Abstract:
The invention features a method of diagnosing an iron disorder, e.g., hemochromatosis, or a genetic susceptibility to developing such a disorder in a mammal by determining the presence of a mutation in exon 2 or in an intron of an HFE nucleic acid.

Description:
RELATED APPLICATIONS 
     This application is a continuation of U.S. Ser. No. 09/277,457, filed Mar. 26, 1999 (now U.S. Pat. No. 6,355,425), the entire contents of which is hereby incorporated by reference. 
    
    
     BACKGROUND OF THE INVENTION 
     Hemochromatosis is the most common progressive (and sometimes fatal) genetic disease in people of European descent. Hemochromatosis is a disease state characterized by an inappropriate increase in intestinal iron absorption. The increase can result in deposition of iron in organs such as the liver, pancreas, heart, and pituitary. Such iron deposition can lead to tissue damage and functional impairment of the organs. 
     In some populations, 60-100% of cases are attributable to homozygosity for a missense mutation at C282Y in the Histocompatibility iron (Fe) loading (HFE) gene, a major histocompatibility (MHC) non-classical class I gene located on chromosome 6p. Some patients are compound heterozygotes for C282Y and another mutation at H63D. 
     SUMMARY OF THE INVENTION 
     The invention is based on the discovery of novel mutations which are associated with aberrant iron metabolims, absorption, or storage, or in advanced cases, clinical hemochromatosis. Accordingly, the invention features a method of diagnosing an iron disorder, e.g., hemochromatosis or a genetic susceptibility to developing such a disorder, in a mammal by determining the presence of a mutation in exon 2 of an HFE nucleic acid. The mutation is not a C→G missense mutation at position 187 of SEQ ID NO:1 which leads to a H63D substitution. The nucleic acid is an RNA or DNA molecule in a biological sample taken from the mammal, e.g. a human patient, to be tested. The presence of the mutation is indicative of the disorder or a genetic susceptibility to developing it. An iron disorder is characterized by an aberrant serum iron level, ferritin level, or percent saturation of transferrin compared to the level associated with a normal control individual. An iron overload disorder is characterized by abnormally high iron absorption compared to a normal control individual. Clinical hemochromatosis is defined by an elevated fasting transferrin saturation level of greater than 45% saturation. 
     For example, the mutation is a missense mutation at nucleotide 314 of SEQ ID NO:1 such as 314C which leads to the expression of mutant HFE gene product with amino acid substitution I105T. The I105T mutation is located in the α1 helix of the HFE protein and participates in a hydrophobic pocket (the “F” pocket). The alpha helix structure of the α1 domain spans residues S80 to N108, inclusive. The I105T mutation is associated with an iron overload disorder. 
     
       
         
               
               
             
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Human HFE cDNA sequence 
                   
               
               
                 (SEQ ID NO:1; GENBANK ® Accession No. U60319) 
               
               
                   
               
             
          
           
               
                  atgggcccg cgagccaggc 
                   
               
               
                 cggcgcttct cctcctgatg cttttgcaga ccgcggtcct gcaggggcgc ttgctgcgtt 
               
               
                 cacactctct gcactacctc ttcatgggtg cctcagagca ggaccttggt ctttccttgt 
               
               
                 ttgaagcttt gggctacgtg gatgaccagc tgttcgtgtt ctatgat cat  gag agt cgcc 
               
               
                                                                    H63D    S65C 
               
               
                 gtgtggagcc ccgaactcca tgggtttcca gtagaatttc aagccagatg tggctgcagc 
               
               
                 tgagtcagag tctgaaa ggg  tgggatcaca tgttcactgt tgacttctgg act att atgg 
               
               
                                   G93R                                     I105T 
               
               
                 aaaatcacaa ccacagcaag gagtcccaca ccctgcaggt catcctgggc tgtgaaatgc 
               
               
                 aagaagacaa cagtaccgag ggctactgga agtacgggta tgatgggcag gaccaccttg 
               
               
                 aattctgccc tgacacactg gattggagag cagcagaacc cagggcctgg cccaccaagc 
               
               
                 tggagtggga aaggcacaag attcgggcca ggcagaacag ggcctacctg gagagggact 
               
               
                 gccctgcaca gctgcagcag ttgctggagc tggggagagg tgttttggac caacaagtgc 
               
               
                 ctcctttggt gaaggtgaca catcatgtga cctcttcagt gaccactcta cggtgtcggg 
               
               
                 ccttgaacta ctacccccag aacatcacca tgaagtggct gaaggataag cagccaatgg 
               
               
                 atgccaagga gttcgaacct aaagacgtat tgcccaatgg ggatgggacc taccagggct 
               
               
                 ggataacctt ggctgtaccc cctggggaag agcagagata tacgtgccag gtggagcacc 
               
               
                 caggcctgga tcagcccctc attgtgatct gggagccctc accgtctggc accctagtca 
               
               
                 ttggagtcat cagtggaatt gctgtttttg tcgtcatctt gttcattgga attttgttca 
               
               
                 taatattaag gaagaggcag ggttcaagag gagccatggg gcactacgtc ttagctgaac 
               
               
                 gtgagtgaca cgcagcctgc agactcactg tgggaaggag acaaaactag agactcaaag 
               
               
                 agggagtgca tttatgagct cttcatgttt caggagagag ttgaacctaa acatagaaat 
               
               
                 tgcctgacga actccttgat tttagccttc tctgttcatt tcctcaaaaa gatttcccca 
               
               
                 tttaggtttc tgagttcctg catgccggtg atccctagct gtgacctctc ccctggaact 
               
               
                 gtctctcatg aacctcaagc tgcatctaga ggcttccttc atttcctccg tcacctcaga 
               
               
                 gacatacacc tatgtcattt catttcctat ttttggaaga ggactcctta aatttggggg 
               
               
                 acttacatga ttcattttaa catctgagaa aagctttgaa ccctgggacg tggctagtca 
               
               
                 taaccttacc agatttttac acatgtatct atgcattttc tggacccgtt caacttttcc 
               
               
                 tttgaatcct ctctctgtgt tacccagtaa ctcatctgtc accaagcctt ggggattctt 
               
               
                 ccatctgatt gtgatgtgag ttgcacagct atgaaggctg tgcactgcac gaatggaaga 
               
               
                 ggcacctgtc ccagaaaaag catcatggct atctgtgggt agtatgatgg gtgtttttag 
               
               
                 caggtaggag gcaaatatct tgaaaggggt tgtgaagagg tgttttttct aattggcatg 
               
               
                 aaggtgtcat acagatttgc aaagtttaat ggtgccttca tttgggatgc tactctagta 
               
               
                 ttccagacct gaagaatcac aataattttc tacctggtct ctccttgttc tgataatgaa 
               
               
                 aattatgata aggatgataa aagcacttac ttcgtgtccg actcttctga gcacctactt 
               
               
                 acatgcatta ctgcatgcac ttcttacaat aattctatga gataggtact attatcccca 
               
               
                 tttctttttt aaatgaagaa agtgaagtag gccgggcacg gtggctcgcg cctgtggtcc 
               
               
                 cagggtgctg agattgcagg tgtgagccac cctgcccagc cgtcaaaaga gtcttaatat 
               
               
                 atatatccag atggcatgtg tttactttat gttactacat gcacttggct gcataaatgt 
               
               
                 ggtacaacca ttctgtcttg aagggcaggt gcttcaggat accatataca gctcagaagt 
               
               
                 ttcttcttta ggcattaaat tttagcaaag atatctcatc tcttctttta aaccattttc 
               
               
                 tttttttgtg gttagaaaag ttatgtagaa aaaagtaaat gtgatttacg ctcattgtag 
               
               
                 aaaagctata aaatgaatac aattaaagct gttatttaat tagccagtga aaaactatta 
               
               
                 acaacttgtc tattacctgt tagtattatt gttgcattaa aaatgcatat actttaataa 
               
               
                 atgtacattg tattgtaaaa aaaaaaa 
               
               
                   
               
             
          
         
       
     
                               TABLE 2                   Human HFE gene product       (SEQ ID NO:2; GENBANK ® Accession No. U60319)                    MGPRARPALLLLMLLQTAVLQG             RLLRSHSLHYLFMGASEQDLGLSLFEALGYVDDQLFVFYDHESRRVEPRT           PWVSSRISSQMWLQLSOSLKGWDHMFTVDFWTIMENHNHSK ESHTLQVIL       GCEMQEDNSTEGYWKYGYDGQDHLEFCPDTLDWRAAEPRAWPTKLEWERH       KIRARQNRAYLERDCPAQLQQLLELGRGVLDQQVPPLVKVTHHVTSSVTT       LRCRALNYYPQNITMKWLKDKQPMDAKEFEPKDVLPNGDGTYQGWITLAV       PPGEEQRYTCQVEHPGLDQPLIVIWEPSPSGTLVIGVISGIAVFVVILFI       GILFIILRKRQGSRGAMGHYVLAERE                    
Residues 1-22 =leader sequence; α1 domain underlined; residues 63, 65, 93, and 105 indicated in bold type)
 
Other mutations include nucleotide 277 of SEQ ID NO: 1, e.g., 277C which leads to expression of mutant HFE gene product G93R and one at nucleotide 193 of SEQ ID NO: 1, e.g., 193T, which leads to expression of mutant HFE gene product S65C.
 
     Any biological sample containing an HFE nucleic acid or gene product is suitable for the diagnostic methods described herein. For example, the biological sample to be analyzed is whole blood, cord blood, serum, saliva, buccal tissue, plasma, effusions, ascites, urine, stool, semen, liver tissue, kidney tissue, cervical tissue, cells in amniotic fluid, cerebrospinal fluid, hair or tears. Prenatal testing can be done using methods used in the art, e.g., amniocentesis or chorionic villa sampling. Preferably, the biological sample is one that can be non-invasively obtained, e.g., cells in saliva or from hair follicles. 
     The assay is also used to screen individuals prior to donating blood to blood banks and to test organ tissue, e.g., a donor liver, prior to transplantation into a recipient patient. Both donors and recipients are screened. 
     In some cases, a nucleic acid is amplified prior to detecting a mutation. The nucleic acid is amplified using a first oligonucleotide primer which is 5′ to exon 2 and a second oligonucleotide primer is 3′ to exon 2. To detect mutation at nucleotide 314 of SEQ ID NO: 1, a first oligonucleotide primer which is 5′ to nucleotide 314 and a second oligonucleotide primer which is 3′ to nucleotide 314 is used in a standard amplification procedure such as polymerase chain reaction (PCR). To amplify a nucleic acid containing nucleotide 277 of SEQ ID NO: 1, a first oligonucleotide primer which is 5′ to nucleotide 277 and a second oligonucleotide primer which is 3′ to nucleotide 277 is used. Similarly, a nucleic acid containing nucleotide 193 of SEQ ID NO:1 is amplified using primers which flank that nucleotide. For example, for nucleotide 277, the first primer has a nucleotide sequence of SEQ ID NO: 3 and said second oligonucleotide primer has a nucleotide sequence of SEQ ID NO: 4, or the first primer has a nucleotide sequence of SEQ ID NO: 15 and said second oligonucleotide primer has a nucleotide sequence of SEQ ID NO: 16. Table 3, below, shows examples of primer pairs for amplification of nucleic acids in exons and introns of the HFE gene. 
     
