Fanconi anemia, chromosome instability and cancer

1 downloads 0 Views 614KB Size Report
"Microglandular hyperplasia--a complicating factor in the diagnosis of cervical intraepithelial neoplasia." Eur J Obstet Gynecol Reprod Biol 17(1): 53-9. Hersey ...
Fanconi anemia, chromosome instability and cancer Alex Lyakhovich, Maria Castellà, and Jordi Surrallés*

1

Group of Mutagenesis, Department of Genetics and Microbiology, Universitat Autònoma de Barcelona, 2Center for Biomedical Research on Rare Diseases (CIBERER), Instituto de Salud Carlos III, Bellaterra, Barcelona, Spain *Corresponding author A.L. and M.C. contributed equally to this chapter.

1

Summary Fanconi Anemia (FA) is an autosomal recessive syndrome characterized by congenital abnormalities, progressive bone marrow failure and cancer predisposition. FA is caused by mutations in at least 13 genes and accordingly is subtyped by 13 complementation groups abbreviated from A to N. For decades all cloned FA genes considered being orphans as they shared no common properties or homological functions either with each other or with any known genes. The discovery that breast cancer susceptibility gene BRCA2 is identical to the Fanconi anemia (FA) gene FANCD1 and that activation and recruitment of FANCD2 is dependent on breast cancer susceptibility gene product BRCA1 made possible to reconsider this orphan status. It was recognized that the defects in the FA pathway occur in subsets of diverse human cancers. Now mutations in FA genes FANCN (PALB2) or FANCJ (BRIP1/BACH1) are considered to be risk factors for the breast cancer. Moreover, accumulating data suggest involvement of FA into other cancer pathways embracing acute myeloid leukaemia, squamous cell carcinoma and solid tumors. Understanding the connection between the FA and cancer seems to hold potential for future cancer research thus making FA an important genetic model to study cancer suppression mechanisms. This review provides an update made recently in the field of FA and its connection to cancer biology.

2

I. Fanconi anemia: the disease I.1. Brief historical introduction: the beginning FA was first identified in 1927 by the Swiss pediatrician Guido Fanconi who described a family of 3 siblings that displayed a number of congenital malformations, severe anemia, recurrent infections and occasional spontaneous bleedings, finally resulted in premature death (Fanconi 1927). This finding received another reincarnation in 1960 when Schroeder discovered the syndrome associated with chromosome fragility and described its recessive autosomic heritability pattern (Schroeder 1966). In 1969, Schuler and co-workers using chromosome fragility as an endpoint provided the first diagnostic test for FA (Schuler et al. 1969), which was later improved and extended by Auerbach and collaborators (Auerbach 1988). The “FA molecular era” started on 1992, when the first FA gene, FANCC, was cloned. From then on, 12 more genes have been identified now comprising a family known as FA/BRCA genes.

I.2. Clinical symptomatology FA affects one out of 1-5 million newborns with the carriers’ frequency of about 1/300 (Joenje and Patel 2001), although it may vary in highly consanguineous ethnic groups, reaching maximum (1/67) incidence rate for the Spanish Gypsies population (Callen et al. 2005). Clinical symptoms and severity of FA patients are very heterogeneous, which complicate diagnostic and treatment. The main clinical features seen in FA patients can be classified in four groups: hematological disorders, congenital defects, endocrinopathologies and tumorigenicity.

3

I.2.1. Hematological disorders A set of hematological disorders is a hallmark of FA and is usually diagnosed upon preliminary checkup of patients. Almost all FA patients display a progressive bone marrow failure (BMF), which implies severe trombocytopenia or pancytopenia in the majority of the cases. Although the time of onset of hematological disease is highly variable, 75% of patients experience medullar failure during the first decade of life, and over 90% of those will develop other hematological disorders by the age of 40. Apart from

cytopenia,

other

common

hematological

defects

in

FA

incorporate

myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) with the incidence rate of 33% by the age of 40 (Butturini et al. 1994; Kutler et al. 2003). Most recently described finding involves appearance of cellular clones in the bone marrow with chromosome 3q gains which was shown to be related to a higher risk of developing MDS and LMD, and is nowadays used as a marker of risk factor for developing these pathologies in FA patients (Tonnies et al. 2003).

I.2.2. Congenital defects From 60 to 75% of FA patients present one or several congenital defects in a large variety of organs. These include skin hyperpigmentation and “café au lait” spots (55%), short stature (51%), upper limbs abnormalities, such us radius hypoplasia or abnormal thumbs (43%), abnormal gonads in males (32%), microcephaly (26%), microphtalmy (23%) and urinary tract malformations (21%). However, these birth defects are not solely attributed to FA phenotypes and may characterize other congenital diseases. Moreover, 25-30% of FA patients do not present any congenital abnormalities at all, suggesting that congenital defects should be considered along other indicators when diagnosing FA (De Kerviler et al. 2000; Sagaseta de Ilurdoz et al. 2003). 4

I.2.3. Endocrinopathologies 80% of FA patients present some endocrinopahology. The most commonly observed features are growth hormone (GH) deficiency (53%), hypothyroidism (37%), abnormal glucose/insulin metabolism (usually seen as impaired glucose tolerance and diabetes mellitus) (39%), obesity (27%) and dyslipidemia (55%). 92% of patients 18 years or older present also osteopenia or osteoporosis and 65% of them have gonadal dysfunction. FA women usually reveal a premature menopause, although the endocrine deficiency has not been described (Wajnrajch et al. 2001; Giri et al. 2007).

I.2.4. Tumorigenicity As extensively described in the third part of this chapter, FA patients have an extremely high risk of developing hematological or solid tumors from their second decade of life. Solid tumors are found in 35% of patients by the age of 40 and the most frequent ones observed are squamous cell carcinomas of head, neck, esophagus, anus and gynecological (42% of all solid tumors). Liver cancer (29% of all solid tumors) is usually related to androgen treatment (Alter 2003; Kutler et al. 2003). Part of the clinical variability seen in FA patients is due to the existence of several complementation groups (see the next section). Therefore, some of the clinical features described above are associated to or more common in FA patients that belong to a specific complementation group. For instance, FA-C and FA-D2 patients display an earlier onset of hematological disease and are overall poorer survivors when compared to FA-A and FA-G patients (Kutler et al. 2003; Kalb et al. 2007). Microcephaly, growth retardation, skin pigmentation, radial-ray defects and microphthalmia are some of the congenital defects that appear more frequently in FA-D2 patients than in the rest of the FA population (Kalb et al. 2007). Thus, all reported FA-D2 patients present one or more 5

congenital malformations, whereas about 30% of the FA patients from other complementation groups are born normal. VACTERL phenotype (vertebral defects, anal atresia, cardiac malformations, tracheoesophageal fistula with esophageal atresia, renal and radial dysplasia and limb malformations) is overrepresented in FA-D1, FA-E and FA-F groups (Faivre et al. 2000). Finally, FA-D1 patients usually present an atypical severe phenotype accompanied by solid tumors (medulloblastoma, Wilms tumor) during early childhood (Hirsch et al. 2004).

I.3. FA cellular phenotype and diagnostics FA cells, or cells deficient in one of the FA genes, are highly sensitive to drugs inducing DNA interstrand crosslinks (ICLs), due to their inability to repair this sort of damage (Sasaki 1975). ICLs are highly deleterious for the cell because they constitute a blocking against the advancing replication fork which apparently stalls the DNA replication and results to the cell death or transformation. Some commonly used ICLinducing agents like mytomicin C (MMC), diepoxybutane (DEB), photoactivated psoralens or cis-platin (CisPt) serve as important drugs in cancer chemotherapy. This hypersensitivity of FA cells to ICL-inducing agents allowed to develop a diagnostic test, still nowadays used as the screening test for FA phenotype (Schuler et al. 1969; German et al. 1987; Auerbach 1988). Upon treating FA cells with DEB or MMC, the incapacity of the cell to properly repair ICLs generates chromatid-type chromosomal breaks, often resulting in tri- or quadri-radial figures which are the outcome of misrejoined broken ends (Figure 1). FA DEB-test is usually performed on peripheral blood, but it can also be done on fibroblasts or amniocytes, if required. The most currently used protocols for this test is the one described by Dr. Auerbach in 1988. After 48h of treatment of whole blood sample with DEB, the number of breaks observed per cell is up to hundred times

6

higher in FA cells compared to non-FA cells, although high variability exist between individuals. MMC is also used to test the chromosome fragility of FA cells giving similar results. Since alteration of the cell cycle in FA cells is common, especially when treated with DNA cross-linking chemicals this serves as an additional indicator for FA diagnoses. As a result of the accumulated damage, the G2 phase of the cell cycle becomes longer than normal as the G2/M checkpoint is activated (Kubbies et al. 1985). Consequently, FA cells are accumulated in G2 phase upon treatment, as seen in a flow cytometry report (Figure 2). Although this assay is being used as a complementary tool for the chromosome fragility test in most laboratories, its simplicity makes this technique to be an ameanable instrument for diagnostic purposes. One common characteristic of the cells from FA upstream subgroup is telomere abnormalities. Cells from FA patients present shorter telomeres compared to those of an age-matched healthy individual (Leteurtre et al. 1999; Callen et al. 2002). Moreover, a high frequency of extra-telomeric TTAGGG repeats (extra- and intra-chromosomic), as well as chromosome ends without a detectable TTAGGG signal are observed in FA cells, indicating that chromosome breaks are as well produced in the telomeric region which may provoke genomic instability (Callen et al. 2002). FA cells also reveal a high sensitivity to oxidative damage (Joenje et al. 1981), which was proposed to complement the mechanism of genomic instability in FA (Pagano et al. 2005). Although presenting high levels of reactive oxygen species (ROS) and DNA oxidation products, such as 8OHdG (Pagano and Korkina 2000), FA cells are proficient in repairing this kinds of DNA damage (Pagano et al. 2004).

7

I.4. Somatic mosaicism Mosaicism is a very interesting phenomenon that affects from 15 to 25% of all FA patients. It appears when a single hematopoietic stem cell reverts the mutation present in one of the two alleles of the affected FA gene, thus reverting the FA cellular phenotype to a “normal” wild-type. Due to proliferation advantage of this reverted cell, it clonally expands and colonizes the bone marrow of the patient. In a number of diseases, mosaicism has only been observed in the hematopoietic compartment, where the high proliferation rate and the ability of the cells to colonize bone marrow makes this phenomenon possible (Kwee et al. 1983; Hirschhorn et al. 1996). FA mosaicism can be observed in peripheral blood by performing chromosome fragility test, where two cell types can be detected: (i) a cell population with several breaks after treatment, which corresponds to the non-reverted cells and (ii) another cell population without breaks, which corresponds to the reverted cells (Lo Ten Foe et al. 1997). Since different levels of bone marrow colonization by the reverted cells in FA mosaic patients can reach from 10 to 100% of the corrected cells, diagnostic sometimes is rather complicated. Somatic cell mosaicism may have relevant clinical implications. The original mutation reversion can occur in a hematopoietic stem cell or in an early progenitor. Depending on the stage of cell differentiation, the reversion will involve either all hematopoietic cell lineages or only part of them. When all cell lineages are reverted, patient blood counts may come back to normal level, a process known as “natural gene therapy” (Youssoufian 1996; Lo Ten Foe et al. 1997). In this manner, several molecular mechanisms can originate the genotype correction. This includes gene conversion, intragenic recombination or reverse point mutation. Consecutively, partial restitution of protein function can be observed due to compensating mutations (Lo Ten Foe et al. 1997; Gross et al. 2002).

8

Diagnostics of mosaic patients As mentioned above, the level of bone marrow repopulation by corrected cells is highly variable in mosaic patients and may be almost 100%. Such FA patients can be easily overlooked in a regular chromosome fragility test performed in peripheral blood cells. FA mosaic patients usually appear to have a resistant phenotype in a viability or G2 arrest test upon treatment with MMC or other ICL-inducing agents, which may lead to a false non-FA diagnostic. A small fraction of mosaic patients have, however, hypersentitivity to MMC comparable to a “classical” FA patient (Casado et al. 2006). When clinical suspicion of FA exists in a patient with a negative chromosome fragility test, more rigorous analysis should be done to disregard a possible highly reverted mosaicism. As mosaicism only affects hematopoietic tissue, FA diagnostics, such as chromosome fragility test or G2 block assay, are usually performed on fibroblasts from the same patient. These tests will unequivocally lead to a final diagnostic (Gross et al. 2002). Given the clinical implications of mosaicism, repeated chromosome fragility tests are advisable in FA patients without hematological phenotype or with consistently stable or improved blood counts.

9

II. FA/BRCA pathway: molecular aspects

II.1.

FA genes and complementation groups

To date, 13 genes are known to be responsible for FA, each of them represents a different complementation group, known as FA-A, -B, -C,-D1,-D2,-E,-F,-G,-I,-J,-L, -M and -N. The frequencies of each complementation group (Figure 3) show that FA-A is the most frequent one, accounting for almost 60% of the FA patients in USA, whereas FA-C, FA-G represent 10-15% and FA-D1, FA-D2 represent 5% for each one. The rest subgroups are much rare (Kennedy and D'Andrea 2005, Casado et al. 2007). In some populations, complementation group frequencies vary due to conservation of a given mutation in highly consanguineous ethnic groups. Some good examples illustrating this fact are: (i) population of Spanish gypsies, where all FA patients share the 295C>T mutation in FANCA gene, thus contributing to an unusually high FA-A frequency (80%) patients in Spain (Callen et al. 2005, Casado et al. 2007) and (ii) the population of Ashkenazi jews, where all FA patients are homozygous for the IVS4+4 A>T mutation in FANCC gene, which makes FA-C patients more frequent in Germany and some other countries (Whitney et al. 1993). A list of all known 13 FA genes is provided in Table 1. The first gene identified in 1992 was FANCC, and the last one, FANCI, was discovered in 2007. Although many scientists think the set of FA genes is almost close to completion, there are some patients that can not be attributed to any known complementation groups suggesting that more FA genes should exist. Due to significant rareness of FA cases and therefore very few cases for the patients that belong to these not-yet-identified complementation groups the identification of new FA genes continue to be a very hard task.

10

The large number of FA-causative genes make this syndrome very complex, although with each discovery of a new FA gene we are getting closer to understanding their yet cryptic functions in the frame of a whole FA pathway. A summary of molecular functions and gene ontology for each FA gene is provided in Table 2.

II.2.

FA/BRCA pathway

FA proteins function in a common DNA repair pathway known as the FA/breast cancer susceptibility (FA/BRCA) pathway (Figure 5). The FA/BRCA pathway can be divided into three stages: (1) “upstream”, that precedes FANCD2/FANCI activation (monoubiquitination),

(2)

formation

of

FANCD2/FANCI

complex

and

(3)

“downstream” step that occurs after monoubiquitinylation of the FANCD2/FANCI - the key event in this pathway. “Upstream” genes seem to be predominantly involved in detection and signaling of DNA damage, whereas “downstream” genes may act on the DNA damage repair process itself.

II.2.1. “Upstream” genes: The FA core complex and FANCI These include most of the FA proteins (FANCA, B, C, E, F, G, L and M). Upon DNA damage this pathway gets activated following by formation of such a called FA core complex, which main function is the monoubiquitinylation and subsequent activation of FANCD2 and FANCI.

