Spectrum ofFANCA mutations in Italian Fanconi anemia patients: Identification of six novel alleles and phenotypic characterization of the S858R variant

Savino, Maria; Borriello, Adriana; D’Apolito, Maria; Criscuolo, Maria; Vecchio, Maria Del; Bianco, Anna Monica; Perna, Martin; Calzone, Rita; Nobili, Brúno; Zatterale, Adriana; Zelante, Leopoldo; Joenje, Hans; Ragione, Fulvio Della; Savoia, Anna

doi:10.1002/humu.9180

Cited by 28 publications

(28 citation statements)

References 26 publications

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“…[26][27][28][29] Genotype-phenotype correlation studies suggest that null mutations resulting in complete loss of the FANCA protein are the most severe, with earlier onset of anemia and a higher incidence of leukemia. 26,30 We show here that absence of FANCA results in negligible amounts of FAAP20.…”

Section: Discussionmentioning

confidence: 99%

FAAP20: a novel ubiquitin-binding FA nuclear core-complex protein required for functional integrity of the FA-BRCA DNA repair pathway

Ali

Pradhan

Singh

et al. 2012

Blood

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Fanconi anemia (FA) nuclear core complex is a multiprotein complex required for the functional integrity of the FA-BRCA pathway regulating DNA repair. This pathway is inactivated in FA, a devastating genetic disease, which leads to hematologic defects and cancer in patients. Here we report the isolation and characterization of a novel 20-kDa FANCA-associated protein (FAAP20). We show that FAAP20 is an integral component of the FA nuclear core complex. We identify a region on FANCA that physically interacts with FAAP20, and show that FANCA regulates stability of this protein. IntroductionFanconi anemia (FA) is characterized by developmental defects, bone marrow (BM) failure, and higher predisposition to both hematologic and nonhematologic cancers. 1 The primary reason for morbidity and mortality in FA patients is progressive BM failure because of the depletion of hematopoietic stem cells. 1,2 Although BM transplant significantly reduces hematologic deficiencies and improves outcomes, FA patients still have a greater risk of developing myelodysplastic syndrome, acute myeloid leukemia, and other solid tumors, such as squamous cell carcinomas. [1][2][3] Diagnostic features of the disease are increased chromosomal breaks and hypersensitivity of FA cells to DNA interstrand cross-linking (ICL) agents. 1 FAis a genetically heterogeneous disease, comprising 15 complementation groups; the genes mutated in these groups have been identified. 3 Eight of the FA proteins (FANCA, -B,-C, -E, -F, -G, -L, and -M) and 5 associated factors (FAAP100, FAAP24, HES1, MHF1, and MHF2) form the FA nuclear core complex. The core complex is required for mono-ubiquitination of FANCD2-FANCI dimer on DNA damage, which results in activation of downstream DNA repair and tolerance reactions. 3,4 The downstream FA proteins include FANCD1/BRCA2, FANCJ/BACH1, FANCN/PALB2, FANCO/RAD51C, and FANCP/ SLX4, along with FA-associated proteins FAN1, RAD18, and RAD51. 3 Together, these proteins function in the "FA-BRCA" pathway, which facilitates DNA cross-link repair and coordinates other DNA damageresponsive events, thereby stabilizing stalled replication forks, conveying signals to DNA checkpoint pathways, and facilitating recovery of replication forks. 3,4 Discovery of several new members of the core complex in the past decade contributed much to the understanding of this pathway. Despite the isolation and characterization of several core complex members, a clear understanding of the core complex is far from clear. To better understand the functions of the core complex, it is necessary to isolate and characterize all core-complex proteins and associated subcomplexes. Our previous attempt to better define the composition of the core complex led to the discovery of FANCB, FANCL, FANCM, FAAP100, MHF1, and MHF2. [5][6][7][8][9] In this study, we report the isolation and characterization of a novel core complex protein, FAAP20. Methods Cloning and constructsThe pMIEG3 retroviral vector was used for protein expression in mammalian cells. 9 The pMYFP retroviral...

