A novel ENU-generated truncation mutation lacking the spectrin-binding and C-terminal regulatory domains of Ank1 models severe hemolytic hereditary spherocytosis.

Abstract

OBJECTIVE:

Hereditary spherocytosis (HS) is a heterogeneous group of spontaneously arising and inherited red blood cell disorders ranging from very mild subclinical cases to severe and life-threatening cases, with symptoms linked directly to the severity of the mutation at the molecular level. We investigated a novel mouse model in which the heterozygotes present with the diagnostic hallmarks of mild HS and surviving homozygotes phenocopy severe hemolytic HS.

MATERIALS AND METHODS:

We used N-ethyl-N-nitrosourea mutagenesis to generate random point mutations in the mouse genome and a dominant screen to identify mouse models of human hematopoietic disease. Gene mapping of the HS strain revealed a unique in-frame nonsense mutation arising from a single base transversion in exon 27 of Ank1 (strain designation: Ank1(E924X)). Employing conventional hematopoietic, pathological, biochemical, and cell biology assays, we characterized heterozygous and homozygous Ank1(E924X) mice at the biochemical, cellular, and pathophysiological levels.

RESULTS:

Although Ank1(E924X/E924X) red blood cell ghosts lack abundant full-length ankyrin-1 isoforms, N-terminal epitope ankyrin-1 antibodies reveal a band consistent with the theoretical size of a truncated mutant ankyrin-1. Using domain-specific antibodies, we further show that this protein lacks both a spectrin-binding domain and a C-terminal regulatory domain. Finally, using antisera that detect C-terminal residues of the products of alternative Ank1 transcripts, we find unique immunoreactive bands not observed in red blood cell ghosts from wild-type or Ank1(E924X) heterozygous mice, including a band similar in size to full-length ankyrin-1.

CONCLUSIONS:

The Ank1(E924X) strain provides a novel tool to study Ank1 and model HS.

• Authors: Michael R Hughes, Nicole Anderson, Steven Maltby, Justin Wong, Zorana Berberovic, Connie S Birkenmeier, D James Haddon, Kamal Garcha, Ann Flenniken, Lucy R Osborne, S Lee Adamson, Janet Rossant, Luanne L Peters, Mark D Minden, Robert F Paulson, Chen Wang, Dwayne L Barber, Kelly M McNagny, William L Stanford
• Journal: Experimental hematology (Exp Hematol) Vol. 39 Issue 3 Pg. 305-20, 320.e1-2 (Mar 2011) ISSN: 1873-2399 [Electronic] Netherlands
• PMID: 21193012 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

Chemical References

• Alkylating Agents
• Ank1 protein, mouse
• Ankyrins
• Codon, Nonsense
• Ethylnitrosourea

Topics

• Alkylating Agents (adverse effects, pharmacology)
• Amino Acid Sequence
• Animals
• Ankyrins (genetics, metabolism)
• Codon, Nonsense
• Disease Models, Animal
• Erythrocytes (metabolism)
• Ethylnitrosourea (adverse effects, pharmacology)
• Female
• Humans
• Male
• Mice
• Mice, Mutant Strains
• Protein Binding
• Sequence Deletion
• Spherocytosis, Hereditary (chemically induced, genetics, metabolism)