       
         
               
               
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                 Target DNA 
                 Forward Primer 
                 Reverse Primer 
               
               
                   
               
             
             
               
                   
                 I. PRIMERS USED FOR AMPLIFICATION 
                   
                   
               
               
                 Exon 2 
                 CCTCCTACTACACATGGTTAAGG 
                 GCTCTGACAACCTCAGGAAGG 
               
               
                   
                 (SEQ ID NO: 3) 
                 (SEQ ID NO: 4) 
               
               
                 Exon 3 
                 GGTGGAAATAGGGACCTATTCC 
                 CACTCTGCCACTAGACTATAGG 
               
               
                   
                 (SEQ ID NO: 5) 
                 (SEQ ID NO: 6) 
               
               
                 Exon 4 
                 GTTCCAGTCTTCCTGGCAAGG 
                 AAATGCTTCCCATGGATGCCAG 
               
               
                   
                 (SEQ ID NO: 7) 
                 (SEQ ID NO: 8) 
               
               
                 RT-PCR 
                 AAAGGATCCACCATGGGCCCGCGAGCCAGG 
                 GTGAGTCTGCAGGCTGCGTG 
               
               
                   
                 (SEQ ID NO: 9) 
                 (SEQ ID NO: 10) 
               
               
                 Intron 4 
                 GTTCCAGTCTTCCTGGCAAGG 
                 AAATGCTTCCCATGGATGCCAG 
               
               
                   
                 (SEQ ID NO: 11) 
                 (SEQ ID NO: 12) 
               
               
                 Intron 5 
                 GTTCCAGTCTTCCTGGCAAGG 
                 AAATGCTTCCCATGGATGCCAG 
               
               
                   
                 (SEQ ID NO: 13) 
                 (SEQ ID NO: 14) 
               
               
                   
                 II. PRIMERS USED FOR AMPLIFICATION 
               
               
                 Exon 2 
                 GTGTGGAGCCTCAACATCCTG 
                 ACAAGACCTCAGACTTCCAGC 
               
               
                   
                 (SEQ ID NO: 15) 
                 (SEQ ID NO: 16) 
               
               
                 Exon 3 
                 GGTGGAAATAGGGACCTATTCC 
                 CACTCTGCCACTAGAGTATAGG 
               
               
                   
                 (SEQ ID NO: 17) 
                 (SEQ ID NO: 18) 
               
               
                 Exon 4 
                 GTTCCAGTCTTCCTGGCAAGG 
                 TTACCTCCTCAGGCACTCCTC 
               
               
                   
                 (SEQ ID NO: 19) 
                 (SEQ ID NO: 20) 
               
               
                 RT-PCR 
                 AAAGGATCCACCATGGGCCCGCGAGCCAGG 
                 GTGAGTCTGCACGCTGCGTG 
               
               
                   
                 (SEQ ID NO: 21) 
                 (SEQ ID NO: 22) 
               
               
                 Intron 4 
                 TGCCTGAGGAGGTAATTATGG 
                 AAATGCTTCCCATGGATGCCAG 
               
               
                   
                 (SEQ ID NO: 23) 
                 (SEQ ID NO: 24) 
               
               
                 Intron 5 
                 TGCCTGAGGAGGTAATTATGG 
                 AAATGCTTCCCATGGATGCCAG 
               
               
                   
                 (SEQ ID NO: 25) 
                 (SEQ ID NO: 26) 
               
               
                   
               
             
          
         
       
     
     Mutations in introns of the HFE gene have now been associated with iron disorders and/or hemochromatosis. By “exon” is meant a segment of a gene the sequence of which is represented in a mature RNA product, and by “intron” is meant a segment of a gene the sequence of which is not represented in a mature RNA product. An intron is a part of a primary nuclear transcript which is subsequently spliced out to produce a mature RNA product, i.e., a mRNA, which is then transported to the cytoplasm. A method of diagnosing an iron disorder or a genetic susceptibility to developing the disorder is carried out by determining the presence or absence of a mutation in an intron of HFE genomic DNA in a biological sample. The presence of the mutation is indicative of the disorder or a genetic susceptibility to developing the disorder. The presence of a mutation in an intron is a marker for an exon mutation, e.g., a mutation in intron 4, e.g., at nucleotide 6884 of SEQ ID NO:27 is associated with the S65C mutation in exon 2. A mutation in intron 5, e.g., at nucleotide 7055 of SEQ ID NO:27 is associated with hemochromatosis. In some cases, intron mutations may adversely affect proper splicing of exons or may alter regulatory signals. Preferably, the intron 4 mutation is 6884C and the intron 5 mutation is 7055G. To amplify nucleic acid molecule containing nucleotide 6884 or 7055, primers which flank that nucleotide, e.g., those described in Table 3, are used according to standard methods. Nucleic acid-based diagnostic methods may or may not include a step of amplification to increase the number of copies of the nucleic acid to be analyzed. To detect a mutation in intron 4, a patient-derived nucleic acid may be amplified using a first oligonucleotide primer which is 5′ to intron 4 and a second oligonucleotide primer which is 3′ to intron 4, and to detect a mutation in intron 5, the nucleic acid may be amplified using a first oligonucleotide primer which is 5′ to intron 5 and a second oligonucleotide primer which is 3′ to intron 5 (see, e.g., Table 3). 
     In addition to nucleic acid-based diagnostic methods, the invention includes a method of diagnosing an iron overload disorder or a genetic susceptibility thereto by determining the presence of a mutation in a HFE gene product in a biological sample. For example, the mutation results in a decrease in intramolecular salt bridge formation in the mutant HFE gene product compared to salt bridge formation in a wild type HFE gene product. The mutation which affects salt bridge formation is at or proximal to residue 63 of SEQ ID NO:2, but is not amino acid substitution H63D. Preferably, the mutation is between residues 23-113, inclusive of SEQ ID NO:2 (Table 2), more preferably, it is between residues 90-100, inclusive, of SEQ ID NO:2, more preferably, it is between residues 58-68, inclusive, of SEQ ID NO:2, and most preferably, the mutation is amino acid substitution S65C. Alternatively, the mutation which affects salt bridge formation is a mutation, e.g., an amino acid substitution at residue 95 or proximal to residue 95 of SEQ ID NO:2. Preferably, the mutation is G93R. Such an HFE mutation is detected by immunoassay or any other ligand binding assay such as binding of the HFE gene product to a transferrin receptor. Mutations are also detected by amino acid sequencing, analysis of the structural conformation of the protein, or by altered binding to a carbohydrate or peptide mimetope. 
     A mutation indicative of an iron disorder or a genetic susceptibility to developing such a disorder is located in the α1 helix (e.g., which spans residues 80-108, inclusive, of SEQ ID NO:2) of an HFE gene product. The mutation may be an addition, deletion, or substitution of an amino acid in the wild type sequence. For example, the mutant HFE gene product contains the amino acid substitution I105T or G93R or in the loop of the β sheet of the HFE molecule, e.g., mutation S65C 
     Isolated nucleic acids encoding a mutated HFE gene products (and nucleic acids with nucleotide sequences complementary to such coding sequences) are also within the invention. Also included are nucleic acids which are at least 12 but less than 100 nucleotides in length. An isolated nucleic acid molecule is a nucleic acid molecule that is separated from the 5′ and 3′ sequences with which it is immediately contiguous in the naturally occurring genome of an organism. “Isolated” nucleic acid molecules include nucleic acid molecules which are not naturally occurring. For example, an isolated nucleic acid is one that has been amplified in vitro, e.g., by PCR; recombinantly produced; purified, e.g., by enzyme cleavage and gel separation; or chemically synthesized. For example, the restriction enzyme, Bst4C I (Sib Enzyme Limited, Novosibirsk, Russia), can be used to detect the G93R mutation (point mutation 277C); this enzyme cuts the mutated HFE nucleic acid but not the wild type HFE nucleic acid. Such nucleic acids are used as markers or probes for disease states. For example, a marker is a nucleic acid molecule containing a nucleotide polymorphism, e.g., a point mutation, associated with an iron disorder disease state flanked by wild type HFE sequences. The invention also encompasses nucleic acid molecules that hybridize, preferably under stringent conditions, to a nucleic acid molecule encoding a mutated HFE gene product (or a complementary strand of such a molecule). Preferably the hybridizing nucleic acid molecule is 400 nucleotides, more preferably 200 nucleotides, more preferably 100, more preferably 50, more preferably 25 nucleotides, more preferably 20 nucleotides, and most preferably 10-15 nucleotides, in length. For example, the nucleotide probe to detect a mutation is 13-15 nucleotides long. The nucleic acids are also used to produce recombinant peptides for generating antibodies specific for mutated HFE gene products. In preferred embodiments, an isolated nucleic acid molecule encodes an HFE polypeptide containing amino acid substitution I105T, G93R, or S65C, as well as nucleic acids the sequence of which are complementary to such nucleic acid which encode a mutant or wild type HFE gene product. 
     Also within the invention are substantially pure mutant HFE gene products, e.g., an HFE polypeptide containing amino acid substitution I105T, G93R, or S65C. Substantially pure or isolated HFE polypeptides include those that correspond to various functional domains of HFE or fragments thereof, e.g., a fragment of HFE that contains the α1 domain. 
     Wild type HFE binds to the transferrin receptor and regulates the affinity of transferrin receptor binding to transferrin. For example, a C282Y mutation in the HFE gene product reduces binding to the transferrin receptor, thus allowing the transferrin receptor to bind to transferrin (which leads to increased iron absorption). 
     The polypeptides of the invention encompass amino acid sequences that are substantially identical to the amino acid sequence shown in Table 2 (SEQ ID NO:2). Polypeptides of the invention are recombinantly produced, chemically synthesized, or purified from tissues in which they are naturally expressed according to standard biochemical methods of purification. Biologically active or functional polypeptides are those which possess one or more of the biological functions or activities of wild type HFE, e.g., binding to the transferrin receptor or regulation of binding of transferrin to the transferrin receptor. A functional polypeptide is also considered within the scope of the invention if it serves as an antigen for production of antibodies that specifically bind to an HFE epitope. In many cases, functional polypeptides retain one or more domains present in the naturally-occurring form of HFE. 
     The functional polypeptides may contain a primary amino acid sequence that has been altered from those disclosed herein. Preferably, the cysteine residues in exons 3 and 4 remain unchanged. Preferably the modifications consist of conservative amino acid substitutions. The terms “gene product”, “protein”, and “polypeptide” are used herein to describe any chain of amino acids, regardless of length or post-translational modification (for example, glycosylation or phosphorylation). Thus, the term “HFE polypeptide or gene product” includes full-length, naturally occurring HFE protein, as well a recombinantly or synthetically produced polypeptide that correspond to a full-length naturally occurring HFE or to a particular domain or portion of it. 
     The term “purified” as used herein refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Polypeptides are said to be “substantially pure” when they are within preparations that are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. 
     Diagnostic kits for identifying individuals suffering from or at risk of developing an iron disorder are also within the invention. A kit for detecting a nucleotide polymorphism associated with an iron disorder or a genetic susceptibility thereto contains an isolated nucleic acid which encodes at least a portion of the wild type or mutated HFE gene product, e.g., a portion which spans a mutation diagnostic for an iron disorder or hemochromatosis (or a nucleic acid the sequence of which is complementary to such a coding sequence). A kit for the detection of the presence of a mutation in exon 2 of an HFE nucleic acid contains a first oligonucleotide primer which is 5′ to exon 2 and a second oligonucleotide primer is 3′ to exon 2, and a kit for an antibody-based diagnostic assay includes an antibody which preferentially binds to an epitope of a mutant HFE gene product, e.g., an HFE polypeptide containing amino acid substitution I105T, G93R, or S65C, compared to its binding to the wild type HFE polypeptide. An increase in binding of the mutant HFE-specific antibody to a patient-derived sample (compared to the level of binding detected in a wild type sample or sample derived from a known normal control individual) indicates the presence of a mutation which is diagnostic of an iron disorder, i.e., that the patient from which the sample was taken has an iron disorder or is at risk of developing one. The kit may also contain an antibody which binds to an epitope of wild type HFE which contains residue 105, 93, or 65. In the latter case, reduced binding of the antibody to a patient-derived HFE gene product (compared to the binding to a wild type HFE gene product or a gene product derived from a normal control individual) indicates the presence of a mutation which is diagnostic of an iron disorder, i.e., that the patient from which the sample was taken has an iron disorder or is at risk of developing one. 
     Individual mutations and combinations of mutations in the HFE gene are associated with varying severity of iron disorders. For example, the C282Y mutation in exon 4 is typically associated with clinical hemochromatosis, whereas other HFE mutations or combinations of mutations in HFE nucleic acids are associated with disorders of varying prognosis. In some cases, hemochromatosis patients have been identified which do not have a C282Y mutation. The I105T and G93R mutations are each alone associated with an increased risk of iron overload (compared to, e.g., the H63D mutation alone), and the presence of both the I105T and H63D mutation is associated with hemochromatosis. Accordingly, the invention includes a method of determining the prognosis for hemochromatosis in a mammal suffering from or at risk of developing said hemochromatosis by (a) detecting the presence or absence of a first mutation in exon 4 in each allele of an HFE nucleic acid, e.g., patient-derived chromosomal DNA, and (b) detecting the presence of a second mutation in exon 2 in each allele of the nucleic acid. The presence of the first mutation in both chromosomes, i.e. an exon 4 homozygote such as a C282Y homozygote, indicates a more negative prognosis compared to the presence of the second mutation in one or both chromosomes, i.e., an exon 2 heterozygote or homozygote. An exon 4 mutation homozygote is also associated with a more negative prognosis compared to the presence of a first mutation (exon 4) in one allele and the presence of the second mutation (exon 2) in one allele, i.e., a compound heterozygote. 
     Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a diagram of the family of proband 1 (HFE genotype H63D/I105T). □=male, ●=female, ø=deceased, ▪=hemochromatosis phenotype. Proband 1 is indicated by an arrow. Phenotype and genotype data: age in year saturation; % Ftn=serum ferritin concentration. I105 separate chromosomes. The sister of the proband (II, 203) has hyperferritinemia. 
         FIG. 2  is a diagram of the family of proband 2 (HFE genotype C282Y/G93R). Symbols and abbreviations are the same as those described for FIG.  1 . Proband 2 is indicated with an arrow. G93R, C282Y, and wt alleles are known to exist only on separate chromosomes. The father and sister of the proband are being treated for hemochromatosis. 
         FIG. 3  is a diagram of the family of proband 3 (HFE genotype C282Y/S65C). Symbols and abbreviations are the same as those described for FIG.  1 . Proband 3 is indicated with an arrow. S65C, C282Y, and wt alleles are know to exist only on separate chromosomes. Proband 3 also has porphyria cutanea tarda, and her brother (II, 203) has ankylosing spondylitis. 
     
