Juvenile hemochromatosis gene (hfe2a), expression products and uses thereof

Abstract

Polynucleotide and polypeptide sequences for HFE2A, as well as mutations associated with juvenile hemochromatosis, and methods of utilizing these for the screening and identification of agents for the treatment of diseases of iron metabolism, including small organic compounds, are disclosed along with methods of treating and/or ameliorating diseases of iron metabolism, especially in human patients are disclosed. Diagnostic compounds, kits and methods using HFE2A are also described.

1 . A method for identifying an agent that modulates HFE2A gene expression, comprising: 
 (a) contacting a test compound with a genetic construct comprising a reporter gene operably linked to an HFE2A promoter under conditions supporting expression of said reporter gene;    (b) determining a change in expression of said reporter gene as a result of said contacting,    wherein a change in expression identifies said test compound as an agent that modulates HFE2A gene expression.    
     
     
         2 . The method of  claim 1  wherein said modulation is an increase in expression of said reporter gene.  
     
     
         3 . The method of  claim 1  wherein said modulation is a decrease in expression of said reporter gene.  
     
     
         4 . The method of  claim 1  wherein said HFE2A promoter is a mammalian HFE2A promoter.  
     
     
         5 . The method of  claim 4  wherein said mammal is a member selected from the group consisting of mouse, rat and human.  
     
     
         6 . The method of  claim 1  wherein said promoter has the nucleotide sequence of SEQ ID NO: 19.  
     
     
         7 . The method of  claim 1  wherein said genetic construct is in a cell.  
     
     
         8 . The method of  claim 7  wherein said cell is a mammalian cell.  
     
     
         9 . The method of  claim 7  wherein said cell is a recombinant cell engineered to express said gene and wherein said cell does not express said gene absent said engineering.  
     
     
         10 . The method of  claim 8  wherein said mammalian cell is a macrophage, inflammatory cell, liver cell, hepatocyte, intestinal cell, hematopoietic cell, pancreatic cell, skeletal muscle cell or a nervous system cell.  
     
     
         11 . The method of  claim 1  wherein said test compound is useful in controlling iron metabolism.  
     
     
         12 . The method of  claim 1  wherein said test compound is first identified as an agent that modulates HFE2A gene expression using said method.  
     
     
         13 . The method of claim 1 wherein said change is determined by measuring a change in an enzyme activity.  
     
     
         14 . A method for identifying an agent that modulates HFE2A gene expression, comprising: 
 a) contacting a test compound with a cell expressing an HFE2A gene;    b) determining a change in expression of said HFE2A gene as a result of said contacting,    wherein a change in expression identifies said test compound as an agent that modulates HFE2A gene expression.    
     
     
         15 . A method for identifying an agent that modulates the activity of an HFE2A polypeptide, comprising: 
 a) contacting a test compound with an HFE2A polypeptide under conditions supporting an activity of said polypeptide; and    b) determining a change in said activity of said polypeptide as a result of said contacting,    wherein a change in the activity identifies said test compound as an agent that modulates the activity of an HFE2A polypeptide.    
     
     
         16 . The method of  claim 15  wherein said HFE2A polypeptide is a mammalian HFE2A polypeptide.  
     
     
         17 . The method of  claim 16  wherein said mammal is a member selected from the group consisting of mouse, rat and human.  
     
     
         18 . The method of  claim 15  wherein said change is a decrease in said activity.  
     
     
         19 . The method of  claim 15  wherein said change is an increase in said activity.  
     
     
         20 . The method of  claim 15  wherein said polypeptide is part of a cell.  
     
     
         21 . The method of  claim 20  wherein said cell is a mammalian cell.  
     
     
         22 . The method of  claim 20  wherein said cell has been engineered to contain said polypeptide.  
     
     
         23 . The method of  claim 22  wherein said cell was engineered by genetic engineering.  
     
