A rapid method for retrovirus-mediated identification of complementation groups in Fanconi anemia patients.
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.
Chandra
Saurabh
S
Division of Experimental Hematology and Fanconi Anemia Comprehensive Care Center, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229, USA.
Levran
Orna
O
Jurickova
Ingrid
I
Maas
Chiel
C
Kapur
Rick
R
Schindler
Detlev
D
Henry
Rashida
R
Milton
Kelly
K
Batish
Sat Dev
SD
Cancelas
Jose A
JA
Hanenberg
Helmut
H
Auerbach
Arleen D
AD
Williams
David A
DA
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
D051856:Fanconi Anemia Complementation Group Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins