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Ramanagoudr-Bhojappa R, Tryon R, Lach FP, Donovan FX, Maxwell R, Rosenberg A, MacMillan ML, Wagner JE, Auerbach AD, Smogorzewska A, Chandrasekharappa SC. FANCA c.3624C>T (p.Ser1208=) is a hypomorphic splice variant associated with delayed onset of Fanconi anemia. Blood Adv 2024; 8:899-908. [PMID: 38191666 PMCID: PMC10875269 DOI: 10.1182/bloodadvances.2023011888] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2023] [Revised: 12/28/2023] [Accepted: 12/31/2023] [Indexed: 01/10/2024] Open
Abstract
ABSTRACT Fanconi anemia (FA) is a hereditary, DNA repair deficiency disorder caused by pathogenic variants in any 1 of 22 known genes (FANCA-FANCW). Variants in FANCA account for nearly two-thirds of all patients with FA. Clinical presentation of FA can be heterogeneous and include congenital abnormalities, progressive bone marrow failure, and predisposition to cancer. Here, we describe a relatively mild disease manifestation among 6 individuals diagnosed with FA, each compound heterozygous for 1 established pathogenic FANCA variant and 1 FANCA exon 36 variant, c.3624C>T. These individuals had delayed onset of hematological abnormalities, increased survival, reduced incidence of cancer, and improved fertility. Although predicted to encode a synonymous change (p.Ser1208=), the c.3624C>T variant causes a splicing error resulting in a FANCA transcript missing the last 4 base pairs of exon 36. Deep sequencing and quantitative reverse transcription polymerase chain reaction analysis revealed that 6% to 10% of the FANCA transcripts included the canonical splice product, which generated wild-type FANCA protein. Consistently, functional analysis of cell lines from the studied individuals revealed presence of residual FANCD2 ubiquitination and FANCD2 foci formation, better cell survival, and decreased late S/G2 accumulation in response to DNA interstrand cross-linking agent, indicating presence of residual activity of the FA repair pathway. Thus, the c.3624C>T variant is a hypomorphic allele, which contributes to delayed manifestation of FA disease phenotypes in individuals with at least 1 c.3624C>T allele.
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Affiliation(s)
- Ramanagouda Ramanagoudr-Bhojappa
- Cancer Genomics Unit, Cancer Genetics and Comparative Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD
| | - Rebecca Tryon
- Department of Genetics, University of Minnesota, Minneapolis, MN
| | - Francis P. Lach
- Laboratory of Genome Maintenance, The Rockefeller University, New York, NY
| | - Frank X. Donovan
- Cancer Genomics Unit, Cancer Genetics and Comparative Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD
| | - Rochelle Maxwell
- Laboratory of Genome Maintenance, The Rockefeller University, New York, NY
| | - Allana Rosenberg
- Laboratory of Genome Maintenance, The Rockefeller University, New York, NY
| | - Margaret L. MacMillan
- Division of Pediatric Blood and Marrow Transplantation, Department of Pediatrics, University of Minnesota Medical School, Minneapolis, MN
| | - John E. Wagner
- Division of Pediatric Blood and Marrow Transplantation, Department of Pediatrics, University of Minnesota Medical School, Minneapolis, MN
| | - Arleen D. Auerbach
- Human Genetics and Hematology Program, The Rockefeller University, New York, NY
| | - Agata Smogorzewska
- Laboratory of Genome Maintenance, The Rockefeller University, New York, NY
| | - Settara C. Chandrasekharappa
- Cancer Genomics Unit, Cancer Genetics and Comparative Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD
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Coleman J, Green AJ, Bradley L. The utility of multiple genomic technologies for interpretation of modern next generation sequencing: A novel case of three FANCA gene variants resulting in autosomal recessive Fanconi anaemia. Blood Cells Mol Dis 2023; 102:102762. [PMID: 37276838 DOI: 10.1016/j.bcmd.2023.102762] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2023] [Revised: 05/24/2023] [Accepted: 05/26/2023] [Indexed: 06/07/2023]
Abstract
Fanconi anaemia (FA) is a rare autosomal recessive condition resulting in changes in the FANC gene family. This report describes a case of Fanconi anaemia in a family with complex biallelic variants. The patient is a 32-year-old female diagnosed with FA on cascade testing during childhood with chromosome breakage studies. On examination she had a fixed deformity of the right thumb and the proximal interphalangeal joint was immobile. Her brother shared this radial abnormality and had FA, requiring a bone marrow transplant. She presented in adulthood seeking further BRCA advice and had next generation sequencing that showed three variants in the FANCA gene. One allele a known pathogenic change, the other had two sequence variants in tandem that have been reported as variants of uncertain significance. There is one other unrelated case of these two variants occurring together in cis, resulting in Fanconi anaemia. This case is an interesting example of three variants in the FANCA gene, one allele with a pathogenic deletion and the other with a single complex allele made up of two missense variants of uncertain significance, likely manifesting with FA. It highlights the utility of different genetic technologies in the interpretation of next generation sequencing.
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Affiliation(s)
- J Coleman
- Department of Clinical Genetics, Children's Health Ireland at Crumlin Children's Hospital, Dublin, Ireland.
| | - A J Green
- Department of Clinical Genetics, Children's Health Ireland at Crumlin Children's Hospital, Dublin, Ireland; School of Medicine, University College Dublin, Ireland
| | - L Bradley
- Department of Clinical Genetics, Children's Health Ireland at Crumlin Children's Hospital, Dublin, Ireland
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3
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Sora V, Laspiur AO, Degn K, Arnaudi M, Utichi M, Beltrame L, De Menezes D, Orlandi M, Stoltze UK, Rigina O, Sackett PW, Wadt K, Schmiegelow K, Tiberti M, Papaleo E. RosettaDDGPrediction for high-throughput mutational scans: From stability to binding. Protein Sci 2023; 32:e4527. [PMID: 36461907 PMCID: PMC9795540 DOI: 10.1002/pro.4527] [Citation(s) in RCA: 22] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2022] [Revised: 11/25/2022] [Accepted: 11/25/2022] [Indexed: 12/05/2022]
Abstract
Reliable prediction of free energy changes upon amino acid substitutions (ΔΔGs) is crucial to investigate their impact on protein stability and protein-protein interaction. Advances in experimental mutational scans allow high-throughput studies thanks to multiplex techniques. On the other hand, genomics initiatives provide a large amount of data on disease-related variants that can benefit from analyses with structure-based methods. Therefore, the computational field should keep the same pace and provide new tools for fast and accurate high-throughput ΔΔG calculations. In this context, the Rosetta modeling suite implements effective approaches to predict folding/unfolding ΔΔGs in a protein monomer upon amino acid substitutions and calculate the changes in binding free energy in protein complexes. However, their application can be challenging to users without extensive experience with Rosetta. Furthermore, Rosetta protocols for ΔΔG prediction are designed considering one variant at a time, making the setup of high-throughput screenings cumbersome. For these reasons, we devised RosettaDDGPrediction, a customizable Python wrapper designed to run free energy calculations on a set of amino acid substitutions using Rosetta protocols with little intervention from the user. Moreover, RosettaDDGPrediction assists with checking completed runs and aggregates raw data for multiple variants, as well as generates publication-ready graphics. We showed the potential of the tool in four case studies, including variants of uncertain significance in childhood cancer, proteins with known experimental unfolding ΔΔGs values, interactions between target proteins and disordered motifs, and phosphomimetics. RosettaDDGPrediction is available, free of charge and under GNU General Public License v3.0, at https://github.com/ELELAB/RosettaDDGPrediction.
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Affiliation(s)
- Valentina Sora
- Cancer Structural Biology, Danish Cancer Society Research CenterCopenhagenDenmark
- Cancer Systems Biology, Section for Bioinformatics, Department of Health and TechnologyTechnical University of DenmarkLyngbyDenmark
| | - Adrian Otamendi Laspiur
- Cancer Systems Biology, Section for Bioinformatics, Department of Health and TechnologyTechnical University of DenmarkLyngbyDenmark
| | - Kristine Degn
- Cancer Systems Biology, Section for Bioinformatics, Department of Health and TechnologyTechnical University of DenmarkLyngbyDenmark
| | - Matteo Arnaudi
- Cancer Structural Biology, Danish Cancer Society Research CenterCopenhagenDenmark
- Cancer Systems Biology, Section for Bioinformatics, Department of Health and TechnologyTechnical University of DenmarkLyngbyDenmark
| | - Mattia Utichi
- Cancer Structural Biology, Danish Cancer Society Research CenterCopenhagenDenmark
- Cancer Systems Biology, Section for Bioinformatics, Department of Health and TechnologyTechnical University of DenmarkLyngbyDenmark
| | - Ludovica Beltrame
- Cancer Structural Biology, Danish Cancer Society Research CenterCopenhagenDenmark
- Cancer Systems Biology, Section for Bioinformatics, Department of Health and TechnologyTechnical University of DenmarkLyngbyDenmark
| | - Dayana De Menezes
- Cancer Systems Biology, Section for Bioinformatics, Department of Health and TechnologyTechnical University of DenmarkLyngbyDenmark
| | - Matteo Orlandi
- Cancer Systems Biology, Section for Bioinformatics, Department of Health and TechnologyTechnical University of DenmarkLyngbyDenmark
| | - Ulrik Kristoffer Stoltze
- Department of Clinical GeneticsCopenhagen University Hospital RigshospitaletCopenhagenDenmark
- Department of Pediatrics and Adolescent MedicineUniversity Hospital RigshospitaletCopenhagenDenmark
- Institute of Clinical Medicine, Faculty of MedicineUniversity of CopenhagenCopenhagenDenmark
| | - Olga Rigina
- Cancer Systems Biology, Section for Bioinformatics, Department of Health and TechnologyTechnical University of DenmarkLyngbyDenmark
| | - Peter Wad Sackett
- Cancer Systems Biology, Section for Bioinformatics, Department of Health and TechnologyTechnical University of DenmarkLyngbyDenmark
| | - Karin Wadt
- Department of Clinical GeneticsCopenhagen University Hospital RigshospitaletCopenhagenDenmark
- Institute of Clinical Medicine, Faculty of MedicineUniversity of CopenhagenCopenhagenDenmark
| | - Kjeld Schmiegelow
- Department of Pediatrics and Adolescent MedicineUniversity Hospital RigshospitaletCopenhagenDenmark
- Institute of Clinical Medicine, Faculty of MedicineUniversity of CopenhagenCopenhagenDenmark
| | - Matteo Tiberti
- Cancer Structural Biology, Danish Cancer Society Research CenterCopenhagenDenmark
| | - Elena Papaleo
- Cancer Structural Biology, Danish Cancer Society Research CenterCopenhagenDenmark
- Cancer Systems Biology, Section for Bioinformatics, Department of Health and TechnologyTechnical University of DenmarkLyngbyDenmark
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Sharma P, Sharma N, Sharma D. A Narrative Review on Fanconi Anemia: Genetic and Diagnostic Considerations. Glob Med Genet 2022; 9:237-241. [PMID: 36071913 PMCID: PMC9444348 DOI: 10.1055/s-0042-1751303] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2022] [Accepted: 05/19/2022] [Indexed: 11/18/2022] Open
Abstract
Fanconi anemia (FA) is an autosomal recessive disorder, both genetically and phenotypically. It is characterized by chromosomal instability, progressive bone marrow failure, susceptibility to cancer, and various other congenital abnormalities. It involves all the three cell lines of blood. So far, biallelic mutations in 21 genes and one x-linked gene have been detected and found to be associated with FA phenotype. Signs and symptoms start setting in by the age of 4 to 7 years, mainly hematological symptoms. This includes pancytopenia, that is, a reduction in the number of white blood cells (WBCs), red blood cells (RBCs), and platelets. Therefore, the main criteria for diagnosis of FA include skeletal malformations, pancytopenia, hyperpigmentation, short stature, urogenital abnormalities, central nervous system, auditory, renal, ocular, and familial occurrence. Patients showing signs and symptoms of FA should be thoroughly evaluated. A complete blood count will reveal a reduced number of RBC, WBC, and platelets, that is, pancytopenia. Chromosomal breakage study/stress cytogenetics should be done in patients with severe pancytopenia. Momentousness timely diagnosis of current disease, prenatal diagnosis, and genetic counseling should be emphasized.
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Affiliation(s)
- Preksha Sharma
- Department of Anatomy, SMS Medical College and Attached Hospitals, Jaipur, Rajasthan, India
| | - Neha Sharma
- Department of Pharmacology, SMS Medical College and Attached Hospitals, Jaipur, Rajasthan, India
| | - Dhruva Sharma
- Department of Cardiothoracic and Vascular Surgery, SMS Medical College and Attached Hospitals, Jaipur, Rajasthan, India
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Chihanga T, Vicente-Muñoz S, Ruiz-Torres S, Pal B, Sertorio M, Andreassen PR, Khoury R, Mehta P, Davies SM, Lane AN, Romick-Rosendale LE, Wells SI. Head and Neck Cancer Susceptibility and Metabolism in Fanconi Anemia. Cancers (Basel) 2022; 14:cancers14082040. [PMID: 35454946 PMCID: PMC9025423 DOI: 10.3390/cancers14082040] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2022] [Revised: 04/05/2022] [Accepted: 04/11/2022] [Indexed: 02/06/2023] Open
Abstract
Fanconi anemia (FA) is a rare inherited, generally autosomal recessive syndrome, but it displays X-linked or dominant negative inheritance for certain genes. FA is characterized by a deficiency in DNA damage repair that results in bone marrow failure, and in an increased risk for various epithelial tumors, most commonly squamous cell carcinomas of the head and neck (HNSCC) and of the esophagus, anogenital tract and skin. Individuals with FA exhibit increased human papilloma virus (HPV) prevalence. Furthermore, a subset of anogenital squamous cell carcinomas (SCCs) in FA harbor HPV sequences and FA-deficient laboratory models reveal molecular crosstalk between HPV and FA proteins. However, a definitive role for HPV in HNSCC development in the FA patient population is unproven. Cellular metabolism plays an integral role in tissue homeostasis, and metabolic deregulation is a known hallmark of cancer progression that supports uncontrolled proliferation, tumor development and metastatic dissemination. The metabolic consequences of FA deficiency in keratinocytes and associated impact on the development of SCC in the FA population is poorly understood. Herein, we review the current literature on the metabolic consequences of FA deficiency and potential effects of resulting metabolic reprogramming on FA cancer phenotypes.
