# US 7,608,401 B2 · Mutations in the ferroportin 1 gene associated with hereditary haemochromatosis

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- Number: US 7,608,401 B2
- Document ID: US7608401B2
- Status: Granted
- Kind: B2
- Application number: 11/942,653
- Filed: 2007-11-19
- Published: 2008-06-19
- Granted: 2009-10-27
- Earliest claimed: 2000-10-17
- Inventors: Antonello Pietrangelo
- Web page: https://patentgrep.com/patent/US7608401B2

## CPC

- G01N 33/68: involving proteins, peptides or amino acids {(involving lipoproteins G01N33/92)}
- A61P 3/02: Nutrients, e.g. vitamins, minerals
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 14/47: from mammals
- C12Q 1/6883: for diseases caused by alterations of genetic material
- G01N 33/5008: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics}
- G01N 33/502: {for testing non-proliferative effects}
- G01N 33/5091: {for testing the pathological state of an organism}
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- C12Q 2600/156: Polymorphic or mutational markers
- C12Q 2600/158: Expression markers
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

The present invention relates to mutations in the gene coding for ferroportin 1 associated with hereditary haemochromatosis and methods for the diagnosis of hereditary haemochromatosis based on the identification of such mutations.

## Claims

### Claim 1

A method of determining a predisposition to hereditary haemochromatosis in a human, the method comprising:
a) isolating a nucleic acid encoding a ferroportin 1 protein from the human, wherein the ferroportin 1 protein comprises SEQ ID NO:2; and
b) detecting if a codon sequence in the nucleic acid encodes aspartic acid at position 77 of SEQ ID NO:2 to determine if the human is predisposed to suffer from hereditary haemochromatosis;
wherein it is determined that the human is predisposed to suffer from hereditary haemochromatosis when the codon sequence in the nucleic acid is detected to encode aspartic acid at position 77 of SEQ ID NO:2.

### Claim 2 (depends on 1)

The method of claim 1, wherein step (a) comprises obtaining a biological sample from the human, wherein the biological sample comprises the nucleic acid encoding ferroportin 1 protein and the biological sample is a sample of blood, plasma, saliva, urine, faeces, amniotic fluid or tissue.

### Claim 3 (depends on 1)

The method of claim 1, wherein the isolated nucleic acid is genomic DNA.

### Claim 4 (depends on 3)

The method of claim 3, wherein step (b) comprises amplifying the nucleic acid by PCR using a pair of oligonucleotides suitable for the amplification of a segment of the nucleic acid comprising the codon encoding the amino acid at position 77 of SEQ ID NO: 2.

### Claim 5 (depends on 4)

The method of claim 4, wherein said pair of oligonucleotides has the nucleotide sequences of SEQ.ID NO: 5 and SEQ.ID NO: 6.

### Claim 6 (depends on 1)

The method of claim 1, wherein the isolated nucleic acid is RNA.

### Claim 7 (depends on 6)

The method of claim 6, wherein step (b) comprises reverse transcribing the RNA to produce cDNA.

### Claim 8 (depends on 7)

The method of claim 7, further comprising amplifying the cDNA by PCR using a pair of oligonucleotides suitable for the amplification of a segment of said cDNA comprising the codon encoding the amino acid at position 77 of SEQ ID NO: 2.

### Claim 9 (depends on 8)

The method of claim 8 wherein said pair of oligonucleotides has the nucleotide sequences of SEQ.ID NO: 3 and SEQ.ID NO: 4.

### Claim 10 (depends on 1)

The method of claim 1 wherein said detecting in step (b) comprises using a technique selected from techniques based on the use of restriction enzymes, hybridization techniques with allele-specific oligonucleotide probes, allele-specific PCR, mismatch repair detection, single-strand conformational polymorphism analysis, gel electrophoresis in denaturant gradient, Hot Cleavage, DNAse and RNAse protection assay, allele specific primer extension, genetic bit analysis oligonucleotide-ligation assay, allele specific ligation chain reaction and sequencing techniques.

### Claim 11 (depends on 10)

The method of claim 10 wherein said technique used is selected from techniques based on the use of restriction enzymes, allele specific PCR, hybridization or sequencing techniques.

### Claim 12 (depends on 1)

The method of claim 1 wherein said detecting in step (b) comprises:
amplifying the isolated nucleic acid with oligonucleotides suitable for the selective amplification of a segment of said nucleic acid including the codon encoding the amino acid at position 77 of SEQ ID NO: 2 to produce an amplification product, wherein the amplification product comprises a consensus sequence for a restriction site when the codon encoding the amino acid at position 77 of SEQ ID NO:2 encodes an aspartic acid, and wherein the consensus sequence is absent when the codon encoding the amino acid at position 77 of SEQ ID NO:2 encodes an alanine;
incubating the amplification product with a restriction enzyme which is able to recognize said restriction site; and
analyzing of the size of the restriction enzyme digestion products.

### Claim 13 (depends on 12)

The method of claim 12, wherein said pair of oligonucleotides has the nucleotide sequences of SEQ ID NO: 6 and SEQ ID NO: 7, and wherein said enzyme is Mbo II.