Chromosome Instability in Fanconi Anemia: From Breaks to Phenotypic Consequences

Fanconi anemia (FA), a chromosomal instability syndrome, is caused by inherited pathogenic variants in any of 22 FANC genes, which cooperate in the FA/BRCA pathway. This pathway regulates the repair of DNA interstrand crosslinks (ICLs) through homologous recombination. In FA proper repair of ICLs is impaired and accumulation of toxic DNA double strand breaks occurs. To repair this type of DNA damage, FA cells activate alternative error-prone DNA repair pathways, which may lead to the formation of gross structural chromosome aberrations of which radial figures are the hallmark of FA, and their segregation during cell division are the origin of subsequent aberrations such as translocations, dicentrics and acentric fragments. The deficiency in DNA repair has pleiotropic consequences in the phenotype of patients with FA, including developmental alterations, bone marrow failure and an extreme risk to develop cancer. The mechanisms leading to the physical abnormalities during embryonic development have not been clearly elucidated, however FA has features of premature aging with chronic inflammation mediated by pro-inflammatory cytokines, which results in tissue attrition, selection of malignant clones and cancer onset. Moreover, chromosomal instability and cell death are not exclusive of the somatic compartment, they also affect germinal cells, as evidenced by the infertility observed in patients with FA.

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Chromosome Instability in Fanconi Anemia: From Breaks to Phenotypic Consequences

10.20944/preprints202011.0563.v2 ◽

2020 ◽

Author(s):

Benilde García-de-Teresa ◽

Alfredo Rodríguez ◽

Sara Frias

Keyword(s):

Dna Repair ◽

Fanconi Anemia ◽

Chromosomal Instability ◽

Bone Marrow Failure ◽

Chromosome Instability ◽

Premature Aging ◽

Dna Double Strand Breaks ◽

Interstrand Crosslinks ◽

Pathogenic Variants ◽

Extreme Risk

Abstract: Fanconi anemia (FA), a chromosomal instability syndrome, is caused by inherited pathogenic variants in any of 22 FANC genes, that cooperate in the FA/BRCA pathway. This pathway regulates the repair of DNA interstrand crosslinks (ICLs) through homologous recombination. In FA proper repair of ICLs is impaired, and accumulation of toxic DNA double strand breaks occurs. In order to repair this type of DNA damage, FA cells activate alternative error-prone DNA repair pathways, that may lead to the formation of gross structural chromosome aberrations of which radial figures are the hallmark of FA and their segregation during cell division are the origin of subsequent aberrations like translocations, dicentrics and acentric fragments. The deficiency in DNA repair has pleiotropic consequences in the phenotype of patients with FA, including developmental alterations, bone marrow failure and an extreme risk to develop cancer. The mechanisms leading to the physical abnormalities during embryonic development have not been clearly elucidated, however FA has features of premature aging with chronic inflammation mediated by pro-inflammatory cytokines, that results in tissue attrition, selection of malignant clones and cancer onset. Moreover, the effect of the FA/BRCA pathway in germinal cells, evidenced by infertility in patients with FA attests of chromosomal instability and cell death also occurring in the germinal compartment.

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Causes and Consequences of Chromosomal Instability in Fanconi Anemia. Alterations at the Cellular and Organism Level

10.20944/preprints202011.0563.v1 ◽

2020 ◽

Author(s):

Benilde García-de-Teresa ◽

Alfredo Rodríguez ◽

Sara Frias

Keyword(s):

Dna Repair ◽

Fanconi Anemia ◽

Chromosomal Instability ◽

Bone Marrow Failure ◽

Chromosome Instability ◽

Premature Aging ◽

Dna Double Strand Breaks ◽

Interstrand Crosslinks ◽

Pathogenic Variants ◽

Extreme Risk

Abstract: Fanconi anemia (FA), a chromosome instability syndrome, is caused by inherited pathogenic variants in any of 22 FANC genes, that cooperate in the FA/BRCA pathway. This pathway regulates the repair of DNA interstrand crosslinks (ICLs) through homologous recombination. In FA proper repair of ICLs is impaired, and accumulation of toxic DNA double strand breaks occurs. In order to repair this type of DNA damage, FA cells activate alternative error-prone DNA repair pathways, that may lead to the formation of gross structural chromosome aberrations of which radial figures are the epitome and origin of subsequent aberrations like translocations, dicentrics and acentric fragments. The deficiency in DNA repair has pleiotropic consequences in the phenotype of patients with FA, including developmental alterations, bone marrow failure and an extreme risk to develop cancer. The mechanisms leading to the physical abnormalities during embryonic development have not been clearly elucidated, however FA has features of premature aging with chronic inflammation mediated by pro-inflammatory cytokines, that results in tissue attrition, selection of malignant clones and cancer onset. Moreover, the effect of the FA/BRCA pathway in germinal cells, evidenced by infertility in patients with FA attests of chromosomal instability and cell death also occurring in the germinal compartment.

