A New Model of Hereditary Spherocytosis Demonstrates Profound Homeostatic Compensation in Severely Anemic Mice.

Michael R. Hughes, Nicole Anderson, Steven Maltby, Justin Wong, Zorana Milenkovic, Chen Wang, William L. Stanford and Kelly M. McNagny
This article has an Erratum 111(9):4830


As part of a concerted program to generate new models of human disease we have generated multiple heritable mutant mouse strains using ethylnitrosourea (ENU) random mutagenesis combined with a dominant blood screen. Here we describe a mutant strain with dominantly-inherited red blood cell (RBC) low mean corpuscular volume. Preliminary phenotypic analysis of affected mice demonstrated spherical RBC morphology, increased osmotic fragility, mild reticulocytosis and a reduction in circulating lifetime - features characteristic of human hereditary spherocytosis. Gene mapping revealed the causative point mutation to be in the mouse ankyrin-1 gene (Ank1) locus at exon 26 - a G to T transversion causing substitution of a glutamate codon with a premature stop (Ank1*895). The resulting gene product is a truncated ankyrin-1 protein (terminating at amino acid 894) consisting primarily of the N-terminal band 3-binding domain without a functional ZU5 domain (spectrin-binding) or regulatory/death domain (Fig.1). The Ank1*895 allele results in a hypomorph such that stably-expressed protein isolated from RBC ghost preparations is undetectable by Coomasie stain and only barely detectable by Western. Our ENU-generated mutation is distinct from the spontaneous mutation identified in the normoblastosis (nb) mouse (Peters et al. 1991. J. Cell Biol; Birkenmeier et al. 2003. Hematol J.) which yields a functional protein product with intact band 3- and spectrin-binding domains (Fig.1).

Figure 1.

Schematic representation of mouse ankyrin-1 peptide showing sites of truncation products encoded by the ENU-generated nonsense mutation Ank1*895 (Glu → stop) and the normoblastosis (nb) mouse (Ank1nb). While the phenotype of the heterozygous (Ank1*895/+) mutant line on the C3H background is mild, intercross breeding of mutant mice did not yield pups homozygous for the mutant allele - suggesting an embryonic lethal phenotype. Surprisingly, when the C3H-Ank1*895 line was bred with the SvImJ/129 strain we were able to obtain viable homozygous Ank1*895/*895 offspring from intercross of the Ank1*895/+ 129xC3H hybrid mutant line. Homozygous Ank1*895 mice were obtained at low frequency and displayed a severe phenotype with remarkable splenomegaly. In this study we have generated a novel mouse model of hereditary spherocytosis and examined the compensatory mechanisms that permit the survival of homozygous Ank1*895 mice from embryo to adults. In addition, we determined the stability of Ank1*895 protein in homozygous mice and its effect on the assembly of RBC membrane structural complexes in the absence of full-length ankyrin-1. MRH and SM are fellows of the CIHR/HSFC Strategic Training Program in Transfusion Science at the UBC Centre for Blood Research (CBR). KMM is a Michael Smith Foundation for Health Research Scholar and CBR member. This study was supported by a group operating grant from the CIHR (FRN 74611) and fellowships from the Heart & Stroke/Richard Lewar Centre of Excellence.