    
    
     DETAILED DESCRIPTION 
     A proband is the first individual in a family identified to be affected by hemochromatosis. Forward and reverse sequencing of HFE exons 2, 3, 4, and 5, and of portions of HFE introns 2, 4, and 5 was carried out on biological samples taken from twenty hemochromatosis probands who lacked C282Y homozygosity, C282Y/H63D compound heterozygosity, or H63D homozygosity. Four probands had novel HFE coding region mutations. Probands 1 and 2 were heterozygous for previously undescribed mutations: exon 2, nt 314T→C (314C; I105T), and exon 2, nt 277G→C (277C; G93R), respectively; these probands were also heterozygous for H63D and C282Y, respectively. Probands 3 and 4 were heterozygous for an HFE mutation in exon 2, nt 193A→T (193T; S65C). Twelve other probands did not have an exon 2 HFE exon mutation; four were heterozygous for H63D. In probands 1, 2, 3, and 4, the amino acid substitutions I105T, G93R, and S65C (respectively) occurred on separate chromosomes from those with the C282Y or H63D mutations. In 176 normal control subjects, two were heterozygous for S65C; I105T and G93R were not detected in controls. Nine probands were heterozygous and two probands were homozygous for a base-pair change at intron 2, nt 4919T/C (SEQ ID NO:27). Heterozygosity for a base-pair change in intron 4 (nt 6884T→C) was detected only in probands 3 and 4, both of whom also had S65C and HLA-A32. The intron 2 mutation is not diagnostic of an iron disorder and appears randomly in the population. One proband was heterozygous for a base-pair change at intron 5 (nt 7055A→G). 
     The data described herein indicate that, in addition to the C282Y and H63D HFE mutations, the HFE exon and intron 5 mutations described herein are diagnostic (and prognostic) of iron disorders. 
     Pathology of Iron Overload 
     Iron plays an essential role in normal growth and development, but in elevated concentrations, iron is a toxic inorganic molecule and is the leading cause of death in children by poisoning. It has been implicated in the pathophysiology of a number of common diseases, e.g., hepatitis, cancer, heart disease, reperfusion injury, rheumatoid arthritis, diabetes, AIDS, and psychological abnormalities (e.g. depression). 
     The incidence of cancer (especially liver cancer) rises dramatically in the course of hemochromatosis. Iron, acting alone or in synergy with other environmental agents, catalyzes free radical formation. These free radicals which mediate tissue damage also cause DNA double strand breaks and oncogene activation. Iron may also play a role in the pathogenesis of rheumatic diseases and in predisposition to heart disease. High levels of iron can also cause diabetes with 2% of diabetics being hemochromatosis patients. High levels of iron may also affect the disease progression of many viral diseases. Individuals infected with such viruses as hepatitis (e.g., hepatitis B or C) or HIV should be tested for HFE mutations because of the impact increased iron stores have on the treatment and prognosis of such diseases. 
     Excessive iron stores and iron deposition is also a major contributing factor in the pathology and treatment of non-valvular heart disease. These conditions include dilated cardiomyopathy cased by deposition of iron in myocardial fibers; myocardial injury the product of anthracycline cardiomyopathy and re-perfusion injury. Increased iron stores may also be a contributing factor in myocardial infarction due to atherosclerosis. Some evidence suggests a significant increase in the incidence of reported heart disease in probands (cardiac symptoms-32%, insulin-dependent diabetes-18%, cardiac arrhythmia-17%, clinically significant coronary artery atherosclerosis-9%, and congestive heart failure-7%. Cardiac complications have been detected in 30% of patients. These include EKG abnormalities, congestive heart failure and cardiac arrhythmias. An increased frequency of HFE mutations in individuals with porphyria cutanea tarda indicates that HFE mutations may predipose an individual to developing this syndrome. 
     The effect of iron overload is irreparable damage to vital organs and a multiplicity of associated pathologies described above. The multiplicity of clinical symptoms (and associated pathologies) often causes misdiagnosis of hemochromatosis or failure to diagnose hemochromatosis. 
     Untreated hemochromatosis is characterized by iron overload of parenchymal cells, which is toxic and the probable cause of various complications including cirrhosis, and liver cancer, arthropathy, hypogonadotropic hypogonadism, marrow aplasia, skin disorders, diabetes mellitus, and cardiomyopathy. There are 1.5 to 2 million active cases in the U.S. of which 40% have progressive liver disease because they have not been properly diagnosed or treated. 
     In untreated hemochromatosis, iron is universally deposited in the hepatocytes of the liver. The iron is found primarily in the cytoplasm of hepatocytes, and by electron microscopy in lysosomal vacuoles, and in more severe cases iron has also been reported deposited in mitochondria. Other liver toxins such as alcohol, and hepatitis exacerbate the damage caused by the iron deposition. Patients with hemochromatosis are advised not to drink, because of increased liver damage, or to smoke, as iron deposition can also occur in the lungs. 
     Individuals which are homozygous (and to a lesser extent heterozygous) for an HFE mutation are at risk for developing increased levels of blood lead. Thus, it is important to identify heterozygous as well as homozygous patients. 
     Identification and detection of mutations in the HFE gene are critical to understanding the general mechanisms of iron disorders and diagnosing iron-related pathologies. 
     Nucleic Acid-based Assays for HFE Mutations 
     A biological sample containing RNA or DNA is obtained from an individual and the nucleic acid extracted. optionally, the nucleic acid is amplified according to standard procedures such as PCR. A nucleic acid polymorphism, e.g., a single base pair polymorphism, is detected using methods well known in the art of molecular biology. For example, a mutation is detected using a standard sequencing assay, nucleic acid hybridization, e.g., using standard Southern, Northern, or dot blot hybridization assay systems and an HFE-specific oligonucleotide probe, restriction enzyme fragment polymorphism analysis, oligonucleotide ligation assay (OLA; Nikerson et al., 1990, Nucl. Acids Res. 87:8923-8927), primer extension analysis (Nikiforov et al., 1994, Nucl. Acids Res. 22:4167-4175), single strand conformation polymorphism (SSCP) analysis, allele-specific PCR (Rust et al., 1993, Nucl. Acids Res. 6:3623-3629), denaturing gradient gel electrophoresis (DGGE), fluorescent probe melting curve analysis (Bernard et al., 1998, Am. J. Pathol. 153:1055-61), RNA mismatch cleavage assay, capillary hybridization, or TAQMAN™ assay (fluorogenic 5′ nuclease assay) (PE Applied Biosystems, Foster City, Calif.). Nucleic acid hybridization assays are also carried out using a bioelectronic microchip technology known in the art, e.g., that described in Sosnowski et al., 1997, Proc. Natl. Acad. Sci. U.S.A. 94:1119-1123; Cheng et al. 1998, Nature Biotechnology 16:541-546; or Edman et al., 1997, Nucl. Acids Res. 25:4907-4914. 
     Detection of Mutations Using Antibodies and Other HFE Ligands 
     Anti-HFE antibodies are know in the art, e.g., those described by Feder et al., 1997, J. Biol. Chem. 272:14025-14028, or are obtained using standard techniques. Such antibodies can be polyclonal or monoclonal. Polyclonal antibodies can be obtained, for example, by the methods described in Ghose et al., Methods in Enzymology, Vol. 93, 326-327, 1983. An HFE polypeptide, or an antigenic fragment thereof, is used as an immunogen to stimulate the production of HFE-reactive polyclonal antibodies in the antisera of animals such as rabbits, goats, sheep, rodents and the like. HFE antibodies specific for mutated HFE gene products are raised by immunizing animals with a polypeptide spanning the mutation, e.g., a polypeptide which contains the mutations described herein. For example, the entire al domain of a mutant HFE gene product is used as an immunogen. Monoclonal antibodies are obtained by the process described by Milstein and Kohler in Nature, 256:495-97, 1975, or as modified by Gerhard, Monoclonal Antibodies, Plenum Press, 1980, pages 370-371. Hybridomas are screened to identify those producing antibodies that are highly specific for an HFE polypeptide containing a mutation characteristic of an iron metabolism abnormality or clinical hemochromatosis. Preferably, the antibody has an affinity of at least about 10≡liters/mole, preferably at least 10≢liters/mole, more preferably at least 10[liters/mole, and most preferably, an affinity of at least about 10]liters/mole. 