FA core complex assembly

11

Besides this major role, each FA core complex member seems to have its own specific function independent from FANCD2/FANCI activation. However, specific functions of FA core complex proteins are not yet completely understood as all of them share no amino acid or domain structures homology with each other. It is known, however, that most of these proteins are required for nuclear localization and structural stability of the FA complex. Co-immunoprecipitation experiments and yeast-two-hybrid assays support the existence of sub-complexes necessary for sequential assembly of the FA core complex (Kennedy and D'Andrea 2005; Medhurst et al. 2006). FANCA directly binds to FANCG, both of which are required for stabilization and nuclear localization of FA core complex (Garcia-Higuera et al. 2000). FANCE directly binds to FANCC and is required for nuclear localization of the last one (Pace et al. 2002). Finally, FANCB and FANCL directly interact with each other resembling the other two pairs (Medhurst et al. 2006). Once inside the nucleus, FANCB-FANCL pair promotes the nuclear localization of FANCA-FANCG, in turn, their gathering inside the nucleus is mediated by FANCM (Meetei et al. 2005). In parallel, FANCF, which also remains in the nucleus, binds to FANCE/FANCC

pair

(Leveille

et

al.

2004).

Assembly

of

FANCA/FANCG/FANCM/FANCL/FANCB proteins is independent of the assembly of FANCE/FANCC/FANCF supporting the existence of two big sub-complexes with possible different functions (Medhurst et al. 2006).

FANCL and FANCM functions Among all FA core complex proteins, FANCL seems to be the one that possess E3 ubiquitin ligase activity and should be responsible for FANCD2 and FANCI ubiquitinylation. Having RING domain, the characteristic of a E3 ubiquitin ligases that mediate activated ubiquitin molecules to a substrate, FANCL plays a role in ubiquitin

12

transfer (Meetei et al. 2003). The E2 ubiquitin conjugating enzyme for FANCL has been recently identified as the UBE2T protein. Although function of UBE2T is necessary for FANCD2 monoubiquitinylation and to prevent genome instability, no patient with mutations in this gene has been found (Machida et al. 2006). Another FA core complex protein FANCM is a human homolog of the archaeal protein Hef, which has a helicase and endonuclease domains. The last one, being degenerated in humans, seems to be nonfunctional and mutations in this domain do not affect crosslinker sensitivity. The helicase domain of FANCM protein is located at the N-teminal region and shares homology with the DEAH box ATP-dependant helicase subfamily (Mosedale et al. 2005). In vitro, FANCM shows a DNA-dependent ATPase activity with affinity to single-stranded and forked structures, but no helicase activity. These facts leave FANCM function similar to DNA translocase rather than to a helicase. According to existing models, FANCM moves the FA complex along the DNA template, acting as a sensor of stalled replication forks (Kennedy and D'Andrea 2005; Meetei et al. 2005).

FAAPs: Fanconi Anemia Associated Proteins Besides eight known FA core complex proteins described above, two other proteins build up an integral part of the FA core complex: FAAP100 and FAAP24. These two new members are known as FAAPs (FA Associated Proteins), because they coimmunoprecipitate with the rest of the FA core complex but are not yet found to be mutated in any FA patients. FAAP100 is a 100kDa protein which was identified by mass spectrometry as LOC80233, a hypothetical protein of unknown function that maps to 17q25.3. FAAP100 has been shown to bind both FANCL and FANCB, thus forming a sub-complex which is necessary for stabilization of all three proteins. Assembly of

13

the B-L-P100 sub-complex towards nuclear translocation depends on FANCA and FANCM (Ling et al. 2007). FAAP24 was identified as a protein containing an ERCC nuclease domain, which maps to 19q13.11. Together with the rest of the FA core complex, FAAP24 is required for the monoubiquitinylation of FANCD2. It has been shown to interact with the C-terminal part of FANCM and forms a heterodimer thus acting like the XPF-family members. FAAP24 binds to single-stranded DNA and seems to target FANCM (and possibly the rest of FA core complex) to the damaged DNA (Ciccia et al. 2007).

II.2.2. FANCD2- FANCI complex and its regulation by monoubiquitinylation FANCD2 and FANCI proteins seem to bridge the sensor and signaling machineries of the FA/BRCA pathway as they provide a link in-between the FA upstream and the downstream (damage/repair response) complexes. Identified in 2001 FANCD2 quickly became the key FA protein due to its unique function, serving as a liaison among major FA players. FANCI was identified very recently as a FANCD2 homolog that shares 40%

of

amino

acid

sequence

with

FANCD2

and

equally

undergoes

monoubiquitinylation (Sims et al. 2007; Smogorzewska et al. 2007). Upon DNA damage, monoubiquitinylation of FANCD2 at Lys561 and FANCI at Lys523 follow relocation of both proteins to the site of damage, finally making foci together with some other members of DNA repair machinery. The function of FANCD2 and FANCI on chromatin is yet unclear, but is probably related to the repair processes (Garcia-Higuera et al. 2001). Amazingly, not only FANCD2 monoubiquitinylation depends on the presence of FANCI: FANCI monoubiquitinylation is FANCD2dependent as well, making two proteins equally important for each other’s functions (Sims et al. 2007; Smogorzewska et al. 2007).

14

Along with phosphorylation and rybosylation, ubiquitinylation is a part of the cell’s regulation machinery that controls many cellular events. The antagonist process, deubiquitinylation is responsible for a system turnover. In this way, once completed its job, FANCD2/FANCI should be deactivated by deubiquitinylating (E4) enzyme USP1. Inhibition of USP1 leads to accumulation of ubiquitinylated forms of FANCD2 and FANCI and seems to protect cells from ICLs-induced chromosome fragility (Nijman et al. 2005). Monoubiquitinylation of FANCD2 was utilized as an indicator to check the functionality of the “upstream” part of the FA pathway. Monoubiquitinylation changes the molecular weight of the protein by approximately 7 kDa from 155 (FANCD2-Short) to 162 kDa (FANCD2-Long) which can be easily visualized if performing Western blot analyses. As shown in Figure 4A, appearance of the second “activated” band of FANCD2 (FANCD2-L) upon damage or other stress factors indicates that the “upstream” part of the FA pathway works correctly. Appearance of only the low FANCD2 band (FANCD2-S) with no or impaired upper-band serves as an indicator of malfunctioning of the upstream FA pathway (Garcia-Higuera et al. 2001). FANCD2 functionality and therefore, functionality of the whole “upstream” part of the FA pathway can also be checked by ICL-induced FANCD2 foci formation (Garcia-Higuera et al., 2001) or relocation of FANCD2 to UVC-induced local DNA damage spots (Figure 4b) (Bogliolo et al. 2007).

II.2.3. “Downstream” genes: from damage recognition to repair The role of “downstream” part of FA/BRCA pathway is more diffused, although it seems to be well-connected with the homologous repair machinery. Once FANCD2 gets active, it relocates to the DNA damaged sites of chromatin. This step requires the

15

presence of BRCA1, a well known tumor suppressor gene involved in homologous recombination repair and cell cycle control. In BRCA1 deficient cells FANCD2 is unable to relocate to the site of damage. Likewise FA cells, BRCA1 deficient cells are hypersensitive to ICL-inducing agents (Garcia-Higuera et al. 2001). Another requirement for FANCD2 relocation is the presence of fully functional histone H2AX, which is distributed along the chromatin and is known to be phosphorylated upon damage to the vicinity of double strand breaks (DSBs) or at stalled replication forks. Once phosphorylated, H2AX recruits monoubiquitinylated FANCD2. In the H2AX deficient cells or cells expressing non-phosphorylable H2AX, FANCD2 fails to relocate to the sites of damage and the cells are also hypersensitive to ICL inducing agents (Lyakhovich and Surralles, 2007). Once recruited to the sites of damage, FANCD2 molecules

colocalize

with

some

other

DNA

repair

proteins,

including

FANCD1/BRCA2 (Wang et al. 2004), Rad51 (Hussain et al. 2004), BRCA1 (GarciaHiguera et al. 2001), NBS1 (Nakanishi et al. 2002) or PCNA (Howlett et al. 2005), and the DNA repair process begins.

FANCD1, FANCJ and FANCN Till now, 3 FA genes have been identified that work “downstream” FANCD2: FANCD1/BRCA2, FANCJ/BRIP1 and FANCN/PALB2. The discovery of each one provided additional insight into the DNA damage and repair events but also expanded our knowledge of FA/BRCA pathway. The finding that FANCD1 gene was identical to the known breast cancer susceptibility gene BRCA2 changed the view of the FA pathway function completely, as it linked the FA pathway to homologous recombination repair (HRR) and cancer network (Howlett et al., 2002). The main function of BRCA2 in HRR is the loading and removal of RAD51 protein to the single

16

stranded DNA region, so that the strand invasion can occur and terminate at the appropriate time (Shivji and Venkitaraman 2004). A direct interaction between monoubiquitinylated FANCD2 and the C-terminal part of BRCA2 was reported to be important for FANCD2-mediated BRCA2 loading to chromatin (Wang et al. 2004). Finally, BRCA2 can bind FANCG. However, the function of FANCG in this context is not yet clear. FANCG is able to bind both N- and C-terminal domains of BRCA2 through its TPR motifs (Hussain et al. 2003). FANCJ/BRIP1 is a member of the RecQ DEAH helicase family, ATP-dependent 5’-to-3’ helicases that are very efficient in unwinding Holliday junction structures. Unwinding of these structures created at the stalled replication fork allow other repair proteins to access the DNA (Cantor et al. 2004). FANCJ binds to the C-terminal BRCT repeats of BRCA1 upon phosphorylation at Ser990, which seems to be necessary for the correct G2/M checkpoint function (Cantor et al. 2001; Yu et al. 2003). Disruption of this interaction is critical for the DSBs repair process, thus further linking the FA/BRCA pathway to HRR (Cantor et al. 2001). Finally, FANCN was identified as PALB2 gene that encodes a BRCA2 interacting protein. FANCN interacts with the N-terminal domain of BRCA2 and is required for nuclear localization and stabilization of the last one. Therefore, FANCN role is probably to secure BRCA2 functionality during HRR (Xia et al. 2006).

II.3.

FA/BRCA pathway activation

FA/BRCA pathway is activated in response to stalled replication forks and during the Sphase of the cell cycle (Taniguchi et al. 2002). Activation is controlled by ATR (ATMrelated kinase), one of the of the phosphatidyl inositol 3-kinase-like family of serine/threonine protein kinases (PIKKs). Upon binding to ssDNA regions originated at

17

the stalled replication fork, ATR phosphorylates a large number of substrates, triggering checkpoint activation and DNA damage repair (Zou and Elledge 2003). S-phase checkpoint is activated trough phosphorylation of CHK1 (CHeckpoint Kinase 1), one of the principal substrates of ATR. CHK1 itself is responsible for the phosphoylation of at least one of the FA core complex proteins, FANCE. This modification is required for ICL resistance, but its function is not yet understood (Wang et al. 2007). As seen in Figure 5, ATR directly phosphorylates some of the FA pathway members including FANCD2 (Andreassen et al. 2004), H2AX (Ward and Chen 2001), BRCA1 (Tibbetts et al. 2000) and NBS1 (Pichierri and Rosselli 2004), hence activating the DNA damage repair process. FANCD2 monoubiquitinylation and relocation to the site of damage are tightly controlled by ATR: FANCD2 monoubiquitinylation pre-requires its phosphorylation and the ability to relocate to the site of damage is dependent on H2AX phosphorylation (Bogliolo et al. 2007).

II.4.

FA pathway function

Although the actual function of the FA pathway is not yet completely elucidated, it is quite clear that FA proteins are engaged in the repair of ICLs through a connection to HRR. ICLs are the most deleterious types of DNA damage as they involve both DNA strands. Therefore, ICLs repair require activation of multiple pathways, not only HRR, which would act coordinately and in a sequential manner. The current model for this event incorporates HRR, Nucleotide Excision Repair (NER) and Trans-lesion Synthesis (TLS) (Figure 6). Once the ICL is detected, the first incision is done, perhaps by MUS81, on one of the two replicated strands, leaving a DSB (Osman and Whitby 2007). The other DNA double helix remains with the ICL and ssDNA regions at one

18

side. The ssDNA region is coated by RPA which is directly bound to ATR. H2AX is rapidly phosphorylated by ATR at the break point triggering the DNA damage response. Activated ATR results in the assembly of whole FA core complex. It seems likely that FANCM should move the entire FA core complex along the DNA until the ICL is detected. At this point, γH2AX-dependent FANCD2 relocation is accompanied by its activation via FANCL. Simultaneously, a second incision is produced at the other site of the ICL. This step would require ERCC1/XPF, a 5’endonuclease that belongs to the NER system (Niedernhofer et al. 2004). Now, the strand containing the ICL can be copied with a TLS polymerase, a part of an error prone system (Nojima et al. 2005). With an already repaired DNA double helix, the other one can be repaired by HRR. First, MRN complex resects the DSB to generate a single strand overhang. After that, the HRR machinery assembles: FANCD2 loads BRCA2, which in turn loads RAD51 to initiate the strand invasion. Finally, the Holliday junction is resolved and the replication process can restart. It is hypothesized that FANCJ could be involved in the resolution of the Holliday junction, but this point remains unclear.

III.

Cancer in Fanconi Anemia

III.1. Cancer in FA patients Studies of cancers in patients with rare cancer predisposition syndromes have been important for the elucidation of pathways of malignancies in the general population. In this way, FA is not an exception – studying cancer cases in FA patients or FA mutation carriers recently allowed to uncover FA genes that are important for cancer predisposition. For a long time, attempts to match FA gene sequences to any known

19

disease-sensitive genes revealed poor homology and did not suggest a model organism to facilitate this area of research. Not surprisingly, the question of cancer-proneness in FA patients remained to be a paradox for decades. Only with the discovery of identity between FANCD1 and BRCA2 and recent findings of three novel FA genes shed some light to the role of FA in cancer biology. Therefore, the more we learn of FA genetics and the more extensively we accumulate FA cases, the more chances to find out common consequences of the much dispersed FA genes with the fundamental diseases we may get. This part of a chapter summarizes of what is known today in the cancer biology of FA. From overall of more than 2000 cases published in the literature from mid-70th till today we may see that the hallmark neoplastic events in FA patients are various forms of leukemias, solid and liver tumors as well as cases of myelodysplastic syndrome (MDS) (Alter 1996; Alter 2003). The later one can further progress to leukaemia, and despite the fact that the term MDS is often used when cytogenetic marrow clones were observed, regardless of bone marrow morphology, MDS and AML must be distinguished for etiologic research (Alter et al. 2000).

Therefore, we separated all FA cancers into four major groups (Figure 7). We should note that the ages at which FA cancers occur are substantially younger than that ones in the general population and the cumulative incidence of FA-related cancers are likely underestimated because many people with FA do not yet live long enough to develop certain types of tumors. This may not always be so because management of aplastic anemia, MDS, or leukemia may improve, decreasing mortality rate.

