Cynthia Prass

Hereditary hemochromatosis (HH) is a common autosomal recessive disease characterized by increased iron absorption and progressive iron storage that results in damage to major organs in the body. Recently, a candidate gene for HH called HFE encoding a major histocompatibility complex class I-like protein was identified by positional cloning. Nearly 90% of Caucasian HH patients have been found to be homozygous for the same mutation (C282Y) in the HFE gene. To test the hypothesis that the HFE gene is involved in regulation of iron homeostasis, we studied the effects of a targeted disruption of the murine homologue of the HFE gene. The HFE-deficient mice showed profound differences in parameters of iron homeostasis. Even on a standard diet, by 10 weeks of age, fasting transferrin saturation was significantly elevated compared with normal littermates (96 +/- 5% vs. 77 +/- 3%, P < 0.007), and hepatic iron concentration was 8-fold higher than that of wild-type littermates (2,071 +/- 450 vs. 255 +/- 23 microg/g dry wt, P < 0.002). Stainable hepatic iron in the HFE mutant mice was predominantly in hepatocytes in a periportal distribution. Iron concentrations in spleen, heart, and kidney were not significantly different. Erythroid parameters were normal, indicating that the anemia did not contribute to the increased iron storage. This study shows that the HFE protein is involved in the regulation of iron homeostasis and that mutations in this gene are responsible for HH. The knockout mouse model of HH will facilitate investigation into the pathogenesis of increased iron accumulation in HH and provide opportunities to evaluate therapeutic strategies for prevention or correction of iron overload.

We recently reported the positional cloning of a candidate gene for hereditary hemochromatosis (HH), calledHLA-H, which is a novel member of the major histocompatibility complex class I family. A mutation in this gene, cysteine 282 → tyrosine (C282Y), was found to be present in 83% of HH patient DNAs, while a second variant, histidine 63 → aspartate (H63D), was enriched in patients heterozygous for C282Y. The functional relevance of either mutation has not been described. Co-immunoprecipitation 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 β2-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. We recently reported the positional cloning of a candidate gene for hereditary hemochromatosis (HH), calledHLA-H, which is a novel member of the major histocompatibility complex class I family. A mutation in this gene, cysteine 282 → tyrosine (C282Y), was found to be present in 83% of HH patient DNAs, while a second variant, histidine 63 → aspartate (H63D), was enriched in patients heterozygous for C282Y. The functional relevance of either mutation has not been described. Co-immunoprecipitation 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 β2-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) 1The abbreviations used are:HHhereditary hemochromatosisMHCmajor histocompatibility complexPCRpolymerase chain reactionβ-COPβ-coatomer proteinERendoplasmic reticulumPAGEpolyacrylamide gel electrophoresisPVDFpolyvinyl difluoride. 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 (1Dadone M.M. Kushner J.P. Edwards C.Q. Bishop D.T. Skolnick M.H. Am. J. Clin. Pathol. 1982; 78: 196-207Crossref PubMed Scopus (157) Google Scholar, (2Edwards C.Q. Griffen L.M. Goldgar D. Drummond C. Skolnick M.H. Kushner J.P. N. Engl. J. Med. 1988; 318: 1355-1362Crossref PubMed Scopus (567) Google Scholar). 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 (3Feder J.N. Gnirke A. Thomas W. Tsuchihashi Z. Ruddy D.A. Basava A. Dormishian F. Domingo R. Ellis M.C. Fullan A. Hinton L.M. Jones N.L. Kimmel B.E. Kronmal G.S. Lauer P. Lee V.K. Loeb D.B. Mapa F.A. McClelland E.E. Meyer N.C. Mintier G.A. Moeller N.N. Moore T. Morikang E. Prass C.E. Quintana L. Starnes S.M. Schatzman R.C. Brunke K.J. Drayna D.T. Risch N.J. Bacon B.R. Wolff R.K. Nat. Genet. 