Skip to main content

Advertisement

Log in

Screen for alterations of iron related parameters in N-ethyl-N-nitrosourea-treated mice identified mutant lines with increased plasma ferritin levels

  • Published:
BioMetals Aims and scope Submit manuscript

Abstract

Iron is essential for numerous cellular processes. For diagnostic purposes iron-related parameters in patients are assessed by clinical chemical blood analysis including the analysis of ferritin, transferrin and iron levels. Here, we retrospectively evaluated the use of these parameters in the phenotype-driven Munich N-ethyl-N-nitrosourea mouse mutagenesis project for the generation of novel animal models for human diseases. The clinical chemical blood analysis was carried out on more than 10,700 G1 and G3 offspring of chemically mutagenized inbred C3H mice to detect dominant and recessive mutations leading to deviations in the plasma levels of iron-related plasma parameters. We identified animals consistently exhibiting altered plasma ferritin or transferrin values. Transmission of the phenotypic deviations to the subsequent generations led to the successful establishment of three mutant lines with increased plasma ferritin levels. For two of these lines the causative mutations were identified in the Fth1gene and the Ireb2 gene, respectively. Thus, novel mouse models for the functional analysis of iron homeostasis were established by a phenotype-driven screen for mutant mice.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Aigner B et al (2009a) Generation of N-ethyl-N-nitrosourea-induced mouse mutants with deviations in plasma enzyme activities as novel organ-specific disease models. Exp Physiol 94:412–421

    Article  CAS  PubMed  Google Scholar 

  • Aigner B, Rathkolb B, Wolf E (2009b) Generation and analysis of disease-specific mouse models by clinical chemical screening. In: Mitchem BH, Sharnham CL (eds) Clinical chemistry research. Nova Science, Hauppauge, pp 239–259

    Google Scholar 

  • Aigner B et al (2011) Generation of N-ethyl-N-nitrosourea-induced mouse mutants with deviations in hematological parameters. Mamm Genome 22:495–505. doi:10.1007/s00335-011-9328-4

    Article  CAS  PubMed  Google Scholar 

  • Andrews NC (2008) Forging a field: the golden age of iron biology. Blood 112:219–230

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Barbaric I, Wells S, Russ A, Dear TN (2007) Spectrum of ENU-induced mutations in phenotype-driven and gene-driven screens in the mouse. Environ Mol Mutagen 48:124–142

    Article  CAS  PubMed  Google Scholar 

  • Beaumont C et al (1995) Mutation in the iron responsive element of the L ferritin mRNA in a family with dominant hyperferritinaemia and cataract. Nat Genet 11:444–446

    Article  CAS  PubMed  Google Scholar 

  • Benyamin B et al (2009) Common variants in TMPRSS6 are associated with iron status and erythrocyte volume. Nat Genet 41:1173–1175

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Bermejo F, Garcia-Lopez S (2009) A guide to diagnosis of iron deficiency and iron deficiency anemia in digestive diseases. World J Gastroenterol 15:4638–4643

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Beutler E, Felitti V, Ho NJ, Gelbart T (2002) Relationship of body iron stores to levels of serum ferritin, serum iron, unsaturated iron binding capacity and transferrin saturation in patients with iron storage disease. Acta Haematol 107:145–149

    Article  CAS  PubMed  Google Scholar 

  • Champy MF et al (2008) Genetic background determines metabolic phenotypes in the mouse. Mamm Genome 19:318–331

    Article  CAS  PubMed  Google Scholar 

  • Cohen LA et al (2010) Serum ferritin is derived primarily from macrophages through a nonclassical secretory pathway. Blood 116:1574–1584. doi:10.1182/blood-2009-11-253815

    Article  CAS  PubMed  Google Scholar 

  • Cooperman SS, Meyron-Holtz EG, Olivierre-Wilson H, Ghosh MC, McConnell JP, Rouault TA (2005) Microcytic anemia, erythropoietic protoporphyria, and neurodegeneration in mice with targeted deletion of iron-regulatory protein 2. Blood 106:1084–1091

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Cordes SP (2005) N-ethyl-N-nitrosourea mutagenesis: boarding the mouse mutant express. Microbiol Mol Biol Rev 69:426–439

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Cozzi A et al (2013) Human L-ferritin deficiency is characterized by idiopathic generalized seizures and atypical restless leg syndrome. J Exp Med 210:1779–1791. doi:10.1084/jem.20130315

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Du X et al (2008) The serine protease TMPRSS6 is required to sense iron deficiency. Science 320:1088–1092

