Timmers, C. et al. Positional cloning of a novel Fanconi anemia gene, FANCD2. Mol. Cell 7, 241-248

Harvard University, Cambridge, Massachusetts, United States
Molecular Cell (Impact Factor: 14.02). 03/2001; 7(2):241-8. DOI: 10.1016/S1097-2765(01)00172-1
Source: PubMed


Fanconi anemia (FA) is a genetic disease with birth defects, bone marrow failure, and cancer susceptibility. To date, genes for five of the seven known complementation groups have been cloned. Complementation group D is heterogeneous, consisting of two distinct genes, FANCD1 and FANCD2. Here we report the positional cloning of FANCD2. The gene consists of 44 exons, encodes a novel 1451 amino acid nuclear protein, and has two protein isoforms. Similar to other FA proteins, the FANCD2 protein has no known functional domains, but unlike other known FA genes, FANCD2 is highly conserved in A. thaliana, C. elegans, and Drosophila. Retroviral transduction of the cloned FANCD2 cDNA into FA-D2 cells resulted in functional complementation of MMC sensitivity.

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Available from: Markus Grompe, Oct 28, 2015
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    • "Removal of ICLs is a multistep process requiring activation of the FA core complex composed of eight FA proteins (FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, and FANCM) and their interacting factors. A key step in ICL repair is the core-complex-mediated monoubiquitination of FANCD2 and FANCI (Dorsman et al., 2007; Garcia-Higuera et al., 2001; Sims et al., 2007; Smogorzewska et al., 2007; Timmers et al., 2001). Ubiquitin transfer requires the activity of an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin-ligating enzyme (reviewed in Hershko and Ciechanover, 1998). "
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    ABSTRACT: Fanconi anemia (FA) is a rare bone marrow failure and cancer predisposition syndrome resulting from pathogenic mutations in genes encoding proteins participating in the repair of DNA interstrand crosslinks (ICLs). Mutations in 17 genes (FANCA-FANCS) have been identified in FA patients, defining 17 complementation groups. Here, we describe an individual presenting with typical FA features who is deficient for the ubiquitin-conjugating enzyme (E2), UBE2T. UBE2T is known to interact with FANCL, the E3 ubiquitin-ligase component of the multiprotein FA core complex, and is necessary for the monoubiquitination of FANCD2 and FANCI. Proband fibroblasts do not display FANCD2 and FANCI monoubiquitination, do not form FANCD2 foci following treatment with mitomycin C, and are hypersensitive to crosslinking agents. These cellular defects are complemented by expression of wild-type UBE2T, demonstrating that deficiency of the protein UBE2T can lead to Fanconi anemia. UBE2T gene gains an alias of FANCT. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Full-text · Article · Jun 2015 · Cell Reports
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    • "COS-7, HeLa, and IMR90 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 12% v/v FBS, L-glutamine and penicillin/streptomycin. 293FT viral producer cells (Invitrogen) were cultured in DMEM containing 12% v/v FBS, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, L-glutamine, and penicillin/streptomycin. PD20 FA-D2 (FANCD2hy/-) cells were purchased from Coriell Cell Repositories (Catalog ID GM16633). These cells harbor a maternally inherited A-G change at nucleotide 376 that leads to the production of a severely truncated protein, and a paternally inherited missense hypomorphic (hy) mutation leading to a R1236H change [14]. To generate stable lines expressing wild type or mutant FANCD2, FA-D2 cells were infected with pLenti6.2-FANCD2 "
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    ABSTRACT: Fanconi anemia (FA) is a rare recessive disease, characterized by congenital defects, bone marrow failure, and increased cancer susceptibility. FA is caused by biallelic mutation of any one of sixteen genes. The protein products of these genes function cooperatively in the FA-BRCA pathway to repair DNA interstrand crosslinks (ICLs). A central step in the activation of this pathway is the monoubiquitination of the FANCD2 and FANCI proteins. Monoubiquitinated FANCD2 and FANCI localize to discrete chromatin regions where they function in ICL repair. Despite their critical role in ICL repair, very little is known about the structure, function, and regulation of the FANCD2 and FANCI proteins, or how they are targeted to the nucleus and chromatin. In this study, we describe the functional characterization of an amino-terminal FANCD2 nuclear localization signal (NLS). We demonstrate that the amino terminal 58 amino acids of FANCD2 can promote the nuclear expression of GFP and is necessary for the nuclear localization of FANCD2. Importantly, mutation of this FANCD2 NLS reveals that intact FANCD2 is required for the nuclear localization of a subset of FANCI. In addition, the NLS is necessary for the efficient monoubiquitination of FANCD2 and FANCI and, consequently, for their localization to chromatin. As a result, FANCD2 NLS mutants fail to rescue the ICL sensitivity of FA-D2 patient cells. Our studies yield important insight into the domain structure of the poorly characterized FANCD2 protein, and reveal a previously unknown mechanism for the coordinate nuclear import of a subset of FANCD2 and FANCI, a key early step in the cellular ICL response.
    Full-text · Article · Nov 2013 · PLoS ONE
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    • "Therefore, the data appeared unreliable for mutation detection. Subsequent Sanger sequencing of that exon demonstrated a c.376A>G base substitution resulting in another missense mutation p.S126G, that had previously been shown to be pathogenic and affects splicing [23] (Fig. 5A). Western blotting revealed distinct deficiency of the FANCD2 protein (Fig. 5B). "
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    ABSTRACT: Fanconi anemia (FA) is a rare genomic instability syndrome. Disease-causing are biallelic mutations in any one of at least 15 genes encoding members of the FA/BRCA pathway of DNA-interstrand crosslink repair. Patients are diagnosed based upon phenotypical manifestationsand the diagnosis of FA is confirmed by the hypersensitivity of cells to DNA interstrand crosslinking agents. Customary molecular diagnostics has become increasingly cumbersome, time-consuming and expensive the more FA genes have been identified. We performed Whole Exome Sequencing (WES) in four FA patients in order to investigate the potential of this method for FA genotyping. In search of an optimal WES methodology we explored different enrichment and sequencing techniques. In each case we were able to identify the pathogenic mutations so that WES provided both, complementation group assignment and mutation detection in a single approach. The mutations included homozygous and heterozygous single base pair substitutions and a two-base-pair duplication in FANCJ, -D1, or -D2. Different WES strategies had no critical influence on the individual outcome. However, database errors and in particular pseudogenes impose obstacles that may prevent correct data perception and interpretation, and thus cause pitfalls. With these difficulties in mind, our results show that WES is a valuable tool for the molecular diagnosis of FA and a sufficiently safe technique, capable of engaging increasingly in competition with classical genetic approaches.
    Full-text · Article · Dec 2012 · PLoS ONE
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