1996; 13: 399-408Crossref PubMed Scopus (3327) Google Scholar). 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 → tyrosine (C282Y). Subsequent reports have confirmed the high frequency of this founder mutation in other HH patients (4Jaswinska E.C. Cullen L.M. Busfield F. Pyper W.R. Webb S.I. Powell L.W. Morris C.P. Nat. 1996; Scholar, P. J. J. P. P. N. Nat. 1996; PubMed Scopus Google Scholar, E. T. C. Lee P. R. P. C.P. G.S. 1996; PubMed Scopus Google is the HH A second mutation, histidine 63 → aspartate (H63D), was reported that was enriched in with the C282Y mutation of (3Feder J.N. Gnirke A. Thomas W. Tsuchihashi Z. Ruddy D.A. Basava A. Dormishian F. Domingo R. Ellis M.C. Fullan A. Hinton L.M. Jones N.L. Kimmel B.E. Kronmal G.S. Lauer P. Lee V.K. Loeb D.B. Mapa F.A. McClelland E.E. Meyer N.C. Mintier G.A. Moeller N.N. Moore T. Morikang E. Prass C.E. Quintana L. Starnes S.M. Schatzman R.C. Brunke K.J. Drayna D.T. Risch N.J. Bacon B.R. Wolff R.K. Nat. Genet. 1996; 13: 399-408Crossref PubMed Scopus (3327) Google Scholar). The that either of in HLA-H in the of HH has not been HLA-H protein is to MHC class I class I and the human of the cysteine that in the and of MHC class I are present in the HLA-H of cysteine is in the C282Y The of the has been to be for of the and of the protein with β2-microglobulin P. PubMed Scopus Google Scholar). the functional significance of an between β2-microglobulin and an class I-like in HH was by a iron to that in human HH R. R. P. PubMed Scopus Google Scholar, B.E. A. 1996; PubMed Scopus Google Scholar, J. Med. 1996; PubMed Scopus Google Scholar). studies have that mutation of cysteine in the of the MHC class I member of the from the to the J. E. A. PubMed Scopus Google a the of HLA-H in HH we the of the C282Y and H63D on HLA-H In this report we demonstrate that wild-type HLA-H binds to β2-microglobulin and that the C282Y mutation completely abrogates this and protein The for the that the C282Y mutation results in of the HLA-H which leads to HH first to demonstrate an of the HLA-H protein with β2-microglobulin and to the of the C282Y and H63D on that interaction. embryonic kidney cells were transfected with the wild-type HLA-H cDNA or the cDNA with either the C282Y or H63D The was the of a for of the expressed proteins C.E. PubMed Scopus Google Scholar). We cell the of cell lysates with to the to the by with β2-microglobulin a between the HLA-H protein and β2-microglobulin 1 β2-microglobulin was not in from cell the HLA-H protein with the C282Y mutation 1 This to β2-microglobulin was not to of HLA-H protein in the mutant cell the and with to the of HLA-H that the of HLA-H protein in the cell was 1 The results with the H63D mutant were to the wild-type HLA-H β2-microglobulin was with that mutant protein 1 H63D and is of to that the wild-type or H63D HLA-H proteins in the to a of and in the C282Y a single of results were by the in which cell lysates were with β2-microglobulin by with the of HLA-H In this the β2-microglobulin HLA-H protein from the wild-type and H63D mutant cell but to in the C282Y mutant cell 1 the and with β2-microglobulin that of β2-microglobulin protein were from cell 1 results an between wild-type HLA-H protein and β2-microglobulin and demonstrate that the but not the mutation this reports have that of the MHC class I chain with β2-microglobulin is for cell-surface expression Jones E. PubMed Scopus Google Scholar, J. Med. 1988; PubMed Scopus Google Scholar). of the of the HLA-H protein the C282Y mutation to with we this mutation cell-surface of the HLA-H cell and the wild-type HLA-H protein or the C282Y mutant were for cell-surface protein