Cystic fibrosis mutation cluster

• US 5,407,796 A
• Filed: 01/04/1991
• Issued: 04/18/1995
• Est. Priority Date: 01/04/1991
• Status: Expired due to Term

• Current Assignee: Johns Hopkins University
• Original Assignee: Johns Hopkins University
• Inventors: Antonarakis, Stylianos E., Cutting, Garry R., Kazazian, Haig H. Jr.
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): JACOBSON, DIAN COOK
• Application Number:
  US07/637,621
• Time in Patent Office: 1,565 Days
• Field of Search: 435/6, 435/91, 435/91.2, 436/94, 536/27, 536/23.2, 536/24.31, 935/77, 935/78
• US Class Current: 435/6.11
• CPC Class Codes: 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
• CPC Class Codes: C07K 14/4712 Cystic fibrosis
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

Four mutations have been found clustered in exon 11 of the CFTR (cystic fibrosis transmembrane conductance regulator) gene. These mutations occur within a set of amino acids highly conserved among ATP-dependent transport proteins. Humans can be tested to determine whether they carry one of these mutations using a number of methods and/or probes taught herein. Specifically the mutations include: Asn₅₄₉, Asp₅₅₁, Stop₅₅₃, and Thr₅₅₉.

Claims

1. A nucleic acid probe which is complementary to a mutant allele of the CFTR gene said allele being selected from the group consisting of:

Asn₅₄₉, Asp₅₅₁, Stop₅₅₃, and Thr₅₅₉.

2. The probe of claim 1 which is complementary to the Asn₅₄₉ allele.

3. The probe of claim 1 which is complementary to the Thr₅₅₉ allele.

4. The probe of claim 1 which is complementary to the ASP₅₅₁ allele.

5. The probe of claim 1 which is complementary to the Stop₅₅₃ allele.

32. The probe of claim 1 which comprises 18 to 22 nucleotides.

6. A method of testing a DNA sample of a human to determine if the human is a carder of Cystic Fibrosis or if the human is affected with Cystic Fibrosis, comprising:

providing a sample of DNA from a human;

testing the sample for the presence of a mutation in exon 11 of the CFTR gene, said mutation comprising a nucleotide selected from the group consisting of;

an adenine at nucleotide number 1778, 1784, or 1807, and a thymidine at nucleotide 1789, the presence of the mutation indicating that the human is a carder of Cystic Fibrosis or is affected with Cystic Fibrosis.

7. The method of claim 6 wherein the step of testing comprises amplifying exon 11 of said gene in a sample of DNA from the human to form a population of amplified DNA.

8. The method of claim 7 further comprising the step

determining the conformation of single strands of the amplified DNA, a conformation different from that of single strands of amplified exon 11 of wild-type CFTR allele indicating an exon 11 mutation.

9. The method of claim 7 wherein the step of amplifying is performed in the presence of radiolabeled deoxynucleotide triphosphates or radiolabeled primers to form labeled amplified DNA.

10. The method of claim 8 wherein the conformation is determined by electrophoresis on non-denaturing gels.

11. The method of claim 7 further comprising the step of;

determining the melting temperature of double strands of the amplified DNA, the presence of species of amplified DNA in the population of amplified DNA having different melting temperatures from DNA amplified from exon 11 of wild-type CFTR allele indicating a mutation in exon 11 of at least one allele of the CFTR gene in the human.

12. The method of claim 11 wherein the melting temperatures are determined by means of denaturing gradient gel electrophoresis.

13. The method of claim 6 further comprising:

contacting the human DNA sample with a nucleic acid probe complementary to a mutant allele of the CFTR gene, said allele being selected from the group consisting of;

Asn₅₄₉, Asp₅₅₁, Stop₅₅₃, and Thr₅₅₉, under conditions where totally homologous sequences anneal but sequences differing in one nucleotide do not;

detecting whether the human DNA sample anneals to one of said probes, annealing to one of said probes indicating the presence of a mutant CFTR allele which can cause cystic fibrosis if no wild type allele is present in the human.

14. The method of claim 13 wherein the human DNA sample has been amplified to increase the number of copies of exon 11 of the CFTR gene.

15. The method of claim 6 further comprising:

determining the nucleotide sequence of a region of exon 11 of a CFTR allele of a human, said region comprising nucleotides 1778-1807;

inspecting the sequence to determine if there is an adenine at nucleotides number 1778, 1784, or 1807, or a thymidine at nucleotide number 1789, the presence of at least one of said nucleotides indicating a mutation in a CFTR allele which can cause cystic fibrosis if no wild-type CFTR allele is present in the human.

