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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
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FANCONI ANAEMIA Clinical symptoms Progressive pancytopaenia Congenital malformations Predisposition to malignancies
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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
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Genetics Autosomal and X-linked recessive Incidence <1:100,000 live births Genetic heterogeneity
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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
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FA Complementation Groups (241 European patients) D2 D1 C B A L J I G F E Levitus et al, Blood 103:2498, 2004
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Taniguchi and D’Andrea. Blood 107:4223, 2006 Ubiquitin ligase activity DNA-dependent ATPase and 5’-3’ helicase Endonuclease and helicase activity
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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
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(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
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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
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Gene conversion Intragenic mitotic recombination Compensatory secondary mutation in cis Mechanisms for reversion
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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
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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?
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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
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FANCA screening: private mutations and intragenic deletions Savino et al, Hum Mutat 22:338-339, 2003
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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-?
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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
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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
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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
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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
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Low FANCD2 expression level Borriello et al. Leukemia 2006, doi: 10.1038/sj.leu.240446
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Identification of the Leu153Ser mutation Borriello et al. Leukemia 2006, doi: 10.1038/sj.leu.240446
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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
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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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