expression by with to in the of the HLA-H protein and by with cells labeling by with the of HLA-H protein in the 1 cells a of labeling by a of labeling that was the of the cells cells the C282Y mutation labeling and were from the and The of the labeling was by the and with in the labeling in the wild-type HLA-H cells was completely not C282Y mutation in HLA-H cell HLA-H on the cell surface, cell and cells transfected with wild-type HLA-H or HLA-H the C282Y or H63D were first in and with a of and by a with cells were and with and in and to a are with this the that the C282Y mutant protein was expressed in the transfected cells but was of the cells with to of cells for the C282Y mutant HLA-H protein which was in the cells and wild-type HLA-H protein cells with the suggesting that not of the wild-type protein in transfected cells the cell the or the results not results demonstrate that the C282Y mutation cell-surface of the HLA-H the of wild-type and mutant HLA-H proteins the cell in we subcellular fractionations on to the were the the the the a of and We the of subcellular by for to were for protein for and for from of cells from were by and on a were found in the and to a in the were the were found in the The fractionations from of the cell the of HLA-H proteins in the by and with HLA-H with the results the that the wild-type HLA-H protein a of the and The was found in the was in a to that of the the C282Y mutant HLA-H protein of the the wild-type the mutant protein was in a that most that of the suggesting the of or The H63D mutant proteins in a that of the wild-type that this mutation or on HLA-H protein trafficking In other HLA-H protein was in the of of the wild-type or mutant cell suggesting that the C282Y the H63D mutation a of the protein to the not C282Y mutation in HLA-H the subcellular of the from cells the wild-type HLA-H or the C282Y or H63D mutant proteins were by from were on to and the HLA-H protein with a of and HLA-H by wild-type HLA-H cell C282Y HLA-H mutant cell H63D HLA-H mutant cell results demonstrate that the C282Y mutation the HLA-H from with β2-microglobulin and cell-surface 282 is one of cysteine that are in and MHC class I and a in the P. PubMed Scopus Google Scholar). the of this is to the of the of β2-microglobulin and HLA-H and for of the I MHC are between an chain and β2-microglobulin P. PubMed Scopus Google Scholar). The of the chain is to and the of the chain with the and the Lee J. 1988; Google P. PubMed Scopus Google Scholar). that the cysteine in protein cell-surface J. E. A. PubMed Scopus Google Scholar). the mutant the to with to the of HLA-H is or the human (3Feder J.N. Gnirke A. Thomas W. Tsuchihashi Z. Ruddy D.A. Basava A. Dormishian F. Domingo R. Ellis M.C. Fullan A. Hinton L.M. Jones N.L. Kimmel B.E. Kronmal G.S. Lauer P. Lee V.K. Loeb D.B. Mapa F.A. McClelland E.E. Meyer N.C. Mintier G.A. Moeller N.N. Moore T. Morikang E. Prass C.E. Quintana L. Starnes S.M. Schatzman R.C. Brunke K.J. Drayna D.T. Risch N.J. Bacon B.R. Wolff R.K. Nat. Genet. 1996; 13: 399-408Crossref PubMed Scopus (3327) Google Scholar). is that the of the be for β2-microglobulin with proteins for of MHC class I is The chain of a MHC class I is on and in the A. PubMed Scopus Google Scholar). the chain with proteins and β2-microglobulin in the and is the to the and the chain is to a complex to the PubMed Scopus Google Scholar, J. PubMed Google Scholar, D.B. PubMed Scopus Google Scholar). I that to are between the and exclusively in the J. PubMed Scopus Google Scholar). In studies the C282Y mutant of HLA-H is on in a that be with The mutant protein to the protein in subcellular but of the of the we that protein be in the The of in the studies not studies be to the which of the C282Y mutant is to results with the C282Y mutation, we found in the β2-microglobulin or of the H63D mutant of which is enriched in C282Y heterozygous patients (3Feder J.N. Gnirke A. Thomas W. Tsuchihashi Z. Ruddy D.A. Basava A. Dormishian F. Domingo R. Ellis M.C. Fullan A. Hinton L.M. Jones N.L. Kimmel B.E. Kronmal G.S. Lauer P. Lee V.K. Loeb D.B. Mapa F.A. McClelland E.E. Meyer N.C. Mintier G.A. Moeller N.N. Moore T. Morikang E. Prass C.E. Quintana L. Starnes S.M. Schatzman R.C. Brunke K.J. Drayna D.T. Risch N.J. Bacon B.R. Wolff R.K. Nat. Genet. 