There are few better examples of the proposition that studying rare genetic disorders can illuminate important biological pathways than research into Fanconi anemia, which has identified several genes important for DNA repair. Fanconi anemia is characterized by congenital defects, progressive bone marrow failure and cancer susceptibility1. Its distinctive cellular phenotype involves chromosomal instability and hypersensitivity to DNA cross-linking agents. Eleven distinct complementation groups for Fanconi anemia have been defined using somatic cell fusion techniques, and the genes underlying eight of these complementation groups have been identified. Now, Meetei et al.2 on page 1219 describe an X-linked gene defective in individuals with Fanconi anemia complementation group B (FA-B). Because of its unique chromosomal location, the gene encoding this newly discovered component of the Fanconi anemia complex may represent a particularly vulnerable component of the DNA repair pathway responsible for maintaining genomic integrity.

Six of the proteins involved in Fanconi anemia (FANCA, FANCC, FANCE, FANCF, FANCG and FANCL) are part of a protein complex that functions as a ubiquitin ligase, of which FANCL is probably the catalytic subunit3. In response to DNA damage or in S phase of the cell cycle, this complex catalyzes the monoubiquitination of FANCD2 (Fig. 1). A defect in any one of these core proteins results in failure to monoubiquitinate FANCD2, emphasizing the importance of this modification. But exactly how this complex functions in DNA repair is not known. An eighth gene involved in Fanconi anemia, corresponding to complementation group D1, has been identified as the breast cancer susceptibility gene BRCA2 (ref. 4). In BRCA2-defective cells, the core Fanconi anemia complex is intact and FANCD2 is appropriately ubiquitinated, suggesting that BRCA2 acts downstream of the other proteins involved in Fanconi anemia (Fig. 1).

Figure 1: Schematic depiction of the Fanconi anemia DNA repair pathway.
figure 1

DNA damage activates the complex of Fanconi anemia–associated proteins FANCA, FANCB, FANCE, FANCF, FANCG and FANCL1. FANCL5 is a ubiquitin ligase that probably causes the ubiquitination of FANCD2. This protein, together with another Fanconi anemia–associated protein, BRCA2 and RAD51, is involved in the repair of DNA damage.

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From B to X

Meetei et al.2 analyzed proteins associated with the core Fanconi anemia complex5 and identified a new component, FAAP95, encoded by a gene of unknown function. They then examined this protein more closely to see whether it is an important functional component of the complex. They found that knockdown of FAAP95 by short interfering RNA in cultured cells resulted in reduced ubiquitination of FANCD2 and produced the sensitivity to DNA cross-linkers that is characteristic of Fanconi anemia. They also found that FAAP95, like other core proteins, localized to the nucleus under normal conditions but became cytoplasmic in cells defective in FANCA, another core component of the complex. Taken together, these data provided strong evidence that FAAP95 functions in the Fanconi anemia core complex and further suggested that mutations of FAAP95 might underlie some cases of the disease.

Because the gene encoding FAAP95 is located on the X chromosome, the authors suspected that mutations in this gene might cause FA-B, which has only been observed in males. Notably, FA-B cell lines have defective FANCD2 ubiquitination, which is also seen in response to FAAP95 depletion2. The authors confirmed this suspicion by sequencing the gene from two FA-B cell lines and finding that both cell lines carried inactivating mutations in the gene. They then found that two other Fanconi anemia cell lines previously unassigned to complementation groups also carry mutations in this gene. They showed that a cDNA expressing FAAP95 was able to complement the sensitivity of FA-B cell lines to DNA cross-linkers, providing strong evidence that mutations in this gene cause the FA-B phenotype.

Before this study, Fanconi anemia was thought to be inherited exclusively in an autosomal recessive fashion. Thus, the location of FANCB on the X chromosome generates new issues in genetic counseling for the disease. Males who inherit a mutation in FANCB will be severely affected because they carry only a single copy of the gene. The situation is more complicated in females, as X-chromosome inactivation6 would be expected to result in half of the somatic cells of heterozygous individuals who carry the FANCB mutation expressing only the wild-type protein and the other half only the mutant form. Meetei et al. show, however, that blood cells and skin fibroblasts from female carriers express only the wild-type allele, presumably because of selection against cells lacking the wild-type protein2. Nevertheless, some cells selectively expressing the mutant form might remain in particular tissues. Because these cells are likely to manifest genomic instability and be prone to transformation, this should be taken into account in the clinical management of females carrying the mutation in FANCB. Finally, it has been suggested that inactivation of genes involved in Fanconi anemia might have a role in sporadic cancer7,8,9. Because FANCB is expected to be present as a single functional copy in most cells, it could potentially be inactivated by a single mutational event, distinct from the two-hit paradigm typical of most tumor-suppressor genes.

Case closed?

A previous study describing a truncating mutation in BRCA2 in an FA-B cell line led to the suggestion that FANCB and BRCA2 were the same gene4. This BRCA2 mutation, 3033delAAAC, is predicted to result in loss of more than half the protein, including functionally important domains. Moreover, this same mutation confers breast cancer susceptibility in heterozygotes and is predicted to cause Fanconi anemia in biallelic carriers. This raises the puzzling question of why two potentially causative mutations should be present in a single individual. The first possibility is that it is due to chance. But apart from certain founder populations, the frequency of BRCA2 mutations is only ∼1 in 800 (ref. 10), requiring a somewhat extraordinary coincidence. Perhaps we need to consider the possibility, then, that the BRCA2 mutation contributes to the phenotype in this FA-B case. Is there any evidence to support this? No two-generation FA-B family has ever been reported, though admittedly, only four families have been identified with FANCB mutations, and, therefore, this subtype may be very rare. Nevertheless, an affected uncle and nephew pair or affected male cousins would almost certainly be reported, and it may be that such families occur very rarely. One possibility for their absence is that mutations in additional genes are required for expression of the phenotype (e.g., BRCA2 in the case of the reference FA-B cell line). Digenic predisposition has been described in other conditions11 and is a plausible cause of some cases of Fanconi anemia. Thus, it remains possible that the case for BRCA2 involvement in FA-B is not yet closed.