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Section: Discussionmentioning

confidence: 99%

FAAP20: a novel ubiquitin-binding FA nuclear core-complex protein required for functional integrity of the FA-BRCA DNA repair pathway

Ali

Pradhan

Singh

et al. 2012

Blood

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“…Twelve of 22 were characterized functionally from the patient's cells at the RNA level. In addition to 4 mutations (c.283+3A>C; c.893+1G>T; c.1567-20A>G, and c.2504+2T>C) that we have previously described, 10 (Table 1). In the presence of c.793-1G>C, PCR on cDNA revealed two products, a faint band lacking exon 9 and another product containing the last 57 nucleotide of intron 8 due to the usage of an intronic cryptic splice site.…”

Section: © F E R R a T A S T O R T I F O U N D A T I O N Of The Firstmentioning

confidence: 93%

“…10 Six samples were first analyzed using the Ion PGM TM system for next generation sequencing of the 16 FA genes according to the manufacturer's protocols (Life Technologies). Variants with minor allele frequency less than 1% were then confirmed by Sanger sequencing as above.…”

Section: Sequencing Analysis and Multiplex Ligation-dependent Probe Amentioning

confidence: 99%

Molecular analysis of Fanconi anemia: the experience of the Bone Marrow Failure Study Group of the Italian Association of Pediatric Onco-Hematology

et al. 2014

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© F e r r a t a S t o r t i F o u n d a t i o napproximately 80% of FA patients worldwide, will not be successful in at least 20%. Secondly, there is a wide spectrum of mutations, mainly private, spanning the entire genes, including intragenic deletions, as frequently observed in FANCA (http://www.rockefeller.edu/fanconi/). Since mutational screening would largely benefit from a preliminary knowledge of the candidate gene, complementation by cell fusion or by viral transduction, as well as protein analyses, have been established as screening strategies to identify the putative gene that is defective. 6,7 Thirdly, a false negative or inconclusive chromosomal breakage test might occur in patients who develop hematopoietic mosaicism due to reversion of the cellular FA phenotype. 8,9 This phenomenon arises if a spontaneous genetic event reconstitutes normal protein activity of one allele. Since it is unlikely that the same somatic event will also occur in primary skin fibroblasts, these cells are often used for mutational screening.In this paper, we report the molecular data of 100 FA families enrolled into the National Network of the Marrow Failure Study Group of the Italian Association of Pediatric Hematology and Oncology. This cohort consists of 76 new families, as well as 24 families that have been described in previous reports. 10,11 Methods Patients, cell lines, and DNA samplesPatients with a positive chromosomal breakage test were included in this study for molecular screening of FA genes by the National Network of the Marrow Failure Study Group of the Italian Association of Pediatric Hematology and Oncology. All the subjects or their legal guardians gave written informed consent to the investigation, according to the Declaration of Helsinki. Protocols were approved by the ethics review boards of the institutions that enrolled the patients. DNA was extracted from peripheral blood, lymphoblastoid cell lines and/or primary fibroblasts. Complementation and Western blot analysesLymphoblast cell lines, peripheral blood T lymphocytes or primary fibroblasts were transduced with retroviral expressing the cDNAs for FANCA, FANCC or FANCG as previously reported. 6 Complementation was considered to occur when the viability of the transduced cells increased by more than 20% that of controls at at least three different mitomycin C concentrations. Western blot analysis of FANCD2 using a monoclonal antibody (Santa Cruz, CA, USA; diluted 1:500) was performed as previously described. 10 Sequencing analysis and multiplex ligation-dependent probe amplificationThe coding exons of the FA genes and their flanking regions were sequenced using a set of oligonucleotides (the primer sequences are available upon request) according to standard procedures. 10 Six samples were first analyzed using the Ion PGM TM system for next generation sequencing of the 16 FA genes according to the manufacturer's protocols (Life Technologies). Variants with minor allele frequency less than 1% were then confirmed by Sanger sequencing as above. N...

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“…Thus, the number of different pathogenic variants described for the FANCA gene is very high considering the relatively low number of patients. [9][10][11][12][13][14] Mutation type is also heterogeneous, including point mutations, small insertions/ deletions, splicing mutations, and large intragenic deletions. Therefore, it is necessary to combine several methodologies for mutation screening.…”

Section: Introductionmentioning

confidence: 99%

Origin, functional role, and clinical impact of Fanconi anemia FANCA mutations

Castellà

Pujol

Callén

et al. 2011

Blood

114

126

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Spectrum ofFANCA mutations in Italian Fanconi anemia patients: Identification of six novel alleles and phenotypic characterization of the S858R variant

Cited by 28 publications

References 26 publications

FAAP20: a novel ubiquitin-binding FA nuclear core-complex protein required for functional integrity of the FA-BRCA DNA repair pathway

FAAP20: a novel ubiquitin-binding FA nuclear core-complex protein required for functional integrity of the FA-BRCA DNA repair pathway

Molecular analysis of Fanconi anemia: the experience of the Bone Marrow Failure Study Group of the Italian Association of Pediatric Onco-Hematology

Origin, functional role, and clinical impact of Fanconi anemia FANCA mutations

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