Disease model: Fanconi anemia

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transactivation function and DNA-binding activity but inhibits induction of apoptosis in mammalian cells. Cancer Res. 59, 5902–5907 Yang, A. et al. (1999) p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 398, 714–718 Mills, A.A. et al. (1999) p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 398, 708–713 van Bokhoven, H. et al. (1999) Limb mammary syndrome: a new genetic disorder with mammary hypoplasia, ectrodactyly, and other hand/foot anomalies maps to human chromosome 3q27. Am. J. Hum. Genet. 64, 538–546 Eckhold, J.G. and Martens, F.H. (1804) Über eine sehr kompicierte Hasenscharte. Leipzig: Steinacker. Celli, J. et al. (1999) Heterozygous germline mutations in the p53 homolog p63 are the cause of EEC syndrome. Cell 99, 143–153 van Bokhoven, H. et al. (2001) p63 gene mutations in EEC syndrome, limb-mammary syndrome, and isolated split hand-split foot malformation suggest a genotype-phenotype correlation. Am. J. Hum. Genet. 69, 481–492 Ianakiev, P. et al. (2000) Split-hand/split-foot malformation is caused by mutations in the p63 gene on 3q27. Am. J. Hum. Genet. 67, 59–66 Wessagowit, V. et al. (2000) Heterozygous germline missense mutation in the p63 gene underlying EEC syndrome. Clin. Exp. Dermatol. 25, 441–443 Hollstein, M. et al. (1991) p53 mutations in human cancers. Science 253, 49–53 Denissenko, M.F. et al. (1996) Preferential formation of benzo[a]pyrene adducts at lung cancer mutational hotspots in P53. Science 274, 430–432 Tornaletti, S. and Pfeifer, G.P. (1994) Slow repair of pyrimidine dimers at p53 mutation hotspots in skin cancer. Science 263, 1436–1438 Kere, J. et al. (1996) X-linked anhidrotic (hypohidrotic) ectodermal dysplasia is caused by

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mutation in a novel transmembrane protein. Nat. Genet. 13, 409–416 Fraser, F.C. The genetics of cleft lip and cleft palate. (1970) Am. J. Hum. Genet. 22, 336–352 Ferguson, M.W. (1988) Palate development. Development 103, 41–60 Murray, J.C. (1995) Face facts: genes, environment, and clefts. Am. J. Hum. Genet. 57, 227–232 Hay, R.J. and Wells, R.S. (1976) The syndrome of ankyloblepharon, ectodermal defects and cleft lip and palate: an autosomal dominant condition. Br. J. Dermatol. 94, 277–289 Schinzel, A. and Klausler, M. (1986) The Van der Woude syndrome (dominantly inherited lip pits and clefts). J. Med. Genet. 23, 291–294 McGrath, J.A. et al. (2001) Hay-Wells syndrome is caused by heterozygous missense mutations in the SAM domain of p63. Hum. Mol. Genet. 10, 221–229 Propping, P. and Zerres, K. (1993) ADULTsyndrome: an autosomal-dominant disorder with pigment anomalies, ectrodactyly, nail dysplasia, and hypodontia. Am. J. Med. Genet. 45, 642–648 Propping, P. et al. (2000) ADULT syndrome allelic to limb mammary syndrome (LMS)? Am. J. Med. Genet. 90, 179–182 van Bokhoven, H. et al. (2000) p63 mutations in the EEC, Hay–Wells, ADULT syndromes and in split hand/foot malformation reveal a genotype–phenotype correlation. Am. J. Hum. Genet. 67, Supplement 2, abstract 149 Amiel, J. et al. (2001) TP63 gene mutation in ADULT syndrome. Eur. J. Hum. Genet. 9, 642–645 Faiyaz ul Haque, M. et al. (1993) Mapping of the gene for X-chromosomal split-hand/splitfoot anomaly to Xq26-q26.1. Hum. Genet. 91, 17–19 Scherer, S.W. et al. (1994) Physical mapping of the split hand/split foot locus on chromosome 7 and implication in syndromic ectrodactyly. Hum. Mol. Genet. 3, 1345–1354 Crackower, M.A. et al. (1996) Characterization of the split hand/split foot malformation locus

