Molecular pathogenesis and clinical management of Fanconi anemia (original) (raw)
FANCs. To date, 15 FANCs have been identified (FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, FANCN, FANCO, and FANCP; Table 1). The majority of FA genes are located on autosomes, with the exception of FANCB, which is on the X chromosome. FA patients with mutations in any of these FA genes share a characteristic clinical and cellular phenotype, and these 15 gene products appear to function in a common cellular pathway, termed the Fanconi anemia pathway (26). Indeed, mutations in eight FA subtypes (FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, and FANCM) result in loss of FANCD2 and FANCI monoubiquitylation, the central regulatory event in the FA pathway. Mutations in these eight upstream genes account for approximately 90% of patients (Table 1). Identification of the breast cancer susceptibility gene BRCA2 as an FA gene (FANCD1; ref. 27) reaffirm the close cooperative relationship between the FA pathway and the breast and ovarian tumor-suppressive BRCA proteins BRCA1 (28) and BRCA2 in DNA repair mechanisms. Subsequent identification of FANCN (also known as partner and localizer of BRCA2 [PALB2]; refs. 29, 30), FANCJ (also known as BRCA1-interacting helicase1 [BRIP1]; refs. 31, 32), and, more recently, FANCO (RAD51C; a previously known homologous repair factor; refs. 33, 34) further solidify the close association of breast and ovarian susceptibility genes with FA.
Newly diagnosed FA patients should be subtyped because of the clinical variability among subtypes (1). While FA patients assigned to subtype FANCA tend to have milder disease with later onset of bone marrow failure, patients in subtypes FANCC and FANCG tend to have more severe disease, mandating earlier intervention, such as unrelated donor bone marrow transplantation. Patients with FANCD1 have earlier onset and increased incidence of leukemia and solid tumors (35). In general, early diagnosis and identification of FANC mutations is critical for the informed genetic counseling of parents of FA patients with regard to future pregnancies.
Role of the FA pathway in DNA repair during S phase. DNA ICLs are highly toxic lesions that block essential DNA metabolism, such as DNA replication and transcription. Hypersensitivity to ICL in FA is associated with the accumulation of G2/M-arrested cells. The cell cycle arrest at G2/M is due to increased activity of checkpoint kinase CHK1, and depleting CHK1 suppresses the G2/M arrest phenotypes of FA cells (36). These observations suggest that FA cells are inherently defective in repairing DNA damage. Indeed, increasing evidence affirms that the FA proteins are directly involved in repairing damaged DNA (26).
Among the 15 FA gene products identified so far, the eight upstream FA proteins (FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, and FANCM) form a nuclear complex, termed the FA core complex, which possesses intrinsic E3 ubiquitin ligase activity (Figure 2). Most FA proteins in the FA core complex do not contain any recognizable enzymatic domain, which suggests that their primary function is a scaffold (Table 1). The key domain in the complex is the PHD-type RING finger domain at the C terminus of FANCL. The PHD domain interacts with an E2 ubiquitin–conjugating enzyme, UBE2T, depletion of which in cultured cells leads to a characteristic FA phenotype (37). The primary function of the FA core complex is to monoubiquitinate two FA proteins, FANCD2 and FANCI, which are expressed as a constitutive heterodimer (38, 39). A crystal structure of the FANCD2/FANCI heterodimer (also referred to as the ID complex) suggests that this complex can recognize various DNA structures that may arise from stalled replication forks (40); however, how monoubiquitylation affects the activity or conformation of this heterodimer is unknown. Monoubiquitylated FANCD2 serves as a signal for recruiting downstream effector proteins that have affinity for the ubiquitin. Fanconi-associated nuclease 1 (FAN1) is recruited to the monoubiquitylated form of FANCD2 via its ubiquitin-binding UBZ domains (41–44) and is thought to participate in ICL repair through its nuclease activity.
FA pathway. ATR-mediated signal activates the FA pathway at the stalled replication forks, leading to the activation of the FA core complex and monoubiquitination of the FANCD2/FANCI heterodimer. ATR phosphorylates multiple FA proteins for activation. Monoubiquitinated D2/I recruits FAN1 nuclease and FANCP (which associates with several nucleases), which might participate in nucleolytic incision near crosslinked DNA. D2/I might recruit TLS polymerases (not shown) and HR factors, including FANCD1 (BRCA2) and RAD51. FANCO (RAD51C) is known to participate in the HR step. The USP1/UAF1 complex deubiquitinates D2/I to complete the pathway. 15 FA proteins (blue) are shown. Other important factors that crosstalk with the FA pathway, such as PCNA, γ-H2AX, TOPBP1, MSH2, TLS polymerases, and BLM complexes, are not shown for simplicity. A DNA ICL (red) is illustrated between the DNA double strand.
The major DNA repair pathway regulated by the FA proteins is homologous recombination (HR), a process that repairs DNA double strand breaks (DSBs). DSBs are intermediate lesions during ICL repair, and HR is critical to their repair. Cells lacking FA proteins are deficient in promoting HR activities. Although the mechanism remains unclear, monoubiquitylated FANCD2/FANCI is thought to functionally associate with the downstream FA proteins critical for HR, including FANCD1 (i.e., BRCA2; 45, 46), FANCN (PALB2; ref. 47), and possibly FANCO (RAD51C) and FANCJ (BACH1; ref. 32). FANCD2 also colocalizes with key HR factors, such as BRCA1 and RAD51 (48). Although the upstream FA proteins are required for HR activity, the degree of the HR defect is milder compared with defects resulting from loss of the downstream FA proteins. FANCD2/FANCI proteins do not appear to directly regulate chromatin loading of the key HR enzyme RAD51 (49). However, RAD51 activity is directly regulated by FANCD1, FANCN, and FANCO, possibly accounting for the phenotypic differences. These downstream FA proteins are also potentially involved in a wider range of HR repair and not limited to ICL repair.
