THE EMERGING GENETIC AND MOLECULAR BASIS OF FANCONI ANAEMIA

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1 THE EMERGING GENETIC AND MOLECULAR BASIS OF FANCONI ANAEMIA Hans Joenje* and Ketan J. Patel The past few years have witnessed a considerable expansion in our understanding of the pathways that maintain chromosome stability in dividing cells through the identification of genes that are mutated in certain human chromosome instability disorders. Cells that are derived from patients with Fanconi anaemia (FA) show spontaneous chromosomal instability and mutagen hypersensitivity, but FA poses a unique challenge as the nature of the DNAdamage-response pathway thought to be affected by the disease has long been a mystery. However, the recent cloning of most of the FA-associated genes, and the characterization of their protein products, has provided tantalizing clues as to the molecular basis of this disease. APLASTIC ANAEMIA Bone marrow failure. This results in a peripheral deficiency of all bone-marrowderived haematopoietic lineages, such as red blood cells, platelets and leukocytes. There are many causes of this potentially fatal clinical syndrome. *Department of Clinical Genetics and Human Genetics, and Oncology Research Institute, Free University Medical Centre, Van der Boechorststraat 7, NL-1081 BT, Amsterdam, The Netherlands. MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. Correspondence to H.J. H.Joenje.HumGen@med.vu.nl Fanconi anaemia (FA) is an autosomal recessive disease characterized by diverse clinical features that often include developmental anomalies affecting the skeleton (absent or abnormal thumbs and radii), kidneys, heart or any other major organ system (BOX 1). The disease was first reported in 1927 by the Swiss paediatrician Guido Fanconi, who described a family with three affected children who had a peculiar combination of specific somatic abnormalities with progressive APLASTIC ANAEMIA 1. Since then, many cases have accumulated in the literature and hundreds of cases have been included and analysed in international registries 2. The disease has a general, worldwide prevalence of 1 5 per million and is found in all races and ethnic groups, with an estimated heterozygous mutation carrier frequency of between 0.3 and 1% 2,3. The life expectancy of FA patients is reduced to an average of 20 years (range 0 50 years), primarily because these individuals develop life-threatening bone marrow failure and are susceptible to developing malignancies, especially ACUTE MYELOID LEUKAEMIA (AML) 4 and, to a lesser extent, solid tumours, in particular SQUAMOUS CELL CARCINOMAS 5. The considerable clinical variability of the disease is shown by the occurrence of severe congenital abnormalities and death from anaemia or AML in the first decade of life at one extreme of the clinical spectrum, and at the other end of the spectrum by the presentation of mild anaemia (without the other symptoms) and death from oral cancer during the fifth decade of life. However, the classical clinical presentation is progressive bone marrow failure, which first manifests as low platelet counts and which eventually leads to transfusion-dependent anaemia in the first two decades of life. Treatment is essentially supportive, the only chance of a cure for the haematopoietic aspects of the disease being bone marrow transplantation. However, those who survive these haematological deficiencies might well develop solid cancers later in life. Since the recognition of FA as a distinct clinical entity, several milestones stand out in its subsequent characterization. The first was the discovery that cultured lymphocytes from FA patients show spontaneous chromosome instability 6 (BOX 2), a cellular feature that is now thought to be responsible for the cancer-susceptibility aspect of the disease (how the other pathological features of the disorder relate to this phenomenon is still a mystery). The second milestone was the finding that FA cells are hyperresponsive to the chromosome-breaking and antiproliferative effects of CROSSLINKING AGENTS, such as mitomycin C (MMC) and diepoxybutane 7 9. This feature seems to be unique to this class of agents, 446 JUNE 2001 VOLUME 2

2 Box 1 Fanconi anaemia: clinical and cellular phenotype Clinical manifestations Pre- and postnatal growth retardation Malformations of the kidneys, heart and skeleton (absent or abnormal thumbs and radii) A typical elfin facial appearance, caused by a small head, eyes and mouth Hearing loss Hypogonadism and reduced fertility Cutaneous abnormalities (hyperpigmentation, hypopigmentation and café-au-lait spots) Elevated serum α-fetoprotein 113 Life-threatening symptoms Bone marrow failure (aplastic anaemia: a lack of sufficient formation of all blood cell types) Cancer predisposition, primarily to acute myeloid leukaemia and squamous cell carcinoma Cellular features Spontaneous arrest and delay during the G2 (4n post-replication) stage of the cell cycle Spontaneous chromosomal instability (chromatid breaks and interchanges) Hypersensitivity to the chromosome-breaking and antiproliferative effects of DNA crosslinking agents, such as mitomycin C, diepoxybutane, cisplatin, cyclophosphamide and nitrogen mustard ACUTE MYELOID LEUKAEMIA (AML) Also known as acute non-lymphocytic leukaemia (ANLL), this is a heterogeneous group of leukaemias in which the lymphoblast cells that give rise to granulocytes do not mature and become too numerous. These immature blast cells are then found in the blood and the bone marrow. SQUAMOUS CELL CARCINOMAS Cancers that arise from squamous epithelial cells or keratinocytes, which cover the skin and oral cavity. CROSSLINKING AGENTS These agents form a specific type of lesion in doublestranded DNA chemical crosslinks between the two DNA strands (interstrand crosslinks) and between adjacent bases on the same strand (intrastrand crosslinks). These modifications obstruct DNA replication and, because they can modify both strands, specific repair pathways are required to maintain genomic integrity in cells exposed to them. CARETAKER GENES Genes that encode the proteins that function to protect the genome against alterations. although a hypersensitivity to X-rays has also been reported in some FA patients 10,11. Because of the extreme variability of the clinical features of this disease, as well as variations in the level of spontaneous chromosome aberrations, a cytogenetic test that quantifies crosslinker-induced chromosomal breakage has become the gold standard for diagnosing FA 2. The third milestone came with the finding that a high degree of genetic heterogeneity underlies the disease, as shown by cellfusion and complementation studies Finally, a crucial step towards the eventual elucidation of the primary defect has been the development of a complementation cloning method that has allowed most of