     
         24 . The method of  claim 23  wherein said cell does not contain said polypeptide absent said engineering.  
     
     
         25 . The method of  claim 20  wherein said cell is a macrophage, inflammatory cell, liver cell, hepatocyte, intestinal cell, hematopoietic cell, pancreatic cell, skeletal muscle cell or a cell of the nervous system.  
     
     
         26 . The method of  claim 15  wherein said HFE2A polypeptide is highly purified.  
     
     
         27 . The method of  claim 15  wherein said HFE2A polypeptide is a semi-purified polypeptide.  
     
     
         28 . The method of  claim 15  wherein said HFE2A polypeptide is attached to a solid surface.  
     
     
         29 . The method of  claim 28  wherein said solid surface is a resin.  
     
     
         30 . The method of  claim 15  wherein said HFE2A polypeptide is part of an extract.  
     
     
         31 . The method of  claim 15  wherein said polypeptide is encoded by a polynucleotide having the sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 and 9.  
     
     
         32 . The method of  claim 15  wherein said polypeptide comprises an amino acid sequence selected from SEQ ID NO: 10, 11 and 12.  
     
     
         33 . A method for treating a disorder comprising administering to an animal afflicted therewith a therapeutically effective amount of an HFE2A modulator.  
     
     
         34 . The method of  claim 33  wherein said HFE2A modulator exhibits modulating activity in a method of  claim 1 , 14 or 15.  
     
     
         35 . The method of  claim 34  wherein said modulator was first identified as an HFE2A modulator using said method.  
     
     
         36 . The method of  claim 33  wherein said HFE2A modulator is a member selected from the group consisting of a selective HFE2A agonist, a selective HFE2A antagonist, pharmaceutically acceptable salts thereof, and combinations of these.  
     
     
         37 . The method of  claim 36  wherein said member is in a pharmaceutically acceptable carrier.  
     
     
         38 . The method of  claim 33  wherein said HFE2A modulator is selected from an antibody, an anti-sense molecule, a ribozyme and a drug-like small organic molecule.  
     
     
         39 . The method of  claim 33  wherein said disorder is a disease of iron metabolism.  
     
     
         40 . The method of  claim 39  wherein said disease of iron metabolism is a member selected from the group consisting of an iron overload disorder and an iron deficiency disorder.  
     
     
         41 . The method of  claim 39  wherein said disease of iron metabolism is a member selected from the group consisting of hemochromatosis, transfusion iron overload, thalassemia, porphyria, and juvenile hemochromatosis.  
     
     
         42 . The method of  claim 39  wherein said disease of iron metabolism is a member selected from the group consisting of anemia and anemia of chronic disease.  
     
     
         43 . The method of  claim 39  wherein said disease of iron metabolism is a member selected from Type I diabetes, Type II diabetes and insulin resistance.  
     
     
         44 . A method for preventing a disorder comprising administering to an animal, at risk of developing said disorder, a therapeutically effective amount of an HFE2A modulator.  
     
     
         45 . The method of  claim 44  wherein said HFE2A modulator exhibits modulating activity in a method of  claim 1 , 14 or 15.  
     
     
         46 . The method of  claim 45  wherein said modulator was first identified as an HFE2A modulator using said method.  
     
     
         47 . The method of  claim 44  wherein said HFE2A modulator is a member selected from the group consisting of a selective HFE2A agonist, a selective HFE2A antagonist, pharmaceutically acceptable salts thereof, and combinations of these.  
     
     
         48 . The method of  claim 47  wherein said member is in a pharmaceutically acceptable carrier.  
     
     
         49 . The method of  claim 44  wherein said HFE2A modulator is selected from an antibody, an anti-sense molecule, a ribozyme and a drug-like small organic molecule.  
     
     
         50 . The method of  claim 44  wherein said disorder is a disease of iron metabolism.  
     