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Association of clinical severity with FANCB variant type in Fanconi anemia

Blood ◽

10.1182/blood.2019003249 ◽

2020 ◽

Vol 135 (18) ◽

pp. 1588-1602 ◽

Cited By ~ 8

Author(s):

Moonjung Jung ◽

Ramanagouda Ramanagoudr-Bhojappa ◽

Sylvie van Twest ◽

Rasim Ozgur Rosti ◽

Vincent Murphy ◽

...

Keyword(s):

Bone Marrow ◽

Fanconi Anemia ◽

Bone Marrow Failure ◽

Complementation Group ◽

Clinical Severity ◽

Missense Variants ◽

Interstrand Crosslinks ◽

Pathogenic Variants ◽

Enzymatic Activation ◽

Fancd2 Monoubiquitination

Abstract Fanconi anemia (FA) is the most common genetic cause of bone marrow failure and is caused by inherited pathogenic variants in any of 22 genes. Of these, only FANCB is X-linked. We describe a cohort of 19 children with FANCB variants, from 16 families of the International Fanconi Anemia Registry. Those with FANCB deletion or truncation demonstrate earlier-than-average onset of bone marrow failure and more severe congenital abnormalities compared with a large series of FA individuals in published reports. This reflects the indispensable role of FANCB protein in the enzymatic activation of FANCD2 monoubiquitination, an essential step in the repair of DNA interstrand crosslinks. For FANCB missense variants, more variable severity is associated with the extent of residual FANCD2 monoubiquitination activity. We used transcript analysis, genetic complementation, and biochemical reconstitution of FANCD2 monoubiquitination to determine the pathogenicity of each variant. Aberrant splicing and transcript destabilization were associated with 2 missense variants. Individuals carrying missense variants with drastically reduced FANCD2 monoubiquitination in biochemical and/or cell-based assays tended to show earlier onset of hematologic disease and shorter survival. Conversely, variants with near-normal FANCD2 monoubiquitination were associated with more favorable outcome. Our study reveals a genotype-phenotype correlation within the FA-B complementation group of FA, where severity is associated with level of residual FANCD2 monoubiquitination.

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Spectrin and its interacting partners in nuclear structure and function

Experimental Biology and Medicine ◽

10.1177/1535370218763563 ◽

2018 ◽

Vol 243 (6) ◽

pp. 507-524 ◽

Cited By ~ 8

Author(s):

Muriel W Lambert

Keyword(s):

