Molecular Genetic Pathogenesis
TFR2-encoded protein is highly expressed in the hepatocytes and its function is likely related to the hepcidin pathway that regulates iron homeostasis.
The hepatic peptide hepcidin is a circulating hormone that regulates the absorption of dietary iron from the duodenum. Hepcidin expression is inappropriately decreased in hereditary hemochromatosis and is abnormally increased in the anemia of chronic diseases. Other hepatic proteins essential for normal iron homeostasis, including HFE, transferrin receptor protein 2 (TfR2), and hemojuvelin, function at least in part by modulating the expression of hepcidin [Fleming 2005].
Individuals with homozygous TFR2 mutations have increased intestinal iron absorption that causes iron overload. Low/absent levels of urinary hepcidin have been reported in TFR2-related hereditary hemochromatosis [Nemeth et al 2005], suggesting that TFR2 is a modulator of hepcidin. A similar finding of down-regulation (or lack of up-regulation following iron loading) of mRNA of hepcidin in liver has been documented in mice with a tfr2Y245X targeted mutation [Kawabata et al 2005] and in tfr2-knockout mice [Wallace et al 2005].
Figure 1. Schematic representation of the localization of TFR2 mutations. Causal mutations are illustrated in bold above the gene; exonic normal variants are marked below the gene (see Table 2). The two alternatively spliced TFR2 transcripts are also shown. Alternative transcription start sites are indicated by the dotted arrow lines.
Normal allelic variants: TFR2 is 21 kb long and consists of 18 exons. There are two main alternatively spliced variants (see
Figure 1):
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Alpha, corresponding to transcription of all exons. Alpha-TFR2 is prevalently and highly expressed in hepatocytes. Two alpha-TFR2 cDNAs of 2.9 and 2.3 kb are recognized. The first (Genbank accession AF053356) lacks 81 nucleotides in exon 8 and is 18 nucleotides longer in exon 18 [Glockner et al 1998] when compared to the second (Genbank accession AF067864) [Kawabata et al 1999].
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Beta, which has an in-frame transcription start site in exon 4 [Kawabata et al 1999]. Beta-TFR2 cDNA lacks exons 1-3 and has 142 additional bases at its 5' end. Beta TFR2 is expressed ubiquitously at very low levels.
Several exonic normal DNA variants have been described (see Figure 1); they are either silent or missense mutations present also in control individuals [Lee et al 2001, Biasiotto et al 2008]. Two nucleotide normal variants have been identified in the non-coding region of TFR2 [Meregalli et al 2000, Biasiotto et al 2008].
Pathologic allelic variants: Fifteen causal mutations have been reported; most are rare or private [Roetto et al 2002, Lee & Barton 2006, Hsiao et al 2007, Biasiotto et al 2008] (see Table 2):
For more information, see Genomic Databases table and Figure 1.
Normal gene product: TFR2 is a type II transmembrane glycoprotein characterized by short intracellular and transmembrane domains and a large extracellular domain. The TFR2 full-length transcript (the alpha form) originates an 801-amino acid transmembrane protein. Residues 1-80 correspond to the cytoplasmic domain, residues 81-104 correspond to the transmembrane, and amino acids 105-801 correspond to the extracellular domain. Cysteines 89-98 and 108-111 are involved in disulfide bonds, likely responsible for TFR2 homodimerization. A YQRV amino acid motif in the cytoplasmic domain, similar to the internalization signal (YTRF) of transferrin receptor (TFRC), could have the same function.
TFR2 is highly expressed in hepatoma (HEPG2) and in erythroleukemia cell lines (K562) [Kawabata et al 1999]. It is expressed in the liver, especially in the hepatocytes, and at low levels in Kuppfer cells [Zhang et al 2004].
Alpha-TFR2 binds and internalizes transferrin. However, binding occurs at low affinity (25- to 30-fold lower) [Kawabata et al 2000], as compared to that of the transferrin receptor (TFRC). Alpha-TFR2 protein shows significant amino acid homology with TFRC and the prostate-specific membrane antigen (PSMA), especially in the extracellular portion [Kawabata et al 1999].
Beta-TFR2 lacks the cytoplasmic and transmembrane domains and could be an intracellular protein.
TFR2 has no IRE elements in its 5' or 3' UTR and is not transcriptionally regulated by iron. TFR2 does not bind HFE in vitro because residues involved in HFE binding in TFRC are not conserved in TFR2 [West et al 2000]. Analysis of TFR2 distribution pattern in humans revealed by immunohistochemistry showed strong expression in liver and duodenal cells [Deaglio et al 2002]. At this level, an interaction has been documented with the HFE-encoded protein by immunohistochemical techniques [Griffiths & Cox 2003]. TFR2 is also expressed by early erythroid progenitors but not by bone marrow precursors [Calzolari et al 2004].
TFR2-encoded protein in H2 cell lines is stabilized by diferric transferrin, which increases TFR2-encoded protein half-life [Johnson & Enns 2004, Robb & Wessling-Resnick 2004]. In addition, TFR2 mediates a biphasic pattern of transferrin uptake associated with ligand delivery to multivesicular bodies [Robb et al 2004]. Tfr2 stabilization by transferrin reduces Tfr2 lysosomal degradation and directs TFR2 toward the recycling endosome [Johnson et al 2007].
The cytoplasmic domain is that involved in membrane stabilization after TF binding [Chen & Enns 2007].
Two animal models exist: (1) A tfr2-deficient mouse homozygous for tfr2Y245X [Fleming et al 2002], a mutation orthologous to the human p.Tyr250X [Camaschella et al 2000], the tfr2-knockout mouse [Wallace et al 2005], and a conditional hepatic knock out [Wallace et al 2007]. All these mice show iron overload in liver and other organs with the spleen relatively free of iron as in the human disorder [Fleming et al 2002]. (2) The tfr2-null (knockout) shows significant iron overload and is unable to increase hepcidin in response to iron [Wallace et al 2005].
Abnormal gene product: The pathologic variants produce a truncated protein or an abnormally structured protein (e.g., p.Ala621_Gln624del, p.Leu490Arg, p.Gln690Pro). The mutation p.Met172Lys is of interest because it causes a missense in the alpha form but changes the methionine that is the putative initiation codon of betaTFR2 [Roetto et al 2001, Majore et al 2006].