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Ferreira C, Santambrogio P, Martin ME, Andrieu V, Feldmann G, Henin D, Beaumont C (2001) H ferritin knockout mice: a model of hyperferritinemia in the absence of iron overload. Blood 98:525–532

    Article  CAS  PubMed  Google Scholar 

  • Gailus-Durner V et al (2005) Introducing the German mouse clinic: open access platform for standardized phenotyping. Nat Methods 2:403–404

    Article  CAS  PubMed  Google Scholar 

  • Galy B et al (2005) Altered body iron distribution and microcytosis in mice deficient in iron regulatory protein 2 (IRP2). Blood 106:2580–2589

    Article  CAS  PubMed  Google Scholar 

  • Girelli D, Corrocher R, Bisceglia L, Olivieri O, De Franceschi L, Zelante L, Gasparini P (1995) Molecular basis for the recently described hereditary hyperferritinemia-cataract syndrome: a mutation in the iron-responsive element of ferritin L-subunit gene (the “Verona mutation”). Blood 86:4050–4053

    CAS  PubMed  Google Scholar 

  • Greth A et al (2012) A novel ENU-mutation in ankyrin-1 disrupts malaria parasite maturation in red blood cells of mice. PLoS One 7:e38999. doi:10.1371/journal.pone.0038999

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Heruth DP et al (2010) Mutation in erythroid specific transcription factor KLF1 causes Hereditary Spherocytosis in the Nan hemolytic anemia mouse model. Genomics 96:303–307

    Article  CAS  PubMed  Google Scholar 

  • Hrabé de Angelis M et al (2000) Genome-wide, large-scale production of mutant mice by ENU mutagenesis. Nat Genet 25:444–447

    Article  PubMed  Google Scholar 

  • Hrabé de Angelis M, Michel D, Wagner S, Becker S, Beckers J (2007) Chemical mutagenesis in mice. In: Fox JG, Barthold SW, Davisson MT, Newcomer CE, Quimby FW, Smith AL (eds) The mouse in biomedical research, vol. 1. History, wild mice, and genetics, 2 edn edn. Academic Press, Burlington, pp 225–260

    Chapter  Google Scholar 

  • Huang H et al (2013) A deep intronic mutation in the ankyrin-1 gene causes diminished protein expression resulting in hemolytic anemia in mice. G3 (Bethesda) 3:1687–1695. doi:10.1534/g3.113.007013

    Article  Google Scholar 

  • Kannengiesser C et al (2009) A new missense mutation in the L ferritin coding sequence associated with elevated levels of glycosylated ferritin in serum and absence of iron overload. Haematologica 94:335–339. doi:10.3324/haematol.2008.000125

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Keays DA, Clark TG, Campbell TG, Broxholme J, Valdar W (2007) Estimating the number of coding mutations in genotypic and phenotypic driven N-ethyl-N-nitrosourea (ENU) screens: revisited. Mamm Genome 18:123–124

    Article  PubMed  Google Scholar 

  • Klempt M, Rathkolb B, Aigner B, Wolf E (2006) Clinical chemical screen. In: Chambon P, Brown S, Hrabé de Angelis M (eds) Standards of mouse model phenotyping. Wiley-VCH, Weinheim, pp 87–107

    Chapter  Google Scholar 

  • Knovich MA, Storey JA, Coffman LG, Torti SV, Torti FM (2009) Ferritin for the clinician. Blood Rev 23:95–104

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Krieg L, Milstein O, Krebs P, Xia Y, Beutler B, Du X (2011) Mutation of the gastric hydrogen-potassium ATPase alpha subunit causes iron-deficiency anemia in mice. Blood 118:6418–6425. doi:10.1182/blood-2011-04-350082

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Lambe T et al (2009) Identification of a Steap3 endosomal targeting motif essential for normal iron metabolism. Blood 113:1805–1808

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Loeb WF, Quimby FW (1999) The clinical chemistry of laboratory animals, Second edition edn. Taylor & Francis, Philadelphia

    Google Scholar 

  • MacKenzie EL, Iwasaki K, Tsuji Y (2008) Intracellular iron transport and storage: from molecular mechanisms to health implications. Antioxid Redox Signal 10:997–1030. doi:10.1089/ars.2007.1893

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • McLaren CE et al (2011) Genome-wide association study identifies genetic loci associated with iron deficiency. PLoS One 6:e17390. doi:10.1371/journal.pone.0017390

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • McLaren CE et al (2012) Associations between single nucleotide polymorphisms in iron-related genes and iron status in multiethnic populations. PLoS One 7:e38339. doi:10.1371/journal.pone.0038339