the FAassociated genes to be cloned 15. The aim of this article is to provide an overview of the genetics and molecular biology of FA. Now that most of the associated genes have been identified, the various parts of the FA puzzle can be put in place. A long-suspected deficiency in the response of FA cells to DNA damage has become a molecular reality with the recent discovery of a FA gene product (FANCD2 Fanconi anaemia complementation group D2) that interacts with BRCA1 (breast cancer 1, early onset) in microscopically visible subnuclear foci. The field now faces the exciting task of unravelling the mechanism by which the many associated FA proteins support a novel pathway that ensures the maintenance of genomic integrity. Fanconi anaemia: a caretaker-gene disease Hereditary diseases, such as FA, xeroderma pigmentosum (XP) and hereditary non-polyposis colorectal cancer (HNPCC), that feature genomic instability in combination with a strong predisposition to cancer, are called the CARETAKER-GENE diseases (TABLE 1).The common feature of these diseases is an impaired capacity to maintain genomic integrity, which results in the accelerated accumulation of key genetic changes that promote cellular transformation and neoplasia. Cancer predisposition in these diseases is therefore an indirect result of the primary genetic defect. The various caretaker-gene diseases show fascinating phenotypes that in some instances can be readily understood from the molecular function of the affected proteins. For example, in XP the disease-associated genes encode proteins with known complementary functions in NUCLEOTIDE EXCISION REPAIR (NER); defects in this process predispose XP individuals to skin cancer because the mutagenic damage caused by sunlight goes unrepaired. Much less evident is why defects in the mismatch repair genes predispose in particular to colorectal cancer. An example of a monogenic caretaker-gene disease is Bloom syndrome, in which a mutation in a recq DNA helicase which catalyses a DNA unwinding reaction leaves sufferers with a marked predisposition to all cancers. Conversely, in Werner syndrome, a related DNA helicase is defective, causing a disease with a clinical phenotype and spectrum of excessive cancers that is quite different from those observed in Bloom syndrome patients. In FA, patients are predominantly predisposed to AML and squamous cell carcinomas the latter occurring most often in the oral cavity. Like XP, FA is genetically heterogeneous in each case, mutations in at least seven or eight different genes lead to a common phenotype but, unlike XP, the molecular process that is defective in FA is only now beginning to be understood. Genetic heterogeneity of Fanconi anaemia The striking hypersensitivity of cultured FA cells to crosslinking agents, such as MMC, has been exploited by using an approach called complementation analysis to answer whether one or more genes cause FA (FIG. 1). At present, FA patients are assigned to one of seven complementation groups (FA-A to FA-G;see BOX 3 on FA nomenclature) 14, and most (60 80%) fall into group A (FIG. 2).The assumption that each group corresponds to a distinct FA disease gene has been supported by the identification of genes with pathogenic mutations in most of these groups. A notable exception to the one group = one gene rule is group D, which seems to be heterogeneous, as a gene has been cloned recently, FANCD2, that has pathogenic mutations in only a subset of FA-D patients; the other FA-D patients (including the first FA-D patient identified) are therefore expected to be mutated in another gene, provisionally termed FANCD1. This finding indicates that eight FA complementation groups might exist. At present, this heterogeneity is not well understood, although it might be due to erroneous conclusions from the original cell-fusion studies 13. The example set by XP indicates that the FA proteins might function in a common pathway that contributes to genomic stability, either as components of a multiprotein complex or of a signaltransduction cascade. NATURE REVIEWS GENETICS VOLUME 2 JUNE

3 Table 1 Caretaker-gene diseases Disease Number of Genes Molecular Key suspected identified process references genes Single gene Ataxia telangiectasia 1 ATM DNA-damage 117 response Bloom syndrome 1 BLM DNA unwinding 118 Werner syndrome 1 WRN DNA unwinding 119 Multiple genes Xeroderma 7 XPA XPG Nucleotide excision/ 120 pigmentosum transcription-coupled repair Hereditary 5 MSH2, MSH6, Mismatch repair 121 non-polyposis MLH1, PMS1, colorectal cancer PMS2 a Hereditary breast/ 3 BRCA1, BRCA2 DNA-damage 122 ovary cancer b response/repair; recombination; transcription Fanconi anaemia 8 FANCA, -C,? 2 -D2, -E, -F, -G a MSH, MLH, human homologues of Escherichia coli mismatch-repair genes muts and mutl, respectively; PMS, human homologue of a mismatch-repair gene originally described in yeast as postmeiotic segregation increased. b Hereditary breast/ovary cancer is included because of the growing evidence to indicate that BRCA1, as well as BRCA2 (REFS ), are critically involved in the DNA-damage response, and in view of the recently discovered link between BRCA1 and the Fanconi-anaemia pathway. NUCLEOTIDE EXCISION REPAIR A DNA-repair pathway that removes DNA damage (such as ultraviolet-light-induced thymidine dimers) and bulky DNA adducts by excising the region of DNA that contains the damaged base(s). The single-stranded gap is filled in by using the intact strand as a template. MICROCELL An experimentally produced cell-like structure, which contains a micronucleus in which one or a few chromosomes are present. They can be used as a vector to transfer genetic material to a normal cell by cell fusion. Cloning Fanconi anaemia genes The extreme rarity of the disease, together with its genetic heterogeneity, have long been obstacles to the cloning of FA genes. Only after a substantial number of families had been assigned to group A by complementation analysis, was the gene defective in this FA subtype mapped to chromosome 16q24.3 by linkage analysis in 1995 (REF. 16). Complementation cloning, a technique that was originally developed in the laboratory of Manuel Buchwald to clone FANCC (REF. 15), has proved to be the most successful approach to identifying FA genes. This method relies on the capacity of a plasmid that expresses a normal copy of the defective FA gene to correct the MMC-sensitive phenotype of a FA lymphoblast cell line (FIG. 3).The success of this approach requires that: first, the cdna library used to initially transfect the FA cells contains the relevant cdna with its open reading frame intact; second, the FA protein is not toxic when overexpressed; third, the cell line can be transfected (often an obstacle, as human lymphoblasts are typically reluctant to take up exogenous DNA); and last, the transfected cell line does not