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Subtyping Analysis of Fanconi Anemia by Immunoblotting and Retroviral Gene Transfer

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Abstract

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.

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References

  1. Liu J, Buchwald M, Walsh CE, Young NS. (1994) Fanconi anemia and novel strategies for therapy. Blood 84: 3995–4007.

    PubMed  CAS  Google Scholar 

  2. D’Andrea AD, Grompe M. (1997) Molecular biology of Fanconi anemia: Implications for diagnosis and therapy. Blood 90: 1725–1736.

    PubMed  Google Scholar 

  3. Alter BP, Young NS. (1993) The bone marrow failure syndromes. In: Nathan DG, Oski FA (eds). Hematology of Infancy and Childhood, 4th ed., Vol 1. W.B. Saunders; Philadelphia, pp. 216–316.

    Google Scholar 

  4. Davies SM, Khan S, Wagner JE, Arthur DC, Auerbach AD, Ramsay NKC, Weisdorf DJ. (1996) Unrelated donor bone marrow transplantation for Fanconi anemia. Bone Marrow Transplant. 17: 43–47.

    PubMed  CAS  Google Scholar 

  5. Kohli-Kumar M, Shahidi NT, Broxmeyer HE, Masterson M, Delaat C, Sambrano J, Morris C, Auerbach AD, Harris RE. (1993) Haemopoietic stem/progenitor cell transplant in Fanconi anaemia using HLA-matched sibling umbilical cord blood cells. Br. J. Haematol. 85: 419.

    Article  CAS  PubMed  Google Scholar 

  6. Kohli-Kumar M, Morris C, Delaat C, Sambrano J, Masterson M, Mueller R, Shahidi NT, Yanik G, Desantes K, Friedman DJ, Auerbach AD, Harris RE. (1994) Bone marrow transplantation in Fanconi anemia using matched sibling donors. Blood 84: 2050–2054.

    PubMed  CAS  Google Scholar 

  7. Walsh CE, Grompe M, Vanin E, Buchwald M, Young NS, Nienhuis AW, Liu JM. (1994) A functionally active retrovirus vector for gene therapy in Fanconi Anemia Group C. Blood 84: 453–459.

    PubMed  CAS  Google Scholar 

  8. Strathdee CA, Gavish H, Shannon WR, Buchwald M. (1992) Cloning of cDNAs for Fanconi’s anaemia by functional complementation. Nature 356: 763–767.

    Article  CAS  PubMed  Google Scholar 

  9. Lo Ten Foe JR, Rooimans MA, Bosnoyan-Collins L, et al. (1996) Expression cloning of a cDNA for the major Fanconi anemia gene, FAA. Nat. Genet. 14: 320–323.

    Article  CAS  PubMed  Google Scholar 

  10. The Fanconi Anemia/Breast Cancer Consortium. (1996) Positional cloning of the Fanconi Anaemia Group A gene. Nat. Genet. 14: 324–328.

    Article  Google Scholar 

  11. Fu K-L, Lo Ten Foe JR, Joenje H, Rao KW, Liu JM, Walsh CE. (1997) Functional correction of Fanconi Anemia Group A hematopoietic cells by retroviral gene transfer. Blood 90: 3296–3303.

    PubMed  CAS  Google Scholar 

  12. Auerbach AD. (1993) Fanconi anemia diagnosis and the diepoxybutane (DEB) test. Exp. Hematol. 21: 731–733.

    PubMed  CAS  Google Scholar 

  13. Auerbach AD, Adler B, Chaganti RSK. (1981) Prenatal and postnatal diagnosis and carrier detection of Fanconi anemia by a cytogenetic method. Pediatrics 67: 128.

    PubMed  CAS  Google Scholar 

  14. Auerbach AD, Alter BP. (1989) Prenatal and postnatal diagnosis of aplastic anemia. In: Alter BP (ed). Methods in Hematology: Perinatal Hematology. Churchill Livingstone, Edinburgh, p. 225.