FA proteins may promote HR activity by different mechanisms. Recent reports suggest that FA proteins actively suppress nonhomologous end-joining (NHEJ), an error-prone DSB repair mechanism that functions during the G1 phase of the cell cycle (50, 51). Improper activation of NHEJ factors was observed in FA pathway–deficient cells, resulting in chromosomal rearrangements and aberrations during S phase. Several mechanisms are possible: (a) FA proteins may restrict the access of NHEJ factors, such as the Ku70-80 heterodimer, to the DSB ends during the ICL repair process; or (b) cryptic nuclease activity of FANCD2 may generate DNA structures at DSB ends recognized by HR factors (1, 27, 51).
In addition to HR, FA proteins coordinate both nucleolytic incision and DNA translesion synthesis (TLS) during ICL repair. The current model suggests that replication fork stalling by ICL requires subsequent incision of the ICL flanking lesion and unhooking of the ICL by endonuclease activities (26, 52). Recruitment of endonucleases MUS81-EME1 or XPF-ERCC1 is likely involved in this step, although genetic studies indicate that XPF-ERCC1 may be the more relevant enzyme in the FA pathway (53, 54). In addition, FAN1 or SLX4-SLX1 nucleases may also be involved in this step. Incision of ICL is followed by TLS, a damage tolerance mechanism in which specialized low-fidelity polymerases bypass bulky damaged lesions. Cells with disrupted TLS polymerase (e.g., REV3) display FA-like phenotypes, such as hypersensitivity to DNA ICLs (55, 56). A biochemical study using the Xenopus replication system demonstrated that the monoubiquitylated FANCD2/FANCI complex is required for both nucleolytic incision and TLS steps during ICL repair (57). Although the incision step is not required for monoubiquitylation of FANCD2, it may be required for the chromatin recruitment of FANCD2 (58). Interestingly, SLX4, a scaffold protein that binds to MUS81-EME1, XPF-ERCC1, and SLX1 endonucleases, was recently identified as a the fifteenth FA subtype, FANCP (59–61). FANCP is not required for FANCD2 monoubiquitylation, but contains two UBZ domains that are required for recruitment to monoubiquitylated FANCD2 (62). Several additional FA-associated proteins are integral to the pathway. These include FA-associated protein 24 kDa (FAAP24; ref. 63), FAAP100 (64), FANCM-associated histone fold protein 1 (MHF1), and MHF2 (26, 65), all of which are part of the FA core complex. The recently identified FAAP20 is also an integral part of the FA core complex (66–68). Cells rendered deficient in these FA-associated proteins display the classical FA phenotypes, including sensitivity to DNA ICLs and G2/M cell cycle arrest. However, FA patients with these genetic mutations have not been found, so these genes do not have FANC assignments.
In addition to these FA or FA-associated proteins, the FA pathway network broadly includes other regulatory proteins. Ubiquitin-specific peptidase 1 (USP1) and the USP1-associated protein (UAF1) regulate deubiquitination of FANCD2/FANCI and are required for completion of the FA pathway (69, 70). The ATR/ATRIP heterodimer is required for sensing single-strand DNA generated during S phase and for the induction of FANCD2/FANCI monoubiquitylation in response to DNA damage (71). Proteins involved in ATR signaling, such as RPA (single-strand DNA binding protein), RAD17, RAD9 (9-1-1 checkpoint complex), CHK1, and HCLK2, are required for the efficient induction of FANCD2/FANCI monoubiquitylation (36, 72). FA proteins also crosstalk with MSH2/MSH3 mismatch proteins (73) and BLM helicase (74, 75) during ICL repair. Thus, defective DNA repair mechanisms during S phase may render FA cells vulnerable to endogenous damage or oxidative stress.
Role of the FA pathway in cytokinesis. The primary DNA repair function of the FA pathway is exerted during S phase. However, the role of FA proteins may extend to M phase, especially in cytokinesis. FA cells exhibit instability at chromosomal fragile sites, the regions in the mitotic chromosomes that serve as a marker for unrepaired DNA (76, 77). A series of recent studies suggested that the FA proteins provide a surveillance mechanism for monitoring unrepaired DNA during cytokinesis (78–80). Fragile sites were stained with FANCD2 and FANCI, and the absence of these FA proteins in these sites was associated with increased chromosome instability and binucleated cells (cells containing two nucleuses). These binucleated cells underwent apoptosis and are a potential cause of bone marrow failure in FA. The fragile sites are connected with mitotic DNA structures called ultrafine DNA bridges (UFBs). Replication errors during S phase may increase UFB structures during M phase, possibly more so in FA cells, which may cause failure in cytokinesis and generation of binucleated cells. The normal function of the FA proteins at the fragile sites and UFB remains uncertain. Several other DNA repair proteins that are linked to the FA pathway, such as ATR, BRCA1, and RAD51, also regulate the stability of fragile sites, suggestive of a broader role of the FA-BRCA network during cytokinesis.