Naturally variant autosomal and sex-linked loci determine the severity of iron overload in beta 2-microglobulin-deficient mice

Abstract

Hereditary hemochromatosis (HH) is a common chronic human genetic disorder whose hallmark is systemic iron overload. Homozygosity for a mutation in the MHC class I heavy chain paralogue gene HFE has been found to be a primary cause of HH. However, many individuals homozygous for the defective allele of HFE do not develop iron overload, raising the possibility that genetic variation in modifier loci contributes to the HH phenotype. Mice deficient in the product of the beta(2)-microglobulin (beta(2)M) class I light chain fail to express HFE and other MHC class I family proteins, and they have been found to manifest many characteristics of the HH phenotype. To determine whether natural genetic variation plays a role in controlling iron overload, we performed classical genetic analysis of the iron-loading phenotype in beta(2)M-deficient mice in the context of different genetic backgrounds. Strain background was found to be a major determinant in iron loading. Sex played a role that was less than that of strain background but still significant. Resistance and susceptibility to iron overload segregated as complex genetic traits in F(1) and back-cross progeny. These results suggest the existence of naturally variant autosomal and Y chromosome-linked modifier loci that, in the context of mice genetically predisposed by virtue of a beta(2)M deficiency, can profoundly influence the severity of iron loading. These results thus provide a genetic explanation for some of the variability of the HH phenotype.

Figure 1

Influence of strain background on hepatic nonheme iron concentration in β₂M-deficient mice. Iron concentration was determined from ≈1-cm² sections of liver from B2m−/− mice with the following backgrounds: C3H, four female and five male; B6, two female and eight male; AKR, five female and five male; NOD, five female and five male. Data are the mean ± SEM. C3H vs. B6, P < 0.05; C3H vs. AKR, P < 0.001; C3H vs. NOD, P < 0.001; B6 vs. AKR, P < 0.001; AKR vs. NOD, not significant.

Figure 2

Influence of strain background on hepatic iron accumulation in β₂M-deficient mice determined by histopathological analysis. Photomicrographs of liver sections stained with Perls' Prussian blue from representative B2m−/− mice of the indicated ages and backgrounds. Photomicrographs of liver sections from B2m+/+ mice showed no stainable iron (data not shown).

Figure 3

Sex differences in hepatic nonheme iron concentration in β₂M-deficient mice. Iron concentration was determined in whole livers of B2m−/− mice. Data are the mean ± SEM, and the number of animals is shown for each point. (A) Three-month-old female and male C3H, NOD, and (C3H × NOD)F₁ mice. P < 0.01 by two-way ANOVA with Bonferroni posttest analysis: C3H female vs. C3H male, NOD female vs. NOD male, F₁ female vs. F₁ male. (B) Longitudinal analysis of hepatic iron concentration of female and male NOD-B2m−/− mice. Comparisons between age-matched female and male by Student's t test: *, P < 0.05; **, P < 0.01; ***, P < 0.005; NS = not significant.

Figure 4

Back-cross analysis of susceptibility to iron overload in β₂M-deficient mice. Distribution of hepatic iron concentrations among the indicated 3-month-old C3H and NOD parental, F₁, and back-cross B2m−/− mice. Total numbers of mice in each group are indicated. Histogram indicates distribution of numbers of mice at the indicated iron concentration. P < 0.0005 by two-way ANOVA with Bonferroni posttest analysis: C3H female ♀ vs. NOD female, C3H female vs. F₁ female, NOD female vs. F₁ female, C3H male ♂ vs. NOD male, C3H male vs. F₁ male, NOD male vs. F₁ male, back-cross female vs. back-cross male.