Volume 132, Issue 2, Pages 679-686 (February 2007) Genetic Study of Variation in Normal Mouse Iron Homeostasis Reveals Ceruloplasmin as an HFE-Hemochromatosis Modifier Gene  Laurent Gouya, Francoise Muzeau, Anne–Marie Robreau, Philippe Letteron, Eric Couchi, Saïd Lyoumi, Jean–Charles Deybach, Herve Puy, Robert Fleming, Peter Demant, Carole Beaumont, Bernard Grandchamp  Gastroenterology  Volume 132, Issue 2, Pages 679-686 (February 2007) DOI: 10.1053/j.gastro.2006.11.024 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Spleen and liver iron load in the C3H/DiSnA, the B10, and the 27 HcB/Dem strains. Results are the means of assays in 9 to 12 male mice. Sixteen RCS (open circles) do not differ significantly from the C3H/DiSnA background strain. Five RCS have similar spleen iron content but either higher (open triangles) or lower (solid triangles) liver iron; 5 RCS have comparable liver iron but lower spleen iron (solid diamonds); and 1 RCS has a higher iron content in both liver and spleen (solid square). Tissue iron loads were analyzed by analysis of variance, and the differences between the strains were evaluated using Dunnett T3 multiple comparison test. Solid circles represent either the B10 donor strain or the C3H/DiSnA background strain. Gastroenterology 2007 132, 679-686DOI: (10.1053/j.gastro.2006.11.024) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Linkage analysis in (C3H/DiSnA × HcB-2/Dem) F2 hybrids. (A) Likelihood ratio statistic, LOD score, and statistical significance obtained by permutation test, and percentage of the variance explained by the PromCp locus for liver iron, serum iron, and serum transferrin. (B) LOD score plot along chromosome 3, showing the maximum likelihood for the position of the QTL modulating liver iron. The x-axis shows genetic distances in cM. (C–F) Liver iron, spleen iron, serum iron, and serum transferrin of the F2 (C3H/DiSnA × HcB-2/Dem) mice according to their genotype at the PromCp locus. Results are presented as box plots showing the median, the quartiles, the 90th, and the 10th percentile. “C” and “B” indicate that the PromCp alleles originate from the C3H/DiSnA and the B10 strains, respectively. The Mann–Whitney U test was used to compare means. Gastroenterology 2007 132, 679-686DOI: (10.1053/j.gastro.2006.11.024) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Functional consequences of the R435X mutation. (A) Liver Cp mRNA quantifications for 6 inbred strains of mice. We used the comparative Ct method; GAPDH is the reference amplification gene, and the B10 strain is the reference condition. Results are expressed as a percentage of the mRNA value for the B10 strain. Data were obtained from 5 mice in each group. (B) Serum ferroxidase activity was measured in 5 to 10 mice in each group and expressed as a percentage of the activity found in the serum of C3H/HeNrj mice. Mice are identified according to their genotype at the PromCp locus, and Cp+/+ refers to C3H/HeNrj wild-type mice, Cp+/m to C3H mice heterozygous for the R435X mutation, and Cp−/− to C3H/DiSnA CpR435X/R435X mice. (C) Changes in serum iron concentration 4 hours after intravenous infusion of damaged red blood cells in 7 C3H/HeNrj CpR435/R435 (Cp+/+, shaded boxes) and 7 C3H/DiSnA CpR435X/R435X (Cpm/m, open boxes) mice. Results are expressed as a percentage of the serum iron measured at T0. (C and D) Results are presented as box plots showing the median, the quartiles, the 90th, and the 10th percentiles. Gastroenterology 2007 132, 679-686DOI: (10.1053/j.gastro.2006.11.024) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Genetic interactions between Cp and Hfe locus. This figure shows liver iron levels in 10-week-old C3H mice carrying null or wild-type alleles of Hfe and Cp genes in various combinations. Results are expressed as means, with their 95% confidence interval, of 8 to 14 values. Cp+/+ refers to C3HR435/R435 mice, Cp+/m to C3H mice heterozygous for the R435X mutation, and Cpm/m to C3H CpR435X homozygous mice. (A) Effect of CpR435X mutation at heterozygous and homozygous state on liver iron levels in a C3H Hfe−/− background. (B) Effect of Hfe null allele heterozygosity and homozygosity on liver iron levels in C3H CpR435X homozygous mice. Mann–Whitney U test was used to compare means. Gastroenterology 2007 132, 679-686DOI: (10.1053/j.gastro.2006.11.024) Copyright © 2007 AGA Institute Terms and Conditions