     Antibodies specific for the wild type HFE can also be used to diagnose hemochromatosis or iron metabolism abnormalities. Such antibodies are also useful research tools to identify novel mutations indicative of iron disorders or hemochromatosis. A reduction in binding to a wild type HFE-specific antibody indicates the presence of a mutation. Antibody binding is detected using known methods. For example, an ELISA assay involves coating a substrate, e.g., a plastic dish, with an antigen, e.g., a patient-derived biological sample containing an HFE gene product. An antibody preparation is then added to the well. Antibodies specific for a mutant HFE gene product bind or fail to bind to a patient-derived sample in the well. Non-binding material is washed away and a marker enzyme e.g., horse radish peroxidase or alkaline phosphatase, coupled to a second antibody directed against the antigen-specific primary antibody is added in excess and the nonadherent material is washed away. An enzyme substrate is added to the well and the enzyme catalyzed conversion is monitored as indicative of presence of the mutation. Antibodies are also labelled with various sizes of colloidal gold particles or latex particles for detection of binding. 
     The invention employs not only intact monoclonal or polyclonal antibodies, but also an immunologically-active antibody fragment, for example, a Fab or (Fab) 2  fragment; an antibody heavy chain, an antibody light chain; a genetically engineered single-chain Fv molecule (Ladner et al., U.S. Pat. No. 4,946,778). 
     EXAMPLE 1 
     Selection and Characterization of Subjects 
     All individuals studied were Caucasians, 18 years of age or older, and from central Alabama. Twenty probands were identified that were either heterozygous for C282Y or H63D, or lacked these mutations. Hemochromatosis is typically diagnosed by detecting elevated saturation of transferrin, with elevated serum ferritin levels, combined with liver biopsy. Each proband patient described below was previously diagnosed to have hemochromatosis by the working diagnostic criterion for hemochromatosis of the American College of Pathologists (elevated fasting transferrin saturation of greater than 60% saturation for males and greater than 50% saturation for females) on at least two occasions in the absence of other known causes. Probands were interviewed regarding their general medical history, diet (including estimated iron content and ethanol consumption), medicinal iron use, receipt of blood transfusion, prior significant hemorrhage, blood donation for transfusion and/or therapeutic phlebotomy, and pregnancy and lactation. Each proband was also evaluated for viral hepatitis B and C and other hepatic disorders, excess ethanol intake, and hereditary, and acquired anemia. Iron overload was defined as evidence of systemic iron overload demonstrated by otherwise unexplained elevated serum is ferritin concentration (≧300 ng/mL in men, ≧200 ng/mL in women), increased hepatic iron content determined using hepatic biopsy specimens, or iron &gt;4 g mobilized by phlebotomy. Complications of iron overload were evaluated and treated, and therapeutic phlebotomy was performed using standard methods. HFE mutation analysis for C282Y and H63D and human leukocyte antigen (HLA) immunophenotyping or molecular typing were performed using known methods. In some family members, HLA haplotyping had been performed previously for other disease associations, or their HLA type could be deduced from analysis of their kinship and HFE genotyping results. Measurement of serum iron and other clinical laboratory parameters and analysis of hepatic biopsy specimens were performed using routine methods. Control subjects (n=176) who were in apparently good health and were unrelated to the hemochromatosis probands were recruited from the general population. Iron parameters were measured and HLA typing was performed in two control subjects after HFE genotyping revealed that they had the S65C mutation. 
     EXAMPLE 2 
     HFE Gene Analysis 
     PCR amplification was used to detect mutations. Genomic DNA was prepared from peripheral blood buffy coat or saliva using the QIAmpBlood Kit (QIAGEN, Valencia, Calif.) or FTA Paper and FTA purification reagent (Fitzco Inc., Maple Plain, Minn.), respectively. Fragments were amplified from genomic DNA using eLONGase (Life Technologies, Gaithersburg, Md.) or HotStarTaq DNA polymerase (QIAGEN, Valencia, Calif.). Primers used to amplify each exon are shown in Table 3. 
     
       
         
               
               
             
               
               
               
             
           
               
                 TABLE 4 
               
               
                   
               
               
                 Human HFE genomic DNA 
                   
               
               
                 (SEQ ID NO:27; GENBANK ® Accession No. Z92910) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 ggatccttta accgaggaga ttattatagc cggagctctg aagcagcaat ctcagttctt 
                   
               
               
                 61 
                 gtgatagtga gcaaagaact acaaactaac accaaaatgc aagcttaaag caaagtttat 
               
               
                 121 
                 tgaagcacaa taatacactc tgagggacag cgggcttatt tctgcgaagt gaactcagca 
               
               
                 181 
                 cttctttaca gagctcaagg tgcttttatg gggtttgtgg ggaggagttg aggtttgggc 
               
               
                 241 
                 tgtatctgag tgacaggatg atgttatttg attgaagttt atagctatac aatctaaaat 
               
               
                 301 
                 taaactgtgc atggtcttac ctataatttg ttaagaaaag cctcccaggg atgggggggc 
               
               
                 361 
                 aaaactgtat gtaaattcta ttataatgat ggcatgatga acttggggtg aacttgaaga 
               
               
                 421 
                 caggcttttg tgttgttggg catgtgccac cttagggaat ttccacctgt accctccttt 
               
               
                 481 
                 ctctttctcc aggatatttt ggccacagac tttatcataa actccatccc ttagggtggc 
               
               
                 541 
                 attagggtag tcttgggcct gaatttaggt gggccagtgg ctgtcttagt gacagccttt 
               
               
                 601 
                 ccgctctctt ctgtcatccc ctcccaactg ctaatgtcta actacctaac aattacccat 
               
               
                 661 
                 taaatcagtg tgtctggggt taggagcagg cctcaatatg tttaatcatt ctccagataa 
               
               
                 721 
                 tcccaatact gtaaagtttg tgaaacactt gtcagataat tcaattatga aggctgtgga 
               
               
                 781 
                 acgtgtttca gtaggatcta attggttaat gttatgactt aattaatttg aatcaaaaaa 
               
               
                 841 
                 caaaatgaaa aagctttata tttctaagtc aaataagaca taagttggtc taaggttgag 
               
               
                 901 
                 ataaaatttt taaatgtatg attgaatttt gaaaatcata aatatttaaa tatctaaagt 
               
               
                 961 
                 tcagatcaga acattgcgaa gctactttcc ccaatcaaca acaccccttc aggatttaaa 
               
               
                 1021 
                 aaccaagggg gacactggat cacctagtgt ttcacaagca ggtaccttct gctgtaggag 
               
               
                 1081 
                 agagagaact aaagttctga aagacctgtt gcttttcacc aggaagtttt actgggcatc 
               
               
                 1141 
                 tcctgagcct aggcaatagc tgtagggtga cttctggagc catccccgtt tccccgcccc 
               
               
                 1201 
                 ccaaaagaag cggagattta acggggacgt gcggccagag ctggggaaat gggcccgcga 
               
               
                 1261 
                 gccaggccgg cgcttctcct cctgatgctt ttgcagaccg cggtcctgca ggggcgcttg 
               
               
                 1321 
                 ctgcgtgagt ccgagggctg cgggcgaact aggggcgcgg cgggggtgga aaaatcgaaa 
               
               
                 1381 
                 ctagcttttt ctttgcgctt gggagtttgc taactttgga ggacctgctc aacccaatcc 
               
               
                 1441 
                 gcaagcccct ctccctactt tctgcgtcca gaccccgtga gggagtgcct accactgaac 
               
               
                 1501 
                 tgcagatagg ggtccctcgc cccaggacct gccccctccc ccggctgtcc cggctctgcg 
               
               
                 1561 
                 gagtgacttt tggaaccgcc cactcccttc ccccaactag aatgctttta aataaatctc 
               