16. The method of claim 7 further comprising:

digesting an aliquot of the amplified exon 11 DNA with a restriction endonuclease which recognizes a sequence CTNAG which occurs at nucleotide 1778 of the wild-type CFTR allele, to form DNA fragments;

measuring the size of the amplified exon 11 DNA and the DNA fragments, DNA fragments which are the same size as the amplified exon 11 DNA indicating a mutation in a CFTR allele which can cause cystic fibrosis if no wild-type CFTR allele is present in the human.

17. The method of claim 16 wherein the endonuclease is DdeI.

18. The method of claim 7 further comprising:

digesting an aliquot of the amplified exon 11 DNA with a restriction endonuclease which recognizes a sequence GTYRAC which occurs at nucleotide 1784 of the wild-type CFTR allele, to form DNA fragments;

measuring the size of the amplified exon 11 DNA and the DNA fragments, DNA fragments which are the same size as the amplified exon 11 DNA indicating a mutation in a CFTR allele which can cause cystic fibrosis if no wild-type CFTR allele is present in the human.

19. The method of claim 18 wherein the endonuclease is HincII.

20. The method of claim 7 further comprising:

digesting an aliquot of the amplified exon 11 DNA with a restriction endonuclease which recognizes a sequence GTYRAC which occurs at nucleotide 1789 of the wild-type CFTR allele to form DNA fragments;

measuring the size of the amplified exon 11 DNA and the DNA fragments, DNA fragments which are the same size as the amplified exon 11 DNA indicating a mutation in a CFTR allele which can cause cystic fibrosis if no wild-type CFTR allele is present in the human.

21. The method of claim 20 wherein the endonuelease is HineII.

22. The method of claim 7 further comprising:

digesting an aliquot of the amplified exon 11 DNA with a restriction endonuclease which recognizes a sequence GATC which occurs at nucleotide 1784 of a mutant CFTR allele but does nor recognize the sequence in a wild-type CFTR allele, to form DNA fragments;

measuring the size of the amplified exon 11 DNA and the DNA fragments, DNA fragments which are not the same size as the amplified exon 11 DNA indicating a mutation in a CFTR allele which can cause cystic fibrosis if no wild-type CFTR allele is present in the human.

23. The method of claim 22 wherein the endonuclease is MboI.

24. The method of claim 6 further comprising:

digesting DNA of the human with a restriction endonuclease which recognizes a sequence GATC which occurs at nucleotide 1784 of a mutant CFTR allele but not of a wild-type CFTR allele;

separating the digested DNA on a gel matrix;

hybridizing the separated, digested DNA with an exon 11 probe which spans nucleotides 1784 but does not span any other sequence which the endonuclease recognizes;

detecting the DNA which hybridizes to the probe, two hybridizing fragments indicating a mutation in a CFTR allele of the human which can cause cystic fibrosis if no wild-type CFTR allele is present in the human.

25. The method of claim 24 wherein the endonuclease is MboI.

26. The method of claim 6 further comprising:

digesting DNA of the human with a restriction endonuclease which recognizes a sequence CTNAG which occurs at nucleotide 1778 of the wild-type CFTR allele;

separating the digested DNA on a gel matrix;

hybridizing the separated, digested DNA with an exon 11 probe which spans nucleotide 1778 but does not span any other sequence which the endonuclease recognizes;

detecting the DNA which hybridizes to the probe, only one hybridizing fragment indicating a mutation in a CFTR gene of the human which can cause cystic fibrosis if no wild-type CFTR allele is present in the human.

27. The method of claim 26 wherein the endonuclease is DdeI.

28. The method of claim 6 further comprising:

digesting DNA of the human with a restriction endonuclease which recognizes a sequence GTYRAC which occurs at nucleotide 1784 of the wild-type CFTR allele;

separating the digested DNA on a gel matrix;

hybridizing the separated, digested DNA with an exon 11 probe which spans nucleotide 1784 but does not span any other sequence which the endonuclease recognizes;

detecting the DNA which hybridizes to the probe, only one hybridizing fragment indicating a mutation in a CFTR gene of the human which can cause cystic fibrosis if no wild-type CFTR allele is present in the human.

29. The method of claim 28 wherein the endonuclease is HincII.

30. The method of claim 6 further comprising:

digesting DNA of the human with a restriction endonuclease which recognizes a sequence GTYRAC which occurs at nucleotide 1789 of the wild-type CFTR allele;

separating the digested DNA on a gel matrix;

hybridizing the separated, digested DNA with an exon 11 probe which spans nucleotide 1789 but does not span any other sequence which the endonuclease recognizes;

detecting the DNA which hybridizes to the probe, only one hybridizing fragment indicating a mutation in a CFTR gene of the human which can cause cystic fibrosis if no wild-type CFTR allele is present in the human.

31. The method of claim 30 wherein the endonuclease is HincII.

33. The method of claim 13 wherein said nucleic acid probe comprises 18 to 22 nucleotides.