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SHFM1 at 7q21.3-q22.1 and analysis of a candidate gene for its expression during limb development. Hum. Mol. Genet. 5, 571–579 Nunes, M.E. et al. (1995) A second autosomal split hand/split foot locus maps to chromosome 10q24- q25. Hum. Mol. Genet. 4, 2165–2170 Sidow, A. et al. (1999) A novel member of the F-box/WD40 gene family, encoding dactylin, is disrupted in the mouse dactylaplasia mutant. Nat. Genet. 23, 104–107 Sidow, A. et al. (1997) Serrate2 is disrupted in the mouse limb-development mutant syndactylism. Nature 389, 722–725 Pellegrini, G. et al. (2001) p63 identifies keratinocyte stem cells. Proc. Natl. Acad. Sci. U. S. A. 98, 3156–3161 Tickle, C. and Munsterberg, A. (2001) Vertebrate limb development. Curr. Opin. Genet. Dev. 11, 476–481 Watt, F.M. (2001) Stem cell fate and patterning in mammalian epidermis. Curr. Opin. Genet. Dev. 11, 410–417 Thesleff, I. and Sharpe, P. (1997) Signalling networks regulating dental development. Mech. Dev. 67, 111–123 Wilkie, A.O. and Morriss-Kay, G.M. (2001) Genetics of craniofacial development and malformation. Nat. Rev. Genet. 2, 458–468 McGrath, J.A. et al. (1997) Mutations in the plakophilin 1 gene result in ectodermal dysplasia/skin fragility syndrome. Nat. Genet. 17, 240–244 Monreal, A.W. et al. (1999) Mutations in the human homologue of mouse dl cause autosomal recessive and dominant hypohidrotic ectodermal dysplasia. Nat. Genet. 22, 366–369 Lamartine, J. et al. (2000) Mutations in GJB6 cause hidrotic ectodermal dysplasia. Nat. Genet. 26, 142–144 Hibi, K. et al. (2000) AIS is an oncogene amplified in squamous cell carcinoma. Proc. Natl. Acad. Sci. U. S. A. 97, 5462–5467

Fanconi anemia (FA) is a chromosomal instability syndrome characterized by the presence of pancytopenia, congenital malformations and cancer predisposition. Six genes associated with this disorder have been cloned, and mice with targeted disruptions of several of the FA genes have been generated. These mouse models display the characteristic FA feature of cellular hypersensitivity to DNA crosslinking agents. Although they do not develop hematological or developmental abnormalities spontaneously, they mimic FA patients in their reduced fertility. Studies using these animal models provide valuable insights into the involvement of apoptotic pathways in FA, and help characterize the defects in FA hematopoietic cells. In addition, mouse models are also useful for testing treatments for FA.

Fanconi anemia (FA) has been considered an important genetic model for the study of chromosomal instability, hematopoietic failure and cancer susceptibility. FA is inherited in an autosomal http://tmm.trends.com

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Jasmine C.Y. Wong and Manuel Buchwald

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Disease model: Fanconi anemia O DEL S

recessive manner, and patients might be affected by a diverse assortment of congenital malformations, the most common being limb malformations and abnormal skin pigmentation. Progressive bone marrow (BM) failure and its complications are the major cause of death, and bone marrow transplant is currently the best treatment available. FA patients are also predisposed to developing cancer, particularly acute myeloid leukemia. Cells cultured from FA patients display an increased level of spontaneous chromosomal abnormalities when compared to normal cells, and this effect is amplified when the cells are exposed to DNA cross-linking agents such as mitomycin C (MMC). For this reason, FA has been classified as a chromosomal instability

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Table 1. Mouse models for Fanconi anemia Model

Genetics

Similarities to human phenotype

Differences from human phenotype

Refs.