     
         51 . The method of  claim 50  wherein said disease of iron metabolism is a member selected from the group consisting of an iron overload disorder and an iron deficiency disorder.  
     
     
         52 . The method of  claim 50  wherein said disease of iron metabolism is a member selected from the group consisting of hemochromatosis, transfusion iron overload, thalassemia, porphyria, and juvenile hemochromatosis.  
     
     
         53 . The method of  claim 50  wherein said disease of iron metabolism is a member selected from the group consisting of anemia and anemia of chronic disease.  
     
     
         54 . The method of  claim 50  wherein said disease of iron metabolism is a member selected from Type I diabetes, Type II diabetes and insulin resistance.  
     
     
         55 . A method to diagnose individuals affected by or at risk of developing a disease of iron metabolism comprising determining a mutation or polymorphism in the nucleic acid sequence of the HFE2A gene in said individual wherein said mutation or polymorphism in said gene identifies said individual as being affected by or at risk of developing a disease of iron metabolism.  
     
     
         56 . The method of  claim 55  wherein said mutation or polymorphism is a mutation or polymorphism of Table 1.  
     
     
         57 . A method to diagnose individuals affected by or at risk of developing a disease of iron metabolism comprising determining a mutation or polymorphism in the amino acid sequence of HFE2A polypeptide in said individual wherein a mutation or polymorphism of said HFE2A polypeptide identifies said individual as being affected by or at risk of developing a disease of iron metabolism.  
     
     
         58 . A method to diagnose individuals affected by or at risk of developing a disease of iron metabolism comprising determining a decrease change in level or amount of an HFE2A polypeptide in said individual wherein said decreare chance identifies said individual as being affected by or at risk of developing a disease of iron metabolism.  
     
     
         59 . The method of  claim 55 , 56, 57 or 58 wherein said disease of iron metabolism is adult onset hemochromatosis.  
     
     
         60 . The method of  claim 55 , 56, 57 or 58 wherein said disease of iron metabolism is juvenile hemochromatosis.  
     
     
         61 . The method of  claim 55 , 56, 57 or 58 wherein said disease of iron metabolism is selected from Type I diabetes, Type II diabetes and insulin resistance.  
     
     
         62 . A method for identifying a compound capable of modulating a HFE2A activity, comprising: (a) contacting a test compound with a cell that expresses HFE2A; and (b) assaying the ability of said test compound to modulate the transcription of a HFE2A nucleic acid or the activity of HFE2A polypeptide, thereby identifying a compound capable of modulating a HFE2A activity.  
     
     
         63 . The method of  claim 62 , wherein the compound is an anti-HFE2A polypeptide antibody.  
     
     
         64 . The method of  claim 62 , wherein the compound is an antisense HFE2A nucleic acid molecule.  
     
     
         65 . The method of  claim 62 , wherein the compound is a HFE2A ribozyme.  
     
     
         66 . The method of  claim 62 , wherein the compound is a small organic molecule.  
     
     
         67 . An isolated polynucleotide comprising a polynucleotide having a nucleotide sequence with at least 60 percent identity to a sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 19, 20, 21 and 22.  
     
     
         68 . The isolated polynucleotide of  claim 67  wherein said percent identity is at least 70 percent.  
     
     
         69 . The isolated polynucleotide of  claim 67  wherein said percent identity is at least 78 percent.  
     
     
         70 . The isolated polynucleotide of  claim 67  wherein said percent identity is at least 90 percent.  
     
     
         71 . The isolated polynucleotide of  claim 67  wherein said percent identity is at least 95 percent.  
     
     
         72 . The isolated polynucleotide of  claim 67  wherein said percent identity is at least 98 percent.  
     
     
         73 . The isolated polynucleotide of  claim 67  wherein said nucleotide sequence is a member selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 19, 20, 21 and 22.  
     
     
         74 . An isolated polypeptide comprising a polypeptide having an amino acid sequence with at least 90 percent identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 11, 12, 23, 24, 25, 26, 27 and 28.  
     