               
                 1621 
                 gtagttcctc acttgagctg agctaagcct ggggctcctt gaacctggaa ctcgggttta 
               
               
                 1681 
                 tttccaatgt cagctgtgca gttttttccc cagtcatctc caaacaggaa gttcttccct 
               
               
                 1741 
                 gagtgcttgc cgagaaggct gagcaaaccc acagcaggat ccgcacgggg tttccacctc 
               
               
                 1801 
                 agaacgaatg cgttgggcgg tgggggcgcg aaagagtggc gttggggatc tgaattcttc 
               
               
                 1861 
                 accattccac ccacttttgg tgagacctgg ggtggaggtc tctagggtgg gaggctcctg 
               
               
                 1921 
                 agagaggcct acctcgggcc tttccccact cttggcaatt gttcttttgc ctggaaaatt 
               
               
                 1981 
                 aagtatatgt tagttttgaa cgtttgaact gaacaattct cttttcggct aggctttatt 
               
               
                 2041 
                 gatttgcaat gtgctgtgta attaagaggc ctctctacaa agtactgata atgaacatgt 
               
               
                 2101 
                 aagcaatgca ctcacttcta agttacattc atatctgatc ttatttgatt ttcactaggc 
               
               
                 2161 
                 atagggaggt aggagctaat aatacgttta ttttactaga agttaactgg aattcagatt 
               
               
                 2221 
                 atataactct tttcaggtta caaagaacat aaataatctg gttttctgat gttatttcaa 
               
               
                 2281 
                 gtactacagc tgcttctaat cttagttgac agtgattttg ccctgtagtg tagcacagtg 
               
               
                 2341 
                 ttctgtgggt cacacgccgg cctcagcaca gcactttgag ttttggtact acgtgtatcc 
               
               
                 2401 
                 acattttaca catgacaaga atgaggcatg gcacggcctg cttcctggca aatttattca 
               
               
                 2461 
                 atggtacacg gggctttggt ggcagagctc atgtctccac ttcatagcta tgattcttaa 
               
               
                 2521 
                 acatcacact gcattagagg ttgaataata aaatttcatg ttgagcagaa atattcattg 
               
               
                 2581 
                 tttacaagtg taaatgagtc ccagccatgt gttgcactgt tcaagcccca agggagagag 
               
               
                 2641 
                 cagggaaaca agtctttacc ctttgatatt ttgcattcta gtgggagaga tgacaataag 
               
               
                 2701 
                 caaatgagca gaaagatata caacatcagg aaatcatggg tgttgtgaga agcagagaag 
               
               
                 2761 
                 tcagggcaag tcactctggg gctgacactt gagcagagac atgaaggaaa taagaatgat 
               
               
                 2821 
                 attgactggg agcagtattt cccaggcaaa ctgagtgggc ctggcaagtt ggattaaaaa 
               
               
                 2881 
                 gcgggttttc tcagcactac tcatgtgtgt gtgtgtgggg gggggggcgg cgtgggggtg 
               
               
                 2941 
                 ggaaggggga ctaccatctg catgtaggat gtctagcagt atcctgtcct ccctactcac 
               
               
                 3001 
                 taggtgctag gagcactccc ccagtcttga caaccaaaaa tgtctctaaa ctttgccaca 
               
               
                 3061 
                 tgtcacctag tagacaaact cctggttaag aagctcgggt tgaaaaaaat aaacaagtag 
               
               
                 3121 
                 tgctggggag tagaggccaa gaagtaggta atgggctcag aagaggagcc acaaacaagg 
               
               
                 3181 
                 ttgtgcaggc gcctgtaggc tgtggtgtga attctagcca aggagtaaca gtgatctgtc 
               
               
                 3241 
                 acaggctttt aaaagattgc tctggctgct atgtggaaag cagaatgaag ggagcaacag 
               
               
                 3301 
                 taaaagcagg gagcccagcc aggaagctgt tacacagtcc aggcaagagg tagtggagtg 
               
               
                 3361 
                 ggctgggtgg gaacagaaaa gggagtgaca aaccattgtc tcctgaatat attctgaagg 
               
               
                 3421 
                 aagttgctga aggattctat gttgtgtgag agaaagagaa gaattggctg ggtgtagtag 
               
               
                 3481 
                 ctcatgccaa ggaggaggcc aaggagagca gattcctgag ctcaggagtt caagaccagc 
               
               
                 3541 
                 ctgggcaaca cagcaaaacc ccttctctac aaaaaataca aaaattagct gggtgtggtg 
               
               
                 3601 
                 gcatgcacct gtgatcctag ctactcggga ggctgaggtg gagggtattg cttgagccca 
               
               
                 3661 
                 ggaagttgag gctgcagtga gccatgactg tgccactgta cttcagccta ggtgacagag 
               
               
                 3721 
                 caagaccctg tctcccctga ccccctgaaa aagagaagag ttaaagttga ctttgttctt 
               
               
                 3781 
                 tattttaatt ttattggcct gagcagtggg gtaattggca atgccatttc tgagatggtg 
               
               
                 3841 
                 aaggcagagg aaagagcagt ttggggtaaa tcaaggatct gcatttggac atgttaagtt 
               
               
                 3901 
                 tgagattcca gtcaggcttc caagtggtga ggccacatag gcagttcagt gtaagaattc 
               
               
                 3961 
                 aggaccaagg cagggcacgg tggctcactt ctgtaatccc agcactttgg tggctgaggc 
               
               
                 4021 
                 aggtagatca tttgaggtca ggagtttgag acaagcttgg ccaacatggt gaaaccccat 
               
               
                 4081 
                 gtctactaaa aatacaaaaa ttagcctggt gtggtggcgc acgcctatag tcccaggttt 
               
               
                 4141 
                 tcaggaggct taggtaggag aatcccttga acccaggagg tgcaggttgc agtgagctga 
               
               
                 4201 
                 gattgtgcca ctgcactcca gcctgggtga tagagtgaga ctctgtctca aaaaaaaaaa 
               
               
                 4261 
                 aaaaaaaaaa aaaaaaaaaa aactgaagga attattcctc aggatttggg tctaatttgc 
               
               
                 4321 
                 cctgagcacc aactcctgag ttcaactacc atggctagac acaccttaac attttctaga 
               
               
                 4381 
                 atccaccagc tttagtggag tctgtctaat catgagtatt ggaataggat ctgggggcag 
               
               
                 4441 
                 tgagggggtg gcagccacgt gtggcagaga aaagcacaca aggaaagagc acccaggact 
               
               
                 4501 
                 gtcatatgga agaaagacag gactgcaact cacccttcac aaaatgagga ccagacacag 
               
               
                 4561 
                 ctgatggtat gagttgatgc aggtgtgtgg agcctcaaca tcctgctccc ctcctactac 
               
               
                 4621 
                 acatggttaa ggcctgttgc tctgtctcca ggttcacact ctctgcacta cctcttcatg 
               
               
                 4681 
                 ggtgcctcag agcaggacct tggtctttcc ttgtttgaag ctttgggcta cgtggatgac 
               
               
                 4741 
                 cagctgttcg tgttctatga tcatgagagt cgccgtgtgg agccccgaac tccatgggtt 
               
               
                 4801 
                 tccagtagaa tttcaagcca gatgtggctg cagctgagtc agagtctgaa agggtgggat 
               
               
                 4861 
                 cacatgttca ctgttgactt ctggactatt atggaaaatc acaaccacag caagggtatg 
               
               
                 4921 
                 tggagagggg gcctcacctt cctgaggttg tcagagcttt tcatcttttc atgcatcttg 
               
               
                 4981 
                 aaggaaacag ctggaagtct gaggtcttgt gggagcaggg aagagggaag gaatttgctt 
               
               
                 5041 
                 cctgagatca tttggtcctt ggggatggtg gaaataggga cctattcctt tggttgcagt 
               
               
                 5101 
                 taacaaggct ggggattttt ccagagtccc acaccctgca ggtcatcctg ggctgtgaaa 
               
               
                 5161 
                 tgcaagaaga caacagtacc gagggctact ggaagtacgg gtatgatggg caggaccacc 
               
               
                 5221 
                 ttgaattctg ccctgacaca ctggattgga gagcagcaga acccagggcc tggcccacca 
               
               
                 5281 
                 agctggagtg ggaaaggcac aagattcggg ccaggcagaa cagggcctac ctggagaggg 
               
               
                 5341 
                 actgccctgc acagctgcag cagttgctgg agctggggag aggtgttttg gaccaacaag 
               
               
                 5401 
                 gtatggtgga aacacacttc tgcccctata ctctagtggc agagtggagg aggttgcagg 
               
               
                 5461 
                 gcacggaatc cctggttgga gtttcagagg tggctgaggc tgtgtgcctc tccaaattct 
               
               
                 5521 
                 gggaagggac tttctcaatc ctagagtctc taccttataa ttgagatgta tgagacagcc 
               
               
                 5581 
                 acaagtcatg ggtttaattt cttttctcca tgcatatggc tcaaagggaa gtgtctatgg 
               
               
                 5641 
                 cccttgcttt ttatttaacc aataatcttt tgtatattta tacctgttaa aaattcagaa 
               
               
                 5701 
                 atgtcaaggc cgggcacggt ggctcacccc tgtaatccca gcactttggg aggccgaggc 
               
               
                 5761 
                 gggtggtcac aaggtcagga gtttgagacc agcctgacca acatggtgaa acccgtctct 
               
               
                 5821 
                 aaaaaaatac aaaaattagc tggtcacagt catgcgcacc tgtagtccca gctaattgga 
               
               
                 5881 
                 aggctgaggc aggagcatcg cttgaacctg ggaagcggaa gttgcactga gccaagatcg 
               
               
                 5941 
                 cgccactgca ctccagccta ggcagcagag tgagactcca tcttaaaaaa aaaaaaaaaa 
               
               
                 6001 
                 aaaaagagaa ttcagagatc tcagctatca tatgaatacc aggacaaaat atcaagtgag 
               
               
                 6061 
                 gccacttatc agagtagaag aatcctttag gttaaaagtt tctttcatag aacatagcaa 
               
               
                 6121 
                 taatcactga agctacctat cttacaagtc cgcttcttat aacaatgcct cctaggttga 
               
               
                 6181 
                 cccaggtgaa actgaccatc tgtattcaat cattttcaat gcacataaag ggcaatttta 
               
               
                 6241 
                 tctatcagaa caaagaacat gggtaacaga tatgtatatt tacatgtgag gagaacaagc 
               
               
                 6301 
                 tgatctgact gctctccaag tgacactgtg ttagagtcca atcttaggac acaaaatggt 
               