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Affiliation(s)
- Tafadzwa Chihanga
- Division of Oncology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229, USA; (T.C.); (S.R.-T.); (B.P.)
| | - Sara Vicente-Muñoz
- Department of Pathology and Laboratory Medicine, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229, USA; (S.V.-M.); (L.E.R.-R.)
| | - Sonya Ruiz-Torres
- Division of Oncology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229, USA; (T.C.); (S.R.-T.); (B.P.)
| | - Bidisha Pal
- Division of Oncology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229, USA; (T.C.); (S.R.-T.); (B.P.)
| | - Mathieu Sertorio
- Department of Radiation Oncology, College of Medicine, University of Cincinnati, Cincinnati, OH 45229, USA;
| | - Paul R. Andreassen
- Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229, USA;
| | - Ruby Khoury
- Division of Bone Marrow Transplantation and Immune Deficiency, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229, USA; (R.K.); (P.M.); (S.M.D.)
| | - Parinda Mehta
- Division of Bone Marrow Transplantation and Immune Deficiency, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229, USA; (R.K.); (P.M.); (S.M.D.)
| | - Stella M. Davies
- Division of Bone Marrow Transplantation and Immune Deficiency, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229, USA; (R.K.); (P.M.); (S.M.D.)
| | - Andrew N. Lane
- Department of Toxicology and Cancer Biology, University of Kentucky, Lexington, KY 40536, USA;
| | - Lindsey E. Romick-Rosendale
- Department of Pathology and Laboratory Medicine, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229, USA; (S.V.-M.); (L.E.R.-R.)
| | - Susanne I. Wells
- Division of Oncology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229, USA; (T.C.); (S.R.-T.); (B.P.)
- Correspondence: ; Tel.: +1-513-636-5986
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6
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Gueiderikh A, Maczkowiak-Chartois F, Rosselli F. A new frontier in Fanconi anemia: From DNA repair to ribosome biogenesis. Blood Rev 2021; 52:100904. [PMID: 34750031 DOI: 10.1016/j.blre.2021.100904] [Citation(s) in RCA: 24] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2021] [Revised: 10/14/2021] [Accepted: 10/26/2021] [Indexed: 12/27/2022]
Abstract
Described by Guido Fanconi almost 100 years ago, Fanconi anemia (FA) is a rare genetic disease characterized by developmental abnormalities, bone marrow failure (BMF) and cancer predisposition. The proteins encoded by FA-mutated genes (FANC proteins) and assembled in the so-called FANC/BRCA pathway have key functions in DNA repair and replication safeguarding, which loss leads to chromosome structural aberrancies. Therefore, since the 1980s, FA has been considered a genomic instability and chromosome fragility syndrome. However, recent findings have demonstrated new and unexpected roles of FANC proteins in nucleolar homeostasis and ribosome biogenesis, the alteration of which impacts cellular proteostasis. Here, we review the different cellular, biochemical and molecular anomalies associated with the loss of function of FANC proteins and discuss how these anomalies contribute to BMF by comparing FA to other major inherited BMF syndromes. Our aim is to determine the extent to which alterations in the DNA damage response in FA contribute to BMF compared to the consequences of the loss of function of the FANC/BRCA pathway on the other roles of the pathway.
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Affiliation(s)
- Anna Gueiderikh
- CNRS - UMR9019, Équipe labellisée "La Ligue contre le Cancer", 94805 Villejuif, France; Gustave Roussy Cancer Center, 94805 Villejuif, France; Université Paris-Saclay - Paris Sud, Orsay, France.
| | - Frédérique Maczkowiak-Chartois
- CNRS - UMR9019, Équipe labellisée "La Ligue contre le Cancer", 94805 Villejuif, France; Gustave Roussy Cancer Center, 94805 Villejuif, France; Université Paris-Saclay - Paris Sud, Orsay, France.
| | - Filippo Rosselli
- CNRS - UMR9019, Équipe labellisée "La Ligue contre le Cancer", 94805 Villejuif, France; Gustave Roussy Cancer Center, 94805 Villejuif, France; Université Paris-Saclay - Paris Sud, Orsay, France.
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7
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Katsuki Y, Abe M, Park SY, Wu W, Yabe H, Yabe M, van Attikum H, Nakada S, Ohta T, Seidman MM, Kim Y, Takata M. RNF168 E3 ligase participates in ubiquitin signaling and recruitment of SLX4 during DNA crosslink repair. Cell Rep 2021; 37:109879. [PMID: 34706224 PMCID: PMC11388903 DOI: 10.1016/j.celrep.2021.109879] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2020] [Revised: 03/24/2021] [Accepted: 10/01/2021] [Indexed: 12/11/2022] Open
Abstract
SLX4/FANCP is a key Fanconi anemia (FA) protein and a DNA repair scaffold for incision around a DNA interstrand crosslink (ICL) by its partner XPF nuclease. The tandem UBZ4 ubiquitin-binding domains of SLX4 are critical for the recruitment of SLX4 to damage sites, likely by binding to K63-linked polyubiquitin chains. However, the identity of the ubiquitin E3 ligase that mediates SLX4 recruitment remains unknown. Using small interfering RNA (siRNA) screening with a GFP-tagged N-terminal half of SLX4 (termed SLX4-N), we identify the RNF168 E3 ligase as a critical factor for mitomycin C (MMC)-induced SLX4 foci formation. RNF168 and GFP-SLX4-N colocalize in MMC-induced ubiquitin foci. Accumulation of SLX4-N at psoralen-laser ICL tracks or of endogenous SLX4 at Digoxigenin-psoralen/UVA ICL is dependent on RNF168. Finally, we find that RNF168 is epistatic with SLX4 in promoting MMC tolerance. We conclude that RNF168 is a critical component of the signal transduction that recruits SLX4 to ICL damage.
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Affiliation(s)
- Yoko Katsuki
- Laboratory of DNA Damage Signaling, Department of Late Effects Studies, Radiation Biology Center, Graduate School of Biostudies, Kyoto University, Kyoto, Japan.
| | - Masako Abe
- The Core Facility, Radiation Biology Center, Graduate School of Biostudies, Kyoto University, Kyoto, Japan
| | - Seon Young Park
- Department of Biological Sciences, Sookmyung Women's University, Seoul, Republic of Korea
| | - Wenwen Wu
- Department of Translational Oncology, St. Marianna University Graduate School of Medicine, Kawasaki, Japan
| | - Hiromasa Yabe
- Department of Innovative Medical Science, Tokai University School of Medicine, Isehara, Japan
| | - Miharu Yabe
- Department of Innovative Medical Science, Tokai University School of Medicine, Isehara, Japan
| | - Haico van Attikum
- Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands
| | - Shinichiro Nakada
- Department of Bioregulation and Cellular Response, Graduate School of Medicine, Osaka University, Osaka, Japan; Institute for Advanced Co-Creation Studies, Osaka University, Osaka, Japan
| | - Tomohiko Ohta
- Department of Translational Oncology, St. Marianna University Graduate School of Medicine, Kawasaki, Japan
| | - Michael M Seidman
- Laboratory of Molecular Biology and Immunology, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA
| | - Yonghwan Kim
- Department of Biological Sciences, Sookmyung Women's University, Seoul, Republic of Korea
| | - Minoru Takata
- Laboratory of DNA Damage Signaling, Department of Late Effects Studies, Radiation Biology Center, Graduate School of Biostudies, Kyoto University, Kyoto, Japan.
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Structural insight into FANCI-FANCD2 monoubiquitination. Essays Biochem 2021; 64:807-817. [PMID: 32725171 PMCID: PMC7588663 DOI: 10.1042/ebc20200001] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2020] [Revised: 06/10/2020] [Accepted: 07/07/2020] [Indexed: 12/13/2022]
Abstract
The Fanconi anemia (FA) pathway coordinates a faithful repair mechanism for DNA damage that blocks DNA replication, such as interstrand cross-links. A key step in the FA pathway is the conjugation of ubiquitin on to FANCD2 and FANCI, which is facilitated by a large E3 ubiquitin ligase complex called the FA core complex. Mutations in FANCD2, FANCI or FA core complex components cause the FA bone marrow failure syndrome. Despite the importance of these proteins to DNA repair and human disease, our molecular understanding of the FA pathway has been limited due to a deficit in structural studies. With the recent development in cryo-electron microscopy (EM), significant advances have been made in structural characterization of these proteins in the last 6 months. These structures, combined with new biochemical studies, now provide a more detailed understanding of how FANCD2 and FANCI are monoubiquitinated and how DNA repair may occur. In this review, we summarize these recent advances in the structural and molecular understanding of these key components in the FA pathway, compare the activation steps of FANCD2 and FANCI monoubiquitination and suggest molecular steps that are likely to be involved in regulating its activity.
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Structure of the FA core ubiquitin ligase closing the ID clamp on DNA. Nat Struct Mol Biol 2021; 28:300-309. [PMID: 33686268 PMCID: PMC8378520 DOI: 10.1038/s41594-021-00568-8] [Citation(s) in RCA: 29] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2020] [Accepted: 01/29/2021] [Indexed: 01/31/2023]
Abstract
The Fanconi anemia (FA) pathway is essential for the repair of DNA interstrand crosslinks. Central to the pathway is the FA core complex, a ubiquitin ligase of nine subunits that monoubiquitinates the FANCI-FANCD2 (ID) DNA clamp. The 3.1 Å structure of the 1.1-MDa human FA core complex, described here, reveals an asymmetric assembly with two copies of all but the FANCC, FANCE and FANCF subunits. The asymmetry is crucial, as it prevents the binding of a second FANCC-FANCE-FANCF subcomplex that inhibits the recruitment of the UBE2T ubiquitin conjugating enzyme, and instead creates an ID binding site. A single active site then ubiquitinates FANCD2 and FANCI sequentially. We also present the 4.2-Å structures of the human core-UBE2T-ID-DNA complex in three conformations captured during monoubiquitination. They reveal the core-UBE2T complex remodeling the ID-DNA complex, closing the clamp on the DNA before ubiquitination. Monoubiquitination then prevents clamp opening after release from the core.
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Gueiderikh A, Maczkowiak-Chartois F, Rouvet G, Souquère-Besse S, Apcher S, Diaz JJ, Rosselli F. Fanconi anemia A protein participates in nucleolar homeostasis maintenance and ribosome biogenesis. SCIENCE ADVANCES 2021; 7:7/1/eabb5414. [PMID: 33523834 PMCID: PMC7775781 DOI: 10.1126/sciadv.abb5414] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/03/2020] [Accepted: 10/28/2020] [Indexed: 05/22/2023]
Abstract
Fanconi anemia (FA), the most common inherited bone marrow failure and leukemia predisposition syndrome, is generally attributed to alterations in DNA damage responses due to the loss of function of the DNA repair and replication rescue activities of the FANC pathway. Here, we report that FANCA deficiency, whose inactivation has been identified in two-thirds of FA patients, is associated with nucleolar homeostasis loss, mislocalization of key nucleolar proteins, including nucleolin (NCL) and nucleophosmin 1 (NPM1), as well as alterations in ribosome biogenesis and protein synthesis. FANCA coimmunoprecipitates with NCL and NPM1 in a FANCcore complex-independent manner and, unique among the FANCcore complex proteins, associates with ribosomal subunits, influencing the stoichiometry of the translational machineries. In conclusion, we have identified unexpected nucleolar and translational consequences specifically associated with FANCA deficiency that appears to be involved in both DNA damage and nucleolar stress responses, challenging current hypothesis on FA physiopathology.