1996; 13: 399-408Crossref PubMed Scopus (3327) Google Scholar). studies have in of class I by in the of PubMed Scopus Google Scholar). The H63D mutation is localized in the between the and of the is that the of this mutation is to the of the HLA-H protein for an or to the that the mutant protein with other proteins in the cell this mutation a common with or on the of the The to this we the HLA-H iron metabolism in the Hereditary hemochromatosis (HH) 1The abbreviations used are:HHhereditary hemochromatosisMHCmajor histocompatibility complexPCRpolymerase chain reactionβ-COPβ-coatomer proteinERendoplasmic reticulumPAGEpolyacrylamide gel electrophoresisPVDFpolyvinyl difluoride. 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 (1Dadone M.M. Kushner J.P. Edwards C.Q. Bishop D.T. Skolnick M.H. Am. J. Clin. Pathol. 1982; 78: 196-207Crossref PubMed Scopus (157) Google Scholar, (2Edwards C.Q. Griffen L.M. Goldgar D. Drummond C. Skolnick M.H. Kushner J.P. N. Engl. J. Med. 1988; 318: 1355-1362Crossref PubMed Scopus (567) Google Scholar). 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 (3Feder J.N. Gnirke A. Thomas W. Tsuchihashi Z. Ruddy D.A. Basava A. Dormishian F. Domingo R. Ellis M.C. Fullan A. Hinton L.M. Jones N.L. Kimmel B.E. Kronmal G.S. Lauer P. Lee V.K. Loeb D.B. Mapa F.A. McClelland E.E. Meyer N.C. Mintier G.A. Moeller N.N. Moore T. Morikang E. Prass C.E. Quintana L. Starnes S.M. Schatzman R.C. Brunke K.J. Drayna D.T. Risch N.J. Bacon B.R. Wolff R.K. Nat. Genet. 1996; 13: 399-408Crossref PubMed Scopus (3327) Google Scholar). 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 → tyrosine (C282Y). Subsequent reports have confirmed the high frequency of this founder mutation in other HH patients (4Jaswinska E.C. Cullen L.M. Busfield F. Pyper W.R. Webb S.I. Powell L.W. Morris C.P. Nat. 1996; Scholar, P. J. J. P. P. N. Nat. 1996; PubMed Scopus Google Scholar, E. T. C. Lee P. R. P. C.P. G.S. 1996; PubMed Scopus Google is the HH A second mutation, histidine 63 → aspartate (H63D), was reported that was enriched in with the C282Y mutation of (3Feder J.N. Gnirke A. Thomas W. Tsuchihashi Z. Ruddy D.A. Basava A. Dormishian F. Domingo R. Ellis M.C. Fullan A. Hinton L.M. Jones N.L. Kimmel B.E. Kronmal G.S. Lauer P. Lee V.K. Loeb D.B. Mapa F.A. McClelland E.E. Meyer N.C. Mintier G.A. Moeller N.N. Moore T. Morikang E. Prass C.E. Quintana L. Starnes S.M. Schatzman R.C. Brunke K.J. Drayna D.T. Risch N.J. Bacon B.R. Wolff R.K. Nat. Genet. 1996; 13: 399-408Crossref PubMed Scopus (3327) Google Scholar). The that either of in HLA-H in the of HH has not been hereditary hemochromatosis major histocompatibility complex chain protein gel difluoride. The HLA-H protein is to MHC class I class I and the human of the cysteine that in the and of MHC class I are present in the HLA-H of cysteine is in the C282Y The of the has been to be for of the and of the protein with β2-microglobulin P. PubMed Scopus Google Scholar). the functional significance of an between β2-microglobulin and an class I-like in HH was by a iron to that in human HH R. R. P. PubMed Scopus Google Scholar, B.E. A. 1996; PubMed Scopus Google Scholar, J. Med. 1996; PubMed Scopus Google Scholar). studies have that mutation of cysteine in the of the MHC class I member of the from the to the J. E. A. PubMed Scopus Google Scholar). a the of