Fanca–/–

Replacement of Fanca exons 4–7 with lacZ-neo cassette

Impaired fertility, DNA crosslinker sensitive

Lack of congenital malformations, hematological abnormalities and tumors

[12]

Fancc–/–

Replacement of Fancc exon 8 or exon 9 with neo cassette

Impaired fertility, DNA crosslinker sensitive, Lack of congenital malformations, blunted response to SCF, hypersensitivity to hematological abnormalities and tumors IFN-γ and TNF-α, apoptotic effects blocked by caspase 3 inhibitor

[10,11, 20–22,26]

Fancg–/–

Replacement of Fancg exons 2–9 with neo cassette

Impaired fertility, DNA crosslinker sensitive, increased sensitivity to ionizing radiation, lack of Fancd2-L protein isoform

Lack of congenital malformations, hematological abnormalities and tumors

[13]

Fancc–/– Sod1–/–

Deletion of Fancc exon 8 and Sod1 entire coding sequence

Hematological abnormalities

Fatty liver, lack of congenital malformations

[18]

FANCC Overexpression of human FANCC Protection against apoptosis transgenic FNT

Overexpression of human TNF-α in Fancc–/–

[25]

Decreased BM colony growth

Normal peripheral blood counts and marrow [24] cellularity

Abbreviations: BM, bone marrow; IFN-γ, interferon γ; SCF, stem cell factor; TNF-α, tumor necrosis factor α.

disorder. Cellular sensitivity to DNA cross-linking agents is currently used as a diagnostic test for FA [1]. Somatic cell hybrid studies demonstrate that at least eight genetic complementation groups, each representing a different gene, are associated with the disease [2]. The genes for complementation groups A (FANCA), C (FANCC), D2 (FANCD2), E (FANCE), F (FANCF), and G (FANCG) have been cloned, and the FANCA, -C, -E, -F and -G proteins form a complex [3]. In addition, FA cell lines are defective in the monoubiquitination of FANCD2, suggesting that FA proteins function through a common pathway [4]. Although the basic biochemical defect of FA is still unknown, various hypotheses including defects in DNA repair, oxygen metabolism, cell cycle regulation, apoptosis and growth factor homeostasis have been proposed, and several proteins from these functional categories have been found to interact with FA proteins [2,3]. Fanconi anemia in mice

Jasmine C.Y. Wong Manuel Buchwald* Program in Genetics and Genomics Biology, Research Institute, Dept of Molecular and Medical Genetics, The Hospital for Sick Children, University of Toronto, 555 University Ave, Toronto, Ontario M5G 1X8, Canada. *e-mail: manuel.buchwald @sickkids.ca

Mouse homologs of FANCA and FANCC (Fanca and Fancc) share 65% and 67% amino acid sequence identity with their human counterparts, respectively. Expression of the mouse homolog in human FA lymphoblast cells from the same complementation group corrects hypersensitivity to DNA-crosslinking agents, suggesting functional conservation of the FA proteins between humans and mice [5–7]. Fanca and Fancc transcripts are found in all adult organs examined, as well as during embryonic development. In situ hybridization demonstrates a complex tissue-specific and stage-specific pattern of expression during development. Expression was detected in tissues such as whisker follicles, teeth, brain, kidney, and limbs, primarily in cells of epithelial and mesenchymal origin, and particularly with osteogenic and hematopoietic potential [8,9]. These findings are consistent with the spectrum of congenital abnormalities seen in FA patients, suggesting that the mouse will be a useful model for the study of FA. http://tmm.trends.com