spontaneously revert to MMC resistance at a significant rate. Despite these limitations, this method has enabled the cloning of five FA genes to date: FANCC, FANCA, FANCG, FANCF and FANCE (in their chronological order of identification) 15,17 20 ; FANCA was also independently identified by positional cloning 21. A third method, which led to the cloning of FANCD2, combined features of both the complementation- and positionalcloning approaches. This method is relatively laborious and time consuming, but it is also robust, as it does not suffer from the above-mentioned difficulties. To clone the FANCD2 gene, the chromosome that carries the disease gene was first identified by fusing a panel of MICROCELLS, which contain single human chromosomes, with an immortalized FA-D fibroblast cell line. These experiments showed that chromosome 3 was able to complement the cellular MMC-sensitive phenotype. By repeating the fusion experiments with partially deleted chromosomes (generated by ionizing radiation), the critical region was mapped to 3p22-26 (REF. 22) and eventually narrowed to a 200-kb interval 23,where FANCD2 was identified as the disease gene 24. FA genes: structure and expression The six FA-associated genes identified so far are scattered throughout the genome and do not seem to share any common features (TABLE 2). The number of exons varies from 1 (FANCF) to 44 (FANCD2) and roughly correlates with the size of the encoded proteins. Database searches for all the predicted proteins have revealed that significant conservation only occurs between vertebrate sequences. An exception is FANCD2, which shows highly significant alignment scores with sequences found in lower organisms, such as Drosophila melanogaster, Caenorhabditis elegans and Arabidopsis thaliana. The relatively high degree of evolutionary conservation of FANCD2 indicates that this protein might act in a conserved pathway, on which the action of the other FA genes has been superimposed later in evolution. The evolutionary appearance of non-d2-type FA genes seems to correlate with the introduction of an internal skeleton, which might be significant in view of the skeletal abnormalities found in FA patients. The lack of significant functional motifs in any of the FA proteins has so far precluded predictions about the nature of their molecular functions. Although the expression of some FA genes has been investigated at the mrna level 21,25 28, the distribution of FA proteins in different tissues remains unclear, owing to generally low expression levels and difficulties in generating suitable antibodies. A recent immunochemical study of mouse tissue indicated that Fanca is present at a low level in most tissues, and at a higher level in lymphoid tissues (spleen, thymus and lymph nodes), the testis and the ovary 29. The significance of this association is unclear, but evidence indicates that the presence of FA proteins might be related to cellular proliferation because in human immortalized lymphoblasts and leukaemia cells, FA proteins are readily detectable by immunoprecipitation 30,31, whereas they are undetectable in unstimulated peripheral blood leukocytes from non-leukaemic individuals (J. P. de Winter, unpublished results). Information is also emerging on the relationship between FA protein levels and the cell cycle. In human embryonic kidney cells, FANCC (when overexpressed from a transfected plasmid) shows remarkable fluctuation during the cell cycle, with a peak at the G2/M boundary, followed by rapid proteasome-dependent 448 JUNE 2001 VOLUME 2

4 Box 2 Chromosomal instability in Fanconi anaemia The chromosomal aberrations that occur spontaneously in dividing Fanconi anaemia (FA) cells are of the chromatid type. The aberrations originate during the DNA-replication phase of the cell cycle, primarily affect the newly replicated DNA and are microscopically visible at metaphase as broken chromatids. As shown in the figure, breakpoints (red) in two chromosomes can be misrepaired to generate a chromatid interchange figure, such as a quadriradial. Up to 50% of stimulated FA T cells have 1 3 aberrations per cell. This spontaneous chromosome damage is a probable cause of the spontaneous arrest and delay in the late S or G2 phase of the cell cycle, as is typically observed in FA cells. When challenged with a low dose of a DNA crosslinking agent, such as mitomycin C, FA cells respond by markedly increasing chromatid breaks and interchanges, whereas wild-type cells do not respond to this treatment. The spontaneous chromosomal instability observed in FA cells is unique and distinct from that observed in other conditions: although it resembles that of Bloom syndrome cells, in FA the chromatid interchanges seem to involve random breakpoints, whereas in Bloom syndrome interchanges preferably involve homologous sequences of homologous chromosomes. Chromosomal instability as seen in FA and Bloom syndrome should not be confused with other forms of genomic instability, often collectively described as genetic instability. For example, many tumour cells are genetically unstable in that they have lost the diploid chromosome complement and often have variable numbers of chromosomes and chromosomal rearrangements. Defective mismatch repair underlies the genetic instability observed in hereditary non-polyposis colorectal cancer as variable lengths of microsatellite sequences. Finally, chromosomal instability in lymphocytes from ataxia-telangiectasia patients takes the form of non-random translocations and inversions that preferentially involve the T- and B-cell receptor gene complexes. SERIAL TRANSPLANTATION When a donor tissue is used for another transplant after engraftment in a recipient. Centromere Chromatid break Quadriradial chromosome Misrepair degradation during the G1 phase 32. These data, which indicate that FA proteins might be tightly regulated during the cell cycle, need to be confirmed by studying endogenous FANCC levels and the levels of the other FA proteins. It would also be interesting to document FA protein dynamics in relation to DNA-damage responses. Another important aspect is the subcellular localization of the various FA proteins. As discussed below, most FA proteins seem to accumulate in the nuclear compartment, where they engage in the formation of a multiprotein complex. FANCC takes part in this complex formation, but an estimated 90% of this protein seems to reside in the cytoplasm 33, indicating that the FA pathway might have a functional component in the cytoplasm as well. Genotype phenotype correlations Screening for pathogenic mutations in FA genes has not revealed any mutation hot spots in the FA proteins that might indicate the presence of functional motifs (FIG. 4). Nearly 100 mutations have so far been found in FANCA. A high proportion of these (roughly one-third) are deletions 34,35, owing to