HLA-H in HH we the of the C282Y and H63D on HLA-H In this report we demonstrate that wild-type HLA-H binds to β2-microglobulin and that the C282Y mutation completely abrogates this and protein The for the that the C282Y mutation results in of the HLA-H which leads to HH disease. first to demonstrate an of the HLA-H protein with β2-microglobulin and to the of the C282Y and H63D on that interaction. embryonic kidney cells were transfected with the wild-type HLA-H cDNA or the cDNA with either the C282Y or H63D The was the of a for of the expressed proteins C.E. PubMed Scopus Google Scholar). We cell the of cell lysates with to the to the by with β2-microglobulin a between the HLA-H protein and β2-microglobulin 1 β2-microglobulin was not in from cell the HLA-H protein with the C282Y mutation 1 This to β2-microglobulin was not to of HLA-H protein in the mutant cell the and with to the of HLA-H that the of HLA-H protein in the cell was 1 The results with the H63D mutant were to the wild-type HLA-H β2-microglobulin was with that mutant protein 1 H63D and is of to that the wild-type or H63D HLA-H proteins in the to a of and in the C282Y a single of results were by the in which cell lysates were with β2-microglobulin by with the of HLA-H In this the β2-microglobulin HLA-H protein from the wild-type and H63D mutant cell but to in the C282Y mutant cell 1 the and with β2-microglobulin that of β2-microglobulin protein were from cell 1 results an between wild-type HLA-H protein and β2-microglobulin and demonstrate that the but not the mutation this reports have that of the MHC class I chain with β2-microglobulin is for cell-surface expression Jones E. PubMed Scopus Google Scholar, J. Med. 1988; PubMed Scopus Google Scholar). of the of the HLA-H protein the C282Y mutation to with we this mutation cell-surface of the HLA-H cell and the wild-type HLA-H protein or the C282Y mutant were for cell-surface protein expression by with to in the of the HLA-H protein and by with cells labeling by with the of HLA-H protein in the 1 cells a of labeling by a of labeling that was the of the cells cells the C282Y mutation labeling and were from the and The of the labeling was by the and with in the labeling in the wild-type HLA-H cells was completely not the that the C282Y mutant protein was expressed in the transfected cells but was of the cells with to of cells for the C282Y mutant HLA-H protein which was in the cells and wild-type HLA-H protein cells with the suggesting that not of the wild-type protein in transfected cells the cell the or the results not results demonstrate that the C282Y mutation cell-surface of the HLA-H the of wild-type and mutant HLA-H proteins the cell in we subcellular fractionations on to the were the the the the a of and We the of subcellular by for to were for protein for and for from of cells from were by and on a were found in the and to a in the were the were found in the The fractionations from of the cell the of HLA-H proteins in the by and with HLA-H with the results the that the wild-type HLA-H protein a of the and The was found in the was in a to that of the the C282Y mutant HLA-H protein of the the wild-type the mutant protein was in a that most that of the suggesting the of or The H63D mutant proteins in a that of the wild-type that this mutation or on HLA-H protein trafficking In other HLA-H protein was in the of of the wild-type or mutant cell suggesting that the C282Y the H63D mutation a of the protein to the not C282Y mutation in HLA-H the subcellular of the from cells the wild-type HLA-H or the C282Y or H63D mutant proteins were by from were on to and the HLA-H protein with a of and HLA-H by wild-type HLA-H cell C282Y HLA-H mutant cell H63D HLA-H mutant cell results demonstrate that the C282Y mutation the HLA-H from with β2-microglobulin and