The earliest FA mouse models reported in the literature were two Fancc knockout models generated by targeted gene disruption, and therefore, they have been the most studied to date [10,11]. Cells cultured from Fancc mouse models show increased chromosomal aberrations when exposed to DNA cross-linking agents, confirming their resemblance to the FA phenotype. Knockout mouse models of Fanca and Fancg have subsequently been reported, and initial studies on these models demonstrate similar phenotypes to the Fancc knockout (Table 1) [12,13]. Preliminary reports suggest that the Fancd2 knockout and Fanca/Fancc double knockout are also qualitatively similar to the known models, which is consistent with the idea that FA proteins function cooperatively in the same cellular pathway(s) [14,15]. FA mouse models have no obvious developmental abnormalities, with the exception of a decrease in the size of testes and ovaries [10–13]. FA knockout mice show reduced fertility, also a characteristic of FA patients. Histological analysis reveals that the ovaries have significantly fewer follicles than controls, whereas the testes demonstrate a mosaic pattern with some seminiferous tubules showing normal spermatogenesis, and others showing vacuolar degeneration (Fig. 1). Newborn and embryonic mutant gonads have a reduced number of germ cells as compared with controls, suggestive of a defect in prenatal germ cell development. BrdU analysis indicates a significantly lower proportion of germ cells undergoing proliferation in Fancc−/− mice than littermate controls, implying a role for Fancc in the mitotic proliferation of murine primordial germ cells [16]. Hematopoiesis in FA knockouts

In contrast to FA patients, FA mouse models show no spontaneous hematological abnormalities or tumor development. Peripheral blood counts of FA knockouts remain relatively normal over time, and there is no significant difference in the number of

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Fig. 1. Fanca−/− mice have gonadal abnormalities. Histological sections of testes from 10-week-old (a) control and (b) Fanca−/− mice (hematoxylin and eosin staining). Fanca−/− mice display a mosaic pattern of normal and degenerated seminiferous tubules not seen in age-matched controls. Histological sections of ovaries from 12-week-old (c) control and (d) Fanca−/− mice (hematoxylin and eosin staining). Fanca−/− mice have significantly fewer follicles and corpora lutea.

committed BM progenitor cells when compared with controls [10,11]. However, when Fancc−/− mice are subjected to sequential sublethal doses of MMC that do not affect normal mice, there is a progressive decrease in all peripheral blood parameters, as well as early and committed progenitors, causing death within 3–8 weeks [17]. Peripheral blood bicytopenia can also be observed after disrupting the ability of Fancc knockouts to modulate reactive oxygen species by deletion of cytosolic Cu/Zn superoxide dismutase (Sod1) [18]. Fancc−/−Sod1−/− double knockout mice show a novel phenotype with decreases in peripheral red and white blood cells, as well as decreases in the numbers of lineage-positive progenitors and in colony-forming capacity. Therefore, it appears that the loss of FA genes in mouse models does not jeopardize survival under normal circumstances, but rather, the defect lies in the compromised ability to respond to environmental insults. Part of this could be a result of the disregulation of cellular response pathways that are important for controlling the survival and apoptosis of cells. Clonal growth of hematopoietic progenitor cells from Fancc−/− animals is less responsive to stem cell factor stimulation, confirming earlier reports that use human FA cells, and suggesting that lack of Fancc might cause blunted responses to survival factors [19,20]. Furthermore, and similar to FA-C patients, BM cells from Fancc knockouts show compromised colony growth capacity following treatment with interferon-γ (IFN-γ) at doses that do not suppress normal cells [11,21]. Tumor necrosis factor-α (TNF-α) http://tmm.trends.com