               
                 6361 
                 gtctctcctg tagcttgttt ttttctgaaa agggtatttc cttcctccaa cctatagaag 
               
               
                 6421 
                 gaagtgaaag ttccagtctt cctggcaagg gtaaacagat cccctctcct catccttcct 
               
               
                 6481 
                 ctttcctgtc aagtgcctcc tttggtgaag gtgacacatc atgtgacctc ttcagtgacc 
               
               
                 6541 
                 actctacggt gtcgggcctt gaactactac ccccagaaca tcaccatgaa gtggctgaag 
               
               
                 6601 
                 gataagcagc caatggatgc caaggagttc gaacctaaag acgtattgcc caatggggat 
               
               
                 6661 
                 gggacctacc agggctggat aaccttggct gtaccccctg gggaagagca gagatatacg 
               
               
                 6721 
                 tgccaggtgg agcacccagg cctggatcag cccctcattg tgatctgggg tatgtgactg 
               
               
                 6781 
                 atgagagcca ggagctgaga aaatctattg ggggttgaga ggagtgcctg aggaggtaat 
               
               
                 6841 
                 tatggcagtg agatgaggat ctgctctttg ttaggggatg ggctgagggt ggcaatcaaa 
               
               
                 6901 
                 ggctttaact tgctttttct gttttagagc cctcaccgtc tggcacccta gtcattggag 
               
               
                 6961 
                 tcatcagtgg aattgctgtt tttgtcgtca tcttgttcat tggaattttg ttcataatat 
               
               
                 7021 
                 taaggaagag gcagggttca agtgagtagg aacaaggggg aagtctctta gtacctctgc 
               
               
                 7081 
                 cccagggcac agtgggaaga ggggcagagg ggatctggca tccatgggaa gcatttttct 
               
               
                 7141 
                 catttatatt ctttggggac accagcagct ccctgggaga cagaaaataa tggttctccc 
               
               
                 7201 
                 cagaatgaaa gtctctaatt caacaaacat cttcagagca cctactattt tgcaagagct 
               
               
                 7261 
                 gtttaaggta gtacaggggc tttgaggttg agaagtcact gtggctattc tcagaaccca 
               
               
                 7321 
                 aatctggtag ggaatgaaat tgatagcaag taaatgtagt taaagaagac cccatgaggt 
               
               
                 7381 
                 cctaaagcag gcaggaagca aatgcttagg gtgtcaaagg aaagaatgat cacattcagc 
               
               
                 7441 
                 tggggatcaa gatagccttc tggatcttga aggagaagct ggattccatt aggtgaggtt 
               
               
                 7501 
                 gaagatgatg ggaggtctac acagacggag caaccatgcc aagtaggaga gtataaggca 
               
               
                 7561 
                 tactgggaga ttagaaataa ttactgtacc ttaaccctga gtttgcttag ctatcactca 
               
               
                 7621 
                 ccaattatgc atttctaccc cctgaacatc tgtggtgtag ggaaaagaga atcagaaaga 
               
               
                 7681 
                 agccagctca tacagagtcc aagggtcttt tgggatattg ggttatgatc actggggtgt 
               
               
                 7741 
                 cattgaagga tcctaagaaa ggaggaccac gatctccctt atatggtgaa tgtgttgtta 
               
               
                 7801 
                 agaagttaga tgagaggtga ggagaccagt tagaaagcca ataagcattt ccagatgaga 
               
               
                 7861 
                 gataatggtt cttgaaatcc aatagtgccc aggtctaaat tgagatgggt gaatgaggaa 
               
               
                 7921 
                 aataaggaag agagaagagg caagatggtg cctaggtttg tgatgcctct ttcctgggtc 
               
               
                 7981 
                 tcttgtctcc acaggaggag ccatggggca ctacgtctta gctgaacgtg agtgacacgc 
               
               
                 8041 
                 agcctgcaga ctcactgtgg gaaggagaca aaactagaga ctcaaagagg gagtgcattt 
               
               
                 8101 
                 atgagctctt catgtttcag gagagagttg aacctaaaca tagaaattgc ctgacgaact 
               
               
                 8161 
                 ccttgatttt agccttctct gttcatttcc tcaaaaagat ttccccattt aggtttctga 
               
               
                 8221 
                 gttcctgcat gccggtgatc cctagctgtg acctctcccc tggaactgtc tctcatgaac 
               
               
                 8281 
                 ctcaagctgc atctagaggc ttccttcatt tcctccgtca cctcagagac atacacctat 
               
               
                 8341 
                 gtcatttcat ttcctatttt tggaagagga ctccttaaat ttgggggact tacatgattc 
               
               
                 8401 
                 attttaacat ctgagaaaag ctttgaaccc tgggacgtgg ctagtcataa cttaccaga 
               
               
                 8461 
                 tttttacaca tgtatctatg cattttctgg acccgttcaa cttttccttt gaatcctctc 
               
               
                 8521 
                 tctgtgttac ccagtaactc atctgtcacc aagccttggg gattcttcca tctgattgtg 
               
               
                 8581 
                 atgtgagttg cacagctatg aaggctgtac actgcacgaa tggaagaggc acctgtccca 
               
               
                 8641 
                 gaaaaagcat catggctatc tgtgggtagt atgatgggtg tttttagcag gtaggaggca 
               
               
                 8701 
                 aatatcttga aaggggttgt gaagaggtgt tttttctaat tggcatgaag gtgtcataca 
               
               
                 8761 
                 gatttgcaaa gtttaatggt gccttcattt gggatgctac tctagtattc cagacctgaa 
               
               
                 8821 
                 gaatcacaat aattttctac ctggtctctc cttgttctga taatgaaaat tatgataagg 
               
               
                 8881 
                 atgataaaag cacttacttc gtgtccgact cttctgagca cctacttaca tgcattactg 
               
               
                 8941 
                 catgcacttc ttacaataat tctatgagat aggtactatt atccccattt cttttttaaa 
               
               
                 9001 
                 tgaagaaagt gaagtaggcc gggcacggtg gctcacgcct gtaatcccag cactttggga 
               
               
                 9061 
                 ggccaaagcg ggtggatcac gaggtcagga gatcgagacc atcctggcta acatggtgaa 
               
               
                 9121 
                 accccatctc taataaaaat acaaaaaatt agctgggcgt ggtggcagac gcctgtagtc 
               
               
                 9181 
                 ccagctactc ggaaggctga ggcaggagaa tggcatgaac ccaggaggca gagcttgcag 
               
               
                 9241 
                 tgagccgagt ttgcgccact gcactccagc ctaggtgaca gagtgagact ccatctcaaa 
               
               
                 9301 
                 aaaataaaaat aaaaataaa aaaatgaaaa aaaaaagaaa gtgaagtata gagtatctca 
               
               
                 9361 
                 tagtttgtca gtgatagaaa caggtttcaa actcagtcaa tctgaccgtt tgatacatct 
               
               
                 9421 
                 cagacaccac tacattcagt agtttagatg cctagaataa atagagaagg aaggagatgg 
               
               
                 9481 
                 ctcttctctt gtctcattgt gtttcttctg aatgagcttg aatcacatga aggggaacag 
               
               
                 9541 
                 cagaaaacaa ccaactgatc ctcagctgtc atgtttcctt taaaagtccc tgaaggaagg 
               
               
                 9601 
                 tcctggaatg tgactccctt gctcctctgt tgctctcttt ggcattcatt tctttggacc 
               
               
                 9661 
                 ctacgcaagg actgtaattg gtggggacag ctagtggccc tgctgggctt cacacacggt 
               
               
                 9721 
                 gtcctcccta ggccagtgcc tctggagtca gaactctggt ggtatttccc tcaatgaagt 
               
               
                 9781 
                 ggagtaagct ctctcatttt gagatggtat aatggaagcc accaagtggc ttagaggatg 
               
               
                 9841 
                 cccaggtcct tccatggagc cactggggtt ccggtgcaca ttaaaaaaaa aatctaacca 
               
               
                 9901 
                 ggacattcag gaattgctag attctgggaa atcagttcac catgttcaaa agagtctttt 
               
               
                 9961 
                 tttttttttt gagactctat tgcccaggct ggagtgcaat ggcatgatct cggctcactg 
               
               
                 10021 
                 taacctctgc ctcccaggtt caagcgattc tcctgtctca gcctcccaag tagctgggat 
               
               
                 10081 
                 tacaggcgtg caccaccatg cccggctaat ttttgtattt ttagtagaga cagggtttca 
               
               
                 10141 
                 ccatgttggc caggctggtc tcgaactctc ctgacctcgt gatccgcctg cctcggcctc 
               
               
                 10201 
                 ccaaagtgct gagattacag gtgtgagcca ccctgcccag ccgtcaaaag agtcttaata 
               
               
                 10261 
                 tatatatcca gatggcatgt gtttacttta tgttactaca tgcacttggc tgcataaatg 
               
               
                 10321 
                 tggtacaagc attctgtctt gaagggcagg tgcttcagga taccatatac agctcagaag 
               
               
                 10381 
                 tttcttcttt aggcattaaa ttttagcaaa gatatctcat ctcttctttt aaaccatttt 
               
               
                 10441 
                 ctttttttgt ggttagaaaa gttatgtaga aaaaagtaaa tgtgatttac gctcattgta 
               
               
                 10501 
                 gaaaagctat aaaatgaata caattaaagc tgttatttaa ttagccagtg aaaaactatt 
               
               
                 10561 
                 aacaacttgt ctattacctg ttagtattat tgttgcatta aaaatgcata tactttaata 
               
               
                 10621 
                 aatgtacatt gtattgtata ctgcatgatt ttattgaagt tcttgttcat cttgtgtata 
               
               
                 10681 
                 tacttaatcg ctttgtcatt ttggagacat ttattttgct tctaatttct ttacattttg 
               
               
                 10741 
                 tcttacggaa tattttcatt caactgtggt agccgaatta atcgtgtttc ttcactctag 
               
               
                 10801 
                 ggacattgtc gtctaagttg taagacattg gttattttac cagcaaacca ttctgaaagc 
               
               
                 10861 
                 atatgacaaa ttatttctct cttaatatct tactatactg aaagcagact gctataaggc 
               
               
                 10921 
                 ttcacttact cttctacctc ataaggaata tgttacaatt aatttattag gtaagcattt 
               
               
                 10981 
                 gttttatatt ggttttattt cacctgggct gagatttcaa gaaacacccc agtcttcaca 
               
               
                 11041 
                 gtaacacatt tcactaacac atttactaaa catcagcaac tgtggcctgt taattttttt 
               
