Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-03-26
2002-03-12
Campbell, Eggerton (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S022100
Reexamination Certificate
active
06355425
ABSTRACT:
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 I105 T mutation is located in the &agr;1 helix of the HFE protein and participates in a hydrophobic pocket (the “F” pocket). The alpha helix structure of the &agr;1 domain spans residues S80 to N108, inclusive. The I105T mutation is associated with an iron overload disorder.
TABLE 1
Human HFE cDNA sequence
atgggccg 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 cacgatttgc aaagtttaat ggtgccttca tttgggatgc tactctagta
ttccagacct gaagaatcac aataattttc tacctggtct ctccttgttc tgataatgaa
aattatgata aggatgataa aagcacttac ttcgtgtccg actcttctga gcacctactt
acatgcatta ctgcatgcac ttcttacaat aatttatga gataggtact attatccccca
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
(SEQ ID NO:1l GENBANK® Accession No. U60319)
TABLE 2
Human HFE gene product
MGPRARPALLLLMLLQTAVLQG
RLLRSHSLHYLFMGASEQDLGLSLFEALGYVDDQLFVFYDHESRRVEPRTPWVSSISSQ
MWLQLSQSLKGWDHMFTVDFWTIMENHNHSK
ESHTLQVILGCEMQEDNSTEGYWKYGYDG
QDHLEFCPDTLDWRAAEPRAWPTKLEWERHKIRARQNRAYLERDCPAQLQQLLELGRGVL
DQQVPPLVKVTHHVTSSVTTLRCRALNYYPQNITMKWLKDKQPMDAKEFEPKDVLPNGDG
TYQGWITLAVPPGEEQRYTCQVEHPGLDQPLIVIWEPSPSGTLVIGVISGIAVFVVILFI
GILFIILRKRQGSRGAMGHYVLAERE (SEQ ID NO: 2; GENBANK ® Accession
No. U60319)
Residues 1-22=leader sequence; &agr;1 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
Barton James C.
Rothenberg Barry E.
Sawada-Hirai Ritsuko
Beattie, Ph.D. Ingrid
Billups-Rothenberg, Inc.
Campbell Eggerton
Mintz Levin Cohn Ferris Glovsky and Popeo P.C.
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