Genetic subtyping of Fanconi anemia by comprehensive mutation screening.

Fanconi anemia (FA) is a recessively inherited syndrome with predisposition to bone marrow failure and malignancies. Hypersensitivity to cross-linking agents is a cellular feature used to confirm the diagnosis. The mode of inheritance is autosomal recessive (12 subtypes) as well as X-linked (one subtype). Most genetic subtypes have initially been defined as "complementation groups" by cell fusion studies. Here we report a comprehensive genetic subtyping approach for FA that is primarily based on mutation screening, supplemented by protein expression analysis and by functional assays to test for pathogenicity of unclassified variants. Of 80 FA cases analyzed, 73 (91%) were successfully subtyped. In total, 92 distinct mutations were detected, of which 56 were novel (40 in FANCA, eight in FANCC, two in FANCD1, three in FANCE, one in FANCF, and three in FANCG). All known complementation groups were represented, except D2, J, L, and M. Three patients could not be classified because proliferating cell cultures from the probands were lacking. In cell lines from the remaining four patients, immunoblotting was used to determine their capacity to monoubiquitinate FANCD2. In one case FANCD2 monoubiquitination was normal, indicating a defect downstream. In the remaining three cases monoubiquitination was not detectable, indicating a defect upstream. In the latter four patients, pathogenic mutations in a known FA gene may have been missed, or these patients might represent novel genetic subtypes. We conclude that direct mutation screening allows a molecular diagnosis of FA in the vast majority of patients, even in cases where growing cells from affected individuals are unavailable. Proliferating cell lines are required in a minority (<15%) of the patients, to allow testing for FANCD2 ubiquitination status and sequencing of FANCD2 using cDNA, to avoid interference from pseudogenes.

Evidence for subcomplexes in the Fanconi anemia pathway.

Fanconi anemia (FA) is a genomic instability disorder, clinically characterized by congenital abnormalities, progressive bone marrow failure, and predisposition to malignancy. Cells derived from patients with FA display a marked sensitivity to DNA cross-linking agents, such as mitomycin C (MMC). This observation has led to the hypothesis that the proteins defective in FA are involved in the sensing or repair of interstrand cross-link lesions of the DNA. A nuclear complex consisting of a majority of the FA proteins plays a crucial role in this process and is required for the monoubiquitination of a downstream target, FANCD2. Two new FA genes, FANCB and FANCL, have recently been identified, and their discovery has allowed a more detailed study into the molecular architecture of the FA pathway. We demonstrate a direct interaction between FANCB and FANCL and that a complex of these proteins binds FANCA. The interaction between FANCA and FANCL is dependent on FANCB, FANCG, and FANCM, but independent of FANCC, FANCE, and FANCF. These findings provide a framework for the protein interactions that occur "upstream" in the FA pathway and suggest that besides the FA core complex different subcomplexes exist that may have specific functions other than the monoubiquitination of FANCD2.