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Targeted Mutagenesis of the Murine Transferrin Receptor-2 Gene Produces Hemochromatosis

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Targeted Mutagenesis of the Murine Transferrin Receptor-2 Gene Produces Hemochromatosis Targeted mutagenesis of the murine transferrin receptor-2 gene produces hemochromatosis Robert E. Fleming*†, John R. Ahmann*, Mary C. Migas*, Abdul Waheed†, H. Phillip Koeffler‡, Hiroshi Kawabata‡, Robert S. Britton§, Bruce R. Bacon§, and William S. Sly†¶ *Department of Pediatrics, †Edward A. Doisy Department of Biochemistry and Molecular Biology, and §Department of Internal Medicine, Division of Gastroenterology and Hepatology, Saint Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis, MO 63104; and ‡Department of Medicine, University of California School of Medicine, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, CA 90048 Contributed by William S. Sly, June 17, 2002 Hereditary hemochromatosis (HH) is a common genetic disorder (Y250X) had a clinical picture similar to HFE-associated HH, characterized by excess absorption of dietary iron and progressive including hepatic iron loading (6). This form of iron overload was iron deposition in several tissues, particularly liver. The vast ma- designated HH type 3 (Online Mendelian Inheritance in Man jority of individuals with HH are homozygous for mutations in the 604250) to distinguish it from HFE-associated HH (HH type 1) HFE gene. Recently a second transferrin receptor (TFR2) was dis- and juvenile-onset HH (HH type 2). Several additional TFR2 covered, and a previously uncharacterized type of hemochroma- gene mutations have been described since in patients with iron tosis (HH type 3) was identified in humans carrying mutations in overload, suggesting that the iron homeostasis abnormalities are the TFR2 gene. To characterize the role for TFR2 in iron homeosta- caused by functional loss of TFR2 (7, 8). By contrast, functional sis, we generated mice in which a premature stop codon (Y245X) loss of Tfr1 (in knockout mice) produces an embryonic lethal was introduced by targeted mutagenesis in the murine Tfr2 coding phenotype (9). These observations indicate that the roles for sequence. This mutation is orthologous to the Y250X mutation TFR1 and TFR2 in iron homeostasis are not redundant. Because identified in some patients with HH type 3. The homozygous TFR1 forms a complex with HFE, it seemed plausible that TFR2 Tfr2Y245X mutant mice showed profound abnormalities in param- may associate also with HFE. However, in vitro experiments eters of iron homeostasis. Even on a standard diet, hepatic iron using expressed soluble TFR2 and HFE failed to demonstrate concentration was several-fold higher in the homozygous Tfr2Y245X significant interaction (3). The mechanism by which TFR2 mutant mice than in wild-type littermates by 4 weeks of age. The participates in iron homeostasis thus remains unknown. To study iron deposition in the mutant mice was predominantly hepatocel- the role of TFR2 in iron homeostasis, we generated a murine lular and periportal. The mean splenic iron concentration in the model for the human C250Y TFR2 mutation. Codon 245 is the homozygous Tfr2Y245X mutant mice was significantly less than that murine orthologue to codon 250 in human TFR2 and is located observed in the wild-type mice. The homozygous Tfr2Y245X mutant in a region that is conserved between mouse and human mice also demonstrated elevated transferrin saturations. There genomes. We observed that by 4 weeks of age, mice homozygous were no significant differences in parameters of erythrocyte pro- for the Y245X mutation developed periportal hepatic iron duction including hemoglobin levels, hematocrits, erythrocyte in- loading, splenic iron sparing, and elevated serum transferrin dices, and reticulocyte counts. Heterozygous Tfr2Y245X mice did not saturations. Thus the Tfr2Y245X mutant mouse seems to provide differ in any measured parameter from wild-type mice. This study a faithful model for the abnormalities in iron homeostasis confirms the important role for TFR2 in iron homeostasis and observed in patients with loss of TFR2. provides a tool for investigating the excess iron absorption and abnormal iron distribution in iron-overload disorders. Materials and Methods Gene Targeting in Embryonic Stem (ES) Cells and Generation of Mutant iron ͉ liver ͉ gene targeting Mice. We used a mouse cDNA probe to isolate clones from a 129͞SvJ mouse Lambda Fix II genomic library (Stratagene). ransferrin receptor (TFR)2 is a recently identified type II Restriction mapping and nucleotide sequencing were performed on selected clones. Site-directed mutagenesis (QuikChange, Tmembrane protein with significant homology to the classical Ϸ transferrin receptor (TFR1). Both TFR1 and TFR2 are capable Stratagene) was performed on an 2.9-kb SalI-to-XbaI murine of transporting transferrin-bound iron into the cell (1) and Tfr2 genomic fragment extending from intron 3 to intron 6 to supporting cell growth (2). However, their properties differ in substitute G for C at the third position of codon 245. The several critical ways. TFR2 has a lower affinity for holotrans- mutation changes the tyrosine codon to a stop codon (Y245X). The mutated genomic DNA fragment was inserted into the ferrin than does TFR1 (1, 3). The expression pattern of TFR2 MEDICAL SCIENCES mRNA is quite distinct from TFR1 as well (4, 5). In particular, targeting vector pPNT-Cass lox A. This vector is a modification the TFR2 gene is expressed at much higher levels in liver of the vector pCass lox A, provided by M. Capecchi (University compared with other tissues, whereas the TFR1 gene demon- of Utah, Salt Lake City) by the addition of sequences expressing strates little hepatic expression. TFR1 and TFR2 differ also in thymidine kinase. The vector contains a cassette flanked by loxP their response to changes in cellular iron status. The TFR1 elements including sequences conferring neomycin resistance transcript contains multiple iron-responsive elements in the and expressing cre recombinase under direction of a testes- 3Ј-untranslated region. These elements stabilize the TFR1 tran- specific angiotensin-converting enzyme promoter (10). After script under conditions of low cellular iron. The TFR2 transcript expression of cre recombinase in the germ line of the male does not contain these elements, and TFR2 message and TFR2 chimeric transgenic mice, the cassette is excised from the genome. The mutated Ϸ2.9-kb SalI-to-XbaI genomic fragment protein levels vary little with changes in iron status (4, 5). The Ϸ observation that hepatic expression of Tfr2 persists despite iron was ligated at one end of the cassette, and an 3.6-kb XbaI overload in a murine Hfe gene knockout model of hereditary genomic fragment extending from intron 6 to intron 8 of the hemochromatosis (HH) led us to suggest that Tfr2-mediated iron uptake contributes to the hepatocellular iron loading ob- Abbreviations: TFR, transferrin receptor; HH, hereditary hemochromatosis; ES, embryonic ␤ ␤ served in HH (4). Surprisingly, however, it was discovered that stem; 2m, 2-microglobulin. individuals homozygous for a truncation mutation in TFR2 ¶To whom reprint requests should be addressed. E-mail: [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.162360699 PNAS ͉ August 6, 2002 ͉ vol. 99 ͉ no. 16 ͉ 10653–10658 Downloaded by guest on October 1, 2021 mouse Tfr2 gene was ligated at the opposite end. The targeting using G418. Liver-tissue membrane preparations were per- vector (25 ␮g) was linearized and introduced into the 129͞Sv- formed by homogenization of the tissue in PBS with protease derived ES cell line RW4 (Genome Systems, St. Louis; 1 ϫ 107 inhibitors (156 ␮g/ml benzamidine, 1 ␮g/ml aprotinin, and 50 cells) by electroporation (230 V and 500 ␮F) in a Bio-Rad gene ␮g/ml PMSF) followed by centrifugation at 100,000 ϫ g for 30 pulser. After 24 hr, the cells were placed under selection with 400 min. The pellets were sonicated in PBS with protease inhibitors, ␮g͞ml G418 (GIBCO͞BRL) and 2 ␮M ganciclovir (Syntex, Palo and protein concentrations were determined by using a modified Alto, CA) for 6 days. Southern blot analysis was performed on Bradford reagent (Bio-Rad). SDS͞PAGE was performed under genomic DNA from resistant clones. The DNA was digested with reducing conditions on whole-cell homogenates (20 ␮g) or BglII and hybridized with an external probe (an Ϸ1.2-kb XbaI- liver-membrane preparations (50 ␮g), and the proteins were to-EcoRI genomic fragment extending from intron 1a to intron transferred onto poly(vinylidene difluoride) by using an elec- 3). Two clones demonstrating homologous recombination with troblotting apparatus. The poly(vinylidene difluoride) mem- the targeting vector were identified. One of these clones was used branes then were reacted with a 1:2,000 dilution of rabbit for injection into the blastocysts of C57BL͞6J mice and trans- anti-mouse Tfr2 antiserum followed by a 1:5,000 dilution of ferred into pseudopregnant female mice as described (11). peroxidase-conjugated anti-rabbit IgG (Sigma). Immuno- Chimeric male offspring were bred to C57BL͞6J females. The reactive proteins were detected by using luminol followed by agouti F1 offspring were tested for transmission of the disrupted autoradiography. allele (as well as removal of the selection cassette), by Southern blot analysis of HindIII-digested genomic DNA, and an external Transferrin Saturations. Mice were bled by cardiac puncture. probe (an Ϸ2.3-kb HindIII-to-XbaI genomic fragment extending Serum iron and total iron-binding capacity were measured by from intron 3 to intron 6). In accordance with Mouse Nomen- using the protocol of Fielding (12). Transferrin saturation was clature Rules and Guidelines from The Jackson Laboratories calculated as (serum iron͞total iron-binding capacity) ϫ 100%. (www.informatics.jax.org͞mgihome͞nomen͞table.shtml), the Values from wild-type (n ϭ 10), homozygous mutant (n ϭ 9), mouse line housing the Tfr2Y245X mutation was designated and heterozygous (n ϭ 5) mice were compared by using the tm1Slu Tfr2 . Heterozygous matings of the F1 mice were carried out Kruskal–Wallis test followed by Dunn’s multiple-comparison to produce homozygous F2 mutant mice. The F2 litters were tests. weaned at 3 weeks and fed a standard diet (LM-485 Teklad sterilized mouse diet 7012, Harlan Teklad, Madison, WI), which Tissue Iron Concentrations.
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