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Affiliation(s)
- Anna Gueiderikh
- CNRS-UMR9019, Équipe labellisée "La Ligue contre le Cancer," 94805 Villejuif, France
- Gustave Roussy Cancer Center, 94805 Villejuif, France
- Université Paris-Saclay-Paris Sud, Orsay, France
| | - Frédérique Maczkowiak-Chartois
- CNRS-UMR9019, Équipe labellisée "La Ligue contre le Cancer," 94805 Villejuif, France
- Gustave Roussy Cancer Center, 94805 Villejuif, France
- Université Paris-Saclay-Paris Sud, Orsay, France
| | - Guillaume Rouvet
- CNRS-UMR9019, Équipe labellisée "La Ligue contre le Cancer," 94805 Villejuif, France
- Gustave Roussy Cancer Center, 94805 Villejuif, France
- Université Paris-Saclay-Paris Sud, Orsay, France
| | - Sylvie Souquère-Besse
- Gustave Roussy Cancer Center, 94805 Villejuif, France
- Université Paris-Saclay-Paris Sud, Orsay, France
- CNRS-UMS3655, 94805 Villejuif, France
| | - Sébastien Apcher
- Gustave Roussy Cancer Center, 94805 Villejuif, France
- Université Paris-Saclay-Paris Sud, Orsay, France
- INSERM-UMR1015, 94805 Villejuif, France
| | - Jean-Jacques Diaz
- Université Lyon, Université Claude Bernard Lyon 1, Inserm 1052, CNRS 5286, Centre Léon Bérard, Cancer Research Center of Lyon, 69373 Lyon cedex 08, France
| | - Filippo Rosselli
- CNRS-UMR9019, Équipe labellisée "La Ligue contre le Cancer," 94805 Villejuif, France.
- Gustave Roussy Cancer Center, 94805 Villejuif, France
- Université Paris-Saclay-Paris Sud, Orsay, France
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11
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Lach FP, Singh S, Rickman KA, Ruiz PD, Noonan RJ, Hymes KB, DeLacure MD, Kennedy JA, Chandrasekharappa SC, Smogorzewska A. Esophageal cancer as initial presentation of Fanconi anemia in patients with a hypomorphic FANCA variant. Cold Spring Harb Mol Case Stud 2020; 6:mcs.a005595. [PMID: 33172906 PMCID: PMC7784490 DOI: 10.1101/mcs.a005595] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2020] [Accepted: 10/26/2020] [Indexed: 11/24/2022] Open
Abstract
Fanconi anemia (FA) is a clinically heterogenous and genetically diverse disease with 22 known complementation groups (FA-A to FA-W), resulting from the inability to repair DNA interstrand cross-links. This rare disorder is characterized by congenital defects, bone marrow failure, and cancer predisposition. FANCA is the most commonly mutated gene in FA and a variety of mostly private mutations have been documented, including small and large indels and point and splicing variants. Genotype-phenotype associations in FA are complex, and a relationship between particular FANCA variants and the observed cellular phenotype or illness severity remains unclear. In this study, we describe two siblings with compound heterozygous FANCA variants (c.3788_3790delTCT and c.4199G > A) who both presented with esophageal squamous cell carcinoma at the age of 51. The proband came to medical attention when he developed pancytopenia after a single cycle of low-dose chemotherapy including platinum-based therapy. Other than a minor thumb abnormality, neither patient had prior findings to suggest FA, including normal blood counts and intact fertility. Patient fibroblasts from both siblings display increased chromosomal breakage and hypersensitivity to interstrand cross-linking agents as seen in typical FA. Based on our functional data demonstrating that the c.4199G > A/p.R1400H variant represents a hypomorphic FANCA allele, we conclude that the residual activity of the Fanconi anemia repair pathway accounts for lack of spontaneous bone marrow failure or infertility with the late presentation of malignancy as the initial disease manifestation. This and similar cases of adult-onset esophageal cancer stress the need for chromosome breakage testing in patients with early onset of aerodigestive tract squamous cell carcinomas before platinum-based therapy is initiated.
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Affiliation(s)
- Francis P Lach
- Laboratory of Genome Maintenance, The Rockefeller University, New York, New York, 10065 USA
| | - Sonia Singh
- Laboratory of Genome Maintenance, The Rockefeller University, New York, New York, 10065 USA.,Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, New York, 10065 USA
| | - Kimberly A Rickman
- Laboratory of Genome Maintenance, The Rockefeller University, New York, New York, 10065 USA
| | - Penelope D Ruiz
- Laboratory of Genome Maintenance, The Rockefeller University, New York, New York, 10065 USA
| | - Raymond J Noonan
- Laboratory of Genome Maintenance, The Rockefeller University, New York, New York, 10065 USA
| | - Kenneth B Hymes
- New York University School of Medicine, Division of Hematology and Oncology, Department of Internal Medicine, Laura and Isaac Perlmutter Cancer Center, New York, New York, 10016 USA
| | - Mark D DeLacure
- Department of Otolaryngology-Head and Neck Surgery, New York University School of Medicine, New York, New York 10003, USA.,Departments of Plastic Surgery and Neurosurgery, New York University, New York, New York 10016, USA
| | - Jennifer A Kennedy
- Laboratory of Genome Maintenance, The Rockefeller University, New York, New York, 10065 USA
| | - Settara C Chandrasekharappa
- Cancer Genetics and Comparative Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892, USA
| | - Agata Smogorzewska
- Laboratory of Genome Maintenance, The Rockefeller University, New York, New York, 10065 USA
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12
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The FANC/BRCA Pathway Releases Replication Blockades by Eliminating DNA Interstrand Cross-Links. Genes (Basel) 2020; 11:genes11050585. [PMID: 32466131 PMCID: PMC7288313 DOI: 10.3390/genes11050585] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2020] [Revised: 05/14/2020] [Accepted: 05/21/2020] [Indexed: 12/24/2022] Open
Abstract
DNA interstrand cross-links (ICLs) represent a major barrier blocking DNA replication fork progression. ICL accumulation results in growth arrest and cell death—particularly in cell populations undergoing high replicative activity, such as cancer and leukemic cells. For this reason, agents able to induce DNA ICLs are widely used as chemotherapeutic drugs. However, ICLs are also generated in cells as byproducts of normal metabolic activities. Therefore, every cell must be capable of rescuing lCL-stalled replication forks while maintaining the genetic stability of the daughter cells in order to survive, replicate DNA and segregate chromosomes at mitosis. Inactivation of the Fanconi anemia/breast cancer-associated (FANC/BRCA) pathway by inherited mutations leads to Fanconi anemia (FA), a rare developmental, cancer-predisposing and chromosome-fragility syndrome. FANC/BRCA is the key hub for a complex and wide network of proteins that—upon rescuing ICL-stalled DNA replication forks—allows cell survival. Understanding how cells cope with ICLs is mandatory to ameliorate ICL-based anticancer therapies and provide the molecular basis to prevent or bypass cancer drug resistance. Here, we review our state-of-the-art understanding of the mechanisms involved in ICL resolution during DNA synthesis, with a major focus on how the FANC/BRCA pathway ensures DNA strand opening and prevents genomic instability.
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13
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Jeong E, Lee SG, Kim HS, Yang J, Shin J, Kim Y, Kim J, Schärer OD, Kim Y, Yeo JE, Kim HM, Cho Y. Structural basis of the fanconi anemia-associated mutations within the FANCA and FANCG complex. Nucleic Acids Res 2020; 48:3328-3342. [PMID: 32002546 PMCID: PMC7102982 DOI: 10.1093/nar/gkaa062] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2019] [Revised: 01/17/2020] [Accepted: 01/21/2020] [Indexed: 12/13/2022] Open
Abstract
Monoubiquitination of the Fanconi anemia complementation group D2 (FANCD2) protein by the FA core ubiquitin ligase complex is the central event in the FA pathway. FANCA and FANCG play major roles in the nuclear localization of the FA core complex. Mutations of these two genes are the most frequently observed genetic alterations in FA patients, and most point mutations in FANCA are clustered in the C-terminal domain (CTD). To understand the basis of the FA-associated FANCA mutations, we determined the cryo-electron microscopy (EM) structures of Xenopus laevis FANCA alone at 3.35 Å and 3.46 Å resolution and two distinct FANCA–FANCG complexes at 4.59 and 4.84 Å resolution, respectively. The FANCA CTD adopts an arc-shaped solenoid structure that forms a pseudo-symmetric dimer through its outer surface. FA- and cancer-associated point mutations are widely distributed over the CTD. The two different complex structures capture independent interactions of FANCG with either FANCA C-terminal HEAT repeats, or the N-terminal region. We show that mutations that disturb either of these two interactions prevent the nuclear localization of FANCA, thereby leading to an FA pathway defect. The structure provides insights into the function of FANCA CTD, and provides a framework for understanding FA- and cancer-associated mutations.
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Affiliation(s)
- Eunyoung Jeong
- Department of Life Science, Pohang University of Science and Technology, Pohang, 37673, Republic of Korea
| | - Seong-Gyu Lee
- Center for Biomolecular and Cellular Structure, Institute for Basic Science (IBS), Daejeon, 34141, Republic of Korea.,Graduate School of Medical Science & Engineering, Korea Advanced Institute of Science and Technology, Daejeon, 34141, Republic of Korea
| | - Hyun-Suk Kim
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan, 44919, Republic of Korea
| | - Jihyeon Yang
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan, 44919, Republic of Korea
| | - Jinwoo Shin
- Department of Life Science, Pohang University of Science and Technology, Pohang, 37673, Republic of Korea
| | - Youngran Kim
- Department of Life Science, Pohang University of Science and Technology, Pohang, 37673, Republic of Korea
| | - Jihan Kim
- Department of Life Science, Pohang University of Science and Technology, Pohang, 37673, Republic of Korea
| | - Orlando D Schärer
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan, 44919, Republic of Korea.,Department of Biological Sciences, School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), Ulsan, 44919, Republic of Korea
| | - Youngjin Kim
- Department of Life Science, Pohang University of Science and Technology, Pohang, 37673, Republic of Korea
| | - Jung-Eun Yeo
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan, 44919, Republic of Korea
| | - Ho Min Kim
- Center for Biomolecular and Cellular Structure, Institute for Basic Science (IBS), Daejeon, 34141, Republic of Korea.,Graduate School of Medical Science & Engineering, Korea Advanced Institute of Science and Technology, Daejeon, 34141, Republic of Korea
| | - Yunje Cho
- Department of Life Science, Pohang University of Science and Technology, Pohang, 37673, Republic of Korea
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14
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Engel NW, Schliffke S, Schüller U, Frenzel C, Bokemeyer C, Kubisch C, Lessel D. Fatal Myelotoxicity Following Palliative Chemotherapy With Cisplatin and Gemcitabine in a Patient With Stage IV Cholangiocarcinoma Linked to Post Mortem Diagnosis of Fanconi Anemia. Front Oncol 2019; 9:420. [PMID: 31192125 PMCID: PMC6540739 DOI: 10.3389/fonc.2019.00420] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2019] [Accepted: 05/03/2019] [Indexed: 12/16/2022] Open
Abstract
Unrecognized genome instability syndromes can potentially impede the rational treatment of cancer in rare patients. Identification of cancer patients with a hereditary condition is a compelling necessity for oncologists, giving varying hypersensitivities to various chemotherapeutic agents or radiation, depending on the underlying genetic cause. Omission of genetic testing in the setting of an overlooked hereditary syndrome may lead to unexpected and unbearable toxicity from oncological standard approaches. We present a case of a 33-year-old man with an early-onset stage IV intrahepatic cholangiocarcinoma, who experienced unusual bone marrow failure and neutropenic fever syndrome as a consequence of palliative chemotherapy containing cisplatin and gemcitabine, leading to a fatal outcome on day 25 of his first chemotherapeutic cycle. The constellation of bone marrow failure after exposure to the platinum-based agent cisplatin, the presence of an early-onset solid malignancy and the critical appraisal of further phenotypical features raised suspicion of a hereditary genome instability syndrome. Whole-exome sequencing from buccal swab DNA enabled the post mortem diagnosis of Fanconi anemia, most likely linked to the fatal outcome due to utilization of the DNA crosslinking agent cisplatin. The patient's phenotype was exceptional, as he never displayed significant hematologic abnormalities, which is the hallmark of Fanconi anemia. As such, this case stresses the importance to at least question the possibility of a hereditary basis in cases of relatively early-onset malignancy before defining an oncological treatment strategy.
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Affiliation(s)
- Nils W Engel
- Department of Oncology, Haematology and Bone Marrow Transplantation with Section Pneumology, Hubertus Wald Tumorzentrum, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Simon Schliffke
- Department of Oncology, Haematology and Bone Marrow Transplantation with Section Pneumology, Hubertus Wald Tumorzentrum, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Ulrich Schüller
- Institute of Neuropathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.,Department of Pediatric Hematology and Oncology, University Medical Center, Hamburg-Eppendorf, Germany.,Research Institute Children's Cancer Center Hamburg, Hamburg, Germany
| | - Christian Frenzel
- Department of Oncology, Haematology and Bone Marrow Transplantation with Section Pneumology, Hubertus Wald Tumorzentrum, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Carsten Bokemeyer
- Department of Oncology, Haematology and Bone Marrow Transplantation with Section Pneumology, Hubertus Wald Tumorzentrum, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Christian Kubisch
- Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Davor Lessel
- Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
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15
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Huang JP, Lin J, Tzen CY, Huang WY, Tsai CC, Chen CJ, Lu YJ, Chou KF, Su YW. FANCA D1359Y mutation in a patient with gastric polyposis and cancer susceptibility: A case report and review of literature. World J Gastroenterol 2018; 24:4412-4418. [PMID: 30344425 PMCID: PMC6189845 DOI: 10.3748/wjg.v24.i38.4412] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/19/2018] [Revised: 08/02/2018] [Accepted: 08/24/2018] [Indexed: 02/06/2023] Open
Abstract
Gastric polyposis is a rare disease. Not all polyps progress to cancer. Monoallelic mutation in Fanconi anemia (FA) genes, unlike biallelic gene mutations that causes typical FA phenotype, can increase risks of cancers in a sporadic manner. Aberrations in the FA pathway were reported in all molecular subtypes of gastric cancer. We studied a patient with synchronous gastric cancer from gastric polyposis by conducting a 13-year long-term follow up. Via pathway-driven massive parallel genomic sequencing, a germline mutation at FANCA D1359Y was identified. We identified several recurrent mutations in DNA methylation (TET1, V873I), the β-catenin pathway (CTNNB1, S45F) and RHO signaling pathway (PLEKHG5, R203C) by comparing the genetic events between benign and malignant gastric polyps. Furthermore, we revealed gastric polyposis susceptible genes and genetic events promoting malignant transformation using pathway-driven targeted gene sequencing.
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Affiliation(s)
- Jeffrey Peng Huang
- Division of Hematology and Medical Oncology, Department of Internal Medicine, Mackay Memorial Hospital, Taipei 10491, Taiwan
| | - Johnson Lin
- Division of Hematology and Medical Oncology, Department of Internal Medicine, Mackay Memorial Hospital, Taipei 10491, Taiwan
| | - Chi-Yuan Tzen
- Department of Pathology, Mackay Memorial Hospital, Taipei 10491, Taiwan
| | - Wen-Yu Huang
- Laboratory of Good Clinical Research Center, Mackay Memorial Hospital, Tamsui Branch, New Taipei City 25160, Taiwan
| | - Chia-Chi Tsai
- Department of General Surgery, Mackay Memorial Hospital, Taipei 10491, Taiwan
| | - Chih-Jen Chen
- Division of Gastroenterology, Department of Internal Medicine, Mackay Memorial Hospital, Taipei 10491, Taiwan
| | - Yen-Jung Lu
- ACT Genomics Co., Ltd., Taipei 11494, Taiwan
| | - Kuei-Fang Chou
- Division of Hematology and Medical Oncology, Department of Internal Medicine, Mackay Memorial Hospital, Taipei 10491, Taiwan
| | - Ying-Wen Su
- Division of Hematology and Medical Oncology, Department of Internal Medicine, Mackay Memorial Hospital, Taipei 10491, Taiwan
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16
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Benitez A, Liu W, Palovcak A, Wang G, Moon J, An K, Kim A, Zheng K, Zhang Y, Bai F, Mazin AV, Pei XH, Yuan F, Zhang Y. FANCA Promotes DNA Double-Strand Break Repair by Catalyzing Single-Strand Annealing and Strand Exchange. Mol Cell 2018; 71:621-628.e4. [PMID: 30057198 PMCID: PMC6097932 DOI: 10.1016/j.molcel.2018.06.030] [Citation(s) in RCA: 66] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2017] [Revised: 02/04/2018] [Accepted: 06/20/2018] [Indexed: 01/24/2023]
Abstract
FANCA is a component of the Fanconi anemia (FA) core complex that activates DNA interstrand crosslink repair by monoubiquitination of FANCD2. Here, we report that purified FANCA protein catalyzes bidirectional single-strand annealing (SA) and strand exchange (SE) at a level comparable to RAD52, while a disease-causing FANCA mutant, F1263Δ, is defective in both activities. FANCG, which directly interacts with FANCA, dramatically stimulates its SA and SE activities. Alternatively, FANCB, which does not directly interact with FANCA, does not stimulate this activity. Importantly, five other patient-derived FANCA mutants also exhibit deficient SA and SE, suggesting that the biochemical activities of FANCA are relevant to the etiology of FA. A cell-based DNA double-strand break (DSB) repair assay demonstrates that FANCA plays a direct role in the single-strand annealing sub-pathway (SSA) of DSB repair by catalyzing SA, and this role is independent of the canonical FA pathway and RAD52.
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Affiliation(s)
- Anaid Benitez
- Department of Biochemistry & Molecular Biology, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Wenjun Liu
- Department of Biochemistry & Molecular Biology, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Anna Palovcak
- Department of Biochemistry & Molecular Biology, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Guanying Wang
- Department of Biochemistry & Molecular Biology, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Jaewon Moon
- Department of Biochemistry & Molecular Biology, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Kevin An
- Department of Biochemistry & Molecular Biology, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Anna Kim
- Department of Biochemistry & Molecular Biology, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Kevin Zheng
- Department of Biochemistry & Molecular Biology, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Yu Zhang
- Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Feng Bai
- Department of Surgery, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Alexander V Mazin
- Department of Biochemistry & Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102, USA
| | - Xin-Hai Pei
- Department of Surgery, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Fenghua Yuan
- Department of Biochemistry & Molecular Biology, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Yanbin Zhang
- Department of Biochemistry & Molecular Biology, University of Miami Miller School of Medicine, Miami, FL 33136, USA.
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17
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Kimble DC, Lach FP, Gregg SQ, Donovan FX, Flynn EK, Kamat A, Young A, Vemulapalli M, Thomas JW, Mullikin JC, Auerbach AD, Smogorzewska A, Chandrasekharappa SC. A comprehensive approach to identification of pathogenic FANCA variants in Fanconi anemia patients and their families. Hum Mutat 2018; 39:237-254. [PMID: 29098742 PMCID: PMC5762269 DOI: 10.1002/humu.23366] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2017] [Revised: 10/20/2017] [Accepted: 10/22/2017] [Indexed: 11/11/2022]
Abstract
Fanconi anemia (FA) is a rare recessive DNA repair deficiency resulting from mutations in one of at least 22 genes. Two-thirds of FA families harbor mutations in FANCA. To genotype patients in the International Fanconi Anemia Registry (IFAR) we employed multiple methodologies, screening 216 families for FANCA mutations. We describe identification of 57 large deletions and 261 sequence variants, in 159 families. All but seven families harbored distinct combinations of two mutations demonstrating high heterogeneity. Pathogenicity of the 18 novel missense variants was analyzed functionally by determining the ability of the mutant cDNA to improve the survival of a FANCA-null cell line when treated with MMC. Overexpressed pathogenic missense variants were found to reside in the cytoplasm, and nonpathogenic in the nucleus. RNA analysis demonstrated that two variants (c.522G > C and c.1565A > G), predicted to encode missense variants, which were determined to be nonpathogenic by a functional assay, caused skipping of exons 5 and 16, respectively, and are most likely pathogenic. We report 48 novel FANCA sequence variants. Defining both variants in a large patient cohort is a major step toward cataloging all FANCA variants, and permitting studies of genotype-phenotype correlations.
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Affiliation(s)
- Danielle C Kimble
- Cancer Genetics and Comparative Genomics Branch, National Human Genome Research Institute, Bethesda, Maryland
| | - Francis P Lach
- Laboratory of Genome Maintenance, The Rockefeller University, New York, New York
| | - Siobhan Q Gregg
- Laboratory of Genome Maintenance, The Rockefeller University, New York, New York
| | - Frank X Donovan
- Cancer Genetics and Comparative Genomics Branch, National Human Genome Research Institute, Bethesda, Maryland
| | - Elizabeth K Flynn
- Cancer Genetics and Comparative Genomics Branch, National Human Genome Research Institute, Bethesda, Maryland
| | - Aparna Kamat
- Cancer Genetics and Comparative Genomics Branch, National Human Genome Research Institute, Bethesda, Maryland
| | - Alice Young
- NIH Intramural Sequencing Center, National Human Genome Research Institute, Bethesda, Maryland
| | - Meghana Vemulapalli
- NIH Intramural Sequencing Center, National Human Genome Research Institute, Bethesda, Maryland
| | - James W Thomas
- NIH Intramural Sequencing Center, National Human Genome Research Institute, Bethesda, Maryland
| | - James C Mullikin
- NIH Intramural Sequencing Center, National Human Genome Research Institute, Bethesda, Maryland
| | - Arleen D Auerbach
- Human Genetics and Hematology Program, The Rockefeller University, New York, New York
| | - Agata Smogorzewska
- Laboratory of Genome Maintenance, The Rockefeller University, New York, New York
| | - Settara C Chandrasekharappa
- Cancer Genetics and Comparative Genomics Branch, National Human Genome Research Institute, Bethesda, Maryland
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18
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A germline FANCA alteration that is associated with increased sensitivity to DNA damaging agents. Cold Spring Harb Mol Case Stud 2017; 3:mcs.a001487. [PMID: 28864460 PMCID: PMC5593159 DOI: 10.1101/mcs.a001487] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2016] [Accepted: 04/27/2017] [Indexed: 12/26/2022] Open
Abstract
Defects in genes involved in DNA damage repair (DDR) pathway are emerging as novel biomarkers and targets for new prostate cancer drug therapies. A previous report revealed an association between an exceptional response to cisplatin treatment and a somatic loss of heterozygosity (LOH) of FANCA in a patient with metastatic prostate cancer who also harbored a germline FANCA variant (S1088F). Although germline FANCA mutations are the most frequent alterations in patients with Fanconi anemia, germline alterations are less common in prostate cancer. We hypothesized that the germline S1088F FANCA variant in combination with FANCA LOH was deleterious for FANCA function and contributed to the patient's exceptional response to cisplatin. We show that although it properly localizes to the nucleus, the S1088F FANCA mutant protein disrupts the FANC protein complex resulting in increased sensitivity to DNA damaging agents. Because molecular stratification is emerging as a strategy for treating men with metastatic, castrate-resistant prostate cancer harboring specific DDR gene defects, our findings suggest that more biomarker studies are needed to better define clinically relevant germline and somatic alterations.
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19
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Palovcak A, Liu W, Yuan F, Zhang Y. Maintenance of genome stability by Fanconi anemia proteins. Cell Biosci 2017; 7:8. [PMID: 28239445 PMCID: PMC5320776 DOI: 10.1186/s13578-016-0134-2] [Citation(s) in RCA: 40] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2016] [Accepted: 12/07/2016] [Indexed: 12/20/2022] Open
Abstract
Persistent dysregulation of the DNA damage response and repair in cells causes genomic instability. The resulting genetic changes permit alterations in growth and proliferation observed in virtually all cancers. However, an unstable genome can serve as a double-edged sword by providing survival advantages in the ability to evade checkpoint signaling, but also creating vulnerabilities through dependency on alternative genomic maintenance factors. The Fanconi anemia pathway comprises an intricate network of DNA damage signaling and repair that are critical for protection against genomic instability. The importance of this pathway is underlined by the severity of the cancer predisposing syndrome Fanconi anemia which can be caused by biallelic mutations in any one of the 21 genes known thus far. This review delineates the roles of the Fanconi anemia pathway and the molecular actions of Fanconi anemia proteins in confronting replicative, oxidative, and mitotic stress.
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Affiliation(s)
- Anna Palovcak
- Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Gautier Building Room 311, 1011 NW 15th Street, Miami, FL 33136 USA
| | - Wenjun Liu
- Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Gautier Building Room 311, 1011 NW 15th Street, Miami, FL 33136 USA
| | - Fenghua Yuan
- Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Gautier Building Room 311, 1011 NW 15th Street, Miami, FL 33136 USA
| | - Yanbin Zhang
- Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Gautier Building Room 311, 1011 NW 15th Street, Miami, FL 33136 USA
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20
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Karras GI, Yi S, Sahni N, Fischer M, Xie J, Vidal M, D'Andrea AD, Whitesell L, Lindquist S. HSP90 Shapes the Consequences of Human Genetic Variation. Cell 2017; 168:856-866.e12. [PMID: 28215707 DOI: 10.1016/j.cell.2017.01.023] [Citation(s) in RCA: 81] [Impact Index Per Article: 10.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2016] [Revised: 12/09/2016] [Accepted: 01/19/2017] [Indexed: 11/26/2022]
Abstract
HSP90 acts as a protein-folding buffer that shapes the manifestations of genetic variation in model organisms. Whether HSP90 influences the consequences of mutations in humans, potentially modifying the clinical course of genetic diseases, remains unknown. By mining data for >1,500 disease-causing mutants, we found a strong correlation between reduced phenotypic severity and a dominant (HSP90 ≥ HSP70) increase in mutant engagement by HSP90. Examining the cancer predisposition syndrome Fanconi anemia in depth revealed that mutant FANCA proteins engaged predominantly by HSP70 had severely compromised function. In contrast, the function of less severe mutants was preserved by a dominant increase in HSP90 binding. Reducing HSP90's buffering capacity with inhibitors or febrile temperatures destabilized HSP90-buffered mutants, exacerbating FA-related chemosensitivities. Strikingly, a compensatory FANCA somatic mutation from an "experiment of nature" in monozygotic twins both prevented anemia and reduced HSP90 binding. These findings provide one plausible mechanism for the variable expressivity and environmental sensitivity of genetic diseases.