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Mohr M, Klempt M, Rathkolb B, Hrabé de Angelis M, Wolf E, Aigner B (2004) Hypercholesterolemia in ENU-induced mouse mutants. J Lipid Res 45:2132–2137

    Article  CAS  PubMed  Google Scholar 

  • Muckenthaler M et al (2003) Relationships and distinctions in iron-regulatory networks responding to interrelated signals. Blood 101:3690–3698

    Article  CAS  PubMed  Google Scholar 

  • Muckenthaler MU, Galy B, Hentze MW (2008) Systemic iron homeostasis and the iron-responsive element/iron-regulatory protein (IRE/IRP) regulatory network. Annu Rev Nutr 28:197–213

    Article  CAS  PubMed  Google Scholar 

  • Nicklas W, Baneux P, Boot R, Decelle T, Deeny AA, Fumanelli M, Illgen-Wilcke B (2002) Recommendations for the health monitoring of rodent and rabbit colonies in breeding and experimental units. Lab Anim 36:20–42

    Article  CAS  PubMed  Google Scholar 

  • Nolan PM et al (2000) A systematic, genome-wide, phenotype-driven mutagenesis programme for gene function studies in the mouse. Nat Genet 25:440–443

    Article  CAS  PubMed  Google Scholar 

  • Rank G et al (2009) Novel roles for erythroid Ankyrin-1 revealed through an ENU-induced null mouse mutant. Blood 113:3352–3362

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Rathkolb B et al (2000) The clinical-chemical screen in the Munich ENU mouse mutagenesis project: screening for clinically relevant phenotypes. Mamm Genome 11:543–546

    Article  CAS  PubMed  Google Scholar 

  • Robledo RF et al (2010) Analysis of novel sph (spherocytosis) alleles in mice reveals allele-specific loss of band 3 and adducin in alpha-spectrin-deficient red cells. Blood 115:1804–1814

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Siatecka M, Sahr KE, Andersen SG, Mezei M, Bieker JJ, Peters LL (2010) Severe anemia in the Nan mutant mouse caused by sequence-selective disruption of erythroid Kruppel-like factor. Proc Natl Acad Sci USA 107:15151–15156

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Takahasi KR, Sakuraba Y, Gondo Y (2007) Mutational pattern and frequency of induced nucleotide changes in mouse ENU mutagenesis. BMC Mol Biol 8:52

    Article  PubMed Central  PubMed  Google Scholar 

  • Tanaka T et al (2010) A genome-wide association analysis of serum iron concentrations. Blood 115:94–96. doi:10.1182/blood-2009-07-232496

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Thompson K et al (2003) Mouse brains deficient in H-ferritin have normal iron concentration but a protein profile of iron deficiency and increased evidence of oxidative stress. J Neurosci Res 71:46–63

    Article  CAS  PubMed  Google Scholar 

  • Wang W, Knovich MA, Coffman LG, Torti FM, Torti SV (2010) Serum ferritin: past, present and future. Biochim Biophys Acta 1800:760–769. doi:10.1016/j.bbagen.2010.03.011

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Yanagawa T et al (2004) Nrf2 deficiency causes tooth decolourization due to iron transport disorder in enamel organ. Genes Cells 9:641–651

    Article  CAS  PubMed  Google Scholar 

  • Yuki KE et al (2013) Suppression of hepcidin expression and iron overload mediate Salmonella susceptibility in ankyrin 1 ENU-induced mutant. PLoS One 8:e55331. doi:10.1371/journal.pone.0055331

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Zohn IE et al (2007) The flatiron mutation in mouse ferroportin acts as a dominant negative to cause ferroportin disease. Blood 109:4174–4180

    Article  PubMed Central  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Andreas Mayer, Sandra Hoffmann and Elfi Holupirek and our animal caretaker team for technical support.

Funding

This work was supported by the German Human Genome Project (DHGP) and the National Genome Research Network (NGFN) (MHdA, EW) and by the German Federal Ministry of Education and Research (Infrafrontier Grant 01KX1012).

Conflict of interest

The authors declare that they have no competing interests.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Birgit Rathkolb.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Rathkolb, B., Klempt, M., Sabrautzki, S. et al. Screen for alterations of iron related parameters in N-ethyl-N-nitrosourea-treated mice identified mutant lines with increased plasma ferritin levels. Biometals 28, 293–306 (2015). https://doi.org/10.1007/s10534-015-9824-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10534-015-9824-1

Keywords

Navigation