Diagnostic method, diagnostic reagent and therapeutic preparation for diseases caused by variation in LKB1 gene

• US 6,800,436 B1
• Filed: 05/25/2000
• Issued: 10/05/2004
• Est. Priority Date: 11/27/1997
• Status: Expired due to Fees

• Current Assignee: Chugai Seiyaku Kabushiki Kaisha (Roche Holding AG)
• Original Assignee: Chugai Seiyaku Kabushiki Kaisha (Roche Holding AG)
• Inventors: Nezu, Jun-ichi, Jenne, Dieter E.
• Primary Examiner(s): Helms, Larry R.
• Assistant Examiner(s): Yu, Misook
• Application Number:
  US09/555,166
• Time in Patent Office: 1,594 Days
• Field of Search: 536/24.3, 536/23.1, 536/24.32, 536/23.5, 436/503, 436/504, 436/64, 435/6
• US Class Current: 435/6.11
• 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: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• 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

With respect to a range of 1.5 Mbp and more in the chromosome 19p13.3 region containing Peutz-Jeghers gene, a continuous cosmid contig is constructed and a restriction map is prepared. Next, genes mapped with this region are searched by using EST database and the locations of these genes are accurately determined. Based on the evaluation of biological data, etc., several highly likely candidates for Peutz-Jeghers genes are specified from the genes thus found. After successively analyzing variations in these genes in DNAs of patients with Peutz-Jeghers syndrome, it is found that one of these genes, i.e., “LKB1” has been specifically varied in these patients. Thus, the diseases caused by the variation in the LKB1 gene can be diagnosed and treated by using the LKB1 gene, primers and probes based on its base sequence, LKB1 protein, an antibody biding to this protein, etc.

Claims

1. A method of diagnosing Peutz-Jeghers syndrome, the method comprising the step of detecting one or more mutations in an LKB1 gene of an individual using a probe or