20

III.1.1. Leukemia All FA patients have a much greater risk of developing leukemia, than people without FA. Among all cancers identified in FA patients, leukaemia occupies roughly 10% of all clinical cases, where males have higher chance of occurrence than females (Wada et al. 1989; Yetgin et al. 1994; Tezcan et al. 1998; Janik-Moszant et al. 1998; Sugita et al. 2000; Tischkowitz and Dokal 2004; Kook 2005; Ross et al. 2005; Velez-Ruelas et al. 2006; Bagby and Alter 2006; Rosenberg et al. 2003). Leukemia is a malignancy of the blood system in which the bone marrow produces vast quantities of immature white cells called "blasts." The blasts can proliferate rapidly and suppress the development of healthy blood cells needed for effective functioning of the patient's body. If untreated, leukemia results in uncontrollable infections, bleeding and death. Almost 90% of all types of leukemia that FA patients are likely to develop belong to AML. AML is difficult to treat successfully, especially in FA patients. This is because FA cells are hypersensitive to the most common chemotherapeutic compounds which decrease the chance of patients’ survival, if treated with the normal drug doses. Administrating lower doses has no or very little effect to cure AML in FA. Some other forms of FA leukaemia include acute lymphocytic leukaemia and MDS-progressed AML. Recent survey of North American FA patients revealed that the hazard of developing AML subsequent to MDS was 9.4%/y, suggesting that in FA not all clones would necessarily progress (Rosenberg et al. 2003). In vitro models provide plausible mechanisms for conversion of MDS to AML in some subsets of patients, but the cytogenetic and clinical heterogeneity of MDS in FA suggest that the natural history may well differ in other subsets.

21

III.1.2. Solid tumors Since the median age at onset of the leukemias (11 years) is significantly lower than that at the solid tumors (29 years), later ones comprise the major neoplastic risk among the older FA patients. Since FA patients die from aplastic anemia at early ages, but solid tumors are more common for adults, it is impossible to extrapolate the time-dependence of incidence rate. FA patients have an extremely high risk of developing squamous cell cancers in areas of the body in which cells normally reproduce rapidly, such as the oral cavity, esophagus, the gastrointestinal tract, the anus and vulva. Normally, FA patients develop these cancers at a much earlier age than people without FA. Patients who have had a successful bone marrow transplant and thus, are cured of the blood problems associated with FA, still have a very high risk of developing solid tumors which is even higher than that of a non-transplanted patients as a result of a radiotherapy requirements before bone marrow transplantation (Soulier et al. 2005). Overall cancer incidence among solid tumors comprise of: head, neck, and esophagus tumors (52%), cervix tumor (4,9%), brain tumor (9%), skin cancer (9%), breast cancer (13%), the rest are gastric and colon cancers, sarcomas and lymphomas (McDonough 1970; Swift et al. 1971; Dosik et al. 1979; Arnold et al. 1980; Schofield and Worth 1980; Vaitiekaitis and Grau 1980; Nara et al. 1980; Kozarek and Sanowski 1981; Kennedy and Hart 1982; Hersey et al. 1982; Hill et al. 1981; Jacobs and Karabus 1984; Helmerhorst et al. 1984; Helmerhorst et al. 1984; Kaplan et al. 1985; de Chadarevian et al. 1985; van Niekerk et al. 1987; Fukuoka et al. 1989; Alter et al. 1989; Snow et al. 1991; Friedman and Chesney 1981; Puig et al. 1993; Alter and Tenner 1994; Lustig et al. 1995; Zatterale et al. 1995; Koo et al. 1996; Doerr et al. 1998; Verbeek et al. 1997; Goldsby et al. 1999; Marcou et al. 2001; Ferro et al. 2001; Ruud and Wesenberg 2001;

22

Sjarif et al. 2001; Unal and Gumruk 2006; Koubik et al. 2006; Gasparini et al. 2006; Tischkowitz et al. 2003). We also included here cases with combinations of malignancies in the FA. Interestingly, in contrast to the patterns seen for AML or BMT, the hazard of a solid tumors is relatively low during childhood as it was shown from the recent study made in North American cohort of FA patients (Rosenberg et al. 2003). The hazard of a solid tumor was approximately 1%/y by age of 17 years, 2%/y by age of 24 years, and 4%/y by age of 30 years. The hazard may be close to 8%/y by age of 40 years, although this estimate is uncertain because relatively few subjects remained by the time this age was reached, and consequently the confidence limits are broad.

III.1.3. Liver tumors Liver tumors are turned out to be quite common among FA cancers (Mulvihill et al. 1975; Sarna et al. 1975; Shapiro et al. 1977; Obeid et al. 1980; Garel et al. 1981; Abbondazo et al. 1995; LeBrun et al. 1991; Linares et al. 1991; Touraine et al. 1993; MacMillan et al. 2000; Kumar et al. 2004; Velazquez and Alter 2004; Zhu and Dutta 2006; Nuamah et al. 2006). These occurrences are not spontaneous as most of FA patients develop liver tumors in response to androgen exposure. However, development of such cancers in FA patients require much smaller and briefer androgen treatment than that for non-FA individuals, although some other factors of development of liver tumors may be necessary to consider. Hepatocellular carcinomas and adenomas are major forms of tumors reported in FA liver cancer patients who received parenteral androgens.

23

III.1.4. MDS As mentioned earlier, MDS cases of FA cancers should be separated from AML. Only 30% of MDS cases are progressed to AML at an average of 30 years, as shown by the study of North American FA patients (Rosenberg et al. 2003). However, in a French registry, MDS did not have a paediatric peak and the incidence rose very slowly until past the age of 50 years (Maynadie et al. 1996). MDS in FA patients may be different from primary MDS in non-FA adults (Alter et al. 2000). Patients with FA may have MDS for long periods of time, with clonal fluctuations and without leukemic transformation. The age distribution of the literature cases with MDS resembled that of the leukemia group with a normal distribution mode to be around 15 years. From the other hand, MDS can progress to other types of tumors thus increasing cumulative incidence rate since age.

III.2. Cancer in FA mutation carriers Now when most of the FA genes and their proteins have been identified, it is important to connect FA to cancer. Circumstantial evidence points towards a role of the FA pathway in cancer predisposition and development. In many recent publications, however, this link was quite diffuse embracing either repair of stalled DNA replication forks or cell cycle malfunctioning. Until recently FANCD1 remains to be the only protein for which a clear function in HR has been described and which was probably needed to reinitiate replication. Thus, the only explanation connecting mutations in FA pathway with cancer was a destabilization of replication forks, probably leading to abnormal repair and resulting in chromosomal instability. Latest discovery that the breast cancer susceptibility gene BRCA2 is identical to the FA gene FANCD1 made possible for the first time to link FA genetics and cancer susceptibility incidences 24

together (Howlett et al. 2002). With the facts that activation/recruitment of FANCD2 is dependent on breast cancer susceptibility gene product BRCA1, it became possible to find out more specific role(s) for the mutated FA genes to the phenotype seen in patients (Taniguchi and Dandrea 2002). Although heterozygous mutations in both BRCA1 and BRCA2 genes are characterized by predisposition to breast, ovarian and other cancers, the risk of developing cancer that is associated with BRCA1 and BRCA2 cancerpredisposing mutations is not well defined and appears to be variable even within families of similar ethnic background with the same mutation (Goldberg and Borgen 2006). It was well studied that cancer cells with non-functional BRCA1 or BRCA2 genes show chromosome damage similar to that in FA cells. However, for a long time it was not clear whether genetic mutations that cause FA can also cause breast cancer (Tischkowitz and Hodgson 2003). At the same time, recent progress made in identifying new FA genes suggest that there is a strong, yet unresolved, link between FA and BRCA pathway. If this is true, then any significant mutation in FA core complex that disturbs downstream FA genes or severe mutations in downstream FA genes might be a platform for cancer predisposition. Supporting this notion, we may refer to a latest study describing children who inherited two copies of severely mutated (non-functional) BRCA2-interacting gene product FANCN (PALB2) (Rahman et al. 2007). It was shown that women inheriting one damaged copy of FANCN have double risk of developing breast cancer. Mutations in another FA gene FANCJ (BRIP1or BACH1) is a risk factor for the breast cancer comprising a class with two breast cancer susceptibility genes, CHEK2 and ATM, which upon exposure to other environmental risk factors may predispose a woman to breast cancer (Litman et al. 2005). The question as to whether FA heterozygotes are at increased risk for cancer is of great importance to those at risk for being a carrier. Using the surveillance, epidemiology, and end results registries

25

among the 944 FA subjects, a significantly higher rate of breast cancer than expected was observed among carrier grandmothers (Berwick et al. 2007). In this part we are not going to describe the link of FA mutations to sporadic cancers as it is now reviewed in our recent paper (Lyakhovich and Surralles 2006). However, increasing evidence shows that the FA/BRCA pathway is one of the DNA repair mechanisms that is affected in sporadic cancers, where the epigenetic silencing of a critical genes through methylation of the corresponding promoter regions is thought to be the main mechanism of FA/BRCA disruption. For instance, FANCF methylation was shown to occur in 24% of ovarian granulosa cell tumors, 30% of cervical cancer, 14% of squamous cell head and neck cancers, 6.7% of germ cell tumors of testis, and 15% of non–small-cell lung cancers where it correlates with a worse prognosis (Kennedy and D'Andrea 2006). Although all newly discovered mutations in FA genes are relatively small contributors to the overall incidence of cancers, their cumulative response may be amplified for maintaining the stability of the entire human genome. Following this notion, we are now going to describe in more details some mutations in FA genes that have been particularly found in various types of cancers.

III.3. Mutations of FA genes in leukaemia

Patients with FA have an approximately 800-fold increased risk of leukaemia (Rosenberg et al. 2003). The rate of this complication was found to be higher in patients of FA-G group when compared with FA-A or FA-C ones, and also to be higher in those with null compared with missense mutations in FANCA (Faivre et al. 2000). Several studies of leukemia in FA homozygotes have recently been reported, totally analyzing 26

almost two thousand FA cases both from the literature (Alter 2003) and from the International FA Registry (IFAR) (Kutler et al. 2003). On the basis of these studies, the cumulative incidence of any hematological abnormality in FA is up to 90% and the cumulative incidence of leukemia is around 10% by 25 years of age.

FANCA There have been several studies to determine whether FA genes are mutated in sporadic AML. Studies looking at FANCA found that heterozygous mutations (deletions or point mutations) of the FANCA gene occur in 5–10% of sporadic adult AML (Condie et al. 2002; Tischkowitz et al. 2004). It is not known, however, whether these mutations are causal in promoting leukemogenesis or secondary cancers to increased genomic stability.

FANCC FANCC has been studied predominately in paediatric leukaemia and a number of sequence variants may be implicated (Awan et al. 1998; Rischewski et al. 2000; Barber et al. 2003). The FA pathway can be disrupted in sporadic AML (Xie et al. 2000; Lensch et al. 2003). It is possible that some other FA genes may also play a role in these cases or that other mechanisms of gene inactivation, such as methylation, are important in the development of sporadic haematological and solid malignancies (Lensch et al. 2003; Taniguchi et al. 2003; Tischkowitz et al. 2003).

BRCA2/FANCD1 Patients with biallelic mutations in BRCA2 (IVS7+1G-->A and IVS7+2T-->G) were recently associated with AML, and 886delGT and 6174delT with brain (Alter et al.

27

2007). However, patients with other alleles remained at very high risk of these events. Missense mutations formed a distinct cluster in a highly conserved region of the BRCA2 protein. The small group of patients with biallelic BRCA2 mutations is distinctive in the severity of the phenotype and early onset and high rates of leukaemia, and

may

comprise

an

extreme

variant

of

FA.

Further

examination

of

genotype/leukaemia associations was recently provided by Barber (Barber et al. 2005), who reviewed the rare group of FA patients with mutations in FANCD1/BRCA2, and stated that all of the cases of acute myeloid leukaemia (AML) occurred in patients with BRCA2 mutations involving the splice site for IVS7.

FANCD2 Recent studies of the clinical and molecular features of a patient initially identified as a potential FA case demonstrated a marked reduction of FANCD2 (>95%), but normal levels of FANCA or FANCG (Borriello et al. 2007). Later, this defect was associated with a homozygous missense mutation of FANCD2, resulting in a novel amino-acid substitution (Leu153Ser), which is highly conserved through evolution. The FANCD2(L153S) protein, whose reduced expression was not due to impaired transcription, was detected also in its monoubiquitinylated form in the nucleus, suggesting that the mutation does not affect post-translation modifications or subcellular localization, but rather serves the stability of FANCD2. Therefore, the hypomorphic Leu153Ser mutation represents the first example of a FANCD2 defect that might promote clonal progression of tumors, such as T-ALL and severe chemotherapy toxicity in patients without any clinical manifestations typical of FA. Previous studies identified a novel FANCD2 polymorphism as well as frameshift-like mutations in FANCD2 gene

28

that are associated with genetic conditions conferring a predisposition to leukaemia (Offman et al. 2005).

FANCG While FANCG mutation carrier status does not predispose to sporadic AML, the identification of unrecognised FA patients implies that FA, presenting with primary AML in childhood, is more common than suspected. Recent studies identified connection of several FANCG mutations with leukaemia (Meyer et al. 2006). Two of these carried two variants, including the known IVS2 + 1G>A mutation with the novel missense mutation S588F, and R513Q with the intronic deletion IVS12-38 (-28)del11, implying that these patients might have been undiagnosed FA patients. R513Q, which affects a semi-conserved amino acid, was carried in two additional children with AML. Although not significant, the frequency of R513Q was higher in children with AML than unselected cord blood samples.

III.4. Mutations of FA genes in solid tumors, especially in the breast and ovarian cancer families The FA patients that survive into early adulthood are hundreds of times more susceptible of developing solid tumors compared with the general population. Moreover, almost one third of FA patients will develop a solid tumor by the age of 48 years (Rosenberg et al. 2003). In particular, there is a high risk of liver cancers as well as squamous cell carcinomas of the oesophagus, oropharynx and vulva origins (Alter 2003; Kutler et al. 2003; Rosenberg et al. 2003). Due to rareness of FA disease it is hard to make a large screening for the BRCA family members in FA patients. Probably, some of the BRCA mutations found in specific ethnic groups could involve

29

individuals with FA, however by the time most of the studies were done, it was unnecessary to screen for FA mutations (Rutter et al. 2003). Along with the evidence linking breast cancer and FANCD1 (BRCA2) mutations provided above, we are going to describe some other FA mutations found in patients having breast and ovarian cancers as well as other types of tumors.

FANCA FANCA is also a potential breast and ovarian cancer susceptibility gene. A novel allele having a tandem duplication of a 13 base pair sequence in the promoter region was identified by screening germline DNA from 352 breast cancer patients and was associated with an increased risk of breast or ovarian cancer. Although this allele with the tandem duplication does not appear to modify breast cancer risk, it may act as a low penetrance protective allele for ovarian cancer (Thompson et al. 2005). One of the major studies made so far was done to investigate whether heterozygous variants in other FA genes are high penetrance breast cancer susceptibility alleles. Since monoallelic (heterozygous) BRCA2 mutations confer a high risk of breast cancer and are a major cause of familial breast cancers, researchers screened germ-line DNA from 88 BRCAI/2-negative families, each with at least three cases of breast cancer, for mutations in FANCA. Missense varian (FANCA L1143V) was identified in family of twin sisters and two additional sisters, all affected with breast cancer before age 55 years. The twins and one sister carried FANCA L1143V as did an unaffected brother (Seal et al. 2003).

FANCC

30

A heterozygous germline mutation in FANCC has been identified in two of 421 patients with pancreatic cancer (Couch et al. 2005). Cancer cells taken from these patients exhibited a loss of heterozygosity at the FANCC locus. Other studies have identified human pancreatic tumor lines with biallelic loss of FANCC and FANCG (van der Heijden et al. 2004). Thus, there is a strong indication that that heterozygous carriers of FANCC (and possibly FANCG) mutation have increased pancreatic cancer risk, although at a lower penetrance than BRCA2/FANCD1 mutation.(van der Heijden et al. 2004). Recent studies of FA subjects within Connecticut registery showed that among the grandmothers, those who were carriers of FANCC mutations were found to be at highest risk (SIR, 2.4; 95% CI, 1.1-5.2). Overall, there was no increased risk for cancer among FA heterozygotes in this study of FA relatives, although there is some evidence that FANCC mutations are possibly breast cancer susceptibility alleles (Berwick et al. 2007).

