Diagnostic test and therapy for manganese superoxide dismutate (mNsod) associated diseases

• US 6,265,172 B1
• Filed: 02/08/2000
• Issued: 07/24/2001
• Est. Priority Date: 02/08/1999
• Status: Expired due to Fees

• Current Assignee: University of Kentucky
• Original Assignee: University of Kentucky
• Inventors: Kasarskis, Edward J., St. Clair, Daret K., Urano, Muneyasu
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US09/499,884
• Time in Patent Office: 532 Days
• Field of Search: 435/6, 435/91.2, 514/44, 536/23.1, 536/24.3
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 Expression markers

Abstract

The present invention provides a diagnostic method and a kit for detection of mutations localized within the 5′ promoter region of the MnSOD gene. Such mutations are associated with diseases characterized by decreased MnSOD activity such as certain formes of cancer, and ALS. Accordingly, the diagnostic method this invention provides, comprising RFLP, direct sequencing, or PCR analysis of the region within 3 kb, the transcription initiation site will detect these disorders. This invention also provides a therapeutic method for such disorders comprising transfection of affected cells or tissues with high activity, MnSOD expression vectors, or the administration of exogenous MnSOD enzyme.

Claims

1. A method for detecting the presence of mutation within the MnSOD gene promoter region wherein the mutation is associated with cancer comprising:

(i) carrying out restriction fragment length polymorphism analysis on a DNA test sample;

(ii) obtaining a restriction endonuclease digestion pattern from the procedure in step (i); and

(iii) comparing results obtained in step (ii) with restriction endonuclease digestion pattern of a wild-type MnSOD gene promoter, wherein a difference in the restriction endonuclease digestion pattern indicates the presence of at least one mutation in the MnSOD gene promoter in the DNA test sample wherein the mutation is associated with cancer.

2. The method of claim 1, wherein the MnSOD gene promoter sequence is first amplified by polymerase chain reaction technique before step (i), wherein said DNA test sample is digested with restriction enzyme which cuts at a recognition sequence within the promoter region, and wherein the resulting restriction endonuclease digestion pattern is visualized by electrophoresis, and staining with an appropriate DNA binding dye.

3. The method of claim 1, wherein the restriction endonuclease digestion pattern following restriction enzyme treatment is visualized by Southern Blot analysis wherein after gel electrophoresis, the DNA is adsorbed to an appropriate membrane and hybridized with labeled oligonucleotide probes complementary to MnSOD gene promoter enhancer sequences.

4. A method for detecting MnSOD gene promoter mutation wherein the mutation is associated with cancer comprising:

i) sequencing a test sample DNA comprising the MnSOD gene promoter; and

ii) comparing the sequence obtained in step (i) with a wild-type sequence, wherein a difference in the sequence indicates the presence of at least one mutation in the MnSOD gene promoter in the DNA test sample wherein the mutation is associated with cancer.

5. A method for detecting MnSOD gene promoter mutation by PCR analysis wherein the mutation is associated with cancer comprising:

(i) making oligonucleotide primer complementary to the MnSOD gene promoter sequence containing one or more of the mutations; and

ii) carrying out PCR procedure to amplify a sequence, wherein the presence of PCR amplified product indicates at least one mutation in the MnSOD gene promoter in the DNA wherein the mutation is associated with cancer.

6. A method for detecting MnSOD gene promoter mutation wherein the mutation is associated with cancer comprising:

(i) admixing a test DNA sample comprising MnSOD gene promoter with at least one transcription factor comprising SP-1, AP-2 or NF-KB under conditions that facilitate DNA-protein binding;

(ii) carrying out a DNA footprinting analysis, wherein a footprinting pattern is obtained for the test sample DNA in step (i) and;

(iii) comparing the footprinting pattern obtained in step (ii) with a footprinting pattern of a wild-type MnSOD gene promoter, wherein a difference in the footprinting pattern indicates the presence of at least one mutation in the MnSOD gene promoter in the DNA test sample wherein the mutation is associated with cancer.

7. A method for detecting MnSoD gene promoter mutation wherein the mutation is associated with cancer comprising:

(i) admixing a test DNA sample comprising MnSOD gene promoter with at least one transcription factor comprising SP-1, AP-2 or NF-KB under conditions that facilitate DNA-protein binding;

(ii) carrying out a DNA mobility shift analysis, wherein an electrophoretic mobility pattern is obtained for the test sample DNA in step (i) and;

(iii) comparing the electrophoretic mobility pattern obtained in step (ii) with an electrophoretic mobility pattern of a wild-type MnSOD gene promoter, wherein a difference in the electrophoretic mobility pattern indicates the presence of at least one mutation in the MnSOD gene promoter in the DNA test sample wherein the mutation is associated with cancer.

8. A method for detecting the presence of mutation within the MnSOD gene promoter region wherein the mutation is associated with cancer comprising:

(i) linking a reporter gene 3′

to the MnSOD gene promoter region in the DNA test sample;

(ii) assaying for expression of the reporter gene; and

(iii) comparing results obtained in step (ii) with level of reporter gene expression obtained using wild-type MnSOD gene promoter, wherein a difference in the level of reporter gene expression indicates the presence of at least one mutation in the MnSOD gene promoter in the DNA test sample wherein the mutation is associated with cancer.

9. The method according to claim 8, wherein said reporter gene is a luciferase gene.