Dna Repair ◽

Cancer Progression ◽

Fanconi Anemia ◽

Mechanism Of Action ◽

Bone Marrow Failure ◽

Chromosome Instability ◽

Nuclear Proteins ◽

Structural Protein ◽

Cellular Functions ◽

Structural Functions

Nonerythroid αII-spectrin is a structural protein whose roles in the nucleus have just begun to be explored. αII-spectrin is an important component of the nucleoskelelton and has both structural and non-structural functions. Its best known role is in repair of DNA ICLs both in genomic and telomeric DNA. αII-spectrin aids in the recruitment of repair proteins to sites of damage and a proposed mechanism of action is presented. It interacts with a number of different groups of proteins in the nucleus, indicating it has roles in additional cellular functions. αII-spectrin, in its structural role, associates/co-purifies with proteins important in maintaining the architecture and mechanical properties of the nucleus such as lamin, emerin, actin, protein 4.1, nuclear myosin, and SUN proteins. It is important for the resilience and elasticity of the nucleus. Thus, αII-spectrin’s role in cellular functions is complex due to its structural as well as non-structural roles and understanding the consequences of a loss or deficiency of αII-spectrin in the nucleus is a significant challenge. In the bone marrow failure disorder, Fanconi anemia, there is a deficiency in αII-spectrin and, among other characteristics, there is defective DNA repair, chromosome instability, and congenital abnormalities. One may speculate that a deficiency in αII-spectrin plays an important role not only in the DNA repair defect but also in the congenital anomalies observed in Fanconi anemia , particularly since αII-spectrin has been shown to be important in embryonic development in a mouse model. The dual roles of αII-spectrin in the nucleus in both structural and non-structural functions make this an extremely important protein which needs to be investigated further. Such investigations should help unravel the complexities of αII-spectrin’s interactions with other nuclear proteins and enhance our understanding of the pathogenesis of disorders, such as Fanconi anemia , in which there is a deficiency in αII-spectrin. Impact statement The nucleoskeleton is critical for maintaining the architecture and functional integrity of the nucleus. Nonerythroid α-spectrin (αIISp) is an essential nucleoskeletal protein; however, its interactions with other structural and non-structural nuclear proteins and its functional importance in the nucleus have only begun to be explored. This review addresses these issues. It describes αIISp’s association with DNA repair proteins and at least one proposed mechanism of action for its role in DNA repair. Specific interactions of αIISp with other nucleoskeletal proteins as well as its important role in the biomechanical properties of the nucleus are reviewed. The consequences of loss of αIISp, in disorders such as Fanconi anemia, are examined, providing insights into the profound impact of this loss on critical processes known to be abnormal in FA, such as development, carcinogenesis, cancer progression and cellular functions dependent upon αIISp’s interactions with other nucleoskeletal proteins.

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The Fanconi Anemia DNA Repair Pathway Is Regulated by an Interaction between Ubiquitin and the E2-like Fold Domain of FANCL

Journal of Biological Chemistry ◽

10.1074/jbc.m115.675835 ◽

2015 ◽

Vol 290 (34) ◽

pp. 20995-21006 ◽

Cited By ~ 17

Author(s):

Jennifer A. Miles ◽

Mark G. Frost ◽

Eilis Carroll ◽

Michelle L. Rowe ◽

Mark J. Howard ◽

...

Keyword(s):

Dna Repair ◽

Fanconi Anemia ◽

Bone Marrow Failure ◽

Repair Pathway ◽

Covalent Interaction ◽

Interstrand Crosslinks ◽

Ring E3 Ligase ◽

Fancd2 Monoubiquitination

The Fanconi Anemia (FA) DNA repair pathway is essential for the recognition and repair of DNA interstrand crosslinks (ICL). Inefficient repair of these ICL can lead to leukemia and bone marrow failure. A critical step in the pathway is the monoubiquitination of FANCD2 by the RING E3 ligase FANCL. FANCL comprises 3 domains, a RING domain that interacts with E2 conjugating enzymes, a central domain required for substrate interaction, and an N-terminal E2-like fold (ELF) domain. The ELF domain is found in all FANCL homologues, yet the function of the domain remains unknown. We report here that the ELF domain of FANCL is required to mediate a non-covalent interaction between FANCL and ubiquitin. The interaction involves the canonical Ile44 patch on ubiquitin, and a functionally conserved patch on FANCL. We show that the interaction is not necessary for the recognition of the core complex, it does not enhance the interaction between FANCL and Ube2T, and is not required for FANCD2 monoubiquitination in vitro. However, we demonstrate that the ELF domain is required to promote efficient DNA damage-induced FANCD2 monoubiquitination in vertebrate cells, suggesting an important function of ubiquitin binding by FANCL in vivo.

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Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

10.1101/811968 ◽

2019 ◽

Author(s):

Kimberly A. Rickman ◽

Ray Noonan ◽

Francis P. Lach ◽

Sunandini Sridhar ◽

Anderson T. Wang ◽

...

Keyword(s):

Fanconi Anemia ◽

Essential Gene ◽

Dna Lesions ◽

Replication Forks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Interstrand Crosslinks ◽