cell-surface 282 is one of cysteine that are in and MHC class I and a in the P. PubMed Scopus Google Scholar). the of this is to the of the of β2-microglobulin and HLA-H and for of the I MHC are between an chain and β2-microglobulin P. PubMed Scopus Google Scholar). The of the chain is to and the of the chain with the and the Lee J. 1988; Google P. PubMed Scopus Google Scholar). that the cysteine in protein cell-surface J. E. A. PubMed Scopus Google Scholar). the mutant the to with to the of HLA-H is or the human (3Feder J.N. Gnirke A. Thomas W. Tsuchihashi Z. Ruddy D.A. Basava A. Dormishian F. Domingo R. Ellis M.C. Fullan A. Hinton L.M. Jones N.L. Kimmel B.E. Kronmal G.S. Lauer P. Lee V.K. Loeb D.B. Mapa F.A. McClelland E.E. Meyer N.C. Mintier G.A. Moeller N.N. Moore T. Morikang E. Prass C.E. Quintana L. Starnes S.M. Schatzman R.C. Brunke K.J. Drayna D.T. Risch N.J. Bacon B.R. Wolff R.K. Nat. Genet. 1996; 13: 399-408Crossref PubMed Scopus (3327) Google Scholar). is that the of the be for β2-microglobulin with proteins for of MHC class I is The chain of a MHC class I is on and in the A. PubMed Scopus Google Scholar). the chain with proteins and β2-microglobulin in the and is the to the and the chain is to a complex to the PubMed Scopus Google Scholar, J. PubMed Google Scholar, D.B. PubMed Scopus Google Scholar). I that to are between the and exclusively in the J. PubMed Scopus Google Scholar). In studies the C282Y mutant of HLA-H is on in a that be with The mutant protein to the protein in subcellular but of the of the we that protein be in the The of in the studies not studies be to the which of the C282Y mutant is to results with the C282Y mutation, we found in the β2-microglobulin or of the H63D mutant of which is enriched in C282Y heterozygous patients (3Feder J.N. Gnirke A. Thomas W. Tsuchihashi Z. Ruddy D.A. Basava A. Dormishian F. Domingo R. Ellis M.C. Fullan A. Hinton L.M. Jones N.L. Kimmel B.E. Kronmal G.S. Lauer P. Lee V.K. Loeb D.B. Mapa F.A. McClelland E.E. Meyer N.C. Mintier G.A. Moeller N.N. Moore T. Morikang E. Prass C.E. Quintana L. Starnes S.M. Schatzman R.C. Brunke K.J. Drayna D.T. Risch N.J. Bacon B.R. Wolff R.K. Nat. Genet. 1996; 13: 399-408Crossref PubMed Scopus (3327) Google Scholar). studies have in of class I by in the of PubMed Scopus Google Scholar). The H63D mutation is localized in the between the and of the is that the of this mutation is to the of the HLA-H protein for an or to the that the mutant protein with other proteins in the cell this mutation a common with or on the of the The to this we the HLA-H iron metabolism in the We first to demonstrate an of the HLA-H protein with β2-microglobulin and to the of the C282Y and H63D on that interaction. embryonic kidney cells were transfected with the wild-type HLA-H cDNA or the cDNA with either the C282Y or H63D The was the of a for of the expressed proteins C.E. PubMed Scopus Google Scholar). We cell the of cell lysates with to the to the by with β2-microglobulin a between the HLA-H protein and β2-microglobulin 1 β2-microglobulin was not in from cell the HLA-H protein with the C282Y mutation 1 This to β2-microglobulin was not to of HLA-H protein in the mutant cell the and with to the of HLA-H that the of HLA-H protein in the cell was 1 The results with the H63D mutant were to the wild-type HLA-H β2-microglobulin was with that mutant protein 1 H63D and is of to that the wild-type or H63D HLA-H proteins in the to a of and in the C282Y a single of The results were by the in which cell lysates were with β2-microglobulin by with the of HLA-H In this the β2-microglobulin HLA-H protein from the wild-type and H63D mutant cell but to in the C282Y mutant cell 1 the and with β2-microglobulin that of β2-microglobulin protein were from cell 1 results an between wild-type HLA-H protein and