Linked breast and ovarian cancer susceptibility gene

• US 5,709,999 A
• Filed: 06/07/1995
• Issued: 01/20/1998
• Est. Priority Date: 08/12/1994
• Status: Expired due to Term

• Current Assignee: University of Utah Research Foundation (State of Utah)
• Original Assignee: Cancer Institute, Centre de recherche du CHUL, Myriad Genetics, Inc.
• Inventors: Simard, Jacques, Durocher, Francine, Emi, Mitsuuru, Nakamura, Yusuke, Shattuck-Eidens, Donna M.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): REES, DIANE
• Application Number:
  US08/483,553
• Time in Patent Office: 958 Days
• Field of Search: 435/6, 435/91.2, 435/7.1-7.9, 536/23.1, 536/24.3, 536/32, 536/26.6, 530/388.1
• US Class Current: 435/6.12
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• 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: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C07K 14/4703 Inhibitors; Suppressors
• CPC Class Codes: C07K 14/82 Translation products from oncogenes
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2600/136 Screening for pharmacological compounds
• CPC Class Codes: C12Q 2600/172 Haplotypes

Abstract

The present invention relates generally to the field of human genetics. Specifically, the present invention relates to methods and materials used to isolate and detect a human breast and ovarian cancer predisposing gene (BRCA1), some mutant alleles of which cause susceptibility to cancer, in particular breast and ovarian cancer. More specifically, the invention relates to germline mutations in the BRCA1 gene and their use in the diagnosis of predisposition to breast and ovarian cancer. The present invention further relates to somatic mutations in the BRCA1 gene in human breast and ovarian cancer and their use in the diagnosis and prognosis of human breast and ovarian cancer. Additionally, the invention relates to somatic mutations in the BRCA1 gene in other human cancers and their use in the diagnosis and prognosis of human cancers. The invention also relates to the therapy of human cancers which have a mutation in the BRCA1 gene, including gene therapy, protein replacement therapy and protein mimetics. The invention further relates to the screening of drugs for cancer therapy. Finally, the invention relates to the screening of the BRCA1 gene for mutations, which are useful for diagnosing the predisposition to breast and ovarian cancer.

Claims

1. A method for detecting a germline alteration in a BRCA1 gene, said alteration selected from the group consisting of the alterations set forth in Tables 12A, 14, 18 or 19 in a human which comprises analyzing a sequence of a BRCA1 gene or BRCA1 RNA from a human sample or analyzing a sequence of BRCA1 cDNA made from mRNA from said human sample with the proviso that said germline alteration is not a deletion of 4 nucleotides corresponding to base numbers 4184-4187 of SEQ ID NO:

1.

2. The method of claim 1 which comprises analyzing BRCA1 RNA from the subject.

3. The method of claim 2 wherein a germline alteration is detected by hybridizing a BRCA1 gene probe which specifically hybridizes to nucleic acids containing at least one of said alterations and not to wild-type BRCA1 sequences to RNA isolated from said human sample and detecting the presence of a hybridization product, wherein the presence of said product indicates the presence of said alteration in said RNA and thereby the presence of said germline alteration in said sample.

4. The method of claim 1 wherein a germline alteration is detected by obtaining a first BRCA1 gene fragment from a BRCA1 gene isolated from said human sample and a second BRCA1 gene fragment from a wild-type BRCA1 gene, said second fragment corresponding to said first fragment, forming single-stranded DNA from said first BRCA1 gene fragment and from said second BRCA1 gene fragment, electrophoresing said single-stranded DNAs on a non-denaturing polyacrylamide gel, comparing the mobility of said single-stranded DNAs on said gel to determine if said single-stranded DNA from said first BRCA1 gene fragment is shifted relative to said second BRCA1 gene fragment and sequencing said single-stranded DNA from said first BRCA1 gene fragment having a shift in mobility.

5. The method of claim 1 wherein a germline alteration is detected by hybridizing a BRCA1 probe which specifically hybridizes to nucleic acids containing at least one of said alterations and not to wild-type BRCA1 sequences to genomic DNA isolated from said sample and detecting the presence of a hybridization product, wherein a presence of said product indicates the presence of said germline alteration in the sample.

6. The method of claim 1 wherein a germline alteration is detected by amplifying all or part of a BRCA1 gene in said sample using a set of primers specific for a wild-type BRCA1 gene to produce amplified BRCA1 nucleic acids and sequencing the amplified BRCA1 nucleic acids.

7. The method of claim 1 wherein a germline alteration is detected by amplifying all or part of a BRCA1 gene in said sample using a primer specific for an allele having for one of said alterations and detecting the presence of an amplified product, wherein the presence of said product indicates the presence of said allele in the sample.

8. The method of claim 1 wherein a germline alteration is detected by molecularly cloning all or part of a BRCA1 gene in said sample to produce a cloned nucleic acid and sequencing the cloned nucleic acid.

9. The method of claim 1 wherein a germline alteration is detected by forming a heteroduplex consisting of a first strand of nucleic acid selected from the group consisting of BRCA1 gene genomic DNA fragment isolated from said sample, BRCA1 RNA fragment isolated from said sample and BRCA1 cDNA fragment made from mRNA from said sample and a second strand of a nucleic acid consisting of a corresponding human wild-type BRCA1 gene fragment, analyzing for the presence of a mismatch in said heteroduplex, heteroduplex and sequencing said first strand of nucleic acid having a mismatch.