the frequent occurrence of Alu repeats in this region, which can facilitate intrachromosomal recombinations 36. The second most important mutations are those that cause truncations 2,34,35,37 (FIG. 4). A couple of missense mutations and a microdeletion have been identified in FANCA (H1110P, R951Q and 1263delF, respectively) that allow protein to be expressed. However, these mutant proteins fail to accumulate in the nucleus 30,38, indicating that the mutated residues might have a crucial role in the nucleartranslocation process. Furthermore, in FANCC, a proline-for-leucine substitution at the carboxyl terminus (L554P) blocks nuclear translocation 39,40 and disrupts the complementing activity of the protein 41. A recent study of FA patients from across the various subtypes did not reveal a strong influence of complementation group on clinical phenotype, although statistically meaningful comparisons could only include the larger groups A, C and G (REF. 42). A relatively early occurrence of AML is seen in FA-G patients compared with those in groups A and C. However, this conclusion might be biased by the paucity of FANCG mutations in the sample as different mutations in a FA gene can have strikingly different phenotypic consequences, indicating that certain mutations might generate proteins with residual activity For example, FANCA-null patients tend to have more severe haematological disease and to develop AML more often than non-null patients 42.A frameshift mutation in FANCC (322delG in exon 1) is predicted to generate a severely carboxy-terminally truncated protein, but the use of a downstream methionine generates an amino-terminally truncated protein instead that is partially functional 45. Interestingly, patients carrying this mutation do not have major skeletal abnormalities, indicating that the carboxyl terminus of FANCC might have a role in supporting skeletal development. No biallelic null mutations have so far been found in the three FA-D2 patients known, indicating that such mutations might be lethal. As the information on protein structure and function from pathogenic mutations in FA patients is scarce, especially for the rare complementation groups, systematic targeted mutagenesis studies are required to obtain more information on the functional domains of the various FA proteins. Animal models of Fanconi anaemia Mice have been generated with targeted disruptions of Fancc or Fanca, and the resulting mutants show only part of the human phenotype, namely hypogonadism and reduced fertility In the Fancc -/- mutant, this phenotype correlates with a significantly reduced number of primordial germ cells 49. Haematopoiesis, which is typically compromised in FA patients, seems unaffected in these mice, although the repopulation capacity of Fancc mutant stem cells upon SERIAL TRANSPLANTATION is reduced 50. NATURE REVIEWS GENETICS VOLUME 2 JUNE

5 MITOTIC RECOMBINATION A crossover between two homologous double-stranded DNA molecules that leads to a physical exchange of DNA and genetic information. This recombination occurs frequently during meiosis, but is relatively rare during mitosis. As a consequence of mitotic recombination, cells can undergo a loss of heterozygosity or gene conversion. GENE CONVERSION A specific type of recombination, which results in non-reciprocal genetic exchange, in which the sequence of one DNA strand is used to alter the sequence of the other. Cultured embryonic fibroblasts from both knockout animals show chromosomal instability and hypersensitivity to DNA crosslinkers, indicating that the basic FA defect is expressed at the cellular level. It is somewhat surprising, therefore, that the knockout animals do not show a predisposition to cancer, in contrast to a mouse model of Bloom syndrome 51. The discrepancies between the human and mouse phenotypes are not unique to FA and might be explained by differences in genetic background and physiological parameters, in combination with environmental factors. However, haematopoietic failure can be induced in knockout mice by administering MMC at doses that do not affect wild-type animals 52. This provides an experimental route to using these knockout mice as preclinical models for bone marrow transplantation, and also in reconstitution experiments that use mutant stem cells in which the gene defect has been corrected 53,54. Interestingly, the mouse Fanca and Fancc cdnas are able to fully complement the defect in human cells from the corresponding complementation group, despite their moderate (~75%) amino-acid similarity to the human proteins 25,29. Human FANCC can also correct the cellular defect of Fancc knockout mice 53, which indicates that, despite the many differences at the amino-acid level, the three-dimensional structure of these proteins is likely to be conserved. Animal models allow genetic defects to be combined in double-knockout mutants, an approach that can reveal the existence of redundancy. Double-knockout Fancc/Fanca mice have recently been generated and seem to have a phenotype that resembles that of the single-knockout animals, which indicates a lack of functional redundancy between these two proteins (M. Grompe, personal communication). Reverse mosaicism and gene therapy A proportion of patients who score positively for FA in a chromosomal-breakage test have two populations of T cells, one that shows FA-like high rates of chromosome breakage and one that shows a wild-type response Immortalized B-lymphoblast cell lines established from such patients are almost always MMC-resistant and are consequently unsuitable for complementation analysis. This requires that a patient s subtype be determined by a different approach, such as by screening the FA genes for mutations or by complementing the phenotype in skin fibroblasts using retroviral vectors 58 (as fibroblasts typically do not show mosaicism in patients with lymphocyte mosaicism). In a few cases, the subtype has been identified by finding mutations in FANCA or FANCC,which allows the molecular basis of the reversion to be studied. In these patients, the phenotypic reversion in T cells seems to be based on genetic reversion at the disease locus. Different mechanisms were found to be responsible. First, in compound heterozygous FA-C patients with non-overlapping mutations, MITOTIC RECOMBINATION between the two alleles had generated a wild-type allele, which allowed the segregation and outgrowth of a genetically corrected clone 57. More recently, a case of lymphocyte mosaicism in a FA-A patient has been associated with the disappearance of a pathogenic 19-bp insertion a 100 % Growth b 50 FA-1 2n S 4n S S Concentration of MMC (nm) MMC sensitive No complementation = same group FA-2 2n S Cell fusion in a haematopoietic stem cell, possibly due to back mutation or GENE CONVERSION 59. Remarkably, mosaicism has been found even in patients