montalcino, A zebrafish model for variegate porphyria

Dooley, Kimberly A.; Fraenkel, Paula G.; Langer, Nathaniel B.; Schmid, Bettina; Davidson, Alan J.; Weber, Gerhard; Chiang, Ken; Foott, Helen; Dwyer, Caitlin; Wingert, Rebecca A.; Zhou, Yi; Paw, Barry H.; Zon, Leonard I.; Tübingen 2000 Screen Consortium,

doi:10.1016/j.exphem.2008.04.008

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Volume 36, Issue 9 , Pages 1132-1142, September 2008

montalcino, A zebrafish model for variegate porphyria

Kimberly A. Dooley
- Division of Hematology/Oncology, Children's Hospital and Dana-Farber Cancer Institute and Harvard Medical School, Howard Hughes Medical Institute, Boston, Mass., USA
- Current address: Broad Institute/MIT, Cambridge, MA, USA.
- Offprint requests to: Leonard I. Zon, M.D., Howard Hughes Medical Institute, Children's Hospital of Boston, Boston, MA 02115
Paula G. Fraenkel
- Division of Hematology/Oncology, Children's Hospital and Dana-Farber Cancer Institute and Harvard Medical School, Howard Hughes Medical Institute, Boston, Mass., USA
- Current address: Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Boston, MA, USA.
Nathaniel B. Langer
- Division of Hematology, Brigham and Women's Hospital, Harvard Medical School, Boston, Mass., USA
Bettina Schmid
- Division of Hematology/Oncology, Children's Hospital and Dana-Farber Cancer Institute and Harvard Medical School, Howard Hughes Medical Institute, Boston, Mass., USA
Alan J. Davidson
- Division of Hematology/Oncology, Children's Hospital and Dana-Farber Cancer Institute and Harvard Medical School, Howard Hughes Medical Institute, Boston, Mass., USA
- Current address: Massachusetts General Hospital, Boston, MA, USA.
Gerhard Weber
- Division of Hematology/Oncology, Children's Hospital and Dana-Farber Cancer Institute and Harvard Medical School, Howard Hughes Medical Institute, Boston, Mass., USA
Ken Chiang
- Division of Hematology/Oncology, Children's Hospital and Dana-Farber Cancer Institute and Harvard Medical School, Howard Hughes Medical Institute, Boston, Mass., USA
Helen Foott
- Division of Hematology/Oncology, Children's Hospital and Dana-Farber Cancer Institute and Harvard Medical School, Howard Hughes Medical Institute, Boston, Mass., USA
Caitlin Dwyer
- Division of Hematology/Oncology, Children's Hospital and Dana-Farber Cancer Institute and Harvard Medical School, Howard Hughes Medical Institute, Boston, Mass., USA
Rebecca A. Wingert
- Division of Hematology/Oncology, Children's Hospital and Dana-Farber Cancer Institute and Harvard Medical School, Howard Hughes Medical Institute, Boston, Mass., USA
- Current address: Massachusetts General Hospital, Boston, MA, USA.
Yi Zhou
- Division of Hematology/Oncology, Children's Hospital and Dana-Farber Cancer Institute and Harvard Medical School, Howard Hughes Medical Institute, Boston, Mass., USA
Barry H. Paw
- Division of Hematology, Brigham and Women's Hospital, Harvard Medical School, Boston, Mass., USA
Leonard I. Zon
- Division of Hematology/Oncology, Children's Hospital and Dana-Farber Cancer Institute and Harvard Medical School, Howard Hughes Medical Institute, Boston, Mass., USA
Tübingen 2000 Screen Consortium
- Tübingen 2000 Screen Consortium: F. Bebber van, E. Busch-Nentwich, R. Dahm, H. G. Frohnhöfer, H. Geiger, D. Gilmour, S. Holley, J. Hooge, D. Jülich, H. Knaut, F. Maderspacher, C. Neumann, T. Nicolson, C. Nüsslein-Volhard, H. Roehl, U. Schönberger, C. Seiler, C. Söllner, M. Sonawane, A. Wehner, C. Weiler and B. Schmid at the Max-Planck-Institut für Entwicklungsbiologie, Spemannstrasse 35, 72076 Tübingen, Germany. U. Hagner, E. Hennen, C. Kaps, A. Kirchner, T. I. Koblizek, U. Langheinrich, C. Metzger, R. Nordin, M. Pezzuti, K. Schlombs, J. deSantana-Stamm, T. Trowe, G. Vacun, A. Walker, and C. Weiler at Artemis Pharmaceuticals/Exelixis Deutchland GmbH, Neurather Ring 1, S51063 Köln, Germany.

Received 7 November 2007; received in revised form 27 February 2008; accepted 15 April 2008. published online 12 June 2008.

Objective

Inherited or acquired mutations in the heme biosynthetic pathway leads to a debilitating class of diseases collectively known as porphyrias, with symptoms that can include anemia, cutaneous photosensitivity, and neurovisceral dysfunction. In a genetic screen for hematopoietic mutants, we isolated a zebrafish mutant, montalcino (mno), which displays hypochromic anemia and porphyria. The objective of this study was to identify the defective gene and characterize the phenotype of the zebrafish mutant.

Materials and Methods

Genetic linkage analysis was utilized to identify the region harboring the mno mutation. Candidate gene analysis together with reverse transcriptase polymerase chain reaction was utilized to identify the genetic mutation, which was confirmed via allele-specific oligo hybridizations. Whole mount in situ hybridizations and o-dianisidine staining were used to characterize the phenotype of the mno mutant. mRNA and morpholino microinjections were performed to phenocopy and/or rescue the mutant phenotype.

Results

Homozygous mno mutant embryos have a defect in the protoporphyrinogen oxidase (ppox) gene, which encodes the enzyme that catalyzes the oxidation of protoporphyrinogen. Homozygous mutant embryos are deficient in hemoglobin, and by 36 hours post-fertilization are visibly anemic and porphyric. The hypochromic anemia of mno embryos was partially rescued by human ppox, providing evidence for the conservation of function between human and zebrafish ppox.

Conclusion

In humans, mutations in ppox result in variegate porphyria. At present, effective treatment for acute attacks requires the administration intravenous hemin and/or glucose. Thus, mno represents a powerful model for investigation, and a tool for future screens aimed at identifying chemical modifiers of variegate porphyria.