     
         75 . The isolated polypeptide of  claim 74  wherein said percent identity is at least 95 percent.  
     
     
         76 . The isolated polypeptide of  claim 74  wherein said percent identity is at least 98 percent.  
     
     
         77 . The isolated polypeptide of  claim 74  wherein said amino acid sequence is selected from the group consisting of SEQ ID NO: 10, 11, 12, 23, 24, 25, 26, 27 and 28.  
     
     
         78 . A cell line comprising a recombinant form of the polynucleotide of  claim 67 .  
     
     
         79 . A cell line comprising a recombinant form of the polypeptide of  claim 74 .  
     
     
         80 . Use of a cell line of  claim 78  or 79 in a method for identifying compositions which modulate expression or activity of hemojuvelin.  
     
     
         81 . A composition for treating a disease of iron metabolism comprising a therapeutically effective amount of a polypeptide of  claim 74  in a pharmaceutically acceptable carrier.  
     
     
         82 . A method for treating a disease of iron metabolism comprising administering to a patient in need thereof a therapeutically effective amount of a composition of  claim 81  in a pharmaceutically acceptable carrier  
     
     
         83 . The method according to  claim 82 , wherein said disease is juvenile hemochromatosis.  
     
     
         84 . A method for producing test data with respect to the modulation of HFE2A gene expression by a compound, comprising: 
 (a) a genetic construct comprising a reporter gene operably linked to an HFE2A promoter under conditions supporting expression of said reporter gene;    (b) determining a change in expression of said reporter gene as a result of said contacting, wherein said change shows modulation, and    (c) producing test data with respect to the gene modulating activity of said test compound based on a change in expression of the determined genes indicating gene modulating activity.    
     
     
         85 . A method for producing test data with respect to the modulation of an HFE2A polypeptide activity by a compound, comprising: 
 (a) contacting a test compound with an HFE2A polypeptide under conditions promoting an activity of said HFE2A polypeptide;    (b) determining a change in activity of said polypeptide as a result of said contacting, wherein said change shows modulation, and    (c) producing test data with respect to the HFE2A polypeptide modulating activity of said test compound based on a change in an HFE2A polypeptide activity indicating modulating activity.    
     
     
         86 . An isolated polypeptide, or fragments thereof, wherein said polypeptide comprises an amino acid sequence with at least 90 percent identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 11 and 12 and wherein said fragment comprises at least ten amino acid residues.  
     
     
         87 . The isolated polypeptide, or fragments thereof, of  claim 86 , wherein said percent identity is at least 95 percent.  
     
     
         88 . The isolated polypeptide, or fragments thereof, of  claim 86 , wherein said percent identity is at least 98 percent.  
     
     
         89 . The isolated polypeptide, or fragments thereof, of  claim 86 , wherein said polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10,11 and 12.  
     
     
         90 . A recombinant cell line engineered to express a polypeptide of  claim 86 , 87, 88 or 89.  
     
     
         91 . A method of identifying compositions that modulate expression or activity of hemojuvelin comprising determining compositions that modulate a polypeptide expressed by a cell of the cell line of  claim 90 .  
     
     
         92 . A composition for treating a disease of iron metabolism comprising a therapeutically effective amount of a polypeptide of  claim 86 , 87, 88 or 89 in a pharmaceutically acceptable carrier.  
     
     
         93 . A method for treating a disease of iron metabolism comprising administering to a patient in need thereof a therapeutically effective amount of a composition of  claim 92 .  
     
     
         94 . The method of  claim 93 , wherein said disease is juvenile hemochromatosis.  
     
     
         95 . The method of  claim 93 , wherein said disease is adult onset hemochromatosis.  
     
     
         96 . The method of  claim 93 , wherein said disease is anemia of chronic disease.  
     
     
         97 . The method of  claim 93 , wherein said disease is iron deficiency anemia.  
     