with homozygous frameshift mutations genotypes that preclude mitotic recombination as a mechanism for reversion owing to the absence of wild-type sequence. In such cases, alterations in cis, presumably due to replication errors, were found to restore the reading frame of a functional, although not wild-type, protein 60. Although it is not known whether this type of event is facilitated by the chromosome-instability phenotype of FA cells, the resulting fortuitous gene correction, which presumably occurs in a haematopoietic stem cell, can cause a significant proportion of corrected cells to accumulate in the patient s blood 57,59. In some, but not all, mosaic patients this situation has been correlated with a less severe haematological condition 55,57, which has fuelled hopes for the feasibility of stem-cell gene therapy to cure the bone marrow failure of FA patients. R 4n R MMC resistant Complementation = different groups FA-3 Figure 1 Complementation groups in Fanconi anaemia. a Immortalized B lymphoblasts derived from Fanconi anaemia (FA) patients (S) are typically 10- to 100- fold more sensitive to the antiproliferative action of mitomycin C (MMC) and other crosslinking agents than non-fa or complemented cells (R). b A hybrid cell line, generated by fusing lymphoblasts from unrelated FA patients, might either continue to show the sensitive trait (S) or be corrected (complemented) (R). In this example, FA-1 and FA-2 are in the same complementation group, which is different from the group that FA-3 belongs to. 2n S 450 JUNE 2001 VOLUME 2

6 Table 2 The Fanconi anaemia genes Gene Pathogenic Location Exons Protein Evolutionary Knockout References mutations (amino conservation a phenotype acids) in mice b FANCA ~100 16q Fish a Reduced 2,17,21,48 fertility FANCB c NA FANCC 10 9q Fish a Reduced 2,15,46,47 fertility FANCD1 c,d NA FANCD2 d 5 3p Arabidopsis NA thaliana, Caenorhabditis elegans, Drosophila melanogaster 24 FANCE 3 6p Fish a NA 20 FANCF 6 11p Fish a NA 19 FANCG e 18 9p Fish a NA 18,116 a BLAST searches for homologous regions in the predicted Fanconi-anaemia proteins show no significant hits in non-vertebrate, yeast or bacterial sequences. This does not mean that homologous sequences do not exist in lower organisms, just that the sequence similarities do not reach statistical significance. Animals most distant from humans with significant hits are ray-finned fishes, including Tetraodon nigroviridis (FANCA, FANCE, FANCF), Takifugu rubripes (FANCC) and the zebrafish Danio rerio (FANCG). b NA, data not available. c Remains to be identified. d Out of four patients assigned to the D group by cell-fusion studies, only two carried pathogenic mutations in a gene identified by positional cloning, termed FANCD2. The remaining two patients were assumed to have mutations in a distinct gene, termed FANCD1. e FANCG, identified by complementation cloning, is identical to XRCC9 (X-ray repair complementing defective repair in Chinese hamster cells 4), a known gene isolated on the basis of its capacity to complement the mitomycin-c-sensitive Chinese hamster cell mutant UV40 (REF. 126). Box 3 Fanconi anaemia nomenclature In current gene therapy trials, retroviral vectors that express wild-type FANCC or FANCA are used to transduce the peripheral blood leukocytes of FA patients leukocytes that have been enriched for haematopoietic stem cells 54,61. Once in the patient s bloodstream, gene-corrected stem cells are presumed to initiate and support proficient haematopoiesis. An important obstacle for successful gene therapy is the poor efficiency with which the rare haematopoietic stem cells are transduced. Subsequent obstacles might also include the unstable expression of the transgene due to gene silencing and immunological attack of the transduced cells, which express a protein that the body Complementation group A Fanconi anaemia (FA) patient or cell line assigned to a particular complementation group by functional complementation tests or mutation analysis is described as being FA-A, FA-C, and so on, to indicate the FA subtype. Genes The first FA gene to be cloned (in 1992), by complementation cloning, was termed FACC,for FA complementation group C complementing. It was said to be complementing 15 because, at the time, it was not clear that the true disease gene had been identified, but the isolated cdna complemented the cellular phenotype of FA-C cells. As many more patients were found with pathogenic mutations in this gene, nomenclature was simplified to FAC, later followed by FAA when the group A gene was cloned. With the cloning of the group G gene in 1998, more letters were required for new gene symbols. So, the current nomenclature was adopted, according to which FA genes and loci are referred to as FANCA, FANCC, and so on, from a truncation of the name Fanconi and the letter of the relevant complementation group. FAassociated genes are collectively referred to as FA genes. might recognize as being foreign. Another potential complication is that residual mutant cells might remain predisposed to cytogenetic abnormalities, so interfering with normal haematopoietic function and potentially leading to leukaemia, even after successful gene therapy 59. Molecular interactions of FA proteins Five FA genes (FANCA, FANCC, FANCE, FANCF and FANCG) encode orphan proteins that are thought to form a nuclear complex. Direct protein interactions have been shown between FANCA and FANCG, and between FANCC and FANCE, by yeast two-hybrid assays and by co-immunoprecipitation Indeed, FANCA, -C, -F and -G all seem to purify together with each other when recovered from nuclear extracts. These interactions probably have a functional basis as the intact nuclear complex seems to be disrupted in cell lines from complementation groups A, C, E, F and G. Expression of the complementing FA protein in these cell lines results in a reconstitution of the nuclear complex and correction of the cellular phenotype 30,64,65. An additional level of complexity is introduced by the apparent subcellular localization of some of the FA proteins. FANCC, FANCA and FANCG are nuclear, as well as cytoplasmic molecules 30,39,64,67. Indeed, the nuclear transport of FANCA is complex: it has a putative nuclear localization signal at the amino-terminus; however, an intact carboxyl terminus is also required for its nuclear transport 38,68,69. Certain missense mutations, such as H1110P in FANCA, are unable to accumulate in the nucleus, indicating a region of the protein that might be required for transport. A genetic requirement for the NATURE REVIEWS GENETICS VOLUME 2 JUNE

