Fanconi Anemia Genes and DNA Damage Repair

Cells and cell lines from FA patients are phenotypically similar regard less of the complementation group that they represent. A hypothesis was therefore formulated and subsequently substantiated that the various wild-type FA proteins function in a common response pathway to repair DNA damage incurred during DNA replication. A major function of FA pathway genes is to repair interstrand DNA crosslinks. Many exogenous agents (e.g. cisplatin, nitrogen mustards, and MMC) and endogenous agents (e.g. aldehydes and free oxygen radicals) can induce formation of interstrand DNA crosslinks.

There are three general steps in the FA DNA damage response pathway: (1) Core complex. Nine wild-type FA proteins (FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, FANCM, and FANCT) and additional proteins (e.g., FAAP20, FAAP24, and FAAP100) form a single large nuclear protein “core complex” as the first step. The core complex functions as a ubiquitin ligase of which FANCT, an E2 ubiquitin-conjugating enzyme and FANCL, an E3 ubiquitin ligase, are the catalytic subunits. The FA core complex is recruited to fork-like DNA structures via FAAP24 and FANCM. (2) ID2 complex. The activated core complex converts a downstream heterodimer composed of two proteins, FANCI and FANCD2 (called the “ID2 complex”), from unubiquitinated isoforms to monoubiquitinated isoforms. Monoubiquitination does not occur if the core complex upstream of the ID2 complex is not intact, and there fore FA cells from patients with upstream mutations do not show the monoubiquitinated FANCI/FANCD2. Recently, it has been shown that monoubiquitination occurs after the ID2 complex binds to an arrested replication fork at an ICL. (3) Downstream effector complexes. In normal cells after monoubiquitination of the ID2 complex, it forms a binding interface for single and double stranded DNA and downstream effector complexes with additional FA and other proteins. The scaffold protein FANCP/SLX4 binds first, followed by the endonucleases FANCQ(ERCC4), MUS81, SLX1, and ERCC1 that cleave DNA interstrand crosslinks, resulting in DNA adducts and dsDNA breaks. DNA adducts are resolved by the exonucleases FANCV/REV7, REV1, REV3 after forming a translesion synthesis complex. dsDNA breaks are repaired by homologous recombination. The exonucleases CTIP, MRN, XO1 cut the dsDNA breaks and generate ssDNA with 3′ overhang. Replication protein A (RPA) is recruited to the ssDNA break. FANCR/RAD51 recombinase localizes and loaded to the ssDNA/RPA complex by FANCD1/BRACA2 and FANCS/BRACA1. FANCR/RAD51 removes RPA and prevents it from self-binding. A recombination filament is generated by FANCR/ RAD51 and the additional proteins that bind to the site in parallel or subsequently: FANCD1/BRCA2, FANCS/BRCA1, FANCO/ RAD51C, FANCJ/BRIP1, FANCN/PALB2, and FANCU/XRCC1. Consequently, the recombination filament searches for homologous bases to repair DNA crosslinks.

There are three main DNA repair processes that the FA genes cooperate with: (1) nucleotide excision repair that excises one DNA strand flanking the interstrand crosslink (via interaction between FANCP/SLX4 and MUS81, SLX1, and others) followed by ligation. This process is utilized in quiescent cells; (2) translesion synthesis that involves one strand incisions around the interstrand crosslink (ICL), unhooking of the ICL, and extension of the uncut strand (via recruitment of translesion polymerase); (3) homologous recombination is initiated after nucleolytic incisions by endonucleases (FANCQ/ ERCC4), MUS81, SLX1, ERCC1) and generation of dsDNA break. The dsDNA break is cut by exonucleases to generate a ssDNA break, to which FANCR/RAD51, FANCD1, FANCS, FANCN, FANCJ, FANCO, FANCU, and other proteins are recruited to form a recombination filament that searches for homologous bases for further repair. Other DNA-repair proteins such as MRE11-RAD50-NBS1, PCNA, and BLM are also involved in the later stages of the DNA repair response.

The exact link between the impaired ability to repair interstrand crosslinks and FA phenotype is still to be defined, but may be related to accumulation of DNA adducts, a failure to arrest DNA synthesis in response to DNA damage, impaired homologous recombination, defective nonhomologous end joining, abnormal induction of p53, induction of P53/CHK1 dependent G2/M cell cycle arrest, and increased apoptosis. In addition, homologous recombination and several FA proteins play a role in replication of telomeric G4 structures and possibly prevention of replication induced telomere damage. Loss of these functions may thus lead to short telomeres.