    Google Scholar 

  15. Shipley J, Rodeck CH, et al. (1984) Mitomycin C-induced chromosome damage in fetal blood cultures and prenatal diagnosis of Fanconi’s anaemia. Prenat. Diagn. 4: 217.

    Article  CAS  PubMed  Google Scholar 

  16. Verlander PC, Lin JD, Udono MU, Q. Zhang, Gibson RA, Mathew CG, Auerbach AD. (1994) Mutation analysis of the Fanconi anemia gene FAC. Am. J. Hum. Genet. 54: 595–601.

    CAS  Google Scholar 

  17. Gillio AP, Verlander PC, Batish SD, Giampietro PF, Auerbach AD. (1997) Phenotypic consequences of mutations in the Fanconi anemia FAC gene: An international Fanconi anemia registry study. Blood 90: 105–110.

    PubMed  CAS  Google Scholar 

  18. Levran O, Erlich T, Magdalena N, Gregory JJ, Batish SD, Verlander PC, Auerbach AD. (1997) Sequence variation in the Fanconi anemia gene FAA. Proc. Natl. Acad. Sci. U.S.A. 94: 13051–13056.

    Article  CAS  PubMed  Google Scholar 

  19. Buchwald M. (1995) Complementation groups: One or more per gene. Nat. Genet. 11: 228–230.

    Article  CAS  PubMed  Google Scholar 

  20. Joenje H, Ten FL, Oostra A, Berkel CV, Rooimans M, Schroeder S, Kurth T, Wegner R, Gille J, Buchwald M, Arwert F. (1995) Classification of Fanconi anemia patients by complementation analysis: Evidence for a fifth genetic subtype. Blood 86: 2156–2160.

    PubMed  CAS  Google Scholar 

  21. Joenje H, Oostra AB, Wijker M, di Summa FM, van Berkel CGM, Rooimans MA, Ebell W, van Weel M, Pronk JC, Buchwald M, Arwert F. (1997) Evidence for at least eight Fanconi anemia genes. Am. J. Hum. Genet. 61: 940–944.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Whitney M, Thayer M, Reifsteck C, Olson S, et al. (1995) Microcell mediated chromosome transfer maps the Fanconi anemia group D gene to chromosome 3p. Nat. Genet. 11: 341–343.

    Article  CAS  PubMed  Google Scholar 

  23. Yamashita T, Barber DL, Zhu Y, Wu N, D’Andrea AD. (1994) The Fanconi anemia polypeptide FACC is localized to the cytoplasm. Proc. Natl. Acad. Sci. U.S.A. 91: 6712–6716.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Riviere I, Brose K, Mulligan RC. (1995) Effects of retroviral vectors design on expression of human adenosine deaminase in murine bone marrow transplant recipients engrafted with genetically modified cells. Proc. Natl. Acad. Sci. U.S.A. 92: 6733–6737.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Colicelli J, Goff SP. (1986) Isolation of a recombinant murine leukemia virus utilizing a new primer tRNA. J. Virol. 57: 37–45.

    PubMed  PubMed Central  CAS  Google Scholar 

  26. Ory D, Neugeboren B, Mulligan R. (1996) A stable human-derived packaging cell line for production of high-titer retrovirus/vesicular stomatitis virus G pseudotypes. Proc. Natl. Acad. Sci. U.S.A. 93: 11400–11406.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Hanenberg H, Xiao XL, Dilloo D, Hashino K, Kato I, Williams DA. (1996) Co-locolization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells. Nature Med. 8: 876–882.

    Article  Google Scholar 

  28. Yamashita T, Wu N, Kupfer G, Corless C, Joenje H, Grompe M, D’Andrea AD. (1996) The clinical variability of Fanconi anemia (type C) results from expression of an amino terminal truncated FAC polypeptide with partial activity. Blood 87: 4424–4432.