7 B C D1 E D2 F Figure 2 Relative prevalences of Fanconi anaemia complementation groups. Taken from the first 100 Fanconi anaemia patients classified by the European Fanconi Anaemia Research Programme. Patients were mainly of Euro-Mediterranean ancestry. Worldwide prevalences might strongly vary with ethnic background: for example, owing to founder effects, most Fanconi anaemia (FA) patients in the Afrikaans-speaking population in South Africa belong to group A (REF. 114), whereas in the Ashkenazi-Jewish population, group C is most frequent 115. Numbers of patients in the various groups were: A, 71; B, 1; C, 7; D1, 1; D2, 1; E, 4; F, 2; G, 13. In Italy only FA-A patients have been found, in The Netherlands predominantly group C were present, and in Germany all known groups were represented, except D1. (A G, complementation groups.) transport of FANCA is shown by the observation that in FA-B cells FANCA protein is predominantly localized to the cytoplasm 30,70. This indicates that the defect in group B might be in the nuclear-transport step of FANCA. In addition to defective nuclear accumulation, the FA-B cells also seem to be defective in FANCA phosphorylation 70, but the significance of this correlation is not known. All these studies argue that the FA core complex is actively assembled in the cytoplasmic and nuclear compartments, and that the nuclear complex is then required to execute steps that are essential for genomic stability (FIG. 5) 30. What are the crucial downstream effector molecules in the pathway? G A Cell lines from complementation group D seem to have an intact nuclear core complex 24,30,71, which indicates that FANCD2 and the putative FANCD1 proteins act downstream of the other known FA proteins. Although the amino-acid sequence of FANCD2 does not reveal any clear structural motifs, changes in its dynamic nuclear localization are compelling. The FANCD2 protein exists in two forms: FANCD2 short and FANCD2 long the long version is formed by the addition of a single ubiquitin molecule on Lys 561, a residue that is conserved in FANCD2 homologues from lower organisms. Mutation of this residue to a conservative change results in loss of ubiquitylation and failure to complement sensitivity to crosslinkers in the FA-D2 cell line 71. (The addition of multiple ubiquitin molecules to a protein usually causes it to be targeted for degradation by the proteasome; addition of a single ubiquitin moiety (monoubiquitylation) might target a protein for altered subcellular trafficking; for a recent review, see REF. 72.) This modification step, which is therefore crucial for the activity of FANCD2, occurs in HeLa cells upon exposure to a range of DNA-damaging agents and during genome replication. However, the link to the other FA proteins comes from the crucial observation that FA cell lines from complementation groups A, C, E, F and G express only unmodified FANCD2; however, there is no experimental evidence that FANCD2 physically interacts with the other FA proteins. Indeed, modified FANCD2 is restored upon complementation of these cells lines with their respective cdnas. The clue as to why this modification might be so crucial is that, upon its monoubiquitylation, FANCD2 is targeted to discrete nuclear foci 71. Here, it co-localizes and co-purifies with BRCA1, a protein that is implicated in many DNA-damageresponse pathways 73 (FIG. 6). These characteristic a RSV 3 LTR EBV ori EBV-EBNA1 cdna cloning site SV40 pa prep4 Amp R Hyg R ColE1 ori Figure 3 Complementation cloning. a The shuttle vector, prep4, used for cloning Fanconi anaemia (FA) genes 15. ColE1 ori and EBV (Epstein Barr virus) ori are origins of replication, which allow the vector to replicate in Escherichia coli and mammalian cells, respectively; the ampicillin resistance (Amp R ) marker selects for plasmid-containing bacteria; and the hygromycin resistance (Hyg R ) marker selects for transfected mammalian cells. A library of reverse-transcribed poly(a) + RNA from human lymphoblasts is cloned into the cdna cloning site, and expression is driven by the RSV 3 LTR promoter and a simian virus 40 polyadenylation signal (SV40 pa). EBV nuclear antigen 1 (EBNA1) protein binds to viral DNA and allows the vector to be maintained in B cells as a circular DNA episome. b The cloning scheme. EBVimmortalized B lymphoblasts from a FA patient are transfected with an expression library cloned into the episomal shuttle vector. After selecting for library uptake with hygromycin, surviving cells are selected with MMC, killing the FA-like hypersensitive cells. Episomes extracted from the surviving cells have inserts that are candidates for the defective gene. Candidates are then screened for their capacity to complement in secondary transfections and for mutations in FA patients. b FA gene? Library transfection Hygromycin selection MMC selection Extract episomal DNA 452 JUNE 2001 VOLUME 2

8 FANCF 1 ORF 1125 ORF Mircrodeletion or microinsertion Nonsense mutation Missense mutation Splice site alteration Deletion Polymorphism FANCE delG IVS4 + 4A T ORF FANCC E105X ORF FANCG delF FANCA ORF ORF FANCD RAD51 The human homologue of bacterial RecA. RAD51 is required for homologous recombination, during which it promotes strand invasion. RAD51 forms nucleoprotein filaments around singlestranded DNA. RAD50 RAD50 forms a complex with MRE11 and NBS1 and is essential for double-stranded DNA-break repair by homologous recombination and non-homologous end-joining. RING-FINGER MOTIF A structure seen in many DNA-binding proteins; it is composed of a polypeptide loop held in a hairpin bend bound to a zinc atom. UBIQUITIN LIGASE An enzyme that couples the small (~7 kda) protein ubiquitin to lysine residues on a target protein, thus targeting it for degradation by the proteasome. It is less clear how monoubiquitylation targets proteins to cellular compartments and modifies their activity. Figure 4 Mutations in FANCA, FANCC, FANCD2, FANCE, FANCF and FANCG. The open reading frames with exon structures are shown (not to scale). A high proportion of the mutations in FANCA (Fanconi anaemia complementation group A) are deletions (indicated by the horizontal bars), mainly caused by the frequent occurrence of Alu repeats in this genomic region 36. Most mutations in FANCA are not found in any other Fanconi anaemia (FA) patient. Specific deletions in FANCA (deletions of exons and 12 31, shown in red) are relatively common in Afrikaans-speaking people in South Africa 114. Other relatively common mutations are boxed and include 1263delF in FANCA (a phenylalanine residue missing at position 1263), which occurs in 5% of mutated FANCA alleles 37. In FANCC, the commonest mutations are the splice-site mutation IVS4 + 4A T (80% of all Ashkenazi-Jewish FA patients are homozygous for this mutation) and the frameshift mutation 322delG (most FA patients of Dutch ancestry are homozygous for this mutation). E105X in FANCG, a mutation that changes a glutamic-acid residue at position 105 into a stop, accounts for 44% of mutant FANCG alleles in Germany 116. (Figures adapted from REFS 2,35,116, with permission). For more