Fanconi Anemia Genes, Cell Survival, and Balancing Oxidative Stress

There are important protein-protein interactions between FA proteins and non-FA “binding partners” for cell survival. FANCC and FANCD2 form complexes with members of the signal transducer and activator of transcription (STAT) family of transcription factors in cytokine-mediated biologic responses. Secondly, heat shock proteins provide several cell survival functions, and FANCC protein specifically facilitates the anti-apoptotic role of Hsp70. FANCC also inter acts with cdc2, PKR, and p53, suggesting that FANCC has other roles that are independent of DNA damage recognition and repair. GSTP1 is an enzyme that detoxifies byproducts of redox stress and xenobiotics and FANCC protein enhances GSTP1 activity in cells exposed to apoptosis inducers.

Several studies suggested a role of oxidative stress in the evolution of BM failure and leukemia in FA. Reactive oxygen species (ROS) were shown to be elevated in FA cells and high oxidative stress causes increased DNA damage, increased hematopoietic stem cell (HSC) senescence and a decreased HSC pool, thereby leading to BM failure. Further, in vivo and in vitro studies have demonstrated the ability of the antioxidant N-acetylcysteine to reduce DNA damage, reduce HSC senescence, and improve HSC reconstitution ability. Therefore, it is possible that patients with FA are particularly sensitive to ROS induced DNA damage due to impaired DNA repair mechanisms. This increased sensitivity may be caused, at least in part, by impaired detoxification of ROS and naturally produced aldehydes. A deficiency in superoxide dismutase and poor cell growth at ambient oxygen have also been demonstrated in FA cells.

In FA patients’ skin fibroblasts, N-acetylcysteine was able to reduce ROS levels and apoptosis as measured by activation of caspase-3 and poly(ADP-ribose)polymerase (PARP) cleavage. In fancc−/− mice, N-acetylcysteine rescues hematopoietic colony formation that is impaired by spontaneous secretion of TNFα. It also reduces TNFα mediated hematopoietic colony formation and HSC senescence and HSC reconstitution potential. Using a fancd2−/− mouse model, treatment with the antioxidant drug resveratrol has also been shown to preserve HSC quiescence, partially correct the abnormal cell cycle status, and significantly improve the spleen colony-forming capacity of BM cells. Importantly, treatment of FA mice with N-acetylcysteine has been shown to reduce the accumulation of cytogenetic abnormalities (that are commonly seen in FA patients who transform to MDS/ AML). In one study, the antioxidant tempol delayed cancer in tumor prone fancd2−/−/Trp53+/− mice. However, in another study neither N-acetylcysteine nor the antioxidant resveratrol had this property in this mouse model.

Cell lines from FA patients have also been shown to feature increased autophagy and mitophagy that was attributed to elevated levels of mitochondrial fission caused by high oxidative stress. In another study, interestingly, cells from FA patients showed impairment of mitochondrial functions as evidenced by a high frequency of mtDNA genetic variants, downregulation of mtDNA complex-I and complex-III encoding genes of OXPHOS, and reduced expression of certain mitophagy-related genes (ATG, Beclin-1, and MAP1-LC3) that may lead to reduced ability to clear damaged mitochondria.

The level at which oxidative stress is linked to FA phenotype independently of DNA damage is still to be defined. The high oxidative stress and oxygen sensitivity phenotype of FA cells shorten cell survival. A cardinal phenotype of FA cells is an abnormality in cell cycle distribution with an increased number of cells with 4 N DNA content arising from a delay in the G2 /M or late S phase of the cell cycle. The strongest evidence supporting an oxygen metabolism deficiency in FA is a reduction of FA cells with 4 N DNA content when grown at low oxygen levels and the unexpected appearance of 4 N DNA content when normal cells are grown at high oxygen levels. Of note, some wild-type FA proteins play a role in redox-related functions. FANCC associates with NADPH (nicotinamide adenine dinucleotide phosphate), cytochrome P-450 reductase, and glutathione S-transferase, proteins with redox functions. FANCA and FANCG are redox-sensitive proteins that multimerize after H2 O2 treatment, prompting the notion that the FA pathway may function in oxidative stress management.

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Testing for Germline Pathogenic Variants Causing Hereditary Cancer

Pathogenic Variants in Tumor Suppressor Genes Causing Autosomal Recessive Pediatric Cancer Syndromes

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The Current State of Treatment of Genetic Disease

Diseases Due to the expansion of Unstable Repeat Sequences

Normal Collagen Structure and Its Relationship to Osteogenesis Imperfecta

Cystic Fibrosis

Familial Hypercholesterolemia: A Genetic Hyperlipidemia

A Loss of Glycosylation: Mucolipidosis II or I-Cell Disease

Congenital Disorders of Glycosylation

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