Genetic characterization of FA in Spanish Gypsy patients. (A) FANCD2 immunoblotting revealed 2 isoforms in a wild-type (WT) cell, no signal in a FANCD2-deficient cell line (PD20), and a nonubiquitinated isoform in an FA-A cell line or in cell lines derived from Spanish Gypsy patients nos. 55, 122, and 109, indicating that the genetic defect is upstream of FANCD2 monoubiquitination. Immunoblotting experiments were performed following a standard Western blot method with a commercially available antibody against FANCD2 (Santa Cruz Biotech, Santa Cruz, CA). (B) Subtyping by retrovirus-mediated cDNA gene transduction in a cell line derived from a Spanish Gypsy patient with FA. The complementation group was determined by retroviral transduction of cDNA of FA genes essentially as previously reported.16 Briefly, peripheral blood T cells were proliferated in the presence of monoclonal antibodies anti-CD3 and anti-CD28. Afterward, cells were transduced with retroviral vectors containing FANCA (2 vectors), FANCC, FANG cDNA, and control vector (MOCK) and then evaluated for MMC hypersensitivity to detect phenotypic cellular correction. Only retrovirus-encoding wild-type FANCA cDNA led to reversion of the cellular hypersensitivity to mitomycin C (MMC), indicating that the patient belonged to complementation group FA-A. (C) Spanish Gypsy patients with FA with a 295C>T base change, as detected by DNA sequencing, leading to a stop codon (Q99X). All obligate carriers were heterozygote for this mutation. (D) FANCA immunoblotting with an affinity-purified antibody against the C-terminus of the FANCA protein in 2 Spanish Gypsy patients (no. 55 and no. 122) with FA, and a wild-type cell line. FANCA signal (upper band) is not present in the Gypsy patients, consistent with the truncating nature of the Spanish Gypsy mutation. The lower band is an unspecific signal that serves as an internal control and is equally present in all cell extracts.