10. The method of claim 1 wherein a germline alteration is detected by amplifying BRCA1 gene nucleic acids in said sample, hybridizing the amplified nucleic acids to a BRCA1 DNA probe which specifically hybridizes to nucleic acids containing at least one of said alterations and not to wild-type BRCA1 sequences and detecting the presence of a hybridization product, wherein a presence of said product indicates the presence of said germline alteration.

11. The method of claim 1 wherein a germline alteration is detected by analyzing the sequence of a BRCA1 gene in said sample for one of the deletion mutations set forth in Tables 12A or 14.

12. The method of claim 1 wherein a germline alteration is detected by analyzing the sequence of a BRCA1 gene in said sample for one of the point mutations set forth in Tables 12A or 14 with the proviso that said germline alteration is not a deletion of 4 nucleotides corresponding to base numbers 4184-4187 of SEQ ID NO:

1.

13. The method of claim 1 wherein a germline alteration is detected by analyzing the sequence of a BRCA1 gene in said sample for one of the insertion mutations set forth in Tables 12A or 14 with the proviso that said germline alteration is not a deletion of 4 nucleotides corresponding to base numbers 4184-4187 of SEQ ID NO:

1.

14. The method of claim 1 wherein a germline alteration is detected by obtaining a first BRCA1 gene fragment from a BRCA1 gene isolated from said human sample and a second BRCA1 gene fragment from a BRCA1 allele specific for one of said alterations, said second fragment corresponding to said first fragment, forming single-stranded DNA from said first BRCA1 gene fragment and from said second BRCA1 gene fragment, electrophoresing said single-stranded DNAs on a non-denaturing polyacrylamide gel and comparing the mobility of said single-stranded DNAs on said gel to determine if said single-stranded DNA from said first BRCA1 gene fragment is shifted relative to said second BRCA1 gene fragment, wherein no shift in electrophoretic mobility indicates the presence of said alteration in said sample.

15. The method of claim 1 wherein a germline alteration is detected by obtaining a first BRCA1 gene fragment from (a) BRCA1 gene genomic DNA isolated from said sample, (b) BRCA1 RNA isolated from said sample or (c) BRCA1 cDNA made from mRNA isolated from said sample and a second BRCA1 gene fragment from a BRCA1 allele specific for one of said alterations, said second fragment corresponding to said first fragment, forming single-stranded DNA from said first BRCA1 gene fragment and from said second BRCA1 gene fragment, forming a heteroduplex consisting of single-stranded DNA from said first BRCA1 gene fragment and single-stranded DNA from said second BRCA1 gene fragment and analyzing for the presence of a mismatch in said heteroduplex, wherein no mismatch indicates the presence of said alteration.

16. The method of claim 1 wherein said germline alteration consists of the deletion of AG in codon 23 of a BRCA1 gene.

17. The method of claim 1 wherein said germline alteration comprises an insertion of a nucleotide C at a position corresponding to a base number 5382 in SEQ ID NO1.

18. The method of claim 1 wherein said germline alteration consists of a deletion of 40 nucleotides corresponding to base numbers 1294-1333 in SEQ ID NO:

1.

19. The method of claim 1 wherein said germline alteration comprises a substitution of a G for the T corresponding to a base number 391 in SEQ ID NO:

17.

20. The method of claim 3 wherein said germline alteration consists of a deletion of AG in codon 23.

21. The method of claim 3 wherein said germline alteration comprises an insertion of a nucleotide C at a position corresponding to a base number 5382 in SEQ ID NO1.

22. The method of claim 3 wherein said germline alteration consists of a deletion of 40 nucleotides corresponding to base numbers 1294-1333 in SEQ ID NO:

1.

23. The method of claim 3 wherein said germline alteration comprises a substitution of a G for the T corresponding to a base number 391 in SEQ ID NO:

17.

24. The method of claim 5 wherein said germline alteration consists of a deletion of AG in codon 23.

25. The method of claim 5 wherein said germline alteration comprises an insertion of a nucleotide C at a position corresponding to a base number 5382 in SEQ ID NO1.

26. The method of claim 5 wherein said germline alteration consists of a deletion of 40 nucleotides corresponding to base numbers 1294-1333 in SEQ ID NO:

1.

27. The method of claim 5 wherein said germline alteration comprises a substitution of a G for the T corresponding to a base number 391 in SEQ ID NO:

17.

28. The method of claim 7 wherein said germline alteration consists of a deletion of AG in codon 23.

29. The method of claim 7 wherein said germline alteration comprises an insertion of a nucleotide C at a position corresponding to a base number 5382 in SEQ ID NO1.

30. The method of claim 10 wherein said germline alteration comprises a substitution of a G for the T corresponding to a base number 391 in SEQ ID NO:

17.

31. The method of claim 7 wherein said germline alteration consists of a deletion of 40 nucleotides corresponding to base numbers 1294-1333 in SEQ ID NO:

1.

32. The method of claim 7 wherein said germline alteration comprises a substitution of a G for the T corresponding to a base number 391 in SEQ ID NO:

17.

33. The method of claim 10 wherein said germline alteration consists of a deletion of AG in codon 23.

34. The method of claim 10 wherein said germline alteration comprises an insertion of a nucleotide C at a position corresponding to a base number 5382 in SEQ ID NO1.

35. The method of claim 10 wherein said germline alteration consists of a deletion of 40 nucleotides corresponding to base numbers 1294-1333 in SEQ ID NO:

1.