Fanconis anemia

Fanconi's anemia (FA) is the most common cause of hereditary BMF. Early studies indicated that FA was a genetically heterogeneous disease, a notion confirmed by the identification, through the use of somatic cell hybridization, of as many as eight complementation groups (FA-A, B, C, D1, D2, E, F, and G). Seven FA genes have now been cloned and characterized. The overall frequency of heterozygotes for FA mutant genes in the general population is estimated at 1 in 300. In Ashkenazi Jews and in the Afrikaans population of the Republic of South Africa it is much higher (1 in 100 and 1 in 89, respectively), most likely as a result of founder effects.

Clinical aspects. Gradual onset of BMF (median age, 7 years; range, birth to 31 years), skeletal abnormalities (most commonly of the radius and thumb), skin lesions (hyperpig-mentation, café-au-lait spots), renal and urinary tract malformations, and gonadal dysfunction are the most common clinical manifestations. However, the clinical spectrum is even wider, as it includes congenital defects of the gastrointestinal system, heart and central nervous system. BMF is often heralded by thrombocytopenia, macrocytosis and increased Hb F levels. Patients with FA have an unusually high risk of develop -ing treatment-resistant MDS and AML, estimated at 52% in total by the age of 40 years. Furthermore, the risk of a variety of solid tumors, especially squamous cell carcinomas of the skin, is several times higher than in the general population.

Molecular genetics. FA is an autosomal recessive disorder. The most characteristic cellular feature of FA cells is the formation of DNA double-strand breaks upon exposure to DNA inter- and intrastrand adducting agents (clastogens), such as mitomycin and diepoxybutane. The in vitro response to clas-togens has made it possible to test cells from different patients for their ability to cross-correct each other's defect by somatic cell fusion. As discussed earlier, this has led to the classification of patients in eight complementation groups (FA A-H).

Table 12.6 The identity of the FA genes and proteins.



Relative frequency


Protein (kDa)










380 (BRCA2)










380 (BRCA2)





















Using the technique of complementation cloning, FANCC was identified first. In this approach, a cDNA library, inserted into an EBV-based episomal expression vector, is transfected into immortalized B-cell lines derived from patients with FA. Cells complemented with the correct cDNA survive the treatment with a clastogenic agent; the episomal DNA recovered from the surviving cells contains inserts that are candidates for the disease gene. The disease gene is identified by its capacity to correct the phenotype of deficient cells and by screening for pathogenic mutations in affected individuals and family members. Using the same approach, another four genes were cloned: FANCA, FANCG, FANCF and FANCE (Table 12.6). The recent identification of FANCD2 was achieved through a combination of complementation and positional cloning.

Complementation was achieved using a panel of microcells lacking one specific chromosome. By fusing such cells with fibroblasts derived from FA-D patients, it was found that the gene of interest lay on chromosome 3. Further complementation experiments using cells with partially deleted chromosome 3 led to the identification of a critical area of 200 kb which contained the FANCD2 gene.

A major advance in the clarification of the genetics of FA and also in our understanding of the molecular pathogenesis of the disease was the finding that the breast cancer susceptibility gene BRCA2 is mutated in FA-B and FA-D1 patients (see below).

Molecular pathology and population genetics. (Figure 12.5) Mutations of FANCA account for about 60% of FA cases and



FANCC -1 -2H3H4rn 6 H 7 H^9n,cl1fh2- 13 -14M677


11 13 14

^ Microdeletion or microinsertion ▼ Nonsense mutation • Missense mutation ■ Splice site alteration — Deletion o Polymorphism


1263delF tT



ORF 4368

22 23

26 H27- 28

Fig 12.5 Molecular pathogenesis of FA: a proposed model are spread throughout the gene. None of the mutant alleles is common and few have been encountered more than once. Thus, identifying mutations in newly diagnosed cases of FA is laborious, as one needs to scan the entire coding sequence of several genes, unless the complementation group is known.

Mutations in the FAC gene account for about 10-15% of FA cases. The IVS4+A^T and del322G mutations comprise >75% of FANCC mutations. The IVS4+A^T allele is found in Ashkenazi Jews at a polymorphic frequency (1 in 80), and it is responsible for 85% of the FA cases in this population. Patients with IVS4 or exon 14 mutations tend to have earlier onset of hematological complications (BMF and MDS/AML), and to have a shorter survival time compared with patients with exon 1 mutations or patients with non-FANCC-related FA. Mutations in the FANCG gene are found in 10% of FA cases. The stop codon mutation E105X accounts for 44% of mutations in German FA-G patients. In a handful of FA patients, mutations in the FANCD2 gene and in BRCA2 genes have been identified. BRCA2 mutations affecting the carboxy-terminus of the protein have been found in patients of FA-B and FA-D1 groups; therefore BRCA2 is the mutant gene for these complementation groups.