Pathogenic Variants ◽

Dna Interstrand Crosslinks ◽

Stalled Replication Forks

SummaryDNA interstrand crosslinks (ICLs) are a form of DNA damage that requires the interplay of a number of repair proteins including those of the Fanconi anemia (FA) and the homologous recombination (HR) pathways. Pathogenic variants in the essential gene BRCA2/FANCD1, when monoallelic, predispose to breast and ovarian cancer, and when biallelic, results in a severe subtype of Fanconi anemia. BRCA2 function in the FA pathway is attributed to its role as a mediator of the RAD51 recombinase in HR repair of the programmed DNA double strand breaks (DSB). BRCA2 and RAD51 functions are also required to protect stalled replication forks from nucleolytic degradation during response to hydroxyurea (HU). While RAD51 has been shown to be necessary in the early steps of ICL repair to prevent aberrant nuclease resection, the role of BRCA2 in this process has not been described. Here, based on the analysis of BRCA2 DNA binding domain (DBD) mutants discovered in FA patients presenting with atypical FA-like phenotypes, we establish that BRCA2 is necessary for protection of DNA at an ICL. Cells carrying DBD BRCA2 mutations are sensitive to ICL inducing agents but resistant to HU treatment consistent with relatively high HR repair in these cells. BRCA2 function at an ICL protects against DNA2-WRN nuclease-helicase complex and not the MRE11 nuclease implicated in the resection of HU-stalled replication forks. Our results also indicate that unlike the processing at HU-stalled forks, function of the SNF2 translocases (SMARCAL1, ZRANB3, or HLTF), implicated in fork reversal, are not an integral component of the ICL repair, pointing to a different mechanism of fork protection at different DNA lesions.

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DNA Repair: Exploiting the Fanconi Anemia Pathway As a Potential Therapeutic Target

Physiological Research ◽

10.33549/physiolres.932115 ◽

2011 ◽

pp. 453-465 ◽

Cited By ~ 13

Author(s):

T. HUCL ◽

E. GALLMEIER

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Fanconi Anemia ◽

Cancer Susceptibility ◽

Synthetic Lethality ◽

Bone Marrow Failure ◽

Long Term Effects ◽

Alternative Pathways ◽

Repair Mechanisms ◽

Relationship Of

DNA repair is an active cellular process to respond to constant DNA damage caused by metabolic processes and environmental factors. Since the outcome of DNA damage is generally adverse and long term effects may contribute to oncogenesis, cells have developed a variety of DNA repair mechanisms, which operate depending on the type of DNA damage inflicted. At least 15 Fanconi anemia (FA) proteins interact in a common pathway involved in homologous recombination. Inherited homozygous mutations in any of these FA genes cause a rare disease, Fanconi anemia, characterized by congenital abnormalities, progressive bone-marrow failure and cancer susceptibility. Heterozygous germline FA mutations predispose to various types of cancer. In addition, somatic FA mutations have been identified in diverse cancer types. Evidence exists that cells deficient in the FA pathway become dependent on alternative pathways for survival. Additional inhibition of such alternative pathways is thus expected to result in cell death, creating a relationship of synthetic lethality. Identifying these relationships can reveal yet unknown mechanisms of DNA repair and new targets for therapy.

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Canonical and Noncanonical Roles of Fanconi Anemia Proteins: Implications in Cancer Predisposition

Cancers ◽

10.3390/cancers12092684 ◽

2020 ◽

Vol 12 (9) ◽

pp. 2684 ◽

Cited By ~ 2

Author(s):

Giacomo Milletti ◽

Luisa Strocchio ◽

Daria Pagliara ◽

Katia Girardi ◽

Roberto Carta ◽

...

Keyword(s):

Dna Repair ◽

Fanconi Anemia ◽

Clinical Phenotype ◽

Bone Marrow Failure ◽

Therapeutic Approaches ◽

Dna Repair Mechanisms ◽

Solid Malignancies ◽

Interstrand Cross Links ◽

Repair Mechanisms ◽

Cross Links

Fanconi anemia (FA) is a clinically and genetically heterogeneous disorder characterized by the variable presence of congenital somatic abnormalities, bone marrow failure (BMF), and a predisposition to develop cancer. Monoallelic germline mutations in at least five genes involved in the FA pathway are associated with the development of sporadic hematological and solid malignancies. The key function of the FA pathway is to orchestrate proteins involved in the repair of interstrand cross-links (ICLs), to prevent genomic instability and replication stress. Recently, many studies have highlighted the importance of FA genes in noncanonical pathways, such as mitochondria homeostasis, inflammation, and virophagy, which act, in some cases, independently of DNA repair processes. Thus, primary defects in DNA repair mechanisms of FA patients are typically exacerbated by an impairment of other cytoprotective pathways that contribute to the multifaceted clinical phenotype of this disease. In this review, we summarize recent advances in the understanding of the pathogenesis of FA, with a focus on the cytosolic noncanonical roles of FA genes, discussing how they may contribute to cancer development, thus suggesting opportunities to envisage novel therapeutic approaches.