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Affiliation(s)
- Georgios I Karras
- Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA.
| | - Song Yi
- Center for Cancer Systems Biology (CCSB), Dana-Farber Cancer Institute, Boston, MA 02215, USA; Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
| | - Nidhi Sahni
- Center for Cancer Systems Biology (CCSB), Dana-Farber Cancer Institute, Boston, MA 02215, USA; Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
| | - Máté Fischer
- Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA
| | - Jenny Xie
- Center for DNA Damage and Repair and Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215, USA
| | - Marc Vidal
- Center for Cancer Systems Biology (CCSB), Dana-Farber Cancer Institute, Boston, MA 02215, USA; Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
| | - Alan D D'Andrea
- Center for DNA Damage and Repair and Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215, USA
| | - Luke Whitesell
- Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA.
| | - Susan Lindquist
- Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Howard Hughes Medical Institute, Cambridge, MA 02139, USA
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21
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van Twest S, Murphy VJ, Hodson C, Tan W, Swuec P, O'Rourke JJ, Heierhorst J, Crismani W, Deans AJ. Mechanism of Ubiquitination and Deubiquitination in the Fanconi Anemia Pathway. Mol Cell 2016; 65:247-259. [PMID: 27986371 DOI: 10.1016/j.molcel.2016.11.005] [Citation(s) in RCA: 99] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2016] [Revised: 10/25/2016] [Accepted: 10/31/2016] [Indexed: 11/26/2022]
Abstract
Monoubiquitination and deubiquitination of FANCD2:FANCI heterodimer is central to DNA repair in a pathway that is defective in the cancer predisposition syndrome Fanconi anemia (FA). The "FA core complex" contains the RING-E3 ligase FANCL and seven other essential proteins that are mutated in various FA subtypes. Here, we purified recombinant FA core complex to reveal the function of these other proteins. The complex contains two spatially separate FANCL molecules that are dimerized by FANCB and FAAP100. FANCC and FANCE act as substrate receptors and restrict monoubiquitination to the FANCD2:FANCI heterodimer in only a DNA-bound form. FANCA and FANCG are dispensable for maximal in vitro ubiquitination. Finally, we show that the reversal of this reaction by the USP1:UAF1 deubiquitinase only occurs when DNA is disengaged. Our work reveals the mechanistic basis for temporal and spatial control of FANCD2:FANCI monoubiquitination that is critical for chemotherapy responses and prevention of Fanconi anemia.
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Affiliation(s)
- Sylvie van Twest
- Genome Stability Unit, St. Vincent's Institute of Medical Research, Fitzroy, VIC 3065, Australia
| | - Vincent J Murphy
- Genome Stability Unit, St. Vincent's Institute of Medical Research, Fitzroy, VIC 3065, Australia
| | - Charlotte Hodson
- Genome Stability Unit, St. Vincent's Institute of Medical Research, Fitzroy, VIC 3065, Australia
| | - Winnie Tan
- Genome Stability Unit, St. Vincent's Institute of Medical Research, Fitzroy, VIC 3065, Australia; Department of Medicine (St. Vincent's Health), The University of Melbourne, VIC 3010, Australia
| | - Paolo Swuec
- Architecture and Dynamics of Macromolecular Machines Laboratory, London Research Institute, South Mimms, Hertfordshire EN6 3LD, UK
| | - Julienne J O'Rourke
- Genome Stability Unit, St. Vincent's Institute of Medical Research, Fitzroy, VIC 3065, Australia; Department of Medicine (St. Vincent's Health), The University of Melbourne, VIC 3010, Australia
| | - Jörg Heierhorst
- Molecular Genetics Unit, St. Vincent's Institute of Medical Research, Fitzroy, VIC 3065, Australia; Department of Medicine (St. Vincent's Health), The University of Melbourne, VIC 3010, Australia
| | - Wayne Crismani
- Genome Stability Unit, St. Vincent's Institute of Medical Research, Fitzroy, VIC 3065, Australia
| | - Andrew J Deans
- Genome Stability Unit, St. Vincent's Institute of Medical Research, Fitzroy, VIC 3065, Australia; Department of Medicine (St. Vincent's Health), The University of Melbourne, VIC 3010, Australia.
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22
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Donovan FX, Kimble DC, Kim Y, Lach FP, Harper U, Kamat A, Jones M, Sanborn EM, Tryon R, Wagner JE, MacMillan ML, Ostrander EA, Auerbach AD, Smogorzewska A, Chandrasekharappa SC. Paternal or Maternal Uniparental Disomy of Chromosome 16 Resulting in Homozygosity of a Mutant Allele Causes Fanconi Anemia. Hum Mutat 2016; 37:465-8. [PMID: 26841305 DOI: 10.1002/humu.22962] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2015] [Accepted: 01/10/2016] [Indexed: 12/31/2022]
Abstract
Fanconi anemia (FA) is a rare inherited disorder caused by pathogenic variants in one of 19 FANC genes. FA patients display congenital abnormalities, and develop bone marrow failure, and cancer susceptibility. We identified homozygous mutations in four FA patients and, in each case, only one parent carried the obligate mutant allele. FANCA and FANCP/SLX4 genes, both located on chromosome 16, were the affected recessive FA genes in three and one family respectively. Genotyping with short tandem repeat markers and SNP arrays revealed uniparental disomy (UPD) of the entire mutation-carrying chromosome 16 in all four patients. One FANCA patient had paternal UPD, whereas FA in the other three patients resulted from maternal UPD. These are the first reported cases of UPD as a cause of FA. UPD indicates a reduced risk of having another child with FA in the family and has implications in prenatal diagnosis.
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Affiliation(s)
- Frank X Donovan
- Cancer Genetics and Comparative Genomics Branch, National Human Genome Research Institute, NIH, Bethesda, Maryland
| | - Danielle C Kimble
- Cancer Genetics and Comparative Genomics Branch, National Human Genome Research Institute, NIH, Bethesda, Maryland
| | - Yonghwan Kim
- Laboratory of Genome Maintenance, The Rockefeller University, New York, New York
| | - Francis P Lach
- Laboratory of Genome Maintenance, The Rockefeller University, New York, New York
| | - Ursula Harper
- Cancer Genetics and Comparative Genomics Branch, National Human Genome Research Institute, NIH, Bethesda, Maryland
| | - Aparna Kamat
- Cancer Genetics and Comparative Genomics Branch, National Human Genome Research Institute, NIH, Bethesda, Maryland
| | - MaryPat Jones
- Cancer Genetics and Comparative Genomics Branch, National Human Genome Research Institute, NIH, Bethesda, Maryland
| | - Erica M Sanborn
- Laboratory of Genome Maintenance, The Rockefeller University, New York, New York
| | - Rebecca Tryon
- Blood and Marrow Transplant Program, Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota
| | - John E Wagner
- Blood and Marrow Transplant Program, Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota
| | - Margaret L MacMillan
- Blood and Marrow Transplant Program, Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota
| | - Elaine A Ostrander
- Cancer Genetics and Comparative Genomics Branch, National Human Genome Research Institute, NIH, Bethesda, Maryland
| | - Arleen D Auerbach
- Human Genetics and Hematology Program, The Rockefeller University, New York, New York
| | - Agata Smogorzewska
- Laboratory of Genome Maintenance, The Rockefeller University, New York, New York
| | - Settara C Chandrasekharappa
- Cancer Genetics and Comparative Genomics Branch, National Human Genome Research Institute, NIH, Bethesda, Maryland
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23
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Solanki A, Mohanty P, Shukla P, Rao A, Ghosh K, Vundinti BR. FANCA Gene Mutations with 8 Novel Molecular Changes in Indian Fanconi Anemia Patients. PLoS One 2016; 11:e0147016. [PMID: 26799702 PMCID: PMC4723128 DOI: 10.1371/journal.pone.0147016] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2015] [Accepted: 12/27/2015] [Indexed: 11/18/2022] Open
Abstract
Fanconi anemia (FA), a rare heterogeneous genetic disorder, is known to be associated with 19 genes and a spectrum of clinical features. We studied FANCA molecular changes in 34 unrelated and 2 siblings of Indian patients with FA and have identified 26 different molecular changes of FANCA gene, of which 8 were novel mutations (a small deletion c.2500delC, 4 non-sense mutations c.2182C>T, c.2630C>G, c.3677C>G, c.3189G>A; and 3 missense mutations; c.1273G>C, c.3679 G>C, and c.3992 T>C). Among these only 16 patients could be assigned FA-A complementation group, because we could not confirm single exon deletions detected by MLPA or cDNA amplification by secondary confirmation method and due to presence of heterozygous non-pathogenic variations or heterozygous pathogenic mutations. An effective molecular screening strategy should be developed for confirmation of these mutations and determining the breakpoints for single exon deletions.
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Affiliation(s)
- Avani Solanki
- Department of Cytogenetics, National Institute of Immunohaematology (ICMR), Mumbai, Maharashtra, India
| | - Purvi Mohanty
- Department of Cytogenetics, National Institute of Immunohaematology (ICMR), Mumbai, Maharashtra, India
| | - Pallavi Shukla
- Department of Cytogenetics, National Institute of Immunohaematology (ICMR), Mumbai, Maharashtra, India
| | - Anita Rao
- Department of Cytogenetics, National Institute of Immunohaematology (ICMR), Mumbai, Maharashtra, India
| | - Kanjaksha Ghosh
- Department of Cytogenetics, National Institute of Immunohaematology (ICMR), Mumbai, Maharashtra, India
| | - Babu Rao Vundinti
- Department of Cytogenetics, National Institute of Immunohaematology (ICMR), Mumbai, Maharashtra, India
- * E-mail:
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24
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De Rocco D, Bottega R, Cappelli E, Cavani S, Criscuolo M, Nicchia E, Corsolini F, Greco C, Borriello A, Svahn J, Pillon M, Mecucci C, Casazza G, Verzegnassi F, Cugno C, Locasciulli A, Farruggia P, Longoni D, Ramenghi U, Barberi W, Tucci F, Perrotta S, Grammatico P, Hanenberg H, Della Ragione F, Dufour C, Savoia A. Molecular analysis of Fanconi anemia: the experience of the Bone Marrow Failure Study Group of the Italian Association of Pediatric Onco-Hematology. Haematologica 2014; 99:1022-31. [PMID: 24584348 DOI: 10.3324/haematol.2014.104224] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023] Open
Abstract
Fanconi anemia is an inherited disease characterized by congenital malformations, pancytopenia, cancer predisposition, and sensitivity to cross-linking agents. The molecular diagnosis of Fanconi anemia is relatively complex for several aspects including genetic heterogeneity with mutations in at least 16 different genes. In this paper, we report the mutations identified in 100 unrelated probands enrolled into the National Network of the Italian Association of Pediatric Hematoly and Oncology. In approximately half of these cases, mutational screening was carried out after retroviral complementation analyses or protein analysis. In the other half, the analysis was performed on the most frequently mutated genes or using a next generation sequencing approach. We identified 108 distinct variants of the FANCA, FANCG, FANCC, FANCD2, and FANCB genes in 85, 9, 3, 2, and 1 families, respectively. Despite the relatively high number of private mutations, 45 of which are novel Fanconi anemia alleles, 26% of the FANCA alleles are due to 5 distinct mutations. Most of the mutations are large genomic deletions and nonsense or frameshift mutations, although we identified a series of missense mutations, whose pathogenetic role was not always certain. The molecular diagnosis of Fanconi anemia is still a tiered procedure that requires identifying candidate genes to avoid useless sequencing. Introduction of next generation sequencing strategies will greatly improve the diagnostic process, allowing a rapid analysis of all the genes.
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Affiliation(s)
| | - Roberta Bottega
- Department of Medical Sciences, University of Trieste, Italy
| | - Enrico Cappelli
- Clinical and Experimental Hematology Unit, G. Gaslini Children's Hospital, Genoa, Italy
| | - Simona Cavani
- Human Genetics laboratory, "E.O. Ospedali Galliera", Genoa, Italy
| | - Maria Criscuolo
- Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, Italy
| | - Elena Nicchia
- Department of Medical Sciences, University of Trieste, Italy
| | - Fabio Corsolini
- Clinical and Experimental Hematology Unit, G. Gaslini Children's Hospital, Genoa, Italy
| | - Chiara Greco
- Pediatric Onco-Hematology, "Azienda Ospedaliero Universitaria Pisana", Pisa, Italy
| | - Adriana Borriello
- Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, Italy
| | - Johanna Svahn
- Clinical and Experimental Hematology Unit, G. Gaslini Children's Hospital, Genoa, Italy
| | - Marta Pillon
- Institute for Maternal and Child Health - IRCCS Burlo Garofolo, Trieste, Italy
| | | | | | - Federico Verzegnassi
- Pediatric Onco-Hematology, "Azienda Ospedaliero Universitaria Pisana", Pisa, Italy
| | - Chiara Cugno
- Pediatric Onco-Hematology, "Fondazione IRCCS Policlinico San Matteo", Pavia, Italy
| | - Anna Locasciulli
- Department of Pediatric and Pediatric Hematology, S.Camillo Hospital, Rome, Italy
| | - Piero Farruggia
- Pediatric Onco-Hematology, ARNAS Civico Hospital, Palermo, Italy
| | - Daniela Longoni
- Pediatrics Unit, University of Milano-Bicocca, Fondazione MBBM, Ospedale San Gerardo, Monza, Italy
| | - Ugo Ramenghi
- Department of Pediatric and Public Health Sciences, Sapienza Università di Roma, Firenze, Italy
| | - Walter Barberi
- Dipartimento di Biotecnologia Cellulari ed Ematologia, Sapienza Università di Roma, Firenze, Italy
| | - Fabio Tucci
- Pediatric Onco-Hematology, "Azienda Ospedaliero-Universitaria" Meyer, Firenze, Italy
| | | | - Paola Grammatico
- Department of Molecular Medicine, "La Sapienza" University, Rome, Italy
| | - Helmut Hanenberg
- Department of Otorhinolaryngology & Head/Neck Surgery, Heinrich Heine University School of Medicine, Duesseldorf, Germany Pediatric Hematology/Oncology, Wells Center for Pediatric Research, Department of Pediatrics, The Riley Hospital, Indiana University School of Medicine, Indianapolis, IN, USA Department of Medical & Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA
| | - Fulvio Della Ragione
- Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, Italy
| | - Carlo Dufour
- Clinical and Experimental Hematology Unit, G. Gaslini Children's Hospital, Genoa, Italy
| | - Anna Savoia
- Department of Medical Sciences, University of Trieste, Italy Pediatric Onco-Hematology, "Azienda Ospedaliero Universitaria Pisana", Pisa, Italy
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25
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Qian L, Yuan F, Rodriguez-Tello P, Padgaonkar S, Zhang Y. Human Fanconi anemia complementation group a protein stimulates the 5' flap endonuclease activity of FEN1. PLoS One 2013; 8:e82666. [PMID: 24349332 PMCID: PMC3857783 DOI: 10.1371/journal.pone.0082666] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2013] [Accepted: 10/26/2013] [Indexed: 11/28/2022] Open
Abstract
In eukaryotic cells, Flap endonuclease 1 (FEN1) is a major structure-specific endonuclease that processes 5’ flapped structures during maturation of lagging strand DNA synthesis, long patch base excision repair, and rescue of stalled replication forks. Here we report that fanconi anemia complementation group A protein (FANCA), a protein that recognizes 5’ flap structures and is involved in DNA repair and maintenance of replication forks, constantly stimulates FEN1-mediated incision of both DNA and RNA flaps. Kinetic analyses indicate that FANCA stimulates FEN1 by increasing the turnover rate of FEN1 and altering its substrate affinity. More importantly, six pathogenic FANCA mutants are significantly less efficient than the wild-type at stimulating FEN1 endonuclease activity, implicating that regulation of FEN1 by FANCA contributes to the maintenance of genomic stability.
