title: 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.
  
  
  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.affiliation: The Biomedical Research Centre, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Hughes
authors.forename: Nicole
authors.initials: N
authors.lastname: Anderson
authors.forename: Steven
authors.initials: S
authors.lastname: Maltby
authors.forename: Justin
authors.initials: J
authors.lastname: Wong
authors.forename: Zorana
authors.initials: Z
authors.lastname: Berberovic
authors.forename: Connie S
authors.initials: CS
authors.lastname: Birkenmeier
authors.forename: D James
authors.initials: DJ
authors.lastname: Haddon
authors.forename: Kamal
authors.initials: K
authors.lastname: Garcha
authors.forename: Ann
authors.initials: A
authors.lastname: Flenniken
authors.forename: Lucy R
authors.initials: LR
authors.lastname: Osborne
authors.forename: S Lee
authors.initials: SL
authors.lastname: Adamson
authors.forename: Janet
authors.initials: J
authors.lastname: Rossant
authors.forename: Luanne L
authors.initials: LL
authors.lastname: Peters
authors.forename: Mark D
authors.initials: MD
authors.lastname: Minden
authors.forename: Robert F
authors.initials: RF
authors.lastname: Paulson
authors.forename: Chen
authors.initials: C
authors.lastname: Wang
authors.forename: Dwayne L
authors.initials: DL
authors.lastname: Barber
authors.forename: Kelly M
authors.initials: KM
authors.lastname: McNagny
authors.forename: William L
authors.initials: WL
authors.lastname: Stanford
mesh_terms: D000477:Alkylating Agents / Q000009:adverse effects* / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D017487:Ankyrins / Q000235:genetics / Q000378:metabolism; D018389:Codon, Nonsense; D004195:Disease Models, Animal; D004912:Erythrocytes / Q000378:metabolism*; D005038:Ethylnitrosourea / Q000009:adverse effects* / Q000494:pharmacology; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D008817:Mice, Mutant Strains; D011485:Protein Binding; D017384:Sequence Deletion; D013103:Spherocytosis, Hereditary / Q000139:chemically induced / Q000235:genetics / Q000378:metabolism
chemical_list: D000477:Alkylating Agents; C491707:Ank1 protein, mouse; D017487:Ankyrins; D018389:Codon, Nonsense; D005038:Ethylnitrosourea