FANCD2 Studies of seven SNPs in FANCA, FANCL and FANCD2 in a total of 897 consecutive and non-related sporadic breast cancer cases and 1033 unaffected controls from the Spanish population revealed statistically significant association with sporadic breast cancers located on FANCD2 indicating a relationship between FANCD2 and breast cancer risk. Statistically significant association with sporadic breast cancer has been observed for the variant rs2272125 located on FANCD2 (Barroso et al. 2006). It was also hypothesised that germline mutations in FANCD2 may account for some of the unexplained multiple-case breast cancer families. Analysis of FANCD2 in the 33 index cases from 30 breast and ovarian cancer families, and of exons 9 and 19 (containing the ATM phosphorylation site and the FANCD2 monoubiquitinylation site, respectively) in

31

a further 399 non-BRCA1/2 index cases, identified 32 germline sequence alterations (Barroso et al. 2006). And the very last study of breast cancers family members revealed that 19% sporadic breast cancers and 10% BRCA1-related breast cancers were completely FANCD2-negative, indicating that somatic inactivation in FANCD2 may be important in both sporadic and hereditary breast carcinogenesis and that FANCD2 is of independent prognostic value in sporadic breast cancer (van der Groep et al. 2007).

FANCE A conservative missense variant FANCE R365K was identified in family of twin sisters and two additional sisters, all affected with breast cancer before age 55 years (Seal et al. 2003).

FANCJ Some more individuals with breast cancer from BRCA1/BRCA2 mutation-negative families turned out to have FANCJ (BRIP1 helicase) mutation. Inactivation of truncating biallelic mutations of BRIP1 (141ΔC, 2392C

T, 2008insT, 2255ΔAA,

2108ΔAinsTCC), similar to those in BRCA2, cause FA and confer susceptibility to breast cancer in monoallelic carriers (Seal et al. 2006). Later on, FANCJ mutations (658C

T, 718G

2636G

A, 2896C

A, 725T T, 2778G

C, 1396C

T, 1732T

A, 2945T

G, 3552C

G, 2392C

T, 1760A

T,

T, 3968A

C, 4049C

T)

have been found in 21 families with potentially inherited breast/ovarian cancer. It should be noted that all these families were BRCA1/BRCA2 negative, but had at least one case of male breast cancer, two cases of ovarian cancer, or three or more cases of female breast and ovarian cancers again confirming that FANCJ mutations have a moderate risk of breast cancer (Rutter et al. 2003).

32

FANCN PALB2 gene product, later identified as FANCN, interacts with BRCA2, and biallelic mutations in PALB2 (2386G 3116ΔA, 3116ΔA, 3549C

T, 2982insT, 3113G

G, 3549C

G, 3549C

A, 3113G

A, 3116ΔA,

G), similar to biallelic BRCA2

mutations, cause FA. Recent study identified monoallelic truncating PALB2 mutations in individuals with familial breast cancer and showed that such mutations confer approximately 2.3-fold higher risk of breast cancer (Rahman et al. 2007). PALB2 frameshift mutation (c.1592ΔT) is present at significantly elevated frequency in Finnish familial breast cancer cases compared with ancestry-matched population controls (Erkko et al. 2007)

BRCA2/FANCD1 Mutations in BRCA genes play important roles in breast and ovarian cancer. Carriers of mutations in BRCA2/FANCD1 have an 82% lifetime risk of breast cancer and a 54% and 23% risk of ovarian cancer respectively. For BRCA2 gene, researchers have identified more than 450 mutations; many of those are insertions or deletions resulting in an abnormally small, non-functional version of the BRCA2 protein. About a third of mutations identified in BRCA2 sequencing studies are of uncertain clinical significance (Shattuck-Eidens et al. 1997). In addition to breast and ovarian cancers, heterozygosity for a mutation in BRCA2/FANCD1 predisposes to pancreatic cancer. In one study, 19% of families with a history of hereditary pancreatic cancer had either a frameshift mutation or an unclassified variant of BRCA2/FANCD1 (Hahn et al. 2003). Other cancers that have been reported to be associated with heterozygous mutation of

33

BRCA2/FANCD1 include prostate cancer, gastric cancer, and melanoma (Liede et al. 2004).

III.5. Molecular biology of FA cancer predisposition: cancer in FA mutation carriers The mechanisms by which defects in the FA complex increase susceptibility to specific cancers are not completely elucidated. The facts that FA crosstalk with BRCA pathway and that the cell lines homozygous for BRCA1/2 mutations are hypersensitive to mitomycin-C and the cells from homozygous BRCA2 mutant mice reveal FA-like phenotype suggest that BRCA1/2 mutations may occur in FA patients and vise versa. (Howlett et al. 2002; D'Andrea and Grompe 2003; Moynahan et al. 2001; Patel et al. 1998; Connor et al. 1997). Moreover, accumulating data suggest that only synergetic functioning of FA and BRCA pathways reveal truly functional and protective mechanism against cancer. Indeed, disruption of FA/BRCA pathway may serve as a risk factor for cancer predisposition (Lyakhovich and Surralles 2006). The early age at onset of these tumors in FA is similar to what is observed in other cancer predisposition syndromes, such as hereditary breast cancer, hereditary colon cancer, Li-Fraumeni syndrome, xeroderma pigmentosum, ataxia telangiectasia, Bloom syndrome, and so forth. The susceptibility to solid tumors in FA may reflect a "2-hit" process in which the FA defect renders the cell susceptible to genomic instability caused by subsequent somatic events occurring relatively early in life. The specific solid tumors that occur excessively in patients with FA suggest that environmental exposures may play a role.

We also know that cells with mutations DNA repair pathway genes exhibit genomic instability syndromes and other disorders that often result in predisposition to cancer.

34

However, depending on the gene deficiency, cells reveal quite remarkable variations upon exposure to genotoxic stress or DNA damaging factors. Our novel biochemical findings suggest a crosstalk of FA/BRCA pathway with γH2AX, the DNA damage marker. H2AX, BRCA2 and FA deficient cells are known to be hypersensitive to crosslinkers but mildly sensitive to ionizing radiation. (Bassing et al. 2002; Rahden-Staron et al. 2003). We and others have shown that cells depleted or deficient of H2AX exhibit hypersensitivity to DNA cross-linking agents (Bogliolo et al. 2007). Importantly, this hypersensitivity of H2AX-deficient cells was not further increased by FANCD2 RNAi depletion, indicating that H2AX and FANCD2 function in the same pathway in response to DNA damage-induced replication blockage. Similarly, BRCA2 and H2AX deficient cells show growth defective G2/M checkpoint arrest at low radiation doses.

Spontaneous chromosomal aberrations are also common features for the cells deficient in FA core genes, FANCD2, BRCA2 or H2AX. Upon DNA damage cells deficient in any of these genetic backgrounds produce errors in repair of different types of genetic lesion and show chromatid breaks (due to the damage effects sustained during or after replication in S or G2) or chromosomes (arise from damage sustained in G1 before chromatid duplication) (Patel et al. 1998). Detached chromosomes, chromatid-type abnormalities in metaphases and appearance of radial structures upon treatment with DNA damaging factors are common features in H2AX-/-, BRCA2-/-, FANCD2-/- or FA core complex deficient cells. (Patel et al. 1998; Foray et al. 1999).

It should be noticed that several mice models with upstream FA knockout (KO) genes exposed only slight phenotypical similarities to human FA patients (Chen et al. 1996; Cheng et al. 2000; Whitney et al. 1996; Rio et al. 2002). The phenotypes of these

35

knockout mice are essentially indistinguishable (Noll et al. 2002). Possible explanation for that could be that all these FA KO models utilized defectiveness of upstream FA genes with main known function is to signal for FANCD2 activation (ubiquitinylation). Therefore, disruption of one of these genes does not significantly affect activation of downstream genes/proteins carrying many more important functions. In other words, to obtain a FA-phenotype similar to the one of FA patients, it is important to alter any gene located in the proximity to the DNA/chromatin level (Figure. 8). In favour to this hypothesis serves experiments with FANCD2–/– mice exhibiting a more severe FA phenotype than FANCA or FANCC KO mice (Houghtaling et al. 2005). The FANCD2-deficient mice have an increased incidence of breast, ovarian, and liver cancers. Consistent with this theory, no biallelic FANCD2 null mutations have been found in FA patients so far. The most recent data suggest that all FA patients from D2 group have hypomorphic mutations in FANCD2 (Kalb et al. 2007). It is likely, that for the high hyrarcied organisms, like human beings, completely null FANCD2 phenotype is not compatible with living functions. In accordance to our scheme, location of BRCA2 is somewhere downstream from FANCD2 thus, triggering the events leading in HR. Accordingly, BRCA2-deficiency should result in even stronger severance than that in FANCD2–/–. Indeed, it was impossible to establish BRCA2–/– KO mice due to embryo lethality. However, mice having hypomorphic mutation in the BRCA2/FANCD1 gene (BRCA2Δ27/Δ27) reveals extremely severe phenotype resembling many clinical features of FA patients (Navarro et al. 2006). Interestingly, BRCA2 deficient FA-D1 patients and mice exhibit a much higher frequency of chromosomal aberrations than other FA subtypes (Navarro et al. 2006). Moreover, FA patients with a homozygous mutation in FANCD1/BRCA2 have a different cancer spectrum with medulloblastoma and Wihlm’s syndrome being the predominant

36

malignancies (Hirsch et al. 2004; Kutler et al. 2003). This may be because BRCA2 has other functions in HR outside the FA pathway. Overall, these observations support the idea that clinical severances of FA/BRCA deficiency is increased towards approaching the genes required for genome integrity (Lyakhovich and Surralles 2007). This increased severance is accompanied with the increasing number of functions of the same gene products. It seems likely that the final role embracing this pathway is to maintain the integrity of a genome by securing normal HR function.

37

Legends to figures

Figure 1. Metaphase chromosomes from a FA patient upon DEB treatment. Arrows indicate some abnormal structures which are typical for FA patients.

Figure 2. Cell cycle profile of FA and non-FA patient cells before and after MMC treatment.

Figure 3. Worldwide distribution of FA complementation groups.

Figure 4. FANCD2 detection by Western blot. (A) The lower and the upper bands represent non-ubiquitinylated (inactive) and ubiquitinilated (active) isoforms of FANCD2 named FANCD2-S(hort) and FANCD2-L(ong), respectively. Two bands are seen in normal and FANCA corrected cells. (B) Only the lower band is seen in a FANCA deficient cell line and none of FANCD2 bands are present in FANCD2 deficient cells. Colocalization of FANCD2 with a UVC-induced spot in normal and FANCA deficient cells.

Figure 5. Model of FA pathway and its activation by ATR.

Figure 6. Proposed function for FA pathway. (1) Replication fork is blocked by ICL. (2) A nick is produced by MUS81 (3) leaving ssDNA region. (4) RPA recognizes the ssDNA region and ATR binds to RPA activating FA pathway. H2AX is rapidly phosphorylated by ATR at the break point triggering the DNA damage response (5) The FA core complex is assembled and then translocated to the chromatin by FANCM until

38

the ICL is detected. FANCD2 becomes activated by the FA core complex. (6) The complex XPA/ERCC1 produces a second nick at the other side of the ICL, (7) leaving a gap that will be filled by the TLS system generating point mutations. In parallel, the ICL is repaired by the NER system. (8) MRN complex resects one strand of the DSB generating ssDNA overhang. (9) Immediately after that, the HRR machinery is loaded on to the chromatin and the strand invasion starts. (10) Once the sequence is copied and the Holliday junction is resolved, the replication process can restart.

Figure 7 Distribution of malignances among FA cancer cases worldwide as seen from the literature search (1970-present). The early on-set of haematological disorders fall onto age 7, although for some complementation groups (e.g. FANCD1) it may arrive earlier. Almost half of FA patients die by the age of 12 from aplastic anemia or complications after bone marrow transplantation. Those who survived develop various types of malignances. Leukemia is the most common cancer developed in FA patients by the average age of 29. MDS, although separated into different group, normally progress to leukemia (30%) or other cancers. FA patients undergoing androgene treatment develop liver cancers – this normally occurs in a group of 12-45 years old patients. The rest of the cases belong to the group of FA patients developing various types of solid tumors, in general at adult age.

Figure 8 Clinical severeness of FA subgroups is increased towards complexity of FA gene functions. Starting from FA core complex genes FA pathway embraces FANCD2 and H2AX going to BRCA2 (FANCD1) with the main goal to secure normal

39

homologous recombination repair at broken replication forks. Clinical severeness is increased beginning from FA core complex deficiency towards cells with FANCD2 and BRCA2 defective genetic backgrounds. This coincides with the increase number of essential gene functions needed to maintain genomic and cellular integrity (adapted from Lyakhovich and Surralles 2007).

Acknowledgements Fanconi anemia research in Surrallés’ laboratory is funded by the Generalitat de Catalunya (SGR-00197-2002), the La Caixa Fundation Oncology Program (BM05-670), Genoma España, the Spanish Ministry of Health and Consumption (projects FIS061099 and CIBER-ER CB06/07/0023), the Spanish Ministry of Science and Technology (projects SAF2004-20372-E and SAF2006-3440), the Commission of the European Union (project FI6R-CT-2003-508842), and the European Regional Development Funds.

40

Bibliography Abbondazo, S. L., N. S. Irey and G. Frizzera (1995). "Dilantin-associated lymphadenopathy. Spectrum of histopathologic patterns." Am J Surg Pathol 19(6): 675-86. Alter, B. P. (1996). "Fanconi's anemia and malignancies." Am J Hematol 53(2): 99-110. Alter, B. P. (2003). "Cancer in Fanconi anemia, 1927-2001." Cancer 97(2): 425-40. Alter, B. P., J. P. Caruso, R. A. Drachtman, T. Uchida, G. V. Velagaleti and M. T. Elghetany (2000). "Fanconi anemia: myelodysplasia as a predictor of outcome." Cancer Genet Cytogenet 117(2): 125-31. Alter, B. P., P. S. Rosenberg and L. C. Brody (2007). "Clinical and molecular features associated with biallelic mutations in FANCD1/BRCA2." J Med Genet 44(1): 19. Alter, B. P. and M. S. Tenner (1994). "Brain tumors in patients with Fanconi's anemia." Arch Pediatr Adolesc Med 148(6): 661-3. Alter, C. L., P. H. Levine, J. Bennett, C. Kessler, M. Rick, R. G. Washburn, J. I. Gallin, R. W. Miller and A. D. Auerbach (1989). "Dominantly transmitted hematologic dysfunction clinically similar to Fanconi's anemia." Am J Hematol 32(4): 241-7. Andreassen, P. R., A. D. D'Andrea and T. Taniguchi (2004). "ATR couples FANCD2 monoubiquitination to the DNA-damage response." Genes Dev 18(16): 1958-63. Arnold, W. J., C. R. King, J. Magrina and B. J. Masterson (1980). "Squamous cell carcinoma of the vulva and Fanconi anemia." Int J Gynaecol Obstet 18(6): 3957. Auerbach, A. D. (1988). "A test for Fanconi's anemia." Blood 72(1): 366-7.