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Affiliation(s)
- Liangyue Qian
- Department of Biochemistry & Molecular Biology, University of Miami Miller School of Medicine, Miami, Florida, United States of America
| | - Fenghua Yuan
- Department of Biochemistry & Molecular Biology, University of Miami Miller School of Medicine, Miami, Florida, United States of America
| | - Paola Rodriguez-Tello
- Department of Biochemistry & Molecular Biology, University of Miami Miller School of Medicine, Miami, Florida, United States of America
| | - Suyog Padgaonkar
- Department of Biochemistry & Molecular Biology, University of Miami Miller School of Medicine, Miami, Florida, United States of America
| | - Yanbin Zhang
- Department of Biochemistry & Molecular Biology, University of Miami Miller School of Medicine, Miami, Florida, United States of America
- * E-mail:
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26
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Yao H, Chen Y, Zhang L, He X, He X, Lian L, Wu X, Lan P. Carnosol inhibits cell adhesion molecules and chemokine expression by tumor necrosis factor-α in human umbilical vein endothelial cells through the nuclear factor-κB and mitogen-activated protein kinase pathways. Mol Med Rep 2013; 9:476-80. [PMID: 24316968 DOI: 10.3892/mmr.2013.1839] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2013] [Accepted: 11/13/2013] [Indexed: 11/05/2022] Open
Abstract
Inflammatory bowel diseases (IBD) are gastrointestinal disorders associated with chronic inflammatory processes. Carnosol has been demonstrated to possess anti-inflammatory properties. This study examined the suppressive effect of carnosol on the expression of cell adhesion molecules (CAMs) and chemokines in human umbilical vein endothelial cells (HUVECs) and the possible underlying mechanism. The effect of carnosol on CAM and chemokine expression in HUVECs was identified by western blotting and ELISA, respectively. nuclear factor (NF)-κB activation of HUVECs was analyzed using the TransAM NF-κB Family kit. The effect of carnosol on the tumor necrosis factor (TNF)-α-induced activation of the NF-κB and mitogen-activated protein kinase (MAPK) pathways, and was subsequently analyzed using western blotting. Carnosol not only inhibited TNF-α-induced protein expression of intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 and E-selectin in HUVECs, but also suppressed interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1 expression. In addition, carnosol inhibited the TNF-α-induced phosphorylation of p-65 and IκB-α, as well as the activation of NF-κB. The same result was observed in TNF-α-stimulated phosphorylation of ERK1/2 and p-38. It was demonstrated that carnosol inhibited TNF-α-induced CAM and chemokine expression in HUVECs. The underlying mechanism may be associated with the blocking of the NF-κB and MAPK pathways. These results indicate that carnosol may be a novel therapeutic agent for targeting endothelial cells in IBDs.
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Affiliation(s)
- Hui Yao
- Department of Colorectal Surgery, The Sixth Affiliated Hospital of Sun Yat-Sen University, Guangdong Gastrointestinal Hospital, Guangzhou, Guangdong 510655, P.R. China
| | - Yufeng Chen
- Department of Colorectal Surgery, The Sixth Affiliated Hospital of Sun Yat-Sen University, Guangdong Gastrointestinal Hospital, Guangzhou, Guangdong 510655, P.R. China
| | - Longjuan Zhang
- Laboratory of Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510080, P.R. China
| | - Xiaosheng He
- Department of Colorectal Surgery, The Sixth Affiliated Hospital of Sun Yat-Sen University, Guangdong Gastrointestinal Hospital, Guangzhou, Guangdong 510655, P.R. China
| | - Xiaowen He
- Department of Colorectal Surgery, The Sixth Affiliated Hospital of Sun Yat-Sen University, Guangdong Gastrointestinal Hospital, Guangzhou, Guangdong 510655, P.R. China
| | - Lei Lian
- Department of Colorectal Surgery, The Sixth Affiliated Hospital of Sun Yat-Sen University, Guangdong Gastrointestinal Hospital, Guangzhou, Guangdong 510655, P.R. China
| | - Xiaojian Wu
- Department of Colorectal Surgery, The Sixth Affiliated Hospital of Sun Yat-Sen University, Guangdong Gastrointestinal Hospital, Guangzhou, Guangdong 510655, P.R. China
| | - Ping Lan
- Department of Colorectal Surgery, The Sixth Affiliated Hospital of Sun Yat-Sen University, Guangdong Gastrointestinal Hospital, Guangzhou, Guangdong 510655, P.R. China
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27
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Zheng Z, Geng J, Yao RE, Li C, Ying D, Shen Y, Ying L, Yu Y, Fu Q. Molecular defects identified by whole exome sequencing in a child with Fanconi anemia. Gene 2013; 530:295-300. [DOI: 10.1016/j.gene.2013.08.031] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2013] [Revised: 08/01/2013] [Accepted: 08/09/2013] [Indexed: 01/25/2023]
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28
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Shukla P, Rao A, Ghosh K, Vundinti BR. Identification of a novel large intragenic deletion in a family with Fanconi anemia: first molecular report from India and review of literature. Gene 2013; 518:470-5. [PMID: 23370339 DOI: 10.1016/j.gene.2013.01.016] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2012] [Revised: 12/27/2012] [Accepted: 01/10/2013] [Indexed: 11/28/2022]
Abstract
We report here an Indian case with Fanconi anemia (FA) presented with fever, pallor, short stature, hyperpigmentation and upper limb anomaly. Chromosome breakage analysis together with FANCD2 Western blot monoubiquitination assay confirmed the diagnosis as FA. Multiplex ligation-dependent probe amplification (MLPA) revealed a novel homozygous large intragenic deletion (exons 8-27 del) in the FANCA gene in the proband. His sib and parents were also analyzed and found to be heterozygous for the same mutation. We also reviewed the literature of FANCA large intragenic deletions found in FA patients from different countries and the mechanism involved in the formation of these deletions. To the best of our knowledge, this is the first molecular report from India on FA. The finding expands the mutation spectrum of the FANCA gene. Identification of the mutation confirms the diagnosis of FA at DNA level and helps in providing proper genetic counseling to the family.
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Affiliation(s)
- Pallavi Shukla
- Department of Cytogenetics, National Institute of Immunohaematology (ICMR), 13th Floor, New Multistoried Building, K.E.M. Hospital Campus, Parel, Mumbai-400012, India
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29
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Towards a molecular understanding of the fanconi anemia core complex. Anemia 2012; 2012:926787. [PMID: 22675617 PMCID: PMC3364535 DOI: 10.1155/2012/926787] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2011] [Accepted: 03/21/2012] [Indexed: 11/17/2022] Open
Abstract
Fanconi Anemia (FA) is a genetic disorder characterized by the inability of patient cells to repair DNA damage caused by interstrand crosslinking agents. There are currently 14 verified FA genes, where mutation of any single gene prevents repair of DNA interstrand crosslinks (ICLs). The accumulation of ICL damage results in genome instability and patients having a high predisposition to cancers. The key event of the FA pathway is dependent on an eight-protein core complex (CC), required for the monoubiquitination of each member of the FANCD2-FANCI complex. Interestingly, the majority of patient mutations reside in the CC. The molecular mechanisms underlying the requirement for such a large complex to carry out a monoubiquitination event remain a mystery. This paper documents the extensive efforts of researchers so far to understand the molecular roles of the CC proteins with regard to its main function in the FA pathway, the monoubiquitination of FANCD2 and FANCI.
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30
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Fanconi anemia proteins and their interacting partners: a molecular puzzle. Anemia 2012; 2012:425814. [PMID: 22737580 PMCID: PMC3378961 DOI: 10.1155/2012/425814] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2011] [Accepted: 03/13/2012] [Indexed: 11/17/2022] Open
Abstract
In recent years, Fanconi anemia (FA) has been the subject of intense investigations, primarily in the DNA repair research field. Many discoveries have led to the notion of a canonical pathway, termed the FA pathway, where all FA proteins function sequentially in different protein complexes to repair DNA cross-link damages. Although a detailed architecture of this DNA cross-link repair pathway is emerging, the question of how a defective DNA cross-link repair process translates into the disease phenotype is unresolved. Other areas of research including oxidative metabolism, cell cycle progression, apoptosis, and transcriptional regulation have been studied in the context of FA, and some of these areas were investigated before the fervent enthusiasm in the DNA repair field. These other molecular mechanisms may also play an important role in the pathogenesis of this disease. In addition, several FA-interacting proteins have been identified with roles in these “other” nonrepair molecular functions. Thus, the goal of this paper is to revisit old ideas and to discuss protein-protein interactions related to other FA-related molecular functions to try to give the reader a wider perspective of the FA molecular puzzle.
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31
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FAAP20: a novel ubiquitin-binding FA nuclear core-complex protein required for functional integrity of the FA-BRCA DNA repair pathway. Blood 2012; 119:3285-94. [PMID: 22343915 DOI: 10.1182/blood-2011-10-385963] [Citation(s) in RCA: 65] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Abstract
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. FAAP20 contains a conserved ubiquitin-binding zinc-finger domain (UBZ), and binds K-63-linked ubiquitin chains in vitro. The FAAP20-UBZ domain is not required for interaction with FANCA, but is required for DNA-damage-induced chromatin loading of FANCA and the functional integrity of the FA pathway. These findings reveal critical roles for FAAP20 in the FA-BRCA pathway of DNA damage repair and genome maintenance.
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32
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Balduini CL, Pecci A, Savoia A. Recent advances in the understanding and management of MYH9-related inherited thrombocytopenias. Br J Haematol 2011; 154:161-74. [DOI: 10.1111/j.1365-2141.2011.08716.x] [Citation(s) in RCA: 142] [Impact Index Per Article: 10.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
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33
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Castella M, Pujol R, Callén E, Trujillo JP, Casado JA, Gille H, Lach FP, Auerbach AD, Schindler D, Benítez J, Porto B, Ferro T, Muñoz A, Sevilla J, Madero L, Cela E, Beléndez C, de Heredia CD, Olivé T, de Toledo JS, Badell I, Torrent M, Estella J, Dasí A, Rodríguez-Villa A, Gómez P, Barbot J, Tapia M, Molinés A, Figuera A, Bueren JA, Surrallés J. Origin, functional role, and clinical impact of Fanconi anemia FANCA mutations. Blood 2011; 117:3759-69. [PMID: 21273304 PMCID: PMC3083295 DOI: 10.1182/blood-2010-08-299917] [Citation(s) in RCA: 103] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2010] [Accepted: 12/30/2010] [Indexed: 12/17/2022] Open
Abstract
Fanconi anemia is characterized by congenital abnormalities, bone marrow failure, and cancer predisposition. To investigate the origin, functional role, and clinical impact of FANCA mutations, we determined a FANCA mutational spectrum with 130 pathogenic alleles. Some of these mutations were further characterized for their distribution in populations, mode of emergence, or functional consequences at cellular and clinical level. The world most frequent FANCA mutation is not the result of a mutational "hot-spot" but results from worldwide dissemination of an ancestral Indo-European mutation. We provide molecular evidence that total absence of FANCA in humans does not reduce embryonic viability, as the observed frequency of mutation carriers in the Gypsy population equals the expected by Hardy-Weinberg equilibrium. We also prove that long distance Alu-Alu recombination can cause Fanconi anemia by originating large interstitial deletions involving FANCA and 2 adjacent genes. Finally, we show that all missense mutations studied lead to an altered FANCA protein that is unable to relocate to the nucleus and activate the FA/BRCA pathway. This may explain the observed lack of correlation between type of FANCA mutation and cellular phenotype or clinical severity in terms of age of onset of hematologic disease or number of malformations.