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and the macrophage inflammatory protein-1α (MIP-1α) have been found to have similar effects [22]. IFN-γ and TNF-α can upregulate each other’s cellular receptors as well as the fas receptor (CD95) [23]. IFN-γ mediated clonal suppression involves at least in part the fas apoptotic pathway and activation of caspase 3. Increase in CD95 expression has been found in the CD34+ fraction of hematopoietic progenitor cells in Fancc mutant mice [24]. Neutralizing anti-fas antibodies can abrogate the inhibitory effects of low-dose IFN-γ, whereas agonistic anti-fas antibodies augment the clonal suppression effects of IFN-γ and TNF-α [21,24]. Addition of agonistic anti-fas antibody alone is also capable of clonal suppression in Fancc−/− BM by causing increased apoptosis, and this effect can be protected by overexpression of FANCC in a transgenic model [25]. Decreased clonal growth of Fancc−/– mouse BM after IFN-γ treatment can be counteracted by the addition of a caspase 3 inhibitor, and similar results are obtained when CD34+ cells from a FA-C patient are used [26]. The hypersensitivity of Fancc−/− mice to IFN-γ and TNF-α is also mediated through activation of the RNA-dependent protein kinase (PKR) pathway [27]. An elevated level of activated PKR was found in Fancc−/− mouse embryonic fibroblasts, whereas overexpressing a catalytically inactive mutant form of PKR increased the survival of Fancc−/− cells after treatment with double-stranded RNA (dsRNA) and IFN-γ. Furthermore, overexpression of a mutant eukaryotic translation initiation factor-2α protein, a known physiological substrate for PKR, abrogated the cytotoxic effects of dsRNA and IFN-γ treatment. These studies suggest that the hematopoietic abnormalities seen in FA patients might be related to aberrant responses to stimulatory and inhibitory molecules, causing perturbation of the proliferation and survival of hematopoietic stem and progenitor cells. Furthermore, accelerated telomere shortening demonstrated in hematopoietic cells from FA patients has been proposed as another mechanism contributing to the onset of anemia [28]. Whether the absence of spontaneous pancytopenia and tumor development in FA mouse models is related to the radical difference in telomere length between Mus musculus and humans poses an interesting area of investigation [29]. Testing clinical treatments in FA mouse models

The successful use of histocompatible matched sibling donor BM transplant to cure the hematopoietic defect in FA make this disorder an attractive candidate for the development of gene therapy. The FA mouse models are useful for testing possible therapeutic strategies. BM cells from Fancc−/− mice transplanted into lethally irradiated recipients have a decreased ability to reconstitute the hematopoietic system when compared with

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Acknowledgements We thank Jeff Lightfoot for helpful comments on the manuscript. Work in our laboratory has been supported by funds from the Canadian Institutes of Health Research, the National Cancer Institute of Canada (with funds from the Canadian Cancer Society) and the Hospital for Sick Children Foundation. MB holds the Lombard Insurance Chair in Pediatric Research at the Hospital for Sick Children and the University of Toronto

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BM cells from normal mice [30]. Fancc−/− hematopoietic stem cells have a 7–12 fold decrease in repopulating ability compared with Fancc+/+ cells [31]. When a 1:1 mix of wild-type and Fancc−/– BM was transplanted into non-ablated Fancc−/− animals, a modest selective advantage of the wild-type BM was observed that was enhanced upon serial transplantation or treatment with MMC [32]. Therefore, selective pressure can be used to promote in vivo enrichment of cells with a normal Fancc function. Phenotypic correction of Fancc−/− hematopoietic cells has been achieved by retroviral gene therapy. Whereas lethally irradiated mice transplanted with Fancc−/− BM cells did not survive treatment with MMC, Fancc−/− BM cells transduced with human FANCC were found to engraft and restore hematopoiesis in the host at blood counts and colony-forming capacity similar to those transplanted with wild-type BM [33]. The use of non-toxic doses of cyclophosphamide (CPA) and γ-irradiation (IR), agents used for preconditioning bone marrow transplantation in FA patients, also lead to the

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in vivo selection of genetically corrected Fancc mutant cells [34]. Significance of FA mouse models

FA is a complex disorder that involves multiple genetic and environmental components. Despite the fact that FA mouse models do not naturally recapitulate all the characteristic features of the human phenotype, they probably share common molecular mechanisms with FA patients. FA knockouts show the hallmark feature of hypersensitivity to DNA-crosslinking agents, and the mouse and human homologs can rescue the FA phenotype in a cross-species assay. Therefore, FA mouse models offer a versatile system for dissecting the effects of specific molecular pathways in an in vivo setting, as demonstrated by the studies on inhibitory cytokines. These studies help clarify the complicated pathophysiology of FA, and provide insights into the type of insults that might cause BM failure in FA patients. In addition, studies of gene therapy in mouse models provide us with information that has important clinical implications, leading us to a potentially promising cure for this life-threatening disease.

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