41

Awan, A., G. Malcolm Taylor, D. A. Gokhale, S. P. Dearden, A. Will, R. F. Stevens, J. M. Birch and T. Eden (1998). "Increased frequency of Fanconi anemia group C genetic variants in children with sporadic acute myeloid leukemia." Blood 91(12): 4813-4. Baer, R. and W. H. Lee (1998). "Functional domains of the BRCA1 and BRCA2 proteins." J Mammary Gland Biol Neoplasia 3(4): 403-12. Bagby, G. C. and B. P. Alter (2006). "Fanconi anemia." Semin Hematol 43(3): 147-56. Barber, L. M., R. A. Barlow, S. Meyer, D. J. White, A. M. Will, T. O. Eden and G. M. Taylor (2005). "Inherited FANCD1/BRCA2 exon 7 splice mutations associated with acute myeloid leukaemia in Fanconi anaemia D1 are not found in sporadic childhood leukaemia." Br J Haematol 130(5): 796-7. Barber, L. M., H. E. McGrath, S. Meyer, A. M. Will, J. M. Birch, O. B. Eden and G. M. Taylor (2003). "Constitutional sequence variation in the Fanconi anaemia group C (FANCC) gene in childhood acute myeloid leukaemia." Br J Haematol 121(1): 57-62. Barroso, E., R. L. Milne, L. P. Fernandez, P. Zamora, J. I. Arias, J. Benitez and G. Ribas (2006). "FANCD2 associated with sporadic breast cancer risk." Carcinogenesis 27(9): 1930-7. Bassing, C. H., K. F. Chua, J. Sekiguchi, H. Suh, S. R. Whitlow, J. C. Fleming, B. C. Monroe, D. N. Ciccone, C. Yan, K. Vlasakova, D. M. Livingston, D. O. Ferguson, R. Scully and F. W. Alt (2002). "Increased ionizing radiation sensitivity and genomic instability in the absence of histone H2AX." Proc Natl Acad Sci U S A 99(12): 8173-8. Berwick, M., J. M. Satagopan, L. Ben-Porat, A. Carlson, K. Mah, R. Henry, R. Diotti, K. Milton, K. Pujara, T. Landers, S. Dev Batish, J. Morales, D. Schindler, H.

42

Hanenberg, R. Hromas, O. Levran and A. D. Auerbach (2007). "Genetic heterogeneity among Fanconi anemia heterozygotes and risk of cancer." Cancer Res 67(19): 9591-6. Blom, E., H. J. van de Vrugt, Y. de Vries, J. P. de Winter, F. Arwert and H. Joenje (2004). "Multiple TPR motifs characterize the Fanconi anemia FANCG protein." DNA Repair (Amst) 3(1): 77-84. Bogliolo, M., E. Callen, A. Lyakhovich, M. Castellà, E. Cappelli, M. J. Ramirez, A. Creus, R. Marcos, R. Kalb, K. Neveling, D. Schindler and J. Surralles (2006). "Functional connection between histone H2AX and the Fanconi anemia/BRCA pathway in genome stability." Embo J: En revisió. Bogliolo, M., A. Lyakhovich, E. Callen, M. Castella, E. Cappelli, M. J. Ramirez, A. Creus, R. Marcos, R. Kalb, K. Neveling, D. Schindler and J. Surralles (2007). "Histone H2AX and Fanconi anemia FANCD2 function in the same pathway to maintain chromosome stability." Embo J 26(5): 1340-51. Borriello, A., A. Locasciulli, A. M. Bianco, M. Criscuolo, V. Conti, P. Grammatico, S. Cappellacci, A. Zatterale, F. Morgese, V. Cucciolla, D. Delia, F. Della Ragione and A. Savoia (2007). "A novel Leu153Ser mutation of the Fanconi anemia FANCD2 gene is associated with severe chemotherapy toxicity in a pediatric Tcell acute lymphoblastic leukemia." Leukemia 21(1): 72-8. Butturini, A., R. P. Gale, P. C. Verlander, B. Adler-Brecher, A. P. Gillio and A. D. Auerbach (1994). "Hematologic abnormalities in Fanconi anemia: an International Fanconi Anemia Registry study." Blood 84(5): 1650-5. Callen, E., J. A. Casado, M. D. Tischkowitz, J. A. Bueren, A. Creus, R. Marcos, A. Dasi, J. M. Estella, A. Munoz, J. J. Ortega, J. de Winter, H. Joenje, D. Schindler, H. Hanenberg, S. V. Hodgson, C. G. Mathew and J. Surralles (2005). "A

43

common founder mutation in FANCA underlies the world's highest prevalence of Fanconi anemia in Gypsy families from Spain." Blood 105(5): 1946-9. Callen, E., E. Samper, M. J. Ramirez, A. Creus, R. Marcos, J. J. Ortega, T. Olive, I. Badell, M. A. Blasco and J. Surralles (2002). "Breaks at telomeres and TRF2independent end fusions in Fanconi anemia." Hum Mol Genet 11(4): 439-44. Cantor, S., R. Drapkin, F. Zhang, Y. Lin, J. Han, S. Pamidi and D. M. Livingston (2004). "The BRCA1-associated protein BACH1 is a DNA helicase targeted by clinically relevant inactivating mutations." Proc Natl Acad Sci U S A 101(8): 2357-62. Cantor, S. B., D. W. Bell, S. Ganesan, E. M. Kass, R. Drapkin, S. Grossman, D. C. Wahrer, D. C. Sgroi, W. S. Lane, D. A. Haber and D. M. Livingston (2001). "BACH1, a novel helicase-like protein, interacts directly with BRCA1 and contributes to its DNA repair function." Cell 105(1): 149-60. Casado, J. A., E. Callen, et al. (2006). "A rational multidisciplinary strategy for the genetic subtyping of Fanconi anemia patients: Conclusions from the Spanish Fanconi anemia Research Network." J Med Genet: En premsa. Castillo, V., O. Cabre, R. Marcos and J. Surralles (2003). "Molecular cloning of the Drosophila Fanconi anaemia gene FANCD2 cDNA." DNA Repair (Amst) 2(6): 751-8. Celeste, A., S. Petersen, P. J. Romanienko, O. Fernandez-Capetillo, H. T. Chen, O. A. Sedelnikova,

B.

Reina-San-Martin,

V.

Coppola,

E.

Meffre,

M.

J.

Difilippantonio, C. Redon, D. R. Pilch, A. Olaru, M. Eckhaus, R. D. CameriniOtero, L. Tessarollo, F. Livak, K. Manova, W. M. Bonner, M. C. Nussenzweig and A. Nussenzweig (2002). "Genomic instability in mice lacking histone H2AX." Science 296(5569): 922-7.

44

Chen, M., D. J. Tomkins, W. Auerbach, C. McKerlie, H. Youssoufian, L. Liu, O. Gan, M. Carreau, A. Auerbach, T. Groves, C. J. Guidos, M. H. Freedman, J. Cross, D. H. Percy, J. E. Dick, A. L. Joyner and M. Buchwald (1996). "Inactivation of Fac in mice produces inducible chromosomal instability and reduced fertility reminiscent of Fanconi anaemia." Nat Genet 12(4): 448-51. Cheng, N. C., H. J. van de Vrugt, M. A. van der Valk, A. B. Oostra, P. Krimpenfort, Y. de Vries, H. Joenje, A. Berns and F. Arwert (2000). "Mice with a targeted disruption of the Fanconi anemia homolog Fanca." Hum Mol Genet 9(12): 180511. Ciccia, A., C. Ling, R. Coulthard, Z. Yan, Y. Xue, A. R. Meetei, H. Laghmani el, H. Joenje, N. McDonald, J. P. de Winter, W. Wang and S. C. West (2007). "Identification of FAAP24, a Fanconi anemia core complex protein that interacts with FANCM." Mol Cell 25(3): 331-43. Condie, A., R. L. Powles, C. D. Hudson, V. Shepherd, S. Bevan, M. R. Yuille and R. S. Houlston (2002). "Analysis of the Fanconi anaemia complementation group A gene in acute myeloid leukaemia." Leuk Lymphoma 43(9): 1849-53. Connor, F., D. Bertwistle, P. J. Mee, G. M. Ross, S. Swift, E. Grigorieva, V. L. Tybulewicz and A. Ashworth (1997). "Tumorigenesis and a DNA repair defect in mice with a truncating Brca2 mutation." Nat Genet 17(4): 423-30. Couch, F. J., M. R. Johnson, K. Rabe, L. Boardman, R. McWilliams, M. de Andrade and G. Petersen (2005). "Germ line Fanconi anemia complementation group C mutations and pancreatic cancer." Cancer Res 65(2): 383-6. D'Andrea, A. D. and M. Grompe (2003). "The Fanconi anaemia/BRCA pathway." Nat Rev Cancer 3(1): 23-34.

45

de Chadarevian, J. P., M. Vekemans and M. Bernstein (1985). "Fanconi's anemia, medulloblastoma, Wilms' tumor, horseshoe kidney, and gonadal dysgenesis." Arch Pathol Lab Med 109(4): 367-9. De Kerviler, E., A. Guermazi, A. M. Zagdanski, E. Gluckman and J. Frija (2000). "The clinical and radiological features of Fanconi's anaemia." Clin Radiol 55(5): 3405. de Winter, J. P., F. Leveille, C. G. van Berkel, M. A. Rooimans, L. van Der Weel, J. Steltenpool, I. Demuth, N. V. Morgan, N. Alon, L. Bosnoyan-Collins, J. Lightfoot, P. A. Leegwater, Q. Waisfisz, K. Komatsu, F. Arwert, J. C. Pronk, C. G. Mathew, M. Digweed, M. Buchwald and H. Joenje (2000). "Isolation of a cDNA representing the Fanconi anemia complementation group E gene." Am J Hum Genet 67(5): 1306-8. de Winter, J. P., M. A. Rooimans, L. van Der Weel, C. G. van Berkel, N. Alon, L. Bosnoyan-Collins, J. de Groot, Y. Zhi, Q. Waisfisz, J. C. Pronk, F. Arwert, C. G. Mathew, R. J. Scheper, M. E. Hoatlin, M. Buchwald and H. Joenje (2000). "The Fanconi anaemia gene FANCF encodes a novel protein with homology to ROM." Nat Genet 24(1): 15-6. de Winter, J. P., L. van der Weel, J. de Groot, S. Stone, Q. Waisfisz, F. Arwert, R. J. Scheper, F. A. Kruyt, M. E. Hoatlin and H. Joenje (2000). "The Fanconi anemia protein FANCF forms a nuclear complex with FANCA, FANCC and FANCG." Hum Mol Genet 9(18): 2665-74. de Winter, J. P., Q. Waisfisz, M. A. Rooimans, C. G. van Berkel, L. Bosnoyan-Collins, N. Alon, M. Carreau, O. Bender, I. Demuth, D. Schindler, J. C. Pronk, F. Arwert, H. Hoehn, M. Digweed, M. Buchwald and H. Joenje (1998). "The

46

Fanconi anaemia group G gene FANCG is identical with XRCC9." Nat Genet 20(3): 281-3. Doerr, T. D., T. Y. Shibuya and S. C. Marks (1998). "Squamous cell carcinoma of the supraglottic larynx in a patient with Fanconi's anemia." Otolaryngol Head Neck Surg 118(4): 523-5. Dorsman, J. C., M. Levitus, D. Rockx, M. A. Rooimans, A. B. Oostra, A. Haitjema, S. T. Bakker, J. Steltenpool, D. Schuler, S. Mohan, D. Schindler, F. Arwert, G. Pals, C. G. Mathew, Q. Waisfisz, J. P. de Winter and H. Joenje (2007). "Identification of the Fanconi anemia complementation group I gene, FANCI." Cell Oncol 29(3): 211-8. Dosik, H., W. Steier and A. Lubiniecki (1979). "Inherited aplastic anaemia with increased endoreduplications: a new syndrome of Fanconi's anaemia variant?" Br J Haematol 41(1): 77-82. Dutrillaux, B., A. Aurias, A. M. Dutrillaux, D. Buriot and M. Prieur (1982). "The cell cycle of lymphocytes in Fanconi anemia." Hum Genet 62(4): 327-32. Erkko, H., B. Xia, J. Nikkila, J. Schleutker, K. Syrjakoski, A. Mannermaa, A. Kallioniemi, K. Pylkas, S. M. Karppinen, K. Rapakko, A. Miron, Q. Sheng, G. Li, H. Mattila, D. W. Bell, D. A. Haber, M. Grip, M. Reiman, A. JukkolaVuorinen, A. Mustonen, J. Kere, L. A. Aaltonen, V. M. Kosma, V. Kataja, Y. Soini, R. I. Drapkin, D. M. Livingston and R. Winqvist (2007). "A recurrent mutation in PALB2 in Finnish cancer families." Nature 446(7133): 316-9. Fabio, T., N. Crescenzio, P. Saracco, L. Leone, G. Ponzio and U. Ramenghi (2000). "Cell cycle analysis in the diagnosis of Fanconi's anemia." Haematologica 85(4): 431-2.

47

Fanconi, G. (1927). "Familiäre infantile perniziosaartige Anämie (perniziöses Blutbild und Konstitution)." Jahrbuch für Kinderheilkunde und physische 117: 257-280. Faivre, L., P. Guardiola, C. Lewis, I. Dokal, W. Ebell, A. Zatterale, C. Altay, J. Poole, D. Stones, M. L. Kwee, M. van Weel-Sipman, C. Havenga, N. Morgan, J. de Winter, M. Digweed, A. Savoia, J. Pronk, T. de Ravel, S. Jansen, H. Joenje, E. Gluckman and C. G. Mathew (2000). "Association of complementation group and mutation type with clinical outcome in fanconi anemia. European Fanconi Anemia Research Group." Blood 96(13): 4064-70. Ferro, M. T., Y. Vazquez-Mazariego, S. Ramiro, M. C. Sanchez-Hombre, C. Villalon, J. M. Garcia-Sagredo, C. Ulibarrena, J. L. Sastre and C. S. Roman (2001). "Triplication of 1q in Fanconi anemia." Cancer Genet Cytogenet 127(1): 38-41. Foray, N., V. Randrianarison, D. Marot, M. Perricaudet, G. Lenoir and J. Feunteun (1999). "Gamma-rays-induced death of human cells carrying mutations of BRCA1 or BRCA2." Oncogene 18(51): 7334-42. Friedman, A. and R. Chesney (1981). "Fanconi's syndrome in renal transplantation." Am J Nephrol 1(1): 45-7. Fukuoka, K., K. Nishikawa, Y. Mizumoto, T. Shimoyama, K. Mikasa, Y. Kounoike, M. Sawaki, N. Narita, R. Oka, Y. Tsuruta and et al. (1989). "[Fanconi's anemia with squamous cell carcinoma--a case report and a review of literature]." Rinsho Ketsueki 30(11): 1992-6. Garcia-Higuera, I., Y. Kuang, J. Denham and A. D. D'Andrea (2000). "The fanconi anemia proteins FANCA and FANCG stabilize each other and promote the nuclear accumulation of the Fanconi anemia complex." Blood 96(9): 3224-30.