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Fanconi anemia group J mutation abolishes its DNA repair function by uncoupling DNA translocation from helicase activity or disruption of protein-DNA complexes

Blood ◽

10.1182/blood-2009-11-256016 ◽

2010 ◽

Vol 116 (19) ◽

pp. 3780-3791 ◽

Cited By ~ 52

Author(s):

Yuliang Wu ◽

Joshua A. Sommers ◽

Avvaru N. Suhasini ◽

Thomas Leonard ◽

Julianna S. Deakyne ◽

...

Keyword(s):

Dna Repair ◽

Fanconi Anemia ◽

Atp Hydrolysis ◽

Bone Marrow Failure ◽

Dna Helicase ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Dna Complexes ◽

Null Cell ◽

Negative Effect

Abstract Fanconi anemia (FA) is a genetic disease characterized by congenital abnormalities, bone marrow failure, and susceptibility to leukemia and other cancers. FANCJ, one of 13 genes linked to FA, encodes a DNA helicase proposed to operate in homologous recombination repair and replicational stress response. The pathogenic FANCJ-A349P amino acid substitution resides immediately adjacent to a highly conserved cysteine of the iron-sulfur domain. Given the genetic linkage of the FANCJ-A349P allele to FA, we investigated the effect of this particular mutation on the biochemical and cellular functions of the FANCJ protein. Purified recombinant FANCJ-A349P protein had reduced iron and was defective in coupling adenosine triphosphate (ATP) hydrolysis and translocase activity to unwinding forked duplex or G-quadruplex DNA substrates or disrupting protein-DNA complexes. The FANCJ-A349P allele failed to rescue cisplatin or telomestatin sensitivity of a FA-J null cell line as detected by cell survival or γ-H2AX foci formation. Furthermore, expression of FANCJ-A349P in a wild-type background exerted a dominant-negative effect, indicating that the mutant protein interferes with normal DNA metabolism. The ability of FANCJ to use the energy from ATP hydrolysis to produce the force required to unwind DNA or destabilize protein bound to DNA is required for its role in DNA repair.

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Fanconi Anemia FANCG Protein in Mitigating Radiation- and Enzyme-Induced DNA Double-Strand Breaks by Homologous Recombination in Vertebrate Cells

Molecular and Cellular Biology ◽

10.1128/mcb.23.15.5421-5430.2003 ◽

2003 ◽

Vol 23 (15) ◽

pp. 5421-5430 ◽

Cited By ~ 103

Author(s):

Kazuhiko Yamamoto ◽

Masamichi Ishiai ◽

Nobuko Matsushita ◽

Hiroshi Arakawa ◽

Jane E. Lamerdin ◽

...

Keyword(s):

Homologous Recombination ◽

Fanconi Anemia ◽

Cancer Susceptibility ◽

Bone Marrow Failure ◽

Chromosome Breakage ◽

Breast Cancer Susceptibility ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Brca1 And Brca2 ◽

Strand Breaks

ABSTRACT The rare hereditary disorder Fanconi anemia (FA) is characterized by progressive bone marrow failure, congenital skeletal abnormality, elevated susceptibility to cancer, and cellular hypersensitivity to DNA cross-linking chemicals and sometimes other DNA-damaging agents. Molecular cloning identified six causative genes (FANCA, -C, -D2, -E, -F, and -G) encoding a multiprotein complex whose precise biochemical function remains elusive. Recent studies implicate this complex in DNA damage responses that are linked to the breast cancer susceptibility proteins BRCA1 and BRCA2. Mutations in BRCA2, which participates in homologous recombination (HR), are the underlying cause in some FA patients. To elucidate the roles of FA genes in HR, we disrupted the FANCG/XRCC9 locus in the chicken B-cell line DT40. FANCG-deficient DT40 cells resemble mammalian fancg mutants in that they are sensitive to killing by cisplatin and mitomycin C (MMC) and exhibit increased MMC and radiation-induced chromosome breakage. We find that the repair of I-SceI-induced chromosomal double-strand breaks (DSBs) by HR is decreased ∼9-fold in fancg cells compared with the parental and FANCG-complemented cells. In addition, the efficiency of gene targeting is mildly decreased in FANCG-deficient cells, but depends on the specific locus. We conclude that FANCG is required for efficient HR-mediated repair of at least some types of DSBs.

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