The Hemochromatosis Founder Mutation in HLA-H Disrupts β2-Microglobulin Interaction and Cell Surface Expression

Abstract

We recently reported the positional cloning of a candidate gene for hereditary hemochromatosis (HH), called HLA-H, which is a novel member of the major histocompatibility complex class I family. A mutation in this gene, cysteine 282 3 tyrosine (C282Y), was found to be present in 83% of HH patient DNAs, while a second variant, histidine 63 3 aspartate (H63D), was enriched in patients heterozygous for C282Y. The functional relevance of either mutation has not been described. Coimmunoprecipitation studies of cell lysates from human embryonic kidney cells transfected with wild-type or mutant HLA-H cDNA demonstrate that wild-type HLA-H binds ␤ 2 -microglobulin and that the C282Y mutation, but not the H63D mutation, completely abrogates this interaction. Immunofluorescence labeling and subcellular fractionations demonstrate that while the wild-type and H63D HLA-H proteins are expressed on the cell surface, the C282Y mutant protein is localized exclusively intracellularly. This report describes the first functional significance of the C282Y mutation by suggesting that an abnormality in protein trafficking and/or cell-surface expression of HLA-H leads to HH disease.Hereditary hemochromatosis (HH) 1 is an autosomal recessive disorder of iron metabolism and represents one of the most common inherited disorders in individuals of Northern European descent with an estimated carrier frequency between 1 in 8 and 1 in 10 (1, (2). In patients with HH, excessive iron deposition in a variety of organs leads to multi-organ dysfunction. Recently, we reported a mutation in a novel MHC class I-like gene, called HLA-H (3). Eighty-three percent of HH patient DNAs were found to be homozygous for this mutation, which consists of a single base transition of G to A and results in a change of cysteine 282 3 tyrosine (C282Y). Subsequent reports have confirmed the high frequency of this founder mutation in other HH patients (4 -6), providing further support that HLA-H is the primary HH locus. A second missense mutation, histidine 63 3 aspartate (H63D), was also reported that was enriched in heterozygotes with the C282Y mutation (eight of nine cases) (3). The specific role that either of these mutations in HLA-H play in the etiology of HH disease has not been elucidated.The HLA-H protein is similar to MHC class I family molecules including HLA-A2, nonclassical class I molecules such as HLA-G, and the human neonatal Fc receptor (FcRn). All four of the invariant cysteine residues that form disulfide bridges in the ␣ 2 and ␣ 3 domains of MHC class I family members are present in the HLA-H protein. One of these conserved cysteine residues is altered in the C282Y mutation. The integrity of the conserved disulfide linkages has been suggested to be critical for proper maintenance of the secondary and tertiary structure of the protein allowing interactions with accessory molecules such as ␤ 2 -microglobulin (7). Importantly, the functional significance of an interaction between ␤ 2 -microglobulin and an unknown class I-like molecule in HH disease was s...

Defective trafficking and localization of mutated transferrin receptor 2: implications for type 3 hereditary hemochromatosis

“…These include type 1 hemochromatosis, in which the C282Y mutation in the HFE protein affects secondary structure, resulting in its inability to bind to ␤ 2 -microglobulin, causing retention in the ER and loss of expression at the cell surface (8). The majority of mutations in the cystic fibrosis transmembrane conductance regulator prevent its trafficking to the cell surface and affect its ability to regulate chloride transport (36).…”

Hepatocyte-targeted HFE and TFR2 control hepcidin expression in mice

“…8 The C282Y mutation in HFE disrupts a disulfide bond in the ␣3 domain, leading to misfolding, lack of association with ␤2-microglobulin, and failure to traffic to the cell surface. 9 The Hfe knockout mouse (Hfe Ϫ/Ϫ ) also develops iron overload, confirming that the HFE-C282Y mutation confers loss of rather than gain of function. 10 Although the importance of HFE in iron regulation is apparent in patients with HH and murine models, 10,11 the underlying mechanism by which HFE regulates iron metabolism is only beginning to be understood.…”

High prevalence of abnormal glucose homeostasis secondary to decreased insulin secretion in individuals with hereditary haemochromatosis

“…The wild-type HFE protein forms a heterodimer with β 2 -microglobulin. The C282Y mutation of HFE prevents binding of β 2 -microglobulin, resulting in unstable production of HFE protein on the cell surface [4]. It has not been established how this interaction is related to the observed changes in iron balance.…”