β2-microglobulin and demonstrate that the but not the mutation this reports have that of the MHC class I chain with β2-microglobulin is for cell-surface expression Jones E. PubMed Scopus Google Scholar, J. Med. 1988; PubMed Scopus Google Scholar). of the of the HLA-H protein the C282Y mutation to with we this mutation cell-surface of the HLA-H cell and the wild-type HLA-H protein or the C282Y mutant were for cell-surface protein expression by with to in the of the HLA-H protein and by with cells labeling by with the of HLA-H protein in the 1 cells a of labeling by a of labeling that was the of the cells cells the C282Y mutation labeling and were from the and The of the labeling was by the and with in the labeling in the wild-type HLA-H cells was completely not We the that the C282Y mutant protein was expressed in the transfected cells but was of the cells with to of cells for the C282Y mutant HLA-H protein which was in the cells and wild-type HLA-H protein cells with the suggesting that not of the wild-type protein in transfected cells the cell the or the results not results demonstrate that the C282Y mutation cell-surface of the HLA-H the of wild-type and mutant HLA-H proteins the cell in we subcellular fractionations on to the were the the the the a of and We the of subcellular by for to were for protein for and for from of cells from were by and on a were found in the and to a in the were the were found in the The fractionations from of the cell We the of HLA-H proteins in the by and with HLA-H with the results the that the wild-type HLA-H protein a of the and The was found in the was in a to that of the the C282Y mutant HLA-H protein of the the wild-type the mutant protein was in a that most that of the suggesting the of or The H63D mutant proteins in a that of the wild-type that this mutation or on HLA-H protein trafficking In other HLA-H protein was in the of of the wild-type or mutant cell suggesting that the C282Y the H63D mutation a of the protein to the not results demonstrate that the C282Y mutation the HLA-H from with β2-microglobulin and cell-surface 282 is one of cysteine that are in and MHC class I and a in the P. PubMed Scopus Google Scholar). the of this is to the of the of β2-microglobulin and HLA-H and for of the I MHC are between an chain and β2-microglobulin P. PubMed Scopus Google Scholar). The of the chain is to and the of the chain with the and the Lee J. 1988; Google P. PubMed Scopus Google Scholar). that the cysteine in protein cell-surface J. E. A. PubMed Scopus Google Scholar). the mutant the to with to the of HLA-H is or the human (3Feder J.N. Gnirke A. Thomas W. Tsuchihashi Z. Ruddy D.A. Basava A. Dormishian F. Domingo R. Ellis M.C. Fullan A. Hinton L.M. Jones N.L. Kimmel B.E. Kronmal G.S. Lauer P. Lee V.K. Loeb D.B. Mapa F.A. McClelland E.E. Meyer N.C. Mintier G.A. Moeller N.N. Moore T. Morikang E. Prass C.E. Quintana L. Starnes S.M. Schatzman R.C. Brunke K.J. Drayna D.T. Risch N.J. Bacon B.R. Wolff R.K. Nat. Genet. 1996; 13: 399-408Crossref PubMed Scopus (3327) Google Scholar). is that the of the be for β2-microglobulin with proteins for The of MHC class I is The chain of a MHC class I is on and in the A. PubMed Scopus Google Scholar). the chain with proteins and β2-microglobulin in the and is the to the and the chain is to a complex to the PubMed Scopus Google Scholar, J. PubMed Google Scholar, D.B. PubMed Scopus Google Scholar). I that to are between the and exclusively in the J. PubMed Scopus Google Scholar). In studies the C282Y mutant of HLA-H is on in a that be with The mutant protein to the protein in subcellular but of the of the we that protein be in the The of in the studies not studies be to the which of the C282Y mutant is In to results with the C282Y mutation, we found in the β2-microglobulin or of the H63D mutant of which is enriched in C282Y heterozygous patients (3Feder J.N. Gnirke A. Thomas W. Tsuchihashi Z. Ruddy D.A. Basava A. Dormishian F. Domingo R. Ellis M.C. Fullan A. Hinton L.M. Jones N.L. Kimmel B.E. Kronmal G.S. Lauer P. Lee V.K. Loeb D.B. Mapa F.A. McClelland E.E. Meyer N.C. Mintier G.A. Moeller N.N. Moore T. Morikang E. Prass C.E. Quintana L. Starnes S.M. Schatzman R.C. Brunke K.J. Drayna D.T. Risch N.J. Bacon B.R. Wolff R.K. Nat. Genet. 