi: 10.1038/sj.leu.240446

slide23

Identification of the Leu153Ser mutation

Borriello et al. Leukemia 2006, doi: 10.1038/sj.leu.240446

slide24

FA+/-

non BRCA2

FA+/+

FA-/-

BRCA2-/-

FA-/-

FA-/-

FA-/-

BRCA2-/-

Defective FA/BRCA in cancers

Germ cells

Somatic cells

Cancers

n mutations

AML, SCC

n mutations

AML, SCC

brain, Wilms

Pancreas (<1%)

Breast cancers? (<1% for J)

Leukemia?

1 FA mutation

n mutations

Epigenetic silencing

2 FA mutations?

Leukemia

ovary, pancreas

n mutations

Modified from Mathew. Oncogene 25:5875, 2006

slide25

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