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Affiliation(s)
- Maria Castella
- Genome Instability and DNA Repair Group, Department of Genetics and Microbiology, Universitat Autònoma de Barcelona, Campus de Bellaterra s/n, Barcelona, Spain
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Akbari MR, Malekzadeh R, Lepage P, Roquis D, Sadjadi AR, Aghcheli K, Yazdanbod A, Shakeri R, Bashiri J, Sotoudeh M, Pourshams A, Ghadirian P, Narod SA. Mutations in Fanconi anemia genes and the risk of esophageal cancer. Hum Genet 2011; 129:573-82. [DOI: 10.1007/s00439-011-0951-7] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2010] [Accepted: 01/17/2011] [Indexed: 01/06/2023]
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Patient-derived C-terminal mutation of FANCI causes protein mislocalization and reveals putative EDGE motif function in DNA repair. Blood 2010; 117:2247-56. [PMID: 20971953 DOI: 10.1182/blood-2010-07-295758] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
Fanconi anemia (FA) is a rare familial genome instability syndrome caused by mutations in FA genes that results in defective DNA crosslink repair. Activation of the FA pathway requires the FA core ubiquitin ligase complex-dependent monoubiquitination of 2 interacting FA proteins, FANCI and FANCD2. Although loss of either FANCI or FANCD2 is known to prevent monoubiquitination of its respective partner, it is unclear whether FANCI has any additional domains that may be important in promoting DNA repair, independent of its monoubiquitination. Here, we focus on an FA-I patient-derived FANCI mutant protein, R1299X (deletion of 30 residues from its C-terminus), to characterize important structural region(s) in FANCI that is required to activate the FA pathway. We show that, within this short 30 amino acid stretch contains 2 separable functional signatures, a nuclear localization signal and a putative EDGE motif, that is critical for the ability of FANCI to properly monoubiquitinate FANCD2 and promote DNA crosslink resistance. Our study enable us to conclude that, although proper nuclear localization of FANCI is crucial for robust FANCD2 monoubiquitination, the putative FANCI EDGE motif is important for DNA crosslink repair.
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Validation of Fanconi anemia complementation Group A assignment using molecular analysis. Genet Med 2009; 11:183-92. [PMID: 19367192 DOI: 10.1097/gim.0b013e318193ba67] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023] Open
Abstract
PURPOSE Fanconi anemia is a genetically heterogeneous chromosomal breakage disorder exhibiting a high degree of clinical variability. Clinical diagnoses are confirmed by testing patient cells for increased sensitivity to crosslinking agents. Fanconi anemia complementation group assignment, essential for efficient molecular diagnosis of the disease, had not been validated for clinical application before this study. The purpose of this study was (1) confirmation of the accuracy of Fanconi anemia complementation group assignment to Group A (FANCA) and (2) development of a rapid mutation detection strategy that ensures the efficient capture of all FANCA mutations. METHODS Using fibroblasts from 29 patients, diagnosis of Fanconi anemia and assignment to complementation Group A was made through breakage analysis studies. FANCA coding and flanking sequences were analyzed using denaturing high pressure liquid chromatography, sequencing, and multiplex ligation-dependent probe amplification. Patients in which two mutations were not identified were analyzed by cDNA sequencing. Patients with no mutations were sequenced for mutations in FANCC, G, E, and F. RESULTS Of the 56 putative mutant alleles studied, 89% had an identifiable FANCA pathogenic mutation. Eight unique novel mutations were identified. CONCLUSION Complementation assignment to Group A was validated in a clinical laboratory setting using our FANCA rapid molecular testing strategy.
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Ali AM, Kirby M, Jansen M, Lach FP, Schulte J, Singh TR, Batish SD, Auerbach AD, Williams DA, Meetei AR. Identification and characterization of mutations in FANCL gene: a second case of Fanconi anemia belonging to FA-L complementation group. Hum Mutat 2009; 30:E761-70. [PMID: 19405097 DOI: 10.1002/humu.21032] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Fanconi anemia (FA) is a rare autosomal recessive or X-linked disorder characterized by aplastic anemia, cancer susceptibility and cellular sensitivity to DNA crosslinking agents. Eight FA proteins (FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL and FANCM) and three non-FA proteins (FAAP100, FAAP24 and HES1) form an FA nuclear core complex, which is required for monoubiquitination of the FANCD2-FANCI dimer upon DNA damage. FANCL possesses a PHD/RING-finger domain and is a putative E3 ubiquitin ligase subunit of the core complex. In this study, we report an FA patient with an unusual presentation belonging to the FA-L complementation group. The patient lacks an obvious FA phenotype except for the presence of a café-au-lait spot, mild hypocellularity and a family history of leukemia. The molecular diagnosis and identification of the FA subgroup was achieved by FA complementation assay. We identified bi-allelic novel mutations in the FANCL gene and functionally characterized them. To the best of our knowledge, this is the second reported case belonging to the FA-L complementation group.
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Affiliation(s)
- Abdullah Mahmood Ali
- Experimental Hematology and Cancer Biology, Cincinnati Children's Research Foundation, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45229, USA
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de Winter JP, Joenje H. The genetic and molecular basis of Fanconi anemia. Mutat Res 2009; 668:11-19. [PMID: 19061902 DOI: 10.1016/j.mrfmmm.2008.11.004] [Citation(s) in RCA: 143] [Impact Index Per Article: 8.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2008] [Revised: 10/28/2008] [Accepted: 11/06/2008] [Indexed: 05/27/2023]
Abstract
The capacity to maintain genomic integrity is shared by all living organisms. Multiple pathways are distinguished that safeguard genomic stability, most of which have originated in primitive life forms. In human individuals, defects in these pathways are typically associated with cancer proneness. The Fanconi anemia pathway, one of these pathways, has evolved relatively late during evolution and exists - in its fully developed form - only in vertebrates. This pathway, in which thus far 13 distinct proteins have been shown to participate, appears essential for error-free DNA replication. Inactivating mutations in the corresponding genes underlie the recessive disease Fanconi anemia (FA). In the last decade the genetic basis of this disorder has been uncovered by a variety of approaches, including complementation cloning, genetic linkage analysis and protein association studies. Here we review these approaches, introduce the encoded proteins, and discuss their possible role in ensuring genomic integrity.
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Affiliation(s)
- Johan P de Winter
- Department of Clinical Genetics, Section Oncogenetics, VU University Medical Center, Van der Boechorststraat 7, Amsterdam 1081 BT, The Netherlands.
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Neveling K, Endt D, Hoehn H, Schindler D. Genotype-phenotype correlations in Fanconi anemia. Mutat Res 2009; 668:73-91. [PMID: 19464302 DOI: 10.1016/j.mrfmmm.2009.05.006] [Citation(s) in RCA: 107] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2008] [Revised: 03/30/2009] [Accepted: 05/12/2009] [Indexed: 11/30/2022]
Abstract
Although still incomplete, we now have a remarkably detailed and nuanced picture of both phenotypic and genotypic components of the FA spectrum. Initially described as a combination of pancytopenia with a limited number of physical anomalies, it was later recognized that additional features were compatible with the FA phenotype, including a form without detectable malformations (Estren-Dameshek variant). The discovery of somatic mosaicism extended the boundaries of the FA phenotype to cases even without any overt hematological manifestations. This clinical heterogeneity was augmented by new conceptualizations. There was the realization of a constant risk for the development of myelodysplasia and certain malignancies, including acute myelogenous leukemia and squamous cell carcinoma, and there was the emergence of a distinctive cellular phenotype. A striking degree of genetic heterogeneity became apparent with the delineation of at least 12 complementation groups and the identification of their underlying genes. Although functional genetic insights have fostered the interpretation of many phenotypic features, surprisingly few stringent genotype-phenotype connections have emerged. In addition to myriad genetic alterations, less predictable influences are likely to modulate the FA phenotype, including modifier genes, environmental factors and chance effects. In reviewing the current status of genotype-phenotype correlations, we arrive at a unifying hypothesis to explain the remarkably wide range of FA phenotypes. Given the large body of evidence that genomic instability is a major underlying mechanism of accelerated ageing phenotypes, we propose that the numerous FA variants can be viewed as differential modulations and compression in time of intrinsic biological ageing.
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Affiliation(s)
- Kornelia Neveling
- Department of Human and Medical Genetics, University of Wurzburg, Biozentrum, Am Hubland, Wurzburg D-97074, Germany
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40
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ATR-dependent phosphorylation of FANCA on serine 1449 after DNA damage is important for FA pathway function. Blood 2008; 113:2181-90. [PMID: 19109555 DOI: 10.1182/blood-2008-05-154294] [Citation(s) in RCA: 68] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Abstract
Previous work has shown several proteins defective in Fanconi anemia (FA) are phosphorylated in a functionally critical manner. FANCA is phosphorylated after DNA damage and localized to chromatin, but the site and significance of this phosphorylation are unknown. Mass spectrometry of FANCA revealed one phosphopeptide, phosphorylated on serine 1449. Serine 1449 phosphorylation was induced after DNA damage but not during S phase, in contrast to other posttranslational modifications of FA proteins. Furthermore, the S1449A mutant failed to completely correct a variety of FA-associated phenotypes. The DNA damage response is coordinated by phosphorylation events initiated by apical kinases ATM (ataxia telangectasia mutated) and ATR (ATM and Rad3-related), and ATR is essential for proper FA pathway function. Serine 1449 is in a consensus ATM/ATR site, phosphorylation in vivo is dependent on ATR, and ATR phosphorylated FANCA on serine 1449 in vitro. Phosphorylation of FANCA on serine 1449 is a DNA damage-specific event that is downstream of ATR and is functionally important in the FA pathway.
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41
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Rego MA, Kolling FW, Howlett NG. The Fanconi anemia protein interaction network: casting a wide net. Mutat Res 2008; 668:27-41. [PMID: 19101576 DOI: 10.1016/j.mrfmmm.2008.11.018] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2008] [Revised: 11/16/2008] [Accepted: 11/25/2008] [Indexed: 11/26/2022]
Abstract
It has long been hypothesized that a defect in the repair of damaged DNA is central to the etiology of Fanconi anemia (FA). Indeed, an increased sensitivity of FA patient-derived cells to the lethal effects of various forms of DNA damaging agents was described over three decades ago [A.J. Fornace, Jr., J.B. Little, R.R. Weichselbaum, DNA repair in a Fanconi's anemia fibroblast cell strain, Biochim. Biophys. Acta 561 (1979) 99-109; Y. Fujiwara, M. Tatsumi, Repair of mitomycin C damage to DNA in mammalian cells and its impairment in Fanconi's anemia cells, Biochem. Biophys. Res. Commun. 66 (1975) 592-598; A.J. Rainbow, M. Howes, Defective repair of ultraviolet- and gamma-ray-damaged DNA in Fanconi's anaemia, Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 31 (1977) 191-195]. Furthermore, the cytological hallmark of FA, the DNA crosslink-induced radial chromosome formation, exemplifies an innate impairment in the repair of these particularly cytotoxic DNA lesions [A.D. Auerbach, Fanconi anemia diagnosis and the diepoxybutane (DEB) test, Exp. Hematol. 21 (1993) 731-733]. Precisely defining the collective role of the FA proteins in DNA repair, however, continues to be one of the most enigmatic and challenging questions in the FA field. The first six identified FA proteins (A, C, E, F, G, and D2) harbored no recognizable enzymatic features, precluding association with a specific metabolic process. Consequently, our knowledge of the role of the FA proteins in the DNA damage response has been gleaned primarily through biochemical association studies with non-FA proteins. Here, we provide a chronological discourse of the major FA protein interaction network discoveries, with particular emphasis on the DNA damage response, that have defined our current understanding of the molecular basis of FA.
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Affiliation(s)
- Meghan A Rego
- Department of Cell and Molecular Biology, University of Rhode Island, 115 Morrill Hall, 45 Lower College Road, Kingston, RI 02881, USA
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42
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Oda T, Hayano T, Miyaso H, Takahashi N, Yamashita T. Hsp90 regulates the Fanconi anemia DNA damage response pathway. Blood 2007; 109:5016-26. [PMID: 17327415 DOI: 10.1182/blood-2006-08-038638] [Citation(s) in RCA: 50] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
Heat shock protein 90 (Hsp90) regulates diverse signaling pathways. Emerging evidence suggests that Hsp90 inhibitors, such as 17-allylamino-17-demethoxygeldanamycin (17-AAG), enhance DNA damage-induced cell death, suggesting that Hsp90 may regulate cellular responses to genotoxic stress. However, the underlying mechanisms are poorly understood. Here, we show that the Fanconi anemia (FA) pathway is involved in the Hsp90-mediated regulation of genotoxic stress response. In the FA pathway, assembly of 8 FA proteins including FANCA into a nuclear multiprotein complex, and the complex-dependent activation of FANCD2 are critical events for cellular tolerance against DNA cross-linkers. Hsp90 associates with FANCA, in vivo and in vitro, in a 17-AAG-sensitive manner. Disruption of the FANCA/Hsp90 association by cellular treatment with 17-AAG induces rapid proteasomal degradation and cytoplasmic relocalization of FANCA, leading to impaired activation of FANCD2. Furthermore, 17-AAG promotes DNA cross-linker-induced cytotoxicity, but this effect is much less pronounced in FA pathway-defective cells. Notably, 17-AAG enhances DNA cross-linker-induced chromosome aberrations. In conclusion, our results identify FANCA as a novel client of Hsp90, suggesting that Hsp90 promotes activation of the FA pathway through regulation of intracellular turnover and trafficking of FANCA, which is critical for cellular tolerance against genotoxic stress.