     
         98 . The isolated polynucleotide of  claim 67 , wherein said nucleotide sequence selected from the group consisting of SEQ ID NO: 7, 8 and 9, wherein said isolated polynucleotide encodes a 13 kDa to 40 kDa cleavage product of the polypeptide.  
     
     
         99 . The isolated polynucleotide of  claim 68 , wherein said nucleotide sequence selected from the group consisting of SEQ ID NO: 7, 8 and 9, wherein said isolated polynucleotide encodes a cleavage product of an HFE2A polypeptide and said cleavage product being approximately 13 kDa to 40 kDa.  
     
     
         100 . The isolated polynucleotide of  claim 69 , wherein said nucleotide sequence selected from the group consisting of SEQ ID NO: 7, 8 and 9, wherein said isolated polynucleotide encodes a cleavage product of an HFE2A polypeptide and said cleavage product being approximately 13 kDa to 40 kDa.  
     
     
         101 . The isolated polynucleotide of  claim 70 , wherein said nucleotide sequence selected from the group consisting of SEQ ID NO: 7, 8 and 9, wherein said isolated polynucleotide encodes a cleavage product of an HFE2A polypeptide and said cleavage product being approximately 13 kDa to 40 kDa.  
     
     
         102 . The isolated polynucleotide of  claim 71 , wherein said nucleotide sequence selected from the group consisting of SEQ ID NO: 7, 8 and 9, wherein said isolated polynucleotide encodes a cleavage product of an HFE2A polypeptide and said cleavage product being approximately 13 kDa to 40 kDa.  
     
     
         103 . The isolated polynucleotide of  claim 72 , wherein said nucleotide sequence selected from the group consisting of SEQ ID NO: 7, 8 and 9, wherein said isolated polynucleotide encodes a cleavage product of an HFE2A polypeptide and said cleavage product being approximately 13 kDa to 40 kDa.  
     
     
         104 . The isolated polynucleotide of  claim 73 , wherein said nucleotide sequence selected from the group consisting of SEQ ID NO: 7, 8 and 9, wherein said isolated polynucleotide encodes a cleavage product of an HFE2A polypeptide and said cleavage product being approximately 13 kDa to 40 kDa.  
     
     
         105 . The isolated polynucleotide of  claim 98 , 99, 100, 101, 102, 103 or 104, wherein the cleavage product comprises a polypeptide of about 26 kDa.  
     
     
         106 . The method of  claim 58  wherein said change is a decrease.  
     
     
         107 . The method of  claim 58  wherein said change is an increase.  
     
     
         108 . A method for diagnosing anemia of chronic disease comprising determining a change in level or amount of an HFE2A polypeptide in said individual wherein said change identifies said individual as being affected by or at risk of developing anemia of chronic disease.  
     
     
         109 . A method for diagnosing iron-deficiency anemia comprising determining a change in level or amount of an HFE2A polypeptide in said individual wherein said change identifies said individual as being affected by or at risk of developing iron-deficiency anemia.  
     
     
         110 . The method of  claim 108  or 109 wherein said change is a decrease.  
     
     
         111 . The method of  claim 108  or 109 wherein said change is an increase.  
     
     
         112 . The method of  claim 57 , wherein said mutation or polymorphism is determined by determining the presence of a fragment of said polypeptide.  
     
     
         113 . The method of  claim 112 , wherein said diagnosis is based on determining the size of said fragment.  
     
     
         114 . The method of  claim 58 , wherein said change is determined by detecting the presence of a fragment of said polypeptide.  
     
     
         115 . The method of  claim 114 , wherein said diagnosis is based on determining the size of said fragment.  
     
     
         116 . The method of  claim 108  or 109, wherein said change is determined by measuring the level or amount of a fragment of said polypeptide.  
     
     
         117 . The method of  claim 116 , wherein said diagnosis is based on determining the size of said fragment.