               
                 11101 
                 aatagaaatt ttaagtcctc attttctttc ggtgtttttt aagcttaatt tttctggctt 
               
               
                 11161 
                 tattcataaa ttcttaaggt caactacatt tgaaaaatca aagacctgca ttttaaattc 
               
               
                 11221 
                 ttattcacct ctggcaaaac cattcacaaa ccatggtagt aaagagaagg gtgacacctg 
               
               
                 11281 
                 gtggccatag gtaaatgtac cacggtggtc cggtgaccag agatgcagcg ctgagggttt 
               
               
                 11341 
                 tcctgaaggt aaaggaataa agaatgggtg gaggggcgtg cactggaaat cacttgtaga 
               
               
                 11401 
                 gaaaagcccc tgaaaatttg agaaaacaaa caagaaacta cttaccagct atttgaattg 
               
               
                 11461 
                 ctggaatcac aggccattgc tgagctgcct gaactgggaa cacaacagaa ggaaaacaaa 
               
               
                 11521 
                 ccactctgat aatcattgag tcaagtacag caggtgattg aggactgctg agaggtacag 
               
               
                 11581 
                 gccaaaattc ttatgttgta ttataataat gtcatcttat aatactgtca gtattttata 
               
               
                 11641 
                 aaacattctt cacaaactca cacacattta aaaacaaaac actgtctcta aaatccccaa 
               
               
                 11701 
                 atttttcata aactcagttt taaactaact ttttttcaaa ccacaatctg atttaacaat 
               
               
                 11761 
                 gactatcatt taaatatttc tgactttcaa attaaagatt ttcacatgca ggctgatatt 
               
               
                 11821 
                 tgtaattgtg attctctctg taggctttgg gtataatgtg ttcttttcct tttttgcatc 
               
               
                 11881 
                 agcgattaac ttctacactc taacatgtag aatgttacta caatattaaa gtattttgta 
               
               
                 11941 
                 tgacaatttt atttgaaagc ctaggatgcg ttgacatcct gcatgcattt attacttgat 
               
               
                 12001 
                 atgcatgcat tctggtatct caagcattct atttctgagt aattgtttaa ggtgtagaag 
               
               
                 12061 
                 agatagatat ggtggatttg gagttgatac ttatatattt tctatttctt ggatggatga 
               
               
                 12121 
                 atttgtacat taaaagtttt ccatgg 
               
               
                   
               
             
          
         
       
     
     Exon 1 spans nt 1028-1324, inclusive; exon 2 spans nt 4652-4915, inclusive; exon 3 spans nt 5125-5400, inclusive; exon 4 spans nt 6494-6769, inclusive; exon 5 spans nt 6928-7041, inclusive; exon 6 spans nt 7995-9050, inclusive, and exon 7 spans nt 10206-10637, inclusive. Intron 4 spans nt 6770-6927, inclusive, and intron 5 spans nt 7042-7994, inclusive. 
     Total RNA for the RT-PCR was prepared from 1.5 mL of whole blood using the RNeasy Blood Kit (QIAGEN, Valencia, Calif.). Total messenger RNA encoding the HFE gene was transcribed and amplified with the primers shown above using standard methods, e.g., the Superscript ONE-STEP RT- PCR System (Life Technologies, Gaithersburg, MD). The amplified product was directly subcloned into the pCR2.1-TOPO vector and transfected into TOP 10 bacteria (Invitrogen, Carlsbad, Calif.). Plasmid DNAs isolated from the subcloning were prepared with the UltraClean Mini Prep Kit (Mo Bio, Solana Beach, Calif.) and sequenced. 
     DNA sequencing was performed using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems, Foster City, Calif.) and analyzed on an ABI Prism 377. 
     To detect mutations in exon 2 of the HFE gene, the genomic DNA of probands and normal control subjects were amplified and subjected to a dot blot hybridization assay. 1.0 μl of each resulting PCR product was then applied to a Magna Graph nylon membrane (MSI, Westboro, Mass.). The membranes were treated with 0.5 N NaOH/1.5 M NaCl to denature the DNA, neutralized with 0.5 M Tris-HCl (pH 8.0)/1.5 M NaCl, and rinsed with 2×SSC (1 ×SSC=0.15 M NaCl/0.015 M sodium citrate, pH 7.0). The DNAs were fixed on the membrane by UV irradiation using a Stratalinker 1800 (Stratagene, Inc., La Jolla, Calif.). The ECL 3′-oligolabelling and detection system (Amersham, Arlington Heights, Ill.) was used for synthesis of labeled oligonucleotide probes, hybridization, and signal detection. The oligonucleotide sequences used to detect each point mutation were (substituted bases are shown as upper case 
                                   TABLE 5                   Oligonucleotide Probes                Point Mutation   Oligonucleotide                       G93R mutation   gtctgaaaCggtgggat               (SEQ ID NO:28)           I105T mutation   acttctggactaCtatgg               (SEQ ID NO:29)           S65C mutation   atcatgagTgtcgccgt               (SEQ ID NO: 30)                        
For signal detection, each oligonucleotide was labeled with fluorescein-11-dUTP using terminal deoxynucleotidyl transferase according to the manufacturer&#39;s instructions (Amersham Ltd., Arlington Heights, Ill.). The membranes were prehybridized in 5×SSC, 0.1% Hybridization buffer component, 0.02% SDS, 5% LiquidBlock at 42° C. for approximately 2 hours. Labelled oligonucleotide probes were added to individual bags containing the membranes and prehybridization buffer and incubated at 42° C. overnight. The blots were washed twice with 5×SSC, 0.1% SDS for 5 minutes at room temperature. Stringency washes for hybridization with oligonucleotides having the sequence of SEQ ID NO: 30 or 28 were performed twice in 0.2×SSC/0.1% SDS for 15 minutes at 42° C. Membranes probed with an oligonucleotide having the sequence of SEQ ID NO:29 was washed twice under less stringent conditions (0.5×SSC/0.1% SDS, 15 minutes at 42° C.). Detection of a fluorescent signal was performed according to standard methods.
 
     EXAMPLE 3 
     Characterization of Probands 
     The mean age of the twenty probands was 44±11 years (range 27-62 years); thirteen (65.0%) were men and seven (35.0%) were women. Eleven had iron overload. One had hepatic cirrhosis, two had diabetes mellitus, four had arthropathy, and two had hypogonadotrophic hypogonadism. One proband also had hereditary stomatocytosis, another had beta-thalassemia trait, a third had ethanol intake &gt;60 g daily, and a fourth had porphyria cutanea tarda. No proband had evidence of excess oral or parenteral iron intake, or of viral hepatitis B or C. At diagnosis of hemochromatosis, evaluation for common HFE mutations revealed that eleven probands were C282Y heterozygotes, five were H63D heterozygotes, and four did not inherit C282Y or H63D. 
     The mean age of the initial 176 control subjects was 52±15 years (range 18-86 years); 79 (44.9%) were men and 97 (55.1%) were women. There was no significant difference in the mean ages of men and women. Frequencies of HFE genotypes among the control subjects are shown in Table 6. These values are similar to those previously reported from normal persons from the same geographic area. 
     
       
         
               
             
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Frequencies of HFE Genotypes in Alabama Subjects. 
               
             
          
           
               
                   
                 Hemochromatosis Probands with 
                 Normal Control 
               
               
                 HFE Genotype 
                 “Atypical” HFE Genotypes, % (n) 
                 Subjects, % (n) 
               
               
                   
               
               
                 wt/wt 
                 15.00 (3) 
                 60.23 (106) 
               
               
                 C282Y/wt 
                 45.00 (9) 
                 13.06 (23) 
               
               
                 H63D/wt 
                 20.00 (4) 
                 15.34 (27) 
               
               
                 S65C/wt 
                  5.00 (1) 
                  1.14 (2) 
               
               
                 C282Y/S65C 
                  5.00 (1) 
                  0 
               
               
                 C282Y/G93R 
                  5.00 (1) 
                  0 
               
               
                 H63D/1105T 
                  5.00 (1) 
                  0 
               
               
                 H63D/C282Y 
                  0 
                  6.82 (12) 
               
               
                 H63D/H63D 
                  0 
                  3.41 (6) 
               
               
                   
               
               
                 Results are expressed as percentage (n). The wild-type (wt) allele was defined as the HFE configuration in which the mutations C282Y, H63D, S65C, I105T, or G93R were not detected.  
               
             
          
         
       
     
     EXAMPLE 4 
     Identification of Novel HFE Mutations in Hemochromatosis Probands 
     The following novel mutations (missense mutations) were identified in probands 1 and 2: exon 2, nt 314T→C (I105T), and exon 2, nt 277G→C (G93R), respectively (Table 7; FIGS.  1  and  2 ). Probands 3 and 4 had a S65C mutation The S65C mutation has been observed in hemochromatosis patients but has not been deemed to be indicative of a disease state. In contrast, the data presented herein indicate that the S65C mutation is diagnostic of a disease state. This result is surprising in view of earlier observations. Other than C282Y or H63D, no HFE exon mutations were detected in the remaining sixteen of the twenty probands (Table 6). Nine probands were heterozygous for a base-pair change at intron 2, nt 4919T/C (SEQ ID NO:27); two probands were homozygous for this base-pair change. Heterozygosity for a base-pair change in intron 4 (nt 6884T→C) was detected only in probands 3 and 4, both of whom also inherited S65C. One proband was heterozygous for a base-pair change at intron 5, nt 7055A→G. 
     Using dot blot methodology, heterozygosity for the S65C mutation was detected in two of 176 normal control subjects (Table 6). The G93R or I105T mutations were not detected in normal control subjects (Tables 6 and 8). 
     EXAMPLE 5 
     Association of Novel HFE Coding Region Mutations to C282Y and H63D and HFE Intron Alleles 
     In proband 1, two mutations of exon 2 (H63D and I105T) were detected. After subcloning the genomic fragment, the subclones revealed that these mutations occurred on separate chromosomes; this observation was confirmed by family studies indicating segregation of I105T 
     
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                                               Phenotypes and Uncommon HFE Genotype in Alabama Subjects* 
               
             
          
           
               
                   
                 Age (years), 
                 HFE 
                 HLA 
                 Transferrin 
                 Serum Ferritin, 
                 Hepatocyte 
                 Phlebotomy, 
               
               
                 Subject† 
                 Sex 
                 Genotype 
                 Type 
                 Saturation % 
                 ng/mL 
                 Iron Grade 
                 Units 
               
               
                   
               
             
          
           
               
                 Proband 1 
                 52 M 
                 H63D/I105T 
                 A2, 3; B7, 7 
                 62 
                 868 
                 2+ 
                 20 
               
               
                 Proband 2‡ 
                 40 M 
                 C282Y/G93R 
                 A2, 3; B7, 62 
                 78 
                 861 
                 4+ 
                 34 
               
               
                 Proband 3§ 
                 47 F 
                 C282Y/S65C 
                 A2, 32; B8, 44; 
                 90 
                 281 
                 3+ 
                 37 
               
               
                   
                   
                   
                 Bw4, 6; Cw5, 7 
               
               
                 Proband 4** 
                 81 F 
                 S65C/wt 
                 A2, 32; B14, 62 
                 100 
                 5,135 
                 N.D. 
                 37 
               
               
                 Normal Control 1 
                 28 M 
                 S65C/wt 
                 A2, 31; B35, 60 
                 28 
                 141 
                 N.D. 
                 N.D. 
               
