Targeted mutagenesis of the murine transferrin receptor-2 gene produces hemochromatosis

Abstract

Hereditary hemochromatosis (HH) is a common genetic disorder characterized by excess absorption of dietary iron and progressive iron deposition in several tissues, particularly liver. The vast majority of individuals with HH are homozygous for mutations in the HFE gene. Recently a second transferrin receptor (TFR2) was discovered, and a previously uncharacterized type of hemochromatosis (HH type 3) was identified in humans carrying mutations in the TFR2 gene. To characterize the role for TFR2 in iron homeostasis, we generated mice in which a premature stop codon (Y245X) was introduced by targeted mutagenesis in the murine Tfr2 coding sequence. This mutation is orthologous to the Y250X mutation identified in some patients with HH type 3. The homozygous Tfr2(Y245X) mutant mice showed profound abnormalities in parameters of iron homeostasis. Even on a standard diet, hepatic iron concentration was several-fold higher in the homozygous Tfr2(Y245X) mutant mice than in wild-type littermates by 4 weeks of age. The iron deposition in the mutant mice was predominantly hepatocellular and periportal. The mean splenic iron concentration in the homozygous Tfr2(Y245X) mutant mice was significantly less than that observed in the wild-type mice. The homozygous Tfr2(Y245X) mutant mice also demonstrated elevated transferrin saturations. There were no significant differences in parameters of erythrocyte production including hemoglobin levels, hematocrits, erythrocyte indices, and reticulocyte counts. Heterozygous Tfr2(Y245X) mice did not differ in any measured parameter from wild-type mice. This study confirms the important role for TFR2 in iron homeostasis and provides a tool for investigating the excess iron absorption and abnormal iron distribution in iron-overload disorders.

Fig 1.

Tfr2^Y245X allele. The genomic region surrounding exon 6 of the Tfr2^Y245X allele is represented. The position of the single C-to-G nucleotide substitution is demonstrated. The hatched region of intron 6 downstream of the XbaI site represents the retained polylinker sequence and loxP element from the transgene construct. The retained sequence includes two HindIII sites that allow distinguishing the wild-type and mutant alleles by Southern blot using the designated HindIII-to-XbaI genomic fragment as a probe. The mutant allele can be identified also by PCR using oligos at the designated positions, which gives a longer product from the mutant than from the wild-type allele.

Fig 2.

Southern blot analysis of genomic DNA from wild-type (wt) and Tfr2^Y245X mutant mice. A representative Southern blot of HindIII-digested genomic DNA isolated from tail samples from a wild-type (+/+), a Tfr2^Y245X heterozygous (+/m), and a homozygous (m/m) mouse and probed with the ³²P-labeled HindIII-to-XbaI genomic DNA fragment designated in Fig. 1. A 4.3-kb restriction fragment is evident from the wild-type allele and a 2.3-kb fragment from the mutant allele.

Fig 3.

Northern blot analysis of Tfr2 mRNA expression in liver of wild-type and Tfr2^Y245X mutant mice. Fifteen micrograms of total cellular RNA from liver tissue of a wild-type (+/+), a heterozygous Tfr2^Y245X mutant (+/m), and a homozygous mutant (m/m) mouse were hybridized with a probe for mouse Tfr2. The blot was rehybridized with a probe for β-actin (Lower).

Fig 4.

Western blot analysis of Tfr2 protein in hepatic membranes of wild-type and Tfr2^Y245X mutant mice. Whole-cell homogenates from the designated cell lines (lanes 1–3) or hepatic membrane preparations from the designated mice (lanes 4–6) were subjected to SDS/PAGE under reducing conditions, blotted, and reacted with an antibody to the intracellular domain of Tfr2.

Fig 5.

Hepatic iron concentrations in wild-type and Tfr2^Y245X mutant mice. Nonheme iron concentrations (μg/gram, dry weight) from 4-week-old wild-type (+/+), heterozygous Tfr2^Y245X (+/m), and homozygous Tfr2^Y245X (m/m) littermates are presented as mean ± SEM. *, P < 0.001 vs. each other group.

Fig 6.

Perls' staining of liver sections. Representative sections from a homozygous Tfr2^Y245X (m/m) and wild-type (+/+) mouse stained for iron with the Perls' Prussian blue technique are shown. Staining is hepatocellular and predominantly periportal in the homozygous Tfr2^Y245X mouse. CV, central vein (short arrow); PV, portal vein (long arrow).

Fig 7.

Splenic iron concentrations in wild-type and Tfr2^Y245X mutant mice. Nonheme iron concentrations (μg/gram, dry weight) from 4-week-old wild-type (+/+), heterozygous Tfr2^Y245X (+/m), and homozygous Tfr2^Y245X (m/m) littermates are presented as mean ± SEM. *, P < 0.001 vs. each other group.