1996; 13: 399-408Crossref PubMed Scopus (3327) Google Scholar). studies have in of class I by in the of PubMed Scopus Google Scholar). The H63D mutation is localized in the between the and of the is that the of this mutation is to the of the HLA-H protein for an or to the that the mutant protein with other proteins in the cell this mutation a common with or on the of the The to this we the HLA-H iron metabolism in the We A. and E. for of the D. A. Ruddy for D. J. for with the of subcellular protein and for

Most human cancers are characterized by genomic instability, the accumulation of multiple genetic alterations and allelic imbalance throughout the genome. Loss of heterozygosity (LOH) is a common form of allelic imbalance and the detection of LOH has been used to identify genomic regions that harbor tumor suppressor genes and to characterize tumor stages and progression. Here we describe the use of high-density oligonucleotide arrays for genome-wide scans for LOH and allelic imbalance in human tumors. The arrays contain redundant sets of probes for 600 genetic loci that are distributed across all human chromosomes. The arrays were used to detect allelic imbalance in two types of human tumors, and a subset of the results was confirmed using conventional gel-based methods. We also tested the ability to study heterogeneous cell populations and found that allelic imbalance can be detected in the presence of a substantial background of normal cells. The detection of LOH and other chromosomal changes using large numbers of single nucleotide polymorphism (SNP) markers should enable identification of patterns of allelic imbalance with potential prognostic and diagnostic utility.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a negative regulator of the phosphoinositol-3-kinase (PI3K)/AKT signaling pathway that controls cell cycle progression, growth and inhibition of apoptosis. Loss of PTEN protein expression has been associated with tumorigenesis, cancer progression and drug resistance, but conflicting results exist which may be due in part to difficulties inherent in PTEN immunohistochemistry (IHC). We sought a robust PTEN IHC assay. Human tumor cell lines with PTEN status verified by copy number analysis were formalin fixed and paraffin embedded for use as positive and negative controls. PTEN antibodies were optimized on tumor cell lines. Five optimized antibodies were analyzed on 10 molecularly characterized endometrial carcinoma samples. Four antibodies (CST, Millipore, Abcam, Novus) stained 3/10 positive and 7/10 negative, however, all but CST exhibited nonspecific nucleolar staining of negative controls. One antibody (Dako) stained 5/10 positive and 5/10 negative but with areas (≤10%) of positivity. The 4 samples predicted to be negative by sequencing were negative with the CST antibody, however, one was positive with Dako; as a result we chose the CST antibody for our assay. The assay was validated on an automated platform using 50 formalin fixed and paraffin embedded colon, lung, prostate and breast adenocarcinoma cases. Tumor cell lines served as external controls; endothelial cells and peripheral nerves served as internal positive controls. Dichotomous scoring achieved 100% concordance between three independent pathologists. This reproducible PTEN assay (PREZEON) has been implemented in a CLIA certified laboratory.