Cellularphenotype and function of the FA proteins. Chromosomal instability, the most striking cellular phenotypic feature in FA, is due to spontaneous development of chromosomal double-strand breaks during replication, a phenomenon accentuated by clastogenic agents at low concentrations. Chromosomal instability is a feature shared by many single-gene (ataxia-telengiectasia, Bloom syndrome, Werner syndrome) or multiple-gene (xeroderma pigmentosum, hereditary non-polyposis colorectal cancer, hereditary breast/ovary cancer) diseases. They all demonstrate a defect in pathways respon sible for the maintenance of the integrity of the genome. As a result, a number of mutations with cell-transforming potential accumulate, leading eventually to neoplasia. The crucial biochemical evidence linking FA to the DNA repair pathways, in particular to those involving repair through homologous recombination, was unveiled recently. Mono-ubiquinated FANCD2 interacts in the so-called nuclear foci (these nuclear structures appear during DNA replication in response to DNA damage) with BRCA1 and BRCA2, two proteins with a central role in DNA damage repair pathways; as discussed above, BRCA2 is an FA gene, mutated in FA-B and -D1 patients. In the currently evolving model of FA molecular pathophysiol-ogy, it is predicted that the FANCA, B, C, F, G and E proteins form a nuclear complex, the assembly of which commences in the cytoplasm (Figure 12.5). This core complex could function as the sensor of DNA damage that relays the signal for repair to the FANCD2/BRCA1 complex, possibly by activating FANCD2 through mono-ubiquitination. An important observation supporting the above is the observation that FANCD2 is not ubiquitinated in any of the FA A, B, C, F, G and E complementation groups. However, currently it is unclear whether the core complex itself provides the enzymatic activity for the ubiquitination of FANCD2. None of the proteins involved has a domain with recognized ubiquitin ligase activity; instead, BRCA1 contains a domain which is thought to provide this enzymatic function. BRCA2 is the downstream effector that leads to homologous recombination repair of the double-strand break through activation of the RAD51 complex; however it is also likely that BRCA2 acts upstream at the level of the FA protein core complex. Other biochemical defects reported in FA cells include increased sensitivity to reactive oxygen species, defects of the cell cycle and increased,

Fig. 12.6 The exon structure of the FA genes and their pathogenic mutations (see also text)

From Joenje H, Patel KJ. (2001) The emerging genetic and molecular basis of Fanconi anemia. Nature Reviews in Genetics, 2, 446-459.

Fig. 12.6 The exon structure of the FA genes and their pathogenic mutations (see also text)

From Joenje H, Patel KJ. (2001) The emerging genetic and molecular basis of Fanconi anemia. Nature Reviews in Genetics, 2, 446-459.

IFN-y-mediated, susceptibility to apoptosis. How such diverse functional defects relate to each other and fit with the subcellular localization of the FA proteins remains to be elucidated.

Diagnosis. The clastogen test remains the gold standard clinical diagnostic test; however, complementation studies using retroviral vectors containing all seven genes have now entered clinical practice and this now allows rapid assignment of patients to a specific complementation group. This is followed by screening for pathogenic mutations in the corresponding gene. With this approach, genetic counseling and prenatal and preimplantation diagnosis are now feasible for most families with affected children. In areas with a large Ashkenazi Jewish population (e.g. New York City), screening for polymorphic FA-C alleles is feasible on a wider basis and can be offered to all couples at risk.

Treatment. The conventional management of FA focusses on the consequences of BMF and includes hematopoietic growth factors, blood product support and androgens. About half of the patients respond to androgens initially, but often suffer from significant side-effects, including androgen-induced hepatic adenomas. Eventually all patients become refractory.

HSCT from an HLA-identical, unaffected sibling or from alternative sources is currently the only therapeutic approach that can successfully achieve long-term correction of BMF and possible prevention of MDS and AML. Currently, clinical research focusses on the use of reduced-intensity, non-myeloablative conditioning HSCT regimens in order to reduce short- and long-term mortality. Unfortunately, even after HSCT the patient remains at increased risk of developing solid tumors.

The cloning of FA genes has opened the way to gene therapy for FA patients. There is clinical and experimental evidence that HSCs with corrected phenotype have a survival and growth advantage over uncorrected cells and can support long-term hemopoiesis. Successful transfer of FA genes to a small number of autologous HSCs should therefore be adequate to reduce the severity of BMF. Four FA-C patients underwent gene therapy in a pilot study using a retroviral vector. Although a transient improvement in the clonogenic capacity of the bone marrow progenitors was observed, this was not reflected in the clinical parameters. The use of len-tivirus-based vectors holds greater promise and preclinical results are encouraging.

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