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Affiliation(s)
- Tsukasa Oda
- Laboratory of Molecular Genetics, Department of Molecular and Cellular Biology, Institute of Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma 371-8512, Japan
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43
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Larder R, Karali D, Nelson N, Brown P. Fanconi anemia A is a nucleocytoplasmic shuttling molecule required for gonadotropin-releasing hormone (GnRH) transduction of the GnRH receptor. Endocrinology 2006; 147:5676-89. [PMID: 16946016 PMCID: PMC1975762 DOI: 10.1210/en.2006-0383] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
GnRH binds its cognate G protein-coupled GnRH receptor (GnRHR) located on pituitary gonadotropes and drives expression of gonadotropin hormones. There are two gonadotropin hormones, comprised of a common alpha- and hormone-specific beta-subunit, which are required for gonadal function. Recently we identified that Fanconi anemia a (Fanca), a DNA damage repair gene, is differentially expressed within the LbetaT2 gonadotrope cell line in response to stimulation with GnRH. FANCA is mutated in more than 60% of cases of Fanconi anemia (FA), a rare genetically heterogeneous autosomal recessive disorder characterized by bone marrow failure, endocrine tissue cancer susceptibility, and infertility. Here we show that induction of FANCA protein is mediated by the GnRHR and that the protein constitutively adopts a nucleocytoplasmic intracellular distribution pattern. Using inhibitors to block nuclear import and export and a GnRHR antagonist, we demonstrated that GnRH induces nuclear accumulation of FANCA and green fluorescent protein (GFP)-FANCA before exporting back to the cytoplasm using the nuclear export receptor CRM1. Using FANCA point mutations that locate GFP-FANCA to the cytoplasm (H1110P) or functionally uncouple GFP-FANCA (Q1128E) from the wild-type nucleocytoplasmic distribution pattern, we demonstrated that wild-type FANCA was required for GnRH-induced activation of gonadotrope cell markers. Cotransfection of H1110P and Q1128E blocked GnRH activation of the alphaGsu and GnRHR but not the beta-subunit gene promoters. We conclude that nucleocytoplasmic shuttling of FANCA is required for GnRH transduction of the alphaGSU and GnRHR gene promoters and propose that FANCA functions as a GnRH-induced signal transducer.
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Affiliation(s)
- Rachel Larder
- Medical Research Council, Human Reproductive Sciences Unit, Centre for Reproductive Biology, The Queen's Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4JT, Scotland, United Kingdom
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44
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Abstract
Fanconi anaemia (FA) is a rare recessive disorder associated with chromosomal fragility, aplastic anaemia, congenital abnormalities and a high risk of cancer, including acute myeloid leukaemia and squamous cell carcinomas. The identification of 11 different FA genes has revealed a complex web of interacting proteins that are involved in the recognition or repair of DNA interstrand crosslinks and perhaps other forms of DNA damage. Bi-allelic mutations in BRCA2 are associated with a rare and highly cancer-prone form of FA, and the DNA helicase BRIP1 (formerly BACH1) is mutated in FA group J. There is little convincing evidence that FA heterozygotes are at increased risk of cancer, but larger studies are needed to address the possibility of modest risk effects. Somatic inactivation of the FA pathway by mutation or epigenetic silencing has been observed in several different types of sporadic cancer, and this may have important implications for targeted chemotherapy. Inhibition of this pathway represents a possible route to sensitization of tumours to DNA crosslinking drugs such as cisplatin.
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Affiliation(s)
- C G Mathew
- King's College London School of Medicine, Division of Genetics and Molecular Medicine, Guy's Hospital, London, UK.
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45
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Medhurst AL, Laghmani EH, Steltenpool J, Ferrer M, Fontaine C, de Groot J, Rooimans MA, Scheper RJ, Meetei AR, Wang W, Joenje H, de Winter JP. Evidence for subcomplexes in the Fanconi anemia pathway. Blood 2006; 108:2072-80. [PMID: 16720839 PMCID: PMC1895538 DOI: 10.1182/blood-2005-11-008151] [Citation(s) in RCA: 74] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Fanconi anemia (FA) is a genomic instability disorder, clinically characterized by congenital abnormalities, progressive bone marrow failure, and predisposition to malignancy. Cells derived from patients with FA display a marked sensitivity to DNA cross-linking agents, such as mitomycin C (MMC). This observation has led to the hypothesis that the proteins defective in FA are involved in the sensing or repair of interstrand cross-link lesions of the DNA. A nuclear complex consisting of a majority of the FA proteins plays a crucial role in this process and is required for the monoubiquitination of a downstream target, FANCD2. Two new FA genes, FANCB and FANCL, have recently been identified, and their discovery has allowed a more detailed study into the molecular architecture of the FA pathway. We demonstrate a direct interaction between FANCB and FANCL and that a complex of these proteins binds FANCA. The interaction between FANCA and FANCL is dependent on FANCB, FANCG, and FANCM, but independent of FANCC, FANCE, and FANCF. These findings provide a framework for the protein interactions that occur "upstream" in the FA pathway and suggest that besides the FA core complex different subcomplexes exist that may have specific functions other than the monoubiquitination of FANCD2.
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Affiliation(s)
- Annette L Medhurst
- Department of Clinical Genetics and Human Genetics, Vrije Universiteit University Medical Center, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands
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46
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Hamanoue S, Yagasaki H, Tsuruta T, Oda T, Yabe H, Yabe M, Yamashita T. Myeloid lineage-selective growth of revertant cells in Fanconi anaemia. Br J Haematol 2006; 132:630-5. [PMID: 16445838 DOI: 10.1111/j.1365-2141.2005.05916.x] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
Abstract
Fanconi anaemia (FA) is a genetically heterogeneous chromosome instability syndrome characterised by bone marrow failure and congenital anomalies. Although an increasing number of reports suggest that reversion mosaicism noted in peripheral blood lymphocytes (PBLs) is associated with mild haematopoietic failure in FA, myeloid cells are rarely directly examined. We here report a patient with prolonged mild pancytopenia in whom proliferation of revertant cells was detected in mature myeloid cells but not in PBLs. While this patient had inherited heterozygous mutations, 2546delC and 3720-3724del, in the major FA gene FANCA, Epstein-Barr virus-immortalised lymphoblastoid cells from the patient had 2546C > T instead of 2546delC, resulting in expression of a functional missense protein. As the identical reversion was detected in polymorphonuclear granulocytes and mononuclear phagocytes, sustained haematopoiesis in the patient can be attributed to a selective growth advantage of revertant myeloid cells. It is noteworthy that such a myeloid lineage-selective mosaicism is overlooked in routine examination of PBLs. Recognition of this status will expand the role of reversion mosaicism in the pathophysiology of FA.
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Affiliation(s)
- Satoshi Hamanoue
- Division of Genetic Diagnosis, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
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47
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Yagasaki H, Hamanoue S, Oda T, Nakahata T, Asano S, Yamashita T. Identification and characterization of novel mutations of the major Fanconi anemia gene FANCA in the Japanese population. Hum Mutat 2005; 24:481-90. [PMID: 15523645 DOI: 10.1002/humu.20099] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Fanconi anemia (FA) is a rare autosomal recessive disorder of hematopoiesis, with at least 11 complementation groups. FANCA, a gene for group A, accounts for the majority of FA patients. Previous studies of FANCA mutations revealed high allelic heterogeneity, frequent occurrence of large deletions, and interpopulation differences. However, systematic mutational analysis, including gene dosage assay to detect large deletions, has not been documented for Asian populations. A newly developed TaqMan quantitative PCR-based gene dosage assay, combined with sequencing of exons and cDNA fragments, allowed for detection of 48 mutant alleles of FANCA in 27 (77%) of 35 unrelated Japanese FA families with no detectable mutations in FANCC or FANCG. We identified 29 different mutations (21 nucleotide substitutions or small deletions/insertions and eight large deletions), at least 20 of which were novel. The FANCA mutational spectrum of the Japanese was different from that of other ethnic groups so far studied. This is the largest scale of mutation analysis of FANCA in the Japanese population. Characterization of these mutations provided new information regarding the mutagenesis mechanisms and structure-function relationship of FANCA. Specifically, our data suggest that diverse mechanisms including nonhomologous recombination as well as Alu-mediated homologous recombination are involved in the generation of large deletions in FANCA.
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Affiliation(s)
- Hiroshi Yagasaki
- Division of Genetic Diagnosis, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
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48
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Litman R, Peng M, Jin Z, Zhang F, Zhang J, Powell S, Andreassen PR, Cantor SB. BACH1 is critical for homologous recombination and appears to be the Fanconi anemia gene product FANCJ. Cancer Cell 2005; 8:255-65. [PMID: 16153896 DOI: 10.1016/j.ccr.2005.08.004] [Citation(s) in RCA: 289] [Impact Index Per Article: 14.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/29/2005] [Revised: 08/18/2005] [Accepted: 08/22/2005] [Indexed: 12/13/2022]
Abstract
We showed in this study that cells deficient of the BRCA1-associated BACH1 helicase, also known as BRIP1, failed to elicit homologous recombination (HR) after DNA double-stranded breaks (DSBs). BACH1-deficient cells were also sensitive to mitomycin C (MMC) and underwent MMC-induced chromosome instability. Moreover, we identified a homozygous nonsense mutation in BACH1 in a FA-J patient-derived cell line and could not detect BACH1 protein in this cell line. Expression of wild-type BACH1 in this cell line reduced the accumulation of cells at G2/M phases following exposure to DNA crosslinkers, a characteristic of Fanconi anemia (FA) cells. These results support the conclusion that BACH1 is FANCJ.
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Affiliation(s)
- Rachel Litman
- Department of Cancer Biology, University of Massachusetts Medical School, Worcester, 01605, USA
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49
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Gordon SM, Alon N, Buchwald M. FANCC, FANCE, and FANCD2 form a ternary complex essential to the integrity of the Fanconi anemia DNA damage response pathway. J Biol Chem 2005; 280:36118-25. [PMID: 16127171 DOI: 10.1074/jbc.m507758200] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Fanconi anemia (FA) is a genetically heterogeneous disorder characterized by bone marrow failure, cancer predisposition, and increased cellular sensitivity to DNA-cross-linking agents. The products of seven of the nine identified FA genes participate in a protein complex required for monoubiquitination of the FANCD2 protein. Direct interaction of the FANCE protein with both fellow FA complex component FANCC and the downstream FANCD2 protein has been observed in the yeast two-hybrid system. Here, we demonstrate the ability of FANCE to mediate the interaction between FANCC and FANCD2 in the yeast three-hybrid system and confirm the FANCE-mediated association of FANCC with FANCD2 in human cells. A yeast two-hybrid system-based screen was devised to identify randomly mutagenized FANCE proteins capable of interaction with FANCC but not with FANCD2. Exogenous expression of these mutants in an FA-E cell line and subsequent evaluation of FANCD2 monoubiquitination and DNA cross-linker sensitivity indicated a critical role for the FANCE/FANCD2 interaction in maintaining FA pathway integrity. Three-hybrid experiments also demonstrated the ability of FANCE to mediate the interaction between FA core complex components FANCC and FANCF, indicating an additional role for FANCE in complex assembly. Thus, FANCE is shown to be a key mediator of protein interactions both in the architecture of the FA protein complex and in the connection of complex components to the putative downstream targets of complex activity.
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Affiliation(s)
- Susan M Gordon
- Program in Genetics and Genomic Biology, Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
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50
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Ferrer M, Rodríguez JA, Spierings EA, de Winter JP, Giaccone G, Kruyt FAE. Identification of multiple nuclear export sequences in Fanconi anemia group A protein that contribute to CRM1-dependent nuclear export. Hum Mol Genet 2005; 14:1271-81. [PMID: 15790592 DOI: 10.1093/hmg/ddi138] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
The Fanconi anemia (FA) pathway plays an important role in maintaining genomic stability, and defects in this pathway cause cancer susceptibility. The FA proteins have been found to function primarily in a nuclear complex, although a cytoplasmic localization and function for several FA proteins has also been reported. In this study, we investigated the possibility that FANCA, FANCC and FANCG are subjected to active export out of the nucleus. After treatment with leptomycin B, a specific inhibitor of CRM1-mediated nuclear export, the accumulation of epitope-tagged FANCA in the nucleus increased, whereas FANCC was affected to a lesser extent and FANCG showed no response. CRM1-mediated export of FANCA was further confirmed using CRM1 cotransfection, which led to a dramatic relocalization of FANCA to the cytoplasm. Five functional leucine-rich nuclear export sequences (NESs) distributed throughout the FANCA sequence were identified and characterized using an in vivo export assay. Simultaneous inactivation of three of these NESs resulted in a discrete but reproducible increase of FANCA nuclear accumulation. However, these NES mutations did not affect the ability of FANCA to complement the mitomycin C or cisplatin sensitivity of FA-A lymphoblasts. Surprisingly, mutations in the other two NESs resulted in an almost complete relocation of the protein to cytoplasm, suggesting that these motifs overlap with domains that are crucial for nuclear import. Taken together, these findings indicate that FANCA can be actively exported out of the nucleus by CRM1, revealing a new mechanism to regulate the function of the FA protein complex.
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Affiliation(s)
- Miriam Ferrer
- Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands
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