48

Garcia-Higuera, I., T. Taniguchi, S. Ganesan, M. S. Meyn, C. Timmers, J. Hejna, M. Grompe and A. D. D'Andrea (2001). "Interaction of the Fanconi anemia proteins and BRCA1 in a common pathway." Mol Cell 7(2): 249-62. Garel, L., G. Kalifa, D. Buriot and J. Sauvegrain (1981). "Multiple adenomas of the liver and Fanconi's anaemia." Ann Radiol (Paris) 24(1): 53-4. Gasparini, G., G. Longobardi, R. Boniello, A. Di Petrillo and S. Pelo (2006). "Fanconi anemia manifesting as a squamous cell carcinoma of the hard palate: a case report." Head Face Med 2: 1. German, J., S. Schonberg, S. Caskie, D. Warburton, C. Falk and J. H. Ray (1987). "A test for Fanconi's anemia." Blood 69(6): 1637-41. Giri, N., D. L. Batista, B. P. Alter and C. A. Stratakis (2007). "Endocrine abnormalities in patients with Fanconi anemia." J Clin Endocrinol Metab 92(7): 2624-31. Goldberg, J. I. and P. I. Borgen (2006). "Breast cancer susceptibility testing: past, present and future." Expert Rev Anticancer Ther 6(8): 1205-14. Goldsby, R. E., S. L. Perkins, D. M. Virshup, A. R. Brothman and C. S. Bruggers (1999). "Lymphoblastic lymphoma and excessive toxicity from chemotherapy: an unusual presentation for Fanconi anemia." J Pediatr Hematol Oncol 21(3): 240-3. Grompe, M. (2002). "FANCD2: a branch-point in DNA damage response?" Nat Med 8(6): 555-6. Gross, M., H. Hanenberg, S. Lobitz, R. Friedl, S. Herterich, R. Dietrich, B. Gruhn, D. Schindler and H. Hoehn (2002). "Reverse mosaicism in Fanconi anemia: natural gene therapy via molecular self-correction." Cytogenet Genome Res 98(2-3): 126-35.

49

Hahn, S. A., B. Greenhalf, I. Ellis, M. Sina-Frey, H. Rieder, B. Korte, B. Gerdes, R. Kress, A. Ziegler, J. A. Raeburn, D. Campra, R. Grutzmann, H. Rehder, M. Rothmund, W. Schmiegel, J. P. Neoptolemos and D. K. Bartsch (2003). "BRCA2 germline mutations in familial pancreatic carcinoma." J Natl Cancer Inst 95(3): 214-21. Helmerhorst, F. M., D. C. Heaton, P. E. Crossen, A. E. von dem Borne, C. P. Engelfriet and A. T. Natarajan (1984). "Familial thrombocytopenia associated with platelet autoantibodies and chromosome breakage." Hum Genet 65(3): 252-6. Helmerhorst, T. J., G. H. Dijkhuizen, R. W. Veldhuizen and J. G. Stolk (1984). "Microglandular hyperplasia--a complicating factor in the diagnosis of cervical intraepithelial neoplasia." Eur J Obstet Gynecol Reprod Biol 17(1): 53-9. Hersey, P., A. Edwards, R. Lewis, A. Kemp and J. McInnes (1982). "Deficient natural killer cell activity in a patient with Fanconi's anaemia and squamous cell carcinoma. Association with defect in interferon release." Clin Exp Immunol 48(1): 205-12. Hill, L. S., P. M. Dennis and S. A. Fairham (1981). "Adenocarcinoma of the stomach and Fanconi's anaemia." Postgrad Med J 57(668): 404. Hirsch, B., A. Shimamura, L. Moreau, S. Baldinger, M. Hag-alshiekh, B. Bostrom, S. Sencer and A. D. D'Andrea (2004). "Association of biallelic BRCA2/FANCD1 mutations with spontaneous chromosomal instability and solid tumors of childhood." Blood 103(7): 2554-9. Hirschhorn, R., D. R. Yang, J. M. Puck, M. L. Huie, C. K. Jiang and L. E. Kurlandsky (1996). "Spontaneous in vivo reversion to normal of an inherited mutation in a patient with adenosine deaminase deficiency." Nat Genet 13(3): 290-5.

50

Houghtaling, S., A. Newell, Y. Akkari, T. Taniguchi, S. Olson and M. Grompe (2005). "Fancd2 functions in a double strand break repair pathway that is distinct from non-homologous end joining." Hum Mol Genet 14(20): 3027-33. Howlett, N. G., T. Taniguchi, S. G. Durkin, A. D. D'Andrea and T. W. Glover (2005). "The Fanconi anemia pathway is required for the DNA replication stress response and for the regulation of common fragile site stability." Hum Mol Genet 14(5): 693-701. Howlett, N. G., T. Taniguchi, S. Olson, B. Cox, Q. Waisfisz, C. De Die-Smulders, N. Persky, M. Grompe, H. Joenje, G. Pals, H. Ikeda, E. A. Fox and A. D. D'Andrea (2002). "Biallelic inactivation of BRCA2 in Fanconi anemia." Science 297(5581): 606-9. Hussain, S., J. B. Wilson, A. L. Medhurst, J. Hejna, E. Witt, S. Ananth, A. Davies, J. Y. Masson, R. Moses, S. C. West, J. P. de Winter, A. Ashworth, N. J. Jones and C. G. Mathew (2004). "Direct interaction of FANCD2 with BRCA2 in DNA damage response pathways." Hum Mol Genet 13(12): 1241-8. Hussain, S., E. Witt, P. A. Huber, A. L. Medhurst, A. Ashworth and C. G. Mathew (2003). "Direct interaction of the Fanconi anaemia protein FANCG with BRCA2/FANCD1." Hum Mol Genet 12(19): 2503-10. Jacobs, P. and C. Karabus (1984). "Fanconi's anemia. A family study with 20-year follow-up including associated breast pathology." Cancer 54(9): 1850-3. Janik-Moszant, A., H. Bubala, M. Stojewska and D. Sonta-Jakimczyk (1998). "[Acute lymphoblastic leukemia in children with Fanconi anemia]." Wiad Lek 51 Suppl 4: 285-8.

51

Jensen, R. A., M. E. Thompson, T. L. Jetton, C. I. Szabo, R. van der Meer, B. Helou, S. R. Tronick, D. L. Page, M. C. King and J. T. Holt (1996). "BRCA1 is secreted and exhibits properties of a granin." Nat Genet 12(3): 303-8. Joenje, H., F. Arwert, A. W. Eriksson, H. de Koning and A. B. Oostra (1981). "Oxygendependence of chromosomal aberrations in Fanconi's anaemia." Nature 290(5802): 142-3. Joenje, H. and K. J. Patel (2001). "The emerging genetic and molecular basis of Fanconi anaemia." Nat Rev Genet 2(6): 446-57. Kalb, R., K. Neveling, H. Hoehn, H. Schneider, Y. Linka, S. D. Batish, C. Hunt, M. Berwick, E. Callen, J. Surralles, J. A. Casado, J. Bueren, A. Dasi, J. Soulier, E. Gluckman, C. M. Zwaan, R. van Spaendonk, G. Pals, J. P. de Winter, H. Joenje, M. Grompe, A. D. Auerbach, H. Hanenberg and D. Schindler (2007). "Hypomorphic mutations in the gene encoding a key Fanconi anemia protein, FANCD2, sustain a significant group of FA-D2 patients with severe phenotype." Am J Hum Genet 80(5): 895-910. Kaplan, M. J., H. Sabio, H. J. Wanebo and R. W. Cantrell (1985). "Squamous cell carcinoma in the immunosuppressed patient: Fanconi's anemia." Laryngoscope 95(7 Pt 1): 771-5. Kennedy, A. W. and W. R. Hart (1982). "Multiple squamous-cell carcinomas in Fanconi's anemia." Cancer 50(4): 811-4. Kennedy, R. D. and A. D. D'Andrea (2005). "The Fanconi Anemia/BRCA pathway: new faces in the crowd." Genes Dev 19(24): 2925-40. Kennedy, R. D. and A. D. D'Andrea (2006). "DNA repair pathways in clinical practice: lessons from pediatric cancer susceptibility syndromes." J Clin Oncol 24(23): 3799-808.

52

Koo, W. H., L. A. Knight and P. T. Ang (1996). "Fanconi's anaemia and recurrent squamous cell carcinoma of the oral cavity: a case report." Ann Acad Med Singapore 25(2): 289-92. Kook, H. (2005). "Fanconi anemia: current management." Hematology 10 Suppl 1: 108-10. Koubik, A. C., B. H. Franca, O. Ribas Mde, M. R. de Araujo, T. M. Mattioli and A. A. de Lima (2006). "Comparative study of chronological, bone, and dental age in Fanconi's anemia." J Pediatr Hematol Oncol 28(4): 260-2. Kozarek, R. A. and R. A. Sanowski (1981). "Carcinoma of the esophagus associated with Fanconi's anemia." J Clin Gastroenterol 3(2): 171-4. Kumar, A. R., J. E. Wagner, A. D. Auerbach, J. E. Coad, C. A. Dietz, S. J. Schwarzenberg and M. L. MacMillan (2004). "Fatal hemorrhage from androgenrelated hepatic adenoma after hematopoietic cell transplantation." J Pediatr Hematol Oncol 26(1): 16-8. Kutler, D. I., A. D. Auerbach, J. Satagopan, P. F. Giampietro, S. D. Batish, A. G. Huvos, A. Goberdhan, J. P. Shah and B. Singh (2003). "High incidence of head and neck squamous cell carcinoma in patients with Fanconi anemia." Arch Otolaryngol Head Neck Surg 129(1): 106-12. Kutler, D. I., B. Singh, J. Satagopan, S. D. Batish, M. Berwick, P. F. Giampietro, H. Hanenberg and A. D. Auerbach (2003). "A 20-year perspective on the International Fanconi Anemia Registry (IFAR)." Blood 101(4): 1249-56. Kwee, M. L., E. H. Poll, J. J. van de Kamp, H. de Koning, A. W. Eriksson and H. Joenje (1983). "Unusual response to bifunctional alkylating agents in a case of Fanconi anaemia." Hum Genet 64(4): 384-7.

53

LeBrun, D. P., M. M. Silver, M. H. Freedman and M. J. Phillips (1991). "Fibrolamellar carcinoma of the liver in a patient with Fanconi anemia." Hum Pathol 22(4): 396-8. Lensch, M. W., M. Tischkowitz, T. A. Christianson, C. A. Reifsteck, S. A. Speckhart, P. M. Jakobs, M. E. O'Dwyer, S. B. Olson, M. M. Le Beau, S. V. Hodgson, C. G. Mathew, R. A. Larson and G. C. Bagby, Jr. (2003). "Acquired FANCA dysfunction and cytogenetic instability in adult acute myelogenous leukemia." Blood 102(1): 7-16. Leteurtre, S., A. Martinot, et al. (1999). "Development of a pediatric multiple organ dysfunction score: use of two strategies." Med Decis Making 19(4): 399-410. Leveille, F., E. Blom, A. L. Medhurst, P. Bier, H. Laghmani el, M. Johnson, M. A. Rooimans, A. Sobeck, Q. Waisfisz, F. Arwert, K. J. Patel, M. E. Hoatlin, H. Joenje and J. P. de Winter (2004). "The Fanconi anemia gene product FANCF is a flexible adaptor protein." J Biol Chem 279(38): 39421-30. Levitus, M., Q. Waisfisz, B. C. Godthelp, Y. de Vries, S. Hussain, W. W. Wiegant, E. Elghalbzouri-Maghrani, J. Steltenpool, M. A. Rooimans, G. Pals, F. Arwert, C. G. Mathew, M. Z. Zdzienicka, K. Hiom, J. P. De Winter and H. Joenje (2005). "The DNA helicase BRIP1 is defective in Fanconi anemia complementation group J." Nat Genet 37(9): 934-5. Liede, A., B. Y. Karlan and S. A. Narod (2004). "Cancer risks for male carriers of germline mutations in BRCA1 or BRCA2: a review of the literature." J Clin Oncol 22(4): 735-42. Linares, M., E. Pastor, A. Gomez and E. Grau (1991). "Hepatocellular carcinoma and squamous cell carcinoma in a patient with Fanconi's anemia." Ann Hematol 63(1): 54-5.

54

Ling, C., M. Ishiai, A. M. Ali, A. L. Medhurst, K. Neveling, R. Kalb, Z. Yan, Y. Xue, A. B. Oostra, A. D. Auerbach, M. E. Hoatlin, D. Schindler, H. Joenje, J. P. de Winter, M. Takata, A. R. Meetei and W. Wang (2007). "FAAP100 is essential for activation of the Fanconi anemia-associated DNA damage response pathway." Embo J 26(8): 2104-14. Litman, R., M. Peng, Z. Jin, F. Zhang, J. Zhang, S. Powell, P. R. Andreassen and S. B. Cantor (2005). "BACH1 is critical for homologous recombination and appears to be the Fanconi anemia gene product FANCJ." Cancer Cell 8(3): 255-65. Lo Ten Foe, J. R., M. L. Kwee, M. A. Rooimans, A. B. Oostra, A. J. Veerman, M. van Weel, R. M. Pauli, N. T. Shahidi, I. Dokal, I. Roberts, C. Altay, E. Gluckman, R. A. Gibson, C. G. Mathew, F. Arwert and H. Joenje (1997). "Somatic mosaicism in Fanconi anemia: molecular basis and clinical significance." Eur J Hum Genet 5(3): 137-48. Lo Ten Foe, J. R., M. A. Rooimans, L. Bosnoyan-Collins, N. Alon, M. Wijker, L. Parker, J. Lightfoot, M. Carreau, D. F. Callen, A. Savoia, N. C. Cheng, C. G. van Berkel, M. H. Strunk, J. J. Gille, G. Pals, F. A. Kruyt, J. C. Pronk, F. Arwert, M. Buchwald and H. Joenje (1996). "Expression cloning of a cDNA for the major Fanconi anaemia gene, FAA." Nat Genet 14(3): 320-3. Lobitz, S. and E. Velleuer (2006). "Guido Fanconi (1892-1979): a jack of all trades." Nat Rev Cancer 6(11): 893-8. Ludwig, T., D. L. Chapman, V. E. Papaioannou and A. Efstratiadis (1997). "Targeted mutations of breast cancer susceptibility gene homologs in mice: lethal phenotypes of Brca1, Brca2, Brca1/Brca2, Brca1/p53, and Brca2/p53 nullizygous embryos." Genes Dev 11(10): 1226-41.

