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

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

3. Spontaneous chromosomal instability Hypersensitivity to: crosslinking agents (MMC, DEB) oxygen radicals tumor necrosis factor (TNF) interferon-gamma G2 phase prolongation and/or arrest Cellular phenotype DEB test Flow cytometry Diagnosis

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

6. 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 MedulloblastomaWilms tumor Medulloblastoma Wilms tumor AML T-ALL

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

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

9. FA/BRCA pathway: a network of processes Cytoplasm Nucleus Nuclear foci (DNA replication DNA recombination DNA repair) D2 ATM Radiation Crosslinking agents S-phase BLM C A G F P D2 RAD51 BRCA1 RAD50 MRE11 NBS1 D1-BRCA2 L Ub B E USP1 ATR P J-BRIP1 M

10. (3) Molecular diagnosis: genetic heterogeneity At least 11 genes responsible for FA Low correlation between genotype and phenotype (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 Fanconi anemia: diagnostic difficulties

11. Fanconi anemia: somatic mosaicism FORWARD MOSAICISM de novo deleterious mutation Lymphocyte cultures DEB 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

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

13. 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 CfoI G FANCA-393r TGG AGG GAG ACA CTG CCA GAG CCC GCT GCG CTG CCC CGG Trp Arg Glu Thr Leu Pro Glu Pro Ala Ala Leu Pro Arg Ly FiPb1Pb2 FANCA-393r complementation CfoI digestion Waisfisz et al. Nat Genet 22: 379-383, 1999

14. 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?

15. Correlation between genotype-phenotype Significant differences PancytopeniaFA-G > FA-C AMLFA-G > FA-A and FA-C MalformationsFA-A > FA-G > FA-C No significant difference Onset of hemathologic abnormalities Requirement for transfusion Solid tumors Faivre et al, Blood 96:4064, 2000

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

17. Molecular Diagnosis Positive DEB test Screening for mutations Linkage Complementation Mutated gene PROTEIN FANCA FANCB FANCC D1-BRCA2 FANCD2 FANCE FANCF FANCG FANCJ FANCL FANCM Phenotype FA-?

18. FANCA FANCG K562 VU337 VU388 VU223 VU232 VU262 VU263 VU268 VU389 VU338 WT FANCA Del Ex18-21 Del Q264X S947X S858R 3761 ins AG IVS10+1G>T IVS26+2T>C Q772X IVS10+1G>T IVS26+2T>C Western blot analysis of FA lymphoblastoid cell lines Savino et al, Hum Mutat 22:338-339, 2003

19. Integrity of FA complex: FANCD2-Ub B Ub D2 Ub L A G E F C M EUFA232 EUFA262EUFA338 EUFA389 K562 FANCD2 FANCD2-Ub FA-A wt Savino et al, Hum Mutat 22:338-339, 2003

20. Anti- FANCA Defective Anti- FANCD2 Ub Defective Normal Defective Normal Defective FANCD2 nonUb Normal Anti- FANCB FANCC FANCE FANCF FANCG FANCL FANCM Anti- FANCD1 FANCJ FANCX POSITIVE DEB TEST FA protein analysis: prescreening strategy

21. 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

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

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

24. Defective FA/BRCA in cancers Germ cellsSomatic cellsCancers FA-/- n mutations AML, SCC 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 Leukemia ovary, pancreas Epigenetic silencing 2 FA mutations? Modified from Mathew. Oncogene 25:5875, 2006

25. 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