information on FA mutations, see the link to the Fanconi Anaemia Mutation Database. nuclear foci appear in cells after DNA damage and in those cells undergoing DNA replication. Proteins such as the central recombination molecule RAD51, and those in the RAD50 NBS1 MRE11 DNA-repair pathway 73,74 are also present in BRCA1-containing foci (NBS1, Nijmegen breakage syndrome 1; MRE11, meiotic recombination 11). At present, it is unclear which other proteins are present in FANCD2 BRCA1 foci, and further studies are required to address this central question. Evidence for the interaction between FANCD2 and BRCA1 comes from the observation that FANCD2 monoubiquitylation and its localization to foci is reduced in a BRCA1 mutant cell line. Restoration of BRCA1 expression restores nuclear foci formation and a marked increase in monoubiquitylated FANCD2. Intriguingly, BRCA1 has an aminoterminal RING-FINGER MOTIF, which is known to function as an E3 UBIQUITIN LIGASE 75,76 ; so, this domain might participate in FANCD2 modification. Fanconi anaemia and DNA-damage response The broken and rearranged chromosomes in FA cells show the importance of the FA pathway in a response to DNA damage. The oldest clue to a DNA-repair defect in FA comes from the marked sensitivity of FA cells from all complementation groups to agents that can generate crosslinks in DNA. Much is already known about the pathways that repair such lesions in lower organisms, such as Escherichia coli, in which homologous recombination is the main repair mechanism 77. Indeed, mammalian cell lines that are defective in this DNA-repair process are very sensitive to DNA crosslinkers 78,79. However, there is no clear defect in homologous recombination in FA cell lines for example, they are able to induce sister chromatid exchange at normal levels when provoked with DNA damage 80. (This does not exclude a subtle role for the FA pathway in recombination processes, such as in the resolution of DNA structural intermediates.) Some NATURE REVIEWS GENETICS VOLUME 2 JUNE

9 B A? Cytoplasm G A G Sequential assembly C V(D)J RECOMBINATION A specialized form of recombination that assembles the genes that encode lymphocyte antigen receptors from variable (V), diversity (D) and joining (J) gene segments. DNA double-stranded breaks are introduced between the V, D and J segments and DNA repair/recombination proteins then join the segments together. ERROR-PRONE REPAIR PATHWAYS DNA lesions can be repaired by many competing pathways. Some of these pathways repair damaged DNA with reduced fidelity, giving rise to new mutations. TRANSCRIPTION-COUPLED REPAIR A specialized pathway that facilitates the repair of damaged DNA during transcription, in which the transcribed strand of active genes is preferentially repaired. This process is defective in the genetic disease, Cockayne syndrome. DNA-repair transcription D2 C E A F G + BRCA1 D2 Nucleus FA core complex Ub D2 evidence indicates that FA cells are hyper-recombinogenic 81, a situation akin to Bloom syndrome. Indeed, a common phenotypic feature of FA is reduced fertility, which can be indicative of defective meiosis. It is noteworthy that the FANCD2 protein binds to meiotic chromatin and preferentially to the sex chromosomes 71 (see supplementary Figure 1 online). As these chromosomes seem to be excluded from meiotic recombination, could the FA pathway perhaps act to suppress recombination? If this were the case, this would imply that the repair of DNA crosslinks in mitotic cells must occur through a distinct pathway. Evidence that FA proteins function in DNA repair has come from studies into the processing of DNA double-stranded breaks (DSBs) in FA cells. Plasmidbased, damaged substrates and DSBs that are introduced at endogenous loci, such as those generated during V(D)J RECOMBINATION, are repaired with impaired fidelity in FA-D1 and FA-C cells When transfected plasmids that contain a blunt DNA break are recovered and sequenced, the break sites are joined with intervening deletions. Large intragenic deletions also occur in selected mutants at the HGPRT (hypoxanthine phosphoribosyltransferase 1 (Lesch Nyhan syndrome)) locus in FA-D1 cells, and in the immunoglobulin loci in FA-D1 and FA-C lymphoblasts. We know that DSBs can be repaired by non-homologous end-joining (NHEJ), but the core elements of this pathway, such as DNA-PK (DNA protein kinase) and XRCC4 (X-ray repair complement- Ub + + Monoubiquitylation D1? D1? Nuclear foci S-phase checkpoints D1 DNA damage DNA replication Figure 5 A model of the Fanconi anaemia pathway. The model shows the sequential assembly of the Fanconi anaemia (FA) core complex in the cytoplasm and nucleus. The defect in FA-B cells indicates that FANCB might function in an early stage of this assembly process. DNA replication or DNA damage might activate the complex, either directly or indirectly, leading to the activation of FANCD2 by monubiquitylation 72 and its targeting to nuclear foci. The FANCD1 protein might participate at this point. The association of BRCA1 at these sites could lead to as yet undefined downstream events, such as DNA repair or the activation of DNA-synthesis (S) checkpoints. In this model, the FA core complex, which consists of FANCA, FANCC, FANCF, FANCG and FANCE, is a sensor for DNA damage and participates by activating FANCD2 through ubiquitylation. FANCD2 then facilitates repair. ing defective repair in Chinese hamster cells 4), seem to be unaffected in cells from the FA-D1 and FA-C complementation groups 83. These findings implicate the FANCC and the as yet unidentified FANCD1 proteins in the fidelity of DSB repair. In a cell-free assay of DSB repair, in which nuclear extracts were tested for their ability to repair a cut plasmid substrate, the nuclear extracts from all FA cell lines were found to be deficient in their ability to repair such a substrate 85. Indeed, mixing experiments revealed that extracts from the FA-D2 cells could rescue repair activity in FA-A and FA-C extracts. This study provides further support for the role of FA proteins in DSB repair, a function that seems to be distinct from proteins involved in NHEJ. More recently, attention has focused on the alteration of S-phase cell-cycle checkpoints in FA cells to DNA damage. Yeast mutants that are defective at this checkpoint rapidly accumulate chromosomal translocations. This might be due to an inability to repair the spontaneous DNA damage that occurs during genome replication by non-error-prone REPAIR PATHWAYS 86. In two studies, the specific introduction of DNA crosslinks during DNA replication in FA cells resulted in defective S-phase responses 87,88. In particular, FA-C and FA- D1 cells did not seem to slow down or arrest in the S phase when a DNA crosslink was introduced and, when replication was completed, damaged cells arrested at the G2 checkpoint. However, at present we