    PubMed  CAS  Google Scholar 

  29. Kupfer GM, Naf D, Suliman A, Pulsipher M, D’Andrea AD. (1997) The Fanconi anemia proteins, FAA and FAC, interact to form a nuclear complex. Nat. Genet. 17: 487–490.

    Article  CAS  PubMed  Google Scholar 

  30. Moustacchi E, Papadopoulo D, Diatloff-Zito C, Buchwald M. (1987) Two complementation groups of Fanconi’s anemia differ in their phenotypic response to a DNA cross-linking treatment. Hum. Genet. 75: 45–47.

    PubMed  CAS  Google Scholar 

  31. Strathdee CA, Duncan AMV, Buchwald M. (1992) Evidence for at least four Fanconi anemia genes including FACC on chromosome 9. Nat. Genet. 1: 196–198.

    Article  CAS  PubMed  Google Scholar 

  32. Jakobs PM, Fiddler-Odell E, Reifsteck C, Olson S, Moses RE, Grompe M. (1997) Complementation group assignments in Fanconi anemia fibroblast cell lines from North America. Somat. Cell Mol. Genet. 23: 1–7.

    Article  CAS  PubMed  Google Scholar 

  33. Kaiser TN, Lojewski A, Dougherty C, Juergens L, Sahar E, Latt SA. (1982) Flow cytometric characterization of the response of Fanconi’s anemia cells to mitomycin C treatment. Cytometry 2: 291–297.

    Article  CAS  PubMed  Google Scholar 

  34. Kubbies M, Schindler D, Hoehn H, Schinzel A, Rabinovich PS. (1985) Endogenous blockage and delay of the chromosome cycle despite normal recruitment and growth phase explain poor proliferation and frequent edomitosis in Fanconi anemia cells. Am. J. Hum. Genet. 37: 1022–1030.

    PubMed  PubMed Central  CAS  Google Scholar 

  35. Ianzano L, D’Apolito M, Centra M, Savino M, Levran O, Auerbach AD, Cleton-Jansen A-M, Doggett NA, Pronk JC, Tipping AJ, Gibso RA, Mathew CG, Whitmore SA, Apostolou S, Callen DF, Zelante L, Savoia A. (1997) The genomic organization of the Fanconi Anemia Group A (FAA) gene. Genomics 41: 309–314.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by NIH grants R01-HL52725 and P01-HL54785. M.P. is supported by KO8-HLO3751-01 and G.M.K. is supported by K08-H103420. D.N. is supported by a fellowship from the Leukemia Society of America (LSA), and A.D.D. is a Scholar of the LSA.

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Authors and Affiliations

  1. Division of Pediatric Oncology, Dana-Farber Cancer Institute, 44 Binney Street, Mayer Building, Room 640, Boston, Massachusetts, 02115, USA

    Michael Pulsipher, Gary M. Kupfer, Dieter Naf, Ahmed Suliman, Colin Sieff, Eva Guinan &  Alan D. D’Andrea

  2. Department of Pediatrics, Children’s Hospital, Harvard Medical School, Boston, Massachusetts, USA

    Michael Pulsipher, Gary M. Kupfer, Jeng-Shin Lee, Colin Sieff, Eva Guinan &  Alan D. D’Andrea

  3. Department of Genetics, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts, USA

    Richard Mulligan

  4. Department of Pediatrics, Oregon Health Sciences University, Portland, Oregon, USA

    Petra Jakobs & Markus Grompe

  5. Department of Human Genetics, Free University, Amsterdam, The Netherlands

    Hans Joenje

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  1. Michael Pulsipher
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  2. Gary M. Kupfer
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Correspondence to Alan D. D’Andrea.

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Communicated by D. Nathan.

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Pulsipher, M., Kupfer, G.M., Naf, D. et al. Subtyping Analysis of Fanconi Anemia by Immunoblotting and Retroviral Gene Transfer. Mol Med 4, 468–479 (1998). https://doi.org/10.1007/BF03401752

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  • DOI: https://doi.org/10.1007/BF03401752

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