Patched genes and their use for diagnostics

• US 6,027,882 A
• Filed: 05/31/1996
• Issued: 02/22/2000
• Est. Priority Date: 10/07/1994
• Status: Expired due to Term

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University), Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Epstein, Ervin, Oro, Tony, Johnson, Ronald L., Goodrich, Lisa V., Scott, Matthew P.
• Primary Examiner(s): Campell, Bruce R.
• Assistant Examiner(s): Martin, Jill D.
• Application Number:
  US08/656,055
• Time in Patent Office: 1,362 Days
• Field of Search: 435/91.2, 435/6, 435/325, 536/23.5, 536/24.31
• US Class Current: 435/6.12
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: C07K 14/43563 from insects
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748

Abstract

Methods for isolating patched genes, particularly mammalian patched genes, including mouse and human patched genes, as well as invertebrate patched genes and sequences, are provided. Loss-of function of the patched is associated with the occurrence of human cancers, particularly basal cell carcinomas of the skin. The cancers may be familial, having as a component of risk an inherited genetic predisposition, or may be sporadic. Therefore, methods for using the patched gene as a diagnostic for assessing a genetic predisposition to cancer, and to identify specific cancers having mutations in this gene, are disclosed.

Claims

1. A method for assessing a genetic predisposition of an animal for a skin cancer, the method comprising:

detecting, from a sample of nucleic acid isolated from the animal, a loss-of-function mutation in a patched gene in the germline of said animal,

wherein the presence of said loss-of-function mutation indicates that said animal has a genetic predisposition for basal cell carcinoma.

2. The method according to claim 1, wherein said sample of nucleic acid is from a biopsy of cells isolated from the animal.

3. The assay of claim 1, wherein the cell sample is obtained from a human patient.

4. The assay of claim 3, wherein the cell sample is obtained from a biopsy.

5. The assay of claim 3, wherein the biopsy is obtained from a carcinoma, meningiomoa, medulloma or fibroma.

6. A method for determining a patched phenotype of cells of a tumor comprising detecting, from a sample of nucleic acid isolated from the cells, the presence or absence of a loss-of-function mutation of a patched gene in the cells.

7. The method according to claim 6, wherein the tumor is a carcinoma.

8. The method according to claim 7, wherien said carcinoma is a basal cell carcinoma.

9. The method according to claim 6, wherein said nucleic acid is from a biopsy of cells of said tumor.

10. An assay for determining the patched phenotype of a cell, comprising

providing a nucleic acid sample isolated from mammalian cells,

detecting the presence or absence of a patched gene sequence or allelic variant thereof, by hybridization of the nucleic acid sample with one or more nucleic acid probes which hybridize to a mammalian patched gene.

11. The assay of claim 10, wherein hybridization of the probe(s) further comprises subjecting the probe(s) and nucleic acid sample to an amplification process and detecting abnormalities in an amplified product.

12. The assay of claim 11, wherein the amplification process is polymerase chain reaction (PCR).

13. The assay of claim 10, wherein the probe(s) hybridizes to SEQ ID No. 9 or 18 under stringency conditions equivalent to 10×

SCC at 50°

C. to SEQ ID No. 9 or 18.

14. The assay of claim 10, wherein the probe(s) hybridizes to SEQ ID No. 9 or 18 under stringency conditions equivalent to 5×

SSC at 60°

C.

15. The assay of claim 10, wherein the probe(s) hybridizes to SEQ ID No. 9 or 18 under stringency conditions equivalent to 0.1×

SCC at 60°

C.

16. The assay of claim 10, wherein the probe(s) further comprises a label group attached to the nucleic acid and able to be detected.

17. The assay of claim 10, wherein the probe(s) are at least 15 nucleotides in length.

18. The assay of claim 10, wherein the probe(s) are at least 50 nucleotides in length.

19. The assay of claim 10, wherein the probe(s) are 15-100 nucleotides in length.

20. The method of claim 10, wherein the nucleic acid sample is an mRNA sample from the mammalian cells.

21. The method of claim 10, wherein the nucleic acid sample is a cDNA sample reverse transcribed from mRNA of the mammalian cells.

22. The method of claim 10, wherein the nucleic acid sample is genomic DNA from the mammalian cells.

23. The method of claim 10, which method detects loss of heterozygosity in a patched gene of the mammalian cells.

24. The assay of claim 10, wherein the sequence of the detected patched is determined.

25. The assay of claim 24, wherein the presence or absence of a deletion of one or more nucleotides from the patched gene, an addition of one or more nucleotides to the patched gene, or a substitution of one or more nucleotides of the patched gene is determined from the sequence.

26. An assay for detecting mutations of a patched gene, comprising detecting, in a sample of isolated mammalian cells or nucleic acid isolated therefrom, the presence or absence of a deletion of one or more nucleotides from the patched gene, an addition of one or more nucleotides to the patched gene, a substitution of one or more nucleotides of the patched gene, a chromosomal rearrangement of all or a portion of the patched gene, an alteration in the level of an mRNA transcript of the patched gene, or alteration of the splicing pattern of an mRNA transcript of the patched gene.

27. The assay of claim 26, wherein all or a portion of the patched gene is amplified by an amplification process and abnormalities in an amplified product, if any, are detected.

28. The assay of claim 27, wherein the amplification process is polymerase chain reaction (PCR).

29. The assay of claim 26, wherein mutations of the patched gene are detected by single strand conformational polymorphism analysis.

30. The assay of claim 26, wherein mutations of the patched gene are detected by gel electrophoresis.

31. The assay of claim 26, wherein mutations of the patched gene are detected by digestions with one or more endonucleases.

32. An assay for phenotyping the patched status of a cell, comprising detecting, in a sample of isolated mammalian cells, the presence or absence of a genetic lesion of a patched gene characterized by at least one of (i) aberrant mutation of a patched gene resulting in loss of function, and (ii) mis-expression of the patched gene resulting in loss of function.

33. The assay of claim 32, which assay includes:

i. providing one or more nucleic acid probes comprising a region of nucleotide sequence which hybridizes to a sense or antisense sequence of the patched gene, or naturally occurring mutants thereof;

ii. combining the probe(s) with a nucleic acid sample from the cells; and

iii. detecting, by hybridization of the probe(s) to the nucleic acid, the presence or absence of a deletion of one or more nucleotides from the patched gene, an addition of one or more nucleotides to the patched gene, a substitution of one or more nucleotides of the patched gene, a chromosomal rearrangement of all or a portion of the patched gene, an alteration in the level of an mRNA transcript of the patched gene, or alteration of the splicing pattern of an mRNA transcript of the patched gene.

34. The assay of claim 33, wherein the probe(s) hybridizes to a sequence designated by SEQ ID No. 9 or 18 under stringency conditions equivalent to 10×

SSC at 50°

C.

35. The assay of claim 33, wherein the probe(s) hybridizes to a sequence designated by SEQ ID No. 9 or 18 under stringency conditions equivalent to 5×

SSC at 50°

C.

36. The assay of claim 33, wherein the probe further comprises a label group attached to the nucleic acid and able to be detected.

37. The assay of claim 33, wherein all or a portion of the patched gene is amplified by an amplification process and abnormalities in an amplified product, if any, are detected.

38. The assay of claim 37, wherein the amplification process is polymerase chain reaction (PCR).

39. The assay of claim 32, wherein mutations of the patched gene are detected by single strand conformational polymorphism analysis.

40. The assay of claim 32, wherein mutations of the patched gene are detected by gel electrophoresis.

41. The assay of claim 32, wherein mutations of the patched gene are detected by digestions with one or more endonucleases.

42. The assay claim 32, wherein detecting the lesion comprises ascertaining, relative to a wild-type level of hedgehog-dependent patched signal transduction, the ability of cells in cell sample to respond to hedgehog induction.

43. A method for assessing a genetic predisposition of an animal for developing basal cell nevus syndrome, the method comprising:

detecting, from a sample of nucleic acid isolated from the animal, a loss-of-function mutation in a patched gene in the germline of said animal,

wherein the presence of said loss-of-function mutation indicates that said animal has a genetic predisposition for developing basal cell nevus syndrome.

44. The method according to claim 43, wherein said sample of nucleic acid is from a biopsy of cells isolated from the animal.