lack the more refined markers and techniques that would enable us to analyse replication checkpoints in vertebrate cells. This makes it difficult to assess whether there is a true defect in FA cells, but is a promising future line of inquiry. The cloning of FANCD2 and the characterization of its protein is a potential breakthrough in understanding the role of the FA pathway in the DNA damage response. Association of BRCA1 with FANCD2 indicates that the FA pathway might participate in the DNA-damage-response pathways that are common to both proteins, such as the RAD50- or RAD51- dependent repair pathways. However, despite intensive research into the function of BRCA1, it remains unclear as to what this protein does upon DNA damage 89. In cell lines, BRCA1 co-purifies with a large nuclear complex that contains many well-characterized DNA-repair proteins, such as those involved in mismatch repair and NHEJ 90. Furthermore, BRCA1 also associates with the large protein complexes that function in chromatin remodelling 91,92. It is intriguing that FAZF (FA zinc-finger), a protein that binds to FANCC, has features that indicate its involvement in higher-order chromatin modification 33. This raises the possibility that the FA proteins might also be components of similarly large chromatin-remodelling complexes. Defects in homologous recombination, as well as in TRANSCRIPTION-COUPLED REPAIR, have been associated with BRCA1 deficiency 93,94, and BRCA1 might also function in transcriptional regulation and centrosome function 95. Whether FANCD2-deficient cell lines 454 JUNE 2001 VOLUME 2

10 Figure 6 Co-localization of FANCD2 and BRCA1. Eight hours after γ-irradiation of HeLa cells, both BRCA1 (green) and FANCD2 (red) accumulate in the same nuclear foci (yellow in merged picture). (Adapted from REF. 71, with permission.) show similar abnormalities remains to be resolved. Crucially, some discrepancies exist that indicate that the relationship between BRCA1 and the FA pathway is not simple. For example, Brca1 deficiency causes mouse embryonic lethality, and cells in which Brca1 is attenuated or conditionally inactivated show catastrophic chromosome breakage 96,97. By contrast, Fancc / and Fanca / (REFS 46 48) knockout mice are viable and their cells do not show gross spontaneous chromosome damage, and there is no evidence of breast cancer susceptibility in homozygous or heterozygous FA mutation carriers. It has been more than 25 years since the discovery that FA cells are sensitive to DNA crosslinking agents, but the nature of the DNA-repair/damage-response defect in this disease remains to be elucidated. However, exciting potential clues are now provided with the cloning of FANCD2 and the characterization of its protein product. Other branches of the FA pathway Early reports of the FA cellular phenotype noted a hyperresponsiveness of FA cells to chromosomal breakage by atmospheric oxygen 98, so that the spontaneous breakage observed in standard cultures of FA cells might be a laboratory artefact caused by the presence of a non-physiologically high level of oxygen. Similarly, spontaneous arrest and delay in the G2 phase of the cell cycle, as observed in primary FA fibroblast cultures 99, is alleviated by reducing the ambient oxygen concentration from 20% (air) to 5% (REF. 100). How elevated oxygen exacerbates the FA cellular phenotype is not known at present, because the molecular mechanism of oxygen toxicity is complex and involves many potential target processes in the cell 101,102. Interestingly, two-hybrid studies have shown that FANCC and NADPH cytochrome P450 reductase, an enzyme that produces potentially toxic activated oxygen species, interact 103. FANCC attenuates the activity of the reductase and so might serve as an intracellular antioxidant. However, now that the known FA gene products point to a nuclear function in the DNA-damage response, it remains to be established how a diversity of seemingly unrelated observations, such as altered responses of FA cells to cytokines, free radical species and apoptosis-inducing agents fit with this subcellular localization. The future FA research faces some major challenges. For patients, it will be important to improve bone marrow transplant procedures, to implement gene therapy and to find ways to avoid the development of cancer. This will depend on a better understanding of the molecular defect of the disease. It is now known that the FA core complex functions upstream of FANCD2, the modified form of which could participate in a DNA-damageresponse pathway specific to FA. The FA core complex might act as a sensor of DNA damage that leads to activation of FANCD2, which might be a crucial effector molecule in this pathway. The discovery of FANCD2 raises many questions. How does the FA core complex respond to DNA damage? How does it participate in the monoubiquitylation of FANCD2? Is this complex a highly regulated E3 ubiquitin ligase? How does FANCD2 modification target this molecule to BRCA1- containing foci, and finally, what precisely does FANCD2 do upon translocation to these sites? Perhaps the identification of the gene defects in the FA-B and FA-D1 complementation groups, and in any other undiscovered groups or interacting proteins, might shed light on these key questions. The FA pathway might be conserved in genetically tractable lower organisms, such as zebrafish; studies on such model organisms could facilitate the discovery of other proteins that participate in the FA pathway. Finally, the use of genome expression profiling might shed light on the downstream transcriptional targets of the FA pathway. Such future studies on tumour samples from FA patients might help to elucidate the key genetic changes that enable FA cells to undergo transformation. Update added in proof The FA core complex was recently shown to bind to chromatin and the nuclear matrix in a DNA-damage and cell-cycle-dependent manner 127, providing strong evidence for a direct physical interaction of the FA protein complex with DNA or chromatin. Another recent study indicates that metabolically generated oxygen radicals have a role in the pathogenesis of FA by showing that Fancc -/- Sod1 -/- (superoxide dismutase 1, soluble) double-knockout mice have a haematopoietic deficiency; Sod1 -/- and Fancc -/- single knockouts have hardly any haematological symptoms 128. Links DATABASE LINKS FANCD BRCA1 hereditary nonpolyposis colorectal cancer Bloom syndrome Werner syndrome FA-A FA-G FANCC FANCA FANCG FANCF FANCE FA-D2 Fancc Fanca FA-C NBS1 MRE11 FA-D1 HGPRT DNA-PK XRCC4 RAD50 RAD51 FAZF Brca1 NADPH cytochrome P450 reductase Sod1 serum α-fetoprotein ataxiatelangiectasia FURTHER INFORMATION Manuel Buchwald s lab Fanconi Anaemia Mutation Database FA Research Fund, Inc. ENCYCLOPEDIA OF LIFE SCIENCES DNA repair DNA crosslink repair NATURE REVIEWS GENETICS VOLUME 2 JUNE