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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. FANCONI ANAEMIA. Clinical symptoms Progressive pancytopaenia Congenital malformations Predisposition to malignancies.

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ETEROGENEITA’ GENETICA DELL’ANEMIA DI FANCONI Anna Savoia Università di Trieste

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

  2. FANCONI ANAEMIA • Clinical symptoms • Progressive pancytopaenia • Congenital malformations • Predisposition to malignancies

  3. 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

  4. Genetics Autosomal and X-linked recessive Incidence <1:100,000 live births Genetic heterogeneity

  5. 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

  6. FA Complementation Groups (241 European patients) A B C L D1 J I D2 E G F Levitus et al, Blood 103:2498, 2004

  7. DNA-dependent ATPase and 5’-3’ helicase Ubiquitin ligase activity Endonuclease and helicase activity Taniguchi and D’Andrea. Blood 107:4223, 2006

  8. 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

  9. 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

  10. 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

  11. Mechanisms for reversion Gene conversion Intragenic mitotic recombination Compensatory secondary mutation in cis

  12. 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

  13. 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?

  14. 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

  15. FANCA screening: private mutations and intragenic deletions Savino et al, Hum Mutat 22:338-339, 2003

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  21. Low FANCD2 expression level Borriello et al. Leukemia 2006, doi: 10.1038/sj.leu.240446

  22. Identification of the Leu153Ser mutation Borriello et al. Leukemia 2006, doi: 10.1038/sj.leu.240446

  23. 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

  24. 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

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