title: A rapid method for retrovirus-mediated identification of complementation groups in Fanconi anemia patients.
abstract: Fanconi anemia (FA) is a rare autosomal recessive disorder that results from mutations in at least 11 different genes. Recent studies have demonstrated that clinical progression of the disease may be influenced by inter- and intragenic variations, emphasizing the importance of identifying the complementation groups. In the present study we have employed bicistronic retrovirus vectors that coexpress FA-specific cDNAs for complementation groups A, C, F, and G, together with the enhanced green fluorescence protein (EGFP), allowing for specific analysis of transduced EGFP+ cells within bulk cultures by flow cytometry. In addition, the assay relies on the correction of the characteristic FA-associated G2/M arrest after treatment of cells with DNA-damaging agents, which is analyzed by flow cytometry. Results obtained with this assay matched the complementation groups known for 12 control lymphoblast cell lines tested. We report here the results obtained for 48 FA patients with unknown complementation groups using this new assay. Complementation groups were identified for 24 patients. We have identified mutations in the genes corresponding to the assigned complementation group in 23 samples. This assay has now been established in a standardized fashion for complementation assignments in FA patients and the subsequent directing of rapid mutation analysis in those patients.
authors.affiliation: Division of Experimental Hematology and Fanconi Anemia Comprehensive Care Center, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229, USA.
authors.forename: Saurabh
authors.initials: S
authors.lastname: Chandra
authors.forename: Orna
authors.initials: O
authors.lastname: Levran
authors.forename: Ingrid
authors.initials: I
authors.lastname: Jurickova
authors.forename: Chiel
authors.initials: C
authors.lastname: Maas
authors.forename: Rick
authors.initials: R
authors.lastname: Kapur
authors.forename: Detlev
authors.initials: D
authors.lastname: Schindler
authors.forename: Rashida
authors.initials: R
authors.lastname: Henry
authors.forename: Kelly
authors.initials: K
authors.lastname: Milton
authors.forename: Sat Dev
authors.initials: SD
authors.lastname: Batish
authors.forename: Jose A
authors.initials: JA
authors.lastname: Cancelas
authors.forename: Helmut
authors.initials: H
authors.lastname: Hanenberg
authors.forename: Arleen D
authors.initials: AD
authors.lastname: Auerbach
authors.forename: David A
authors.initials: DA
authors.lastname: Williams
mesh_terms: D002460:Cell Line; D004252:DNA Mutational Analysis / Q000379:methods*; D005199:Fanconi Anemia / Q000235:genetics*; D051856:Fanconi Anemia Complementation Group Proteins / Q000235:genetics*; D018014:Gene Transfer Techniques; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D009154:Mutation; D012190:Retroviridae / Q000235:genetics*; D014162:Transfection
chemical_list: D051856:Fanconi Anemia Complementation Group Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins