Complementation analysis in Fanconi anemia: assignment of the reference FA-H patient to group A

Abstract

Fanconi anemia (FA) is an autosomal recessive disorder with diverse clinical symptoms and extensive genetic heterogeneity. Of eight FA genes that have been implicated on the basis of complementation studies, four have been identified and two have been mapped to different loci; the status of the genes supposed to be defective in groups B and H is uncertain. Here we present evidence indicating that the patient who has been the sole representative of the eighth complementation group (FA-H) in fact belongs to group FA-A. Previous exclusion from group A was apparently based on phenotypic reversion to wild-type rather than on genuine complementation in fusion hybrids. To avoid the pitfall of reversion, future assignment of patients with FA to new complementation groups should conform with more-stringent criteria. A new group should be based on at least two patients with FA whose cell lines are excluded from all known groups and that fail to complement each other in fusion hybrids, or, if only one such cell line were available, on a new complementing gene that carries pathogenic mutations in this cell line. On the basis of these criteria, the current number of complementation groups in FA is seven.

Figure 1

Correction of MMC hypersensitivity of EUFA173 cells after retroviral transduction of FANCA cDNA. EUFA173 lymphoblasts were infected with retroviral supernatants carrying either the FANCA cDNA or the empty vector pMMP, essentially according to the study by Pulsipher et al. (1998). After puromycin selection, cells were tested for growth inhibition by MMC. A normal lymphoblastoid cell line (PD7) and the uninfected EUFA173 cells were also included in the assay. Results shown are representative of multiple independent MMC survival assays performed with cells from three separate retroviral infections.

Figure 2

Four FANCA alleles in the fusion hybrid from EUFA173 and HSC72OT cells, with the mutations indicated (2852G→A [Arg951Gln] and E17–31del in EUFA173; and E18–28del [homozygous] in HSC72OT [dotted regions are deletions; drawing is not to scale]). Either mitotic recombination at the “X” or a gene-conversion event would predict the generation of a wild-type allele, which would explain the reverted phenotype of the hybrid cells. PCR primers were chosen as indicated by the arrows, allowing specific amplification of a 200-bp fragment (nucleotides 2748–2947) predicted to have lost the missense mutation after recombination.

Figure 3

Loss of complementation in EUFA173 cells, as a function of time after transfection with FANCA cDNA. Cells were transfected with the episomal vector pDR2 containing the FANCA cDNA (Kruyt et al. 1996) and were grown in the continued presence of hygromycin (200 μg/ml), to select for plasmid-containing cells. Growth inhibition by MMC was tested at 2, 4, and 6 wk after transfection, as indicated.