               
                 Normal Control 2 
                 69 M 
                 S65C/wt 
                 A24, 26; B8, 
                 42 
                 747 
                 2+ 
                 N.D. 
               
               
                   
                   
                   
                 B37; Bw4, 6; 
               
               
                   
                   
                   
                 Cw6, 5 (or 7) 
               
               
                   
               
               
                 *Serum transferrin saturation, serum ferritin concentration, and percutaneous hepatic biopsy were performed before therapeutic phlebotomy was initiated. Reference ranges for these parameters are 15-45%; 20-300 ng/mL (men) and 20-200 ng/mL (women); and 0-1+, respectively. Iron depletion (serum  
               
               
                 #   ferritin ≦ 20 ng/mL) was induced by removing the indicated numbers of units of blood. None of these persons had evidence of hepatic cirrhosis, diabetes mellitus, hemochromatosis-associated  
               
               
                 #   arthropathy, hypogonadotrophic hypogonadism, other endocrinopathy, or cardiomopathy.  
               
               
                 N.D. = not done. The mutations indicated are exon 4, nt 845G→A (C282Y); exon 2, nt 187C→G (H63D); exon 2, nt 314T→C (I105T); exon 2, nt 277G→C (G93R); and exon 2, nt 193A→T (S65C). The wild-type (wt) allele was defined as an HFE allele in which the mutations C282Y, H63D, S65C, I105T, or G93R were not detected.  
               
               
                 †Countries of origin: Probands 1 and 2, England; Proband 3, Wales, England, and Americas (Cherokee); Proband 4, England and Ireland; Normal Control 1, England; Normal Control 2, The Netherlands.  
               
               
                 ‡The father and sister of Proband 2 are presently undergoing therapy for hemochromatosis and iron overload, but their clinical and genetic data were unavailable.  
               
               
                 §Proband 3 had porphyria cutanea tarda alleviated with therapeutic phlebotomy.  
               
               
                 **Proband 4 had hereditary stomatocytosis unaffected by phlebotomy treatments. 37 units of blood were removed by phlebotomy before treatment was discontinued due to stroke apparently unrelated to anemia or iron overload (post-treatment serum ferritin 1,561 ng/mL). Her 59 year-old daughter (who does not have hereditary stomatocytosis) had transferrin saturation 42%, serum ferritin 62 ng/mL, HLA type A1, 32; B14, 15; Bw4, 6; Cw3, 8, and HFE genotype S65C/H63D. These data permitted  
               
               
                 # assignment of the S65C mutation in this family to a haplotype carrying HLA-A32; linkage of S65C and HLA-A32 was also observed in the family of Proband 3.  
               
             
          
         
       
     
                                                                                 TABLE 8                   Frequencies of HFE  Alleles in Alabama Subjects.                wt*   C282Y   H63D   S65C†   I105T   G93R                        Hemochromatosis Probands with   0.500   0.275   0.125   0.050   0.025   0.025       “Atypical” HFE Genotypes (n = 20)       Normal Control Subjects (n = 176)   0.750   0.099   0.145   0.006   ‡   ‡               The wild-type (wt) allele was defined as an HFE allele in which the mutations C282Y, H63D, S65C, I105T, or G93R were not detected.        †S65C was detected in 2 of 22 (0.091) proband chromosomes and in 2 of 266 (0.0075) control chromosomes that did not bear the C282Y, H63D, S65C, I105T, or G93R mutation.        ‡Based on this data set, the frequency of the I105T and G93R HFE alleles is estimated to be &lt; 0.0028, respectively.             
and H63D (FIG.  1 ). In proband 2 (HFE genotype C282Y/G93R), RT-PCR analysis (with subsequent subcloning and sequencing) revealed that these HFE mutations occurred on separate chromosomes. Family studies of proband 3 (HFE genotype C282Y/S65C) indicated that the C282Y and S65C HFE alleles segregated independently, establishing their occurrence on separate chromosomes (Table 7, FIG.  3 ).
 
     In proband 1 (HFE genotype H63D/I105T), the I105T mutation was co-inherited with HLA-A3, B7. In probands 3 and 4 and their respective families, S65C was inherited on the same chromosome as HLA-A32, indicating that HLA-A32 is a marker for chromosomes bearing the S65C mutation, and individuals with HLA-A32 have an increased risk for developing hemochromatosis. The G93R mutation is associated with HLA-A2, and individuals with that haplotype have an increased risk for developing hemochromatosis. The I105T mutation is associated with HLA-A3, e.g., HLA-A3, B7, and individuals with that haplotype have an increased risk for developing hemochromatosis. Among twenty probands tested, the nucleotide polymorphism in intron 4 (nt 6884T→C) was detected in probands 3 and 4, both of whom also had S65C. Subjects that tested positive for the S65C mutation all were found to have the intron 4 (6884T→C) mutation, including two probands (3 and 4), their families, and two normal controls. 
     EXAMPLE 6 
     HFE Coding Region Mutations and Clinical Phenotype 
     The I105T and G93R mutations were associated with a hemochromatosis clinical phenotype in probands 1 and 2 who also inherited H63D and C282Y, respectively. Proband 3 had clinical evidence of hemochromatosis, iron overload, and porphyria cutanea tarda associated with compound heterozygosity for C282Y and S65C. Proband 4 had severe iron overload associated with heterozygosity for S65C and co-inheritance of hereditary stomatocytosis (Table 7). The sister of proband 1 (HFE genotype I105T/wt) was not completely evaluated for hyperferritinemia (FIG.  1 ). Otherwise, family members of probands who were heterozygous for novel HFE mutations described herein had little or no evidence of abnormal iron parameters, a hemochromatosis phenotype, or of iron overload (Table 7 and 9; FIGS.  1  and  3 ). Normal Control 1 who had HFE genotype S65C/wt had a 
                                                       TABLE 9                                          Hemochromatiosis (HC) Family study/patent                            intron 4   Tf sat**   Ftn**   Diagnosis/Hepatocyte       Subject/Age/Sex   HLA Type   exon 2   exon 4   5636 bp   %   ng/ml   Iron grade               Proband 1/57M (201)   A2, 3; B7, 7   H63D/H, 1105T/1   Wt   T   62   868   HC/2+       brother/45M (204)       H63D/H   Wt   T*   31   186       sister/50F (203)   A3, 3: B7, 7   1105T   Wt*   T*   37   576       daughter/31F (301)   A32, 68; B7, 44   1105T/1   Wt*   T*   31    56       son/27M (302)   A2, 68; B7, 44   H63D/H   Wt*   T*   33    44       Proband 2/40M   A2, 3; B7, 62   G93R/G   C282Y/C   T   78   861   HC/4+       Father       Wt   C282Y/Y*   T*           HC       Sister       G93R/G   C282Y/C*   T*           HC       Proband 3/47 (201)   A2, 32; B8, 44   S65C/S   C282Y/C   T/C   90   281   HC/3+       brother/45M (202)   A2, 32; B44, 51   S65C/S   Wt   T/C   33    42       mother/81F (102)   A2, 2; B8, 51   Wt   C282Y/C   T*   NT   NT       sister/33F (204)   A2, 7; B27, 51   Wt   Wt   T*   NT   NT       brother/35M (203)   A2, 7; B27, 51   Wt   Wt*   T*   NT   NT       sister       Wt   C282Y/C*   T*       sister       S65C/S   Wt*   T/C*       Proband 4/81F   A2, 32; B14, 62   S65C/S   Wt   T/C   100    S135   HC + stomatocytosis       daughter/59″   A1, 32; B14, 15   H63D/H, S65C/S   Wt*   T/C   42    62       Control 1/28M   A2, 31; B35, 60   S65C/S   Wt   T/C   28   141       Control 2/69M    A24, 26; B8, 37   S65C/S   Wt   T/C   42   747   2+               *RE cut        **normal (15-45%)        ***20-300 ng/ml (men)        2C-200 ng/ml (women)             
normal iron phenotype (Table 7). Normal Control 2, who also had the HFE genotype S65C/wt, had hyperferritinemia and mildly increased stainable hepatocellular iron deposition, but had no symptoms or other objective findings attributable to iron overload (Table 7). These data indicate that S65C heterozygosity is associated with abnormal iron parameters.
 
     EXAMPLE 7 
     HLA Gene Linkage 
     In the family of proband 1, the I105T mutation was linked to HLA-A3, B7, markers which are often linked to the C282Y mutation and its ancestral haplotype. HLA-A3, B7 is also significantly more common among C282Y-negative hemochromatosis probands than in normal control subjects tested. S65C was linked to HLA-A32 in probands 3 and 4 (and their respective families). The base-pair change in intron 4 (nt 6884T→C) was detected only in probands who inherited the S65C mutation. These data indicate that an intron 4 mutation (nt 6884→C) is a marker for chromosomes bearing the S65C HFE allele. Three of four probands who inherited mutated HFE exon 2 mutations described herein also inherited the C282Y or H63D mutations on separate chromosomes. In a fourth proband, the co-inheritance of S65C heterozygosity and hereditary stomatocytosis was associated with severe iron overload. 
     Altered interactions of transferrin receptor, transferrin, and C282Y and H63D mutant HFE protein contribute to the pathology of hemochromatosis. The S65C, G93R, and I105T mutations are located within the al domain: in the α1 helix of the HFE class I-like heavy chain (I105T and G93R), and at the tip of the A chain loop of the β-pleated sheet (S65C). These mutations affect the overall structure of the HFE gene product, and specifically affect the salt bridge between residues H63 and D95. The I105T substitution also inhibits proper folding of the α1 domain of the HFE gene product, and specifically affects the hydrophobicity of the hydrophobic F pocket. 
     Other embodiments are within the following claims.