Chapter 31

## Abstract

1. Fanconi anemia (FA) is an autosomal recessive disorder that is characterized clinically by diverse congenital abnormalities and a predisposition to bone marrow failure and malignancy, particularly acute myelogenous leukemia (AML). FA patients exhibit extreme clinical heterogeneity and may have abnormalities in any major organ system. It is recognized that the FA phenotype is so variable, with considerable overlap with the phenotypes of a variety of genetic and nongenetic diseases, that diagnosis on the basis of clinical manifestations alone is difficult.

2. FA is found in all races and ethnic groups and has been widely reported to have a carrier frequency of 1 in 300. This estimate was based on the incidence of affected individuals before the full spectrum of the FA phenotype was recognized. The true gene frequency is likely to be considerably higher than this; a low estimate would result from an incomplete ascertainment of cases before the widespread application of chromosomal breakage tests for FA diagnosis. Up to 0.5 percent of the general population may be heterozygous at an FA locus.

3. Hypersensitivity of FA cells to the clastogenic (chromosome-breaking) effect of crosslinking agents provides a unique cellular marker for the disorder. This is used as a diagnostic criterion because of the difficulty of diagnosing FA on the basis of clinical manifestations alone. Comparative studies have led to the choice of diepoxybutane (DEB) as the agent most widely used for FA diagnosis. The crosslinking test can be used to identify preanemic patients as well as patients with aplastic anemia or leukemia who may or may not have the physical stigmata associated with FA.

4. The hypersensitivity of FA cells to crosslinking agents has been used to assess complementation in somatic cell hybrids. Complementation groups usually are considered to represent distinct disease genes, and for FA, at least four groups (A, C, D, and G) represent distinct genes. The first FA gene isolated by expression cloning methodology (FANCC, alias FAC) mapped to chromosome 9q22.3 by in situ hybridization. FANCA (alias FAA) was mapped by linkage of the disease in FA-A families to microsatellite markers positioned close to the telomere of chromosome 16 (16q24.3). FANCD (alias FAD) was mapped to 3p22-26 by microcell-mediated chromosome transfer. FANCG (alias FAG) is identical with the previously isolated human gene XRCC9, which was mapped to 9p13. Considerable variability in the prevalence of the different complementation groups has been observed among various ethnic groups. Overall, FA-A is the most prevalent group, accounting for 60 to 65 percent of all FA cases.

5. A cDNA expression cloning procedure was adapted and used successfully to clone the gene defective in FA-C cells (FANCC). The FANCC coding region contains 14 exons and leads to a predicted protein of 558 amino acids. The predicted structure of FANCC does not resemble that of any known protein and has no obvious functional domains. The protein is found primarily in the cytoplasm, although approximately 10 percent is in the nucleus. FANCC appears to play a direct role in protecting cells against the damage produced by crosslinking agents.

6. Homologous recombination in embryonic stem (ES) cells has been used to target the endogenous Fancc locus, with the consequent removal of exon 8 or exon 9. These cells have been used to derive strains of mice (Fancc−/−) in which no active Fancc protein is produced. The mutant mice show the characteristic FA sensitivity to crosslinkers but do not demonstrate any morphologic or hematopoietic phenotypes up to 1 year of age. In addition to the cellular sensitivity, the principal phenotype of Fancc−/− mice is decreased fertility of both male and female animals. This phenotype appears to be a more severe version of similar disease-related complications in FA patients.

7. FANCA was cloned by two parallel approaches. One was essentially the same as that used to clone FANCC. A cDNA clone was identified that corrected the crosslink hypersensitivity of FA-A cells but not that of FA-C cells. FANCA also was identified through positional cloning of the 16q24.3 region. The defective gene was identified by fine mapping, contig isolation, and exon trapping. FANCA codes for a predicted protein of 1455 amino acids that has no strong homologies to known proteins. On the basis of a predicted nuclear localization signal, the protein may be localized to the nucleus. The gene contains 43 exons spanning approximately 80 kb. More than 70 mutations in FANCA have been described worldwide. The heterogeneity of the mutation spectrum and the frequency of intragenic deletions present a considerable challenge for the molecular diagnosis of FA-A.

8. A cDNA representing the FA-G gene FANCG was isolated from the same expression library used for the functional cloning of FANCC and FANCA. The 2.5-kb complementing cDNA was identified as identical to human XRCC9, a novel gene defined by its capacity to partially complement the MMC-sensitive Chinese hamster mutant UV40. The encoded FAG/XRCC9 protein has no sequence similarities to any other known protein, including FAA and FAC, and has no functional motifs.

9. Transplantation with hematopoietic stem cells from bone marrow or umbilical cord blood currently offers the only possibility for a cure for bone marrow failure in FA as well as a possible cure for or prevention of leukemia. Recent analyses of HLA-matched sibling transplants show that increased survival is associated with younger age, less severe hematologic disease, and absence of malignant transformation.

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