55

Lustig, J. P., G. Lugassy, A. Neder and E. Sigler (1995). "Head and neck carcinoma in Fanconi's anaemia--report of a case and review of the literature." Eur J Cancer B Oral Oncol 31B(1): 68-72. Lyakhovich, A. and Surralles, J. (2007). "New roads to FA/BRCA pathway: H2AX." Cell Cycle 6(9): 1019-1023. Lyakhovich, A. and J. Surralles (2006). "Disruption of the Fanconi anemia/BRCA pathway in sporadic cancer." Cancer Lett 232(1): 99-106. Machida, Y. J., Y. Machida, Y. Chen, A. M. Gurtan, G. M. Kupfer, A. D. D'Andrea and A. Dutta (2006). "UBE2T is the E2 in the Fanconi anemia pathway and undergoes negative autoregulation." Mol Cell 23(4): 589-96. MacMillan, M. L., A. D. Auerbach, S. M. Davies, T. E. Defor, A. Gillio, R. Giller, R. Harris, M. Cairo, K. Dusenbery, B. Hirsch, N. K. Ramsay, D. J. Weisdorf and J. E. Wagner (2000). "Haematopoietic cell transplantation in patients with Fanconi anaemia using alternate donors: results of a total body irradiation dose escalation trial." Br J Haematol 109(1): 121-9. Marcou, Y., A. D'Andrea, P. A. Jeggo and P. N. Plowman (2001). "Normal cellular radiosensitivity in an adult Fanconi anaemia patient with marked clinical radiosensitivity." Radiother Oncol 60(1): 75-9. Maynadie, M., C. Verret, P. Moskovtchenko, F. Mugneret, T. Petrella, D. Caillot and P. M. Carli (1996). "Epidemiological characteristics of myelodysplastic syndrome in a well-defined French population." Br J Cancer 74(2): 288-90. McDonough, E. R. (1970). "Fanconi anemia syndrome." Arch Otolaryngol 92(3): 2845. Medhurst, A. L., H. Laghmani el, J. Steltenpool, M. Ferrer, C. Fontaine, J. de Groot, M. A. Rooimans, R. J. Scheper, A. R. Meetei, W. Wang, H. Joenje and J. P. de

56

Winter (2006). "Evidence for subcomplexes in the Fanconi anemia pathway." Blood 108(6): 2072-80. Meetei, A. R., J. P. de Winter, A. L. Medhurst, M. Wallisch, Q. Waisfisz, H. J. van de Vrugt, A. B. Oostra, Z. Yan, C. Ling, C. E. Bishop, M. E. Hoatlin, H. Joenje and W. Wang (2003). "A novel ubiquitin ligase is deficient in Fanconi anemia." Nat Genet 35(2): 165-70. Meetei, A. R., M. Levitus, Y. Xue, A. L. Medhurst, M. Zwaan, C. Ling, M. A. Rooimans, P. Bier, M. Hoatlin, G. Pals, J. P. de Winter, W. Wang and H. Joenje (2004). "X-linked inheritance of Fanconi anemia complementation group B." Nat Genet 36(11): 1219-24. Meetei, A. R., A. L. Medhurst, C. Ling, Y. Xue, T. R. Singh, P. Bier, J. Steltenpool, S. Stone, I. Dokal, C. G. Mathew, M. Hoatlin, H. Joenje, J. P. de Winter and W. Wang (2005). "A human ortholog of archaeal DNA repair protein Hef is defective in Fanconi anemia complementation group M." Nat Genet 37(9): 95863. Meetei, A. R., Z. Yan and W. Wang (2004). "FANCL replaces BRCA1 as the likely ubiquitin ligase responsible for FANCD2 monoubiquitination." Cell Cycle 3(2): 179-81. Meyer, S., L. M. Barber, D. J. White, A. M. Will, J. M. Birch, J. A. Kohler, K. Ersfeld, E. Blom, H. Joenje, T. O. Eden and G. Malcolm Taylor (2006). "Spectrum and significance of variants and mutations in the Fanconi anaemia group G gene in children with sporadic acute myeloid leukaemia." Br J Haematol 133(3): 284-92. Mosedale, G., W. Niedzwiedz, A. Alpi, F. Perrina, J. B. Pereira-Leal, M. Johnson, F. Langevin, P. Pace and K. J. Patel (2005). "The vertebrate Hef ortholog is a

57

component of the Fanconi anemia tumor-suppressor pathway." Nat Struct Mol Biol 12(9): 763-71. Moynahan, M. E., T. Y. Cui and M. Jasin (2001). "Homology-directed dna repair, mitomycin-c resistance, and chromosome stability is restored with correction of a Brca1 mutation." Cancer Res 61(12): 4842-50. Mulvihill, J. J., R. L. Ridolfi, F. R. Schultz, M. S. Borzy and P. B. Haughton (1975). "Hepatic adenoma in Fanconi anemia treated with oxymetholone." J Pediatr 87(1): 122-4. Nakanishi, K., T. Taniguchi, V. Ranganathan, H. V. New, L. A. Moreau, M. Stotsky, C. G. Mathew, M. B. Kastan, D. T. Weaver and A. D. D'Andrea (2002). "Interaction of FANCD2 and NBS1 in the DNA damage response." Nat Cell Biol 4(12): 913-20. Nara, N., T. Miyamoto, A. Kurisu, H. Tsunemoto, Y. Ohtsu and E. Tanabe (1980). "[A case of cutaneous T cell lymphoma (mycosis fungoides) associated with multiple hematological malignancies in the patient's sibship]." Rinsho Ketsueki 21(7): 1007-12. Navarro, S., N. W. Meza, O. Quintana-Bustamante, J. A. Casado, A. Jacome, K. McAllister, S. Puerto, J. Surralles, J. C. Segovia and J. A. Bueren (2006). "Hematopoietic dysfunction in a mouse model for Fanconi anemia group D1." Mol Ther 14(4): 525-35. Niedernhofer, L. J., H. Odijk, M. Budzowska, E. van Drunen, A. Maas, A. F. Theil, J. de Wit, N. G. Jaspers, H. B. Beverloo, J. H. Hoeijmakers and R. Kanaar (2004). "The structure-specific endonuclease Ercc1-Xpf is required to resolve DNA interstrand cross-link-induced double-strand breaks." Mol Cell Biol 24(13): 5776-87.

58

Nijman, S. M., T. T. Huang, A. M. Dirac, T. R. Brummelkamp, R. M. Kerkhoven, A. D. D'Andrea and R. Bernards (2005). "The deubiquitinating enzyme USP1 regulates the Fanconi anemia pathway." Mol Cell 17(3): 331-9. Nojima, K., H. Hochegger, A. Saberi, T. Fukushima, K. Kikuchi, M. Yoshimura, B. J. Orelli, D. K. Bishop, S. Hirano, M. Ohzeki, M. Ishiai, K. Yamamoto, M. Takata, H. Arakawa, J. M. Buerstedde, M. Yamazoe, T. Kawamoto, K. Araki, J. A. Takahashi, N. Hashimoto, S. Takeda and E. Sonoda (2005). "Multiple repair pathways mediate tolerance to chemotherapeutic cross-linking agents in vertebrate cells." Cancer Res 65(24): 11704-11. Noll, M., K. P. Battaile, R. Bateman, T. P. Lax, K. Rathbun, C. Reifsteck, G. Bagby, M. Finegold, S. Olson and M. Grompe (2002). "Fanconi anemia group A and C double-mutant mice: functional evidence for a multi-protein Fanconi anemia complex." Exp Hematol 30(7): 679-88. Nuamah, N. M., E. Hamaloglu, A. Ozdemir, A. Ozenc, C. Sozseker and C. Sokmensuer (2006). "Hepatic focal nodular hyperplasia developing in a Fanconi anemia patient: a case report and literature review." Haematologica 91(8 Suppl): ECR39. Obeid, D. A., F. G. Hill, D. Harnden, J. R. Mann and B. S. Wood (1980). "Fanconi anemia. Oxymetholone hepatic tumors, and chromosome aberrations associated with leukemic transition." Cancer 46(6): 1401-4. Offman, J., K. Gascoigne, F. Bristow, P. Macpherson, M. Bignami, I. Casorelli, G. Leone, L. Pagano, S. Sica, O. Halil, D. Cummins, N. R. Banner and P. Karran (2005). "Repeated sequences in CASPASE-5 and FANCD2 but not NF1 are targets for mutation in microsatellite-unstable acute leukemia/myelodysplastic syndrome." Mol Cancer Res 3(5): 251-60.

59

Osman, F. and M. C. Whitby (2007). "Exploring the roles of Mus81-Eme1/Mms4 at perturbed replication forks." DNA Repair (Amst) 6(7): 1004-17. Pace, P., M. Johnson, W. M. Tan, G. Mosedale, C. Sng, M. Hoatlin, J. de Winter, H. Joenje, F. Gergely and K. J. Patel (2002). "FANCE: the link between Fanconi anaemia complex assembly and activity." Embo J 21(13): 3414-23. Pagano, G., P. Degan, M. d'Ischia, F. J. Kelly, B. Nobili, F. V. Pallardo, H. Youssoufian and A. Zatterale (2005). "Oxidative stress as a multiple effector in Fanconi anaemia clinical phenotype." Eur J Haematol 75(2): 93-100. Pagano, G., P. Degan, M. d'Ischia, F. J. Kelly, F. V. Pallardo, A. Zatterale, S. S. Anak, E. E. Akisik, G. Beneduce, R. Calzone, E. De Nicola, C. Dunster, A. Lloret, P. Manini, B. Nobili, A. Saviano, E. Vuttariello and M. Warnau (2004). "Genderand age-related distinctions for the in vivo prooxidant state in Fanconi anaemia patients." Carcinogenesis 25(10): 1899-909. Pagano, G. and L. G. Korkina (2000). "Prospects for nutritional interventions in the clinical management of Fanconi anemia." Cancer Causes Control 11(10): 881-9. Patel, K. J., V. P. Yu, H. Lee, A. Corcoran, F. C. Thistlethwaite, M. J. Evans, W. H. Colledge, L. S. Friedman, B. A. Ponder and A. R. Venkitaraman (1998). "Involvement of Brca2 in DNA repair." Mol Cell 1(3): 347-57. Pichierri, P. and F. Rosselli (2004). "The DNA crosslink-induced S-phase checkpoint depends on ATR-CHK1 and ATR-NBS1-FANCD2 pathways." Embo J 23(5): 1178-87. Puig, S., J. Ferrando, F. Cervantes, F. Ballesta, J. Palou, L. Trujillo, C. Herrero and J. M. Mascaro (1993). "Fanconi's anemia with cutaneous amyloidosis." Arch Dermatol 129(6): 788-9.

60

Rahden-Staron, I., M. Szumilo, E. Grosicka, M. Kraakman van der Zwet and M. Z. Zdzienicka (2003). "Defective Brca2 influences topoisomerase I activity in mammalian cells." Acta Biochim Pol 50(1): 139-44. Rahman, N., S. Seal, D. Thompson, P. Kelly, A. Renwick, A. Elliott, S. Reid, K. Spanova, R. Barfoot, T. Chagtai, H. Jayatilake, L. McGuffog, S. Hanks, D. G. Evans, D. Eccles, D. F. Easton and M. R. Stratton (2007). "PALB2, which encodes a BRCA2-interacting protein, is a breast cancer susceptibility gene." Nat Genet 39(2): 165-7. Reid, S., D. Schindler, H. Hanenberg, K. Barker, S. Hanks, R. Kalb, K. Neveling, P. Kelly, S. Seal, M. Freund, M. Wurm, S. D. Batish, F. P. Lach, S. Yetgin, H. Neitzel, H. Ariffin, M. Tischkowitz, C. G. Mathew, A. D. Auerbach and N. Rahman (2007). "Biallelic mutations in PALB2 cause Fanconi anemia subtype FA-N and predispose to childhood cancer." Nat Genet 39(2): 162-4. Rio, P., J. C. Segovia, H. Hanenberg, J. A. Casado, J. Martinez, K. Gottsche, N. C. Cheng, H. J. Van de Vrugt, F. Arwert, H. Joenje and J. A. Bueren (2002). "In vitro phenotypic correction of hematopoietic progenitors from Fanconi anemia group A knockout mice." Blood 100(6): 2032-9. Rischewski, J. R., H. Clausen, V. Leber, C. Niemeyer, J. Ritter, D. Schindler and R. Schneppenheim (2000). "A heterozygous frameshift mutation in the Fanconi anemia C gene in familial T-ALL and secondary malignancy." Klin Padiatr 212(4): 174-6. Rosenberg, P. S., M. H. Greene and B. P. Alter (2003). "Cancer incidence in persons with Fanconi anemia." Blood 101(3): 822-6. Ross, J. A., L. G. Spector and S. M. Davies (2005). "Etiology of childhood cancer: recent reports." Pediatr Blood Cancer 45(3): 239-41.

61

Rutter, J. L., A. M. Smith, M. R. Davila, A. J. Sigurdson, R. M. Giusti, M. A. Pineda, M. M. Doody, M. A. Tucker, M. H. Greene, J. Zhang and J. P. Struewing (2003). "Mutational analysis of the BRCA1-interacting genes ZNF350/ZBRK1 and BRIP1/BACH1 among BRCA1 and BRCA2-negative probands from breastovarian cancer families and among early-onset breast cancer cases and reference individuals." Hum Mutat 22(2): 121-8. Ruud, E. and F. Wesenberg (2001). "Microcephalus, medulloblastoma and excessive toxicity from chemotherapy: an unusual presentation of Fanconi anaemia." Acta Paediatr 90(5): 580-3. Sagaseta de Ilurdoz, M., J. Molina, I. Lezaun, A. Valiente and G. Duran (2003). "[Updating Fanconi's anaemia]." An Sist Sanit Navar 26(1): 63-78. Sarna, G., P. Tomasulo, M. J. Lotz, J. F. Bubinak and N. R. Shulman (1975). "Multiple neoplasms in two siblings with a variant form of Fanconi's anemia." Cancer 36(3): 1029-33. Sasaki, M. S. (1975). "Is Fanconi's anaemia defective in a process essential to the repair of DNA cross links?" Nature 257(5526): 501-3. Schofield, I. D. and A. T. Worth (1980). "Malignant mucosal change in Fanconi's anemia." J Oral Surg 38(8): 619-22. Schroeder, T. M. (1966). "[Cytogenetic and cytologic findings in enzymopenic panmyelopathies and pancytopenias. Familial myelopathy of Fanconi, glutathione-reductase deficiency anemia and megaloblastic B12 deficiency anemia]." Humangenetik 2(3): 287-316. Schuler, D., A. Kiss and F. Fabian (1969). "[Chromosome studies in Fanconi's anemia]." Orv Hetil 110(13): 713-20 passim.

62

Seal, S., R. Barfoot, H. Jayatilake, P. Smith, A. Renwick, L. Bascombe, L. McGuffog, D. G. Evans, D. Eccles, D. F. Easton, M. R. Stratton and N. Rahman (2003). "Evaluation of Fanconi Anemia genes in familial breast cancer predisposition." Cancer Res 63(24): 8596-9. Seal, S., D. Thompson, A. Renwick, A. Elliott, P. Kelly, R. Barfoot, T. Chagtai, H. Jayatilake, M. Ahmed, K. Spanova, B. North, L. McGuffog, D. G. Evans, D. Eccles, D. F. Easton, M. R. Stratton and N. Rahman (2006). "Truncating mutations in the Fanconi anemia J gene BRIP1 are low-penetrance breast cancer susceptibility alleles." Nat Genet 38(11): 1239-41. Seyschab, H., R. Friedl, Y. Sun, D. Schindler, H. Hoehn, S. Hentze and T. SchroederKurth (1995). "Comparative evaluation of diepoxybutane sensitivity and cell cycle blockage in the diagnosis of Fanconi anemia." Blood 85(8): 2233-7. Shapiro, P., R. M. Ikeda, B. H. Ruebner, M. H. Connors, C. C. Halsted and C. F. Abildgaard (1977). "Multiple hepatic tumors and peliosis hepatis in Fanconi's anemia treated with androgens." Am J Dis Child 131(10): 1104-6. Shattuck-Eidens, D., A. Oliphant, M. McClure, C. McBride, J. Gupte, T. Rubano, D. Pruss, S. V. Tavtigian, D. H. Teng, N. Adey, M. Staebell, K. Gumpper, R. Lundstrom, M. Hulick, M. Kelly, J. Holmen, B. Lingenfelter, S. Manley, F. Fujimura, M. Luce, B. Ward, L. Cannon-Albright, L. Steele, K. Offit, A. Thomas and et al. (1997). "BRCA1 sequence analysis in women at high risk for susceptibility mutations. Risk factor analysis and implications for genetic testing." Jama 278(15): 1242-50. Shivji, M. K. and A. R. Venkitaraman (2004). "DNA recombination, chromosomal stability and carcinogenesis: insights into the role of BRCA2." DNA Repair (Amst) 3(8-9): 835-43.

63

Sims, A. E., E. Spiteri, R. J. Sims, 3rd, A. G. Arita, F. P. Lach, T. Landers, M. Wurm, M. Freund, K. Neveling, H. Hanenberg, A. D. Auerbach and T. T. Huang (2007). "FANCI is a second monoubiquitinated member of the Fanconi anemia pathway." Nat Struct Mol Biol 14(6): 564-7. Sjarif, D. R., T. Revesz, T. J. de Koning, M. Duran, F. A. Beemer and B. T. Poll-The (2001). "Isolated glycerol kinase deficiency and Fanconi anemia." Am J Med Genet 99(2): 159-60. Smogorzewska, A., S. Matsuoka, P. Vinciguerra, E. R. McDonald, 3rd, K. E. Hurov, J. Luo, B. A. Ballif, S. P. Gygi, K. Hofmann, A. D. D'Andrea and S. J. Elledge (2007). "Identification of the FANCI protein, a monoubiquitinated FANCD2 paralog required for DNA repair." Cell 129(2): 289-301. Snow, D. G., J. B. Campbell and L. A. Smallman (1991). "Fanconi's anaemia and postcricoid carcinoma." J Laryngol Otol 105(2): 125-7. Soulier, J., T. Leblanc, J. Larghero, H. Dastot, A. Shimamura, P. Guardiola, H. Esperou, C. Ferry, C. Jubert, J. P. Feugeas, A. Henri, A. Toubert, G. Socie, A. Baruchel, F. Sigaux, A. D. D'Andrea and E. Gluckman (2005). "Detection of somatic mosaicism and classification of Fanconi anemia patients by analysis of the FA/BRCA pathway." Blood 105(3): 1329-36. Strathdee, C. A., H. Gavish, W. R. Shannon and M. Buchwald (1992). "Cloning of cDNAs for Fanconi's anaemia by functional complementation." Nature 358(6385): 434. Sugita, K., T. Taki, Y. Hayashi, H. Shimaoka, H. Kumazaki, H. Inoue, Y. Konno, M. Taniwaki, H. Kurosawa and M. Eguchi (2000). "MLL-CBP fusion transcript in a therapy-related acute myeloid leukemia with the t(11;16)(q23;p13) which

64

developed in an acute lymphoblastic leukemia patient with Fanconi anemia." Genes Chromosomes Cancer 27(3): 264-9. Swift, M., D. Zimmerman and E. R. McDonough (1971). "Squamous cell carcinomas in Fanconi's anemia." Jama 216(2): 325-6. Taniguchi, T. and A. D. Dandrea (2002). "Molecular pathogenesis of fanconi anemia." Int J Hematol 75(2): 123-8. Taniguchi, T., I. Garcia-Higuera, P. R. Andreassen, R. C. Gregory, M. Grompe and A. D. D'Andrea (2002). "S-phase-specific interaction of the Fanconi anemia protein, FANCD2, with BRCA1 and RAD51." Blood 100(7): 2414-20. Taniguchi, T., M. Tischkowitz, N. Ameziane, S. V. Hodgson, C. G. Mathew, H. Joenje, S. C. Mok and A. D. D'Andrea (2003). "Disruption of the Fanconi anemiaBRCA pathway in cisplatin-sensitive ovarian tumors." Nat Med 9(5): 568-74. Tezcan, I., M. Tuncer, D. Uckan, M. Cetin, M. Alikasifoglu, F. Ersoy and C. Altay (1998). "Allogeneic bone marrow transplantation in Fanconi anemia from Turkey: a report of four cases." Pediatr Transplant 2(3): 236-9. Thompson, E., R. L. Dragovic, S. A. Stephenson, D. M. Eccles, I. G. Campbell and A. Dobrovic (2005). "A novel duplication polymorphism in the FANCA promoter and its association with breast and ovarian cancer." BMC Cancer 5(1): 43. Tibbetts, R. S., D. Cortez, K. M. Brumbaugh, R. Scully, D. Livingston, S. J. Elledge and R. T. Abraham (2000). "Functional interactions between BRCA1 and the checkpoint kinase ATR during genotoxic stress." Genes Dev 14(23): 2989-3002. Timmers, C., T. Taniguchi, J. Hejna, C. Reifsteck, L. Lucas, D. Bruun, M. Thayer, B. Cox, S. Olson, A. D. D'Andrea, R. Moses and M. Grompe (2001). "Positional cloning of a novel Fanconi anemia gene, FANCD2." Mol Cell 7(2): 241-8.

65

Tischkowitz, M., N. Ameziane, Q. Waisfisz, J. P. De Winter, R. Harris, T. Taniguchi, A. D'Andrea, S. V. Hodgson, C. G. Mathew and H. Joenje (2003). "Bi-allelic silencing of the Fanconi anaemia gene FANCF in acute myeloid leukaemia." Br J Haematol 123(3): 469-71. Tischkowitz, M. and I. Dokal (2004). "Fanconi anaemia and leukaemia - clinical and molecular aspects." Br J Haematol 126(2): 176-91. Tischkowitz, M. D. and S. V. Hodgson (2003). "Fanconi anaemia." J Med Genet 40(1): 1-10. Tischkowitz, M. D., N. V. Morgan, D. Grimwade, C. Eddy, S. Ball, I. Vorechovsky, S. Langabeer, R. Stoger, S. V. Hodgson and C. G. Mathew (2004). "Deletion and reduced expression of the Fanconi anemia FANCA gene in sporadic acute myeloid leukemia." Leukemia 18(3): 420-5. Tonnies, H., S. Huber, J. S. Kuhl, A. Gerlach, W. Ebell and H. Neitzel (2003). "Clonal chromosomal aberrations in bone marrow cells of Fanconi anemia patients: gains of the chromosomal segment 3q26q29 as an adverse risk factor." Blood 101(10): 3872-4. Touraine, R. L., Y. Bertrand, P. Foray, J. Gilly and N. Philippe (1993). "Hepatic tumours during androgen therapy in Fanconi anaemia." Eur J Pediatr 152(8): 691-3. Unal, S. and F. Gumruk (2006). "Fanconi anemia patient with bilaterally hypoplastic scapula and unilateral winging associated with scoliosis and rib abnormality." J Pediatr Hematol Oncol 28(9): 616-7. Vaitiekaitis, A. S. and W. H. Grau (1980). "Squamous cell carcinoma of the mandible in Franconi anemia: report of case." J Oral Surg 38(5): 372-3.

66

van der Groep, P., M. Hoelzel, H. Buerger, H. Joenje, J. P. de Winter and P. J. van Diest (2007). "Loss of expression of FANCD2 protein in sporadic and hereditary breast cancer." Breast Cancer Res Treat. van der Heijden, M. S., J. R. Brody, E. Gallmeier, S. C. Cunningham, D. A. Dezentje, D. Shen, R. H. Hruban and S. E. Kern (2004). "Functional defects in the fanconi anemia pathway in pancreatic cancer cells." Am J Pathol 165(2): 651-7. van Niekerk, C. H., C. Jordaan and P. N. Badenhorst (1987). "Pancytopenia secondary to primary malignant lymphoma of bone marrow as the first hematologic manifestation of the Estren-Dameshek variant of Fanconi's anemia." Am J Pediatr Hematol Oncol 9(4): 344-9. Velazquez, I. and B. P. Alter (2004). "Androgens and liver tumors: Fanconi's anemia and non-Fanconi's conditions." Am J Hematol 77(3): 257-67. Velez-Ruelas, M. A., G. Martinez-Jaramillo, R. M. Arana-Trejo and H. Mayani (2006). "Hematopoietic changes during progression from Fanconi anemia into acute myeloid leukemia: case report and brief review of the literature." Hematology 11(5): 331-4. Verbeek, W., D. Haase, C. Schoch, W. Hiddemann and B. Wormann (1997). "Induction of a hematological and cytogenetic remission in a patient with a myelodysplastic syndrome secondary to Fanconi's anemia employing the S-HAM regimen." Ann Hematol 74(6): 275-7. Wada, E., M. Murata and S. Watanabe (1989). "Acute lymphoblastic leukemia following treatment with human growth hormone in a boy with possible preanemic Fanconi's anemia." Jpn J Clin Oncol 19(1): 36-9. Wajnrajch, M. P., J. M. Gertner, Z. Huma, J. Popovic, K. Lin, P. C. Verlander, S. D. Batish, P. F. Giampietro, J. G. Davis, M. I. New and A. D. Auerbach (2001).

67

"Evaluation of growth and hormonal status in patients referred to the International Fanconi Anemia Registry." Pediatrics 107(4): 744-54. Wang, X., P. R. Andreassen and A. D. D'Andrea (2004). "Functional interaction of monoubiquitinated FANCD2 and BRCA2/FANCD1 in chromatin." Mol Cell Biol 24(13): 5850-62. Wang, X., R. D. Kennedy, K. Ray, P. Stuckert, T. Ellenberger and A. D. D'Andrea (2007). "Chk1-mediated phosphorylation of FANCE is required for the Fanconi anemia/BRCA pathway." Mol Cell Biol 27(8): 3098-108. Ward, I. M. and J. Chen (2001). "Histone H2AX is phosphorylated in an ATRdependent manner in response to replicational stress." J Biol Chem 276(51): 47759-62. Whitney, M. A., G. Royle, M. J. Low, M. A. Kelly, M. K. Axthelm, C. Reifsteck, S. Olson, R. E. Braun, M. C. Heinrich, R. K. Rathbun, G. C. Bagby and M. Grompe (1996). "Germ cell defects and hematopoietic hypersensitivity to gamma-interferon in mice with a targeted disruption of the Fanconi anemia C gene." Blood 88(1): 49-58. Whitney, M. A., H. Saito, P. M. Jakobs, R. A. Gibson, R. E. Moses and M. Grompe (1993). "A common mutation in the FACC gene causes Fanconi anaemia in Ashkenazi Jews." Nat Genet 4(2): 202-5. Xia, B., Q. Sheng, K. Nakanishi, A. Ohashi, J. Wu, N. Christ, X. Liu, M. Jasin, F. J. Couch and D. M. Livingston (2006). "Control of BRCA2 cellular and clinical functions by a nuclear partner, PALB2." Mol Cell 22(6): 719-29. Xie, Y., J. P. de Winter, Q. Waisfisz, A. W. Nieuwint, R. J. Scheper, F. Arwert, M. E. Hoatlin, G. J. Ossenkoppele, G. J. Schuurhuis and H. Joenje (2000). "Aberrant

68

Fanconi anaemia protein profiles in acute myeloid leukaemia cells." Br J Haematol 111(4): 1057-64. Yetgin, S., M. Tuncer, E. Guler, F. Duru and M. Ali Kasifolu (1994). "Acute lymphoblastic leukemia in Fanconi's anemia." Am J Hematol 45(1): 94. Youssoufian, H. (1994). "Localization of Fanconi anemia C protein to the cytoplasm of mammalian cells." Proc Natl Acad Sci U S A 91(17): 7975-9. Youssoufian, H. (1996). "Natural gene therapy and the Darwinian legacy." Nat Genet 13(3): 255-6. Yu, X., C. C. Chini, M. He, G. Mer and J. Chen (2003). "The BRCT domain is a phospho-protein binding domain." Science 302(5645): 639-42. Zatterale, A., R. Calzone, S. Renda, L. Catalano, C. Selleri, R. Notaro and B. Rotoli (1995). "Identification and treatment of late onset Fanconi's anemia." Haematologica 80(6): 535-8. Zhu, W. and A. Dutta (2006). "Activation of fanconi anemia pathway in cells with rereplicated DNA." Cell Cycle 5(20): 2306-9. Zou, L. and S. J. Elledge (2003). "Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes." Science 300(5625): 1542-8.

69

Table 1. FA genes description

Comple mentatio n group A B C D1

Gene

Chromoso me maping 16q24.3 Xp22.31 9q22.3 13q12.3

D2 E F G I

FANCA FANCB FANCC FANCD1/BRCA 2 FANCD2 FANCE FANCF FANCG FANCI

J L M N

FANCJ/BRIP1 FANCL FANCM FANCN/PALB2

17q23.2 2p16 14q21.2 16p12.1

Gene exons

Reference

42-43* 8 14

(Lo Ten Foe et al. 1996) (Meetei et al. 2004) (Strathdee et al. 1992) (Howlett et al. 2002)

26

3p25.3 6p21.3 11p15 9p13 15q26.1

42-43* 10 1 14 36 19 14 23 13

70

(Timmers et al. 2001) (de Winter et al. 2000) (de Winter et al. 2000) (de Winter et al. 1998) (Dorsman et al. 2007; Smogorzewska et al. 2007) (Levitus et al. 2005) (Meetei et al. 2003) (Meetei et al. 2005) (Reid et al. 2007)

Table 2. FA proteins description

Gene

Protei n (kDa)

Protein length (aa) 14551668*

FANCA

163

FANCB

95

859

FANCC 63

558

FANCD 1 / 380 BRCA2

3418

FANCD 155,16 2 2

14711451*

FANCE

60

536

FANCF

42

374

FANCG 68

622

FANCI

150

FANCJ 130 / BRIP1

Known protein domains No known domains No known domains No known domains

Conservati on in evolution Nuclear and From lower cytoplasmic vertebrates Nuclear and From lower cytoplasmic vertebrates Nuclear and From lower cytoplasmic vertebrates Nuclear, BRC repeats cytoplasmic, From lower OB1, OB2 vertebrates secretory and OB3 granule? Subcellular localization

No known Nuclear domains No known domains No known domains Tetratricopept ide repeat motifs (TPRs)

Nuclear Nuclear

References

(Lo Ten Foe et al. 1996) (Meetei et al. 2004) (Youssoufia n 1994) (Jensen et al. 1996; Baer and Lee 1998) (Timmers et From al. 2001; invertebrat Castillo et al. es 2003) From lower (Pace et al. vertebrates 2002) From lower (de Winter et vertebrates al. 2000)

Nuclear and From lower (Blom et al. cytoplasmic vertebrates 2004)

1268

No known Nuclear domains

(Smogorzew From lower ska et al. vertebrates 2007)

1249

DEAH helicase domain

From lower (Cantor et al. vertebrates 2001)

FANCL

52

375

FANC M

250

2048

FANCN 131 /PALB2

1186

Nuclear

Nuclear, Ring Znnucleus finger domain envelope (Ub-ligase and motif) cytoplasmic DEAH-box helicase domain, ERCC4 Nucelar nuclease domain (not functional) No known Nuclear domains

71

From invertebrat es

(Meetei et al. 2004)

From archaea

(Meetei et al. 2005)

From lower (Xia et al. vertebrates 2006)

Figures Figure 1

72

Figure 2

73

Figure 3

D1 5%

F E 1% D2 1% 5%

G 9%

I 1%

L M J 1% 1% 1%

N 1%

A 59%

C 14% B 1%

74

Figure 4

A

B

- /D2 NC FA

A -/ NC FA

te d rr e c Co

No

rm

al

FANCA-/-

FANCD2-L FANCD2-S

75

Normal

Figure 5

A

C

G P24 C F A M E P100 L B

C F E

D2 I

Nucleus

USP

P100 L B

G P24 M P100 L B A

P

G

Ub

D2 I

Ub

P BRCA

ATR

P

P

BRCA

J

G

RAD51 H2AX D2 D1 I G N

Ub

Ub

Chromatin

Cytoplasm

76

Figure 6

77

Figure 7

78

Figure 8

79