Subtyping analysis of Fanconi anemia by immunoblotting and retroviral gene transfer.
Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least eight complementation groups (A-H). Two of the FA genes (FAA and FAC) have been cloned, and mutations in these genes account for approximately 80% of FA patients. Subtyping of FA patients is an important first step toward identifying candidates for FA gene therapy. In the current study, we analyzed a reference group of 26 FA patients of known subtype. Most of the patients (18/26) were confirmed as either type A or type C by immunoblot analysis with anti-FAA and anti-FAC antisera. In order to resolve the subtype of the remaining patients, we generated retroviral constructs expressing FAA and FAC for transduction of FA cell lines (pMMP-FAA and pMMP-FAC). The pMMP-FAA construct specifically complemented the abnormal phenotype of cell lines from FA-A patients, while pMMP-FAC complemented FA-C cells. In summary, the combination of immunoblot analysis and retroviral-mediated phenotypic correction of FA cells allows a rapid method of FA subtyping.
Pulsipher
M
M
Division of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.
Kupfer
G M
GM
Naf
D
D
Suliman
A
A
Lee
J S
JS
Jakobs
P
P
Grompe
M
M
Joenje
H
H
Sieff
C
C
Guinan
E
E
Mulligan
R
R
D'Andrea
A D
AD
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western / Q000379:methods; D002453:Cell Cycle / Q000187:drug effects; D018797:Cell Cycle Proteins; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D004268:DNA-Binding Proteins; D005199:Fanconi Anemia / Q000145:classification* / Q000175:diagnosis / Q000235:genetics*; D051856:Fanconi Anemia Complementation Group Proteins; D005347:Fibroblasts / Q000187:drug effects; D018014:Gene Transfer Techniques; D005816:Genetic Complementation Test; D015316:Genetic Therapy; D006801:Humans; D007106:Immune Sera; D008214:Lymphocytes / Q000187:drug effects; D016685:Mitomycin / Q000494:pharmacology; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D011506:Proteins / Q000032:analysis* / Q000235:genetics; D012190:Retroviridae / Q000235:genetics
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D051856:Fanconi Anemia Complementation Group Proteins; D007106:Immune Sera; D009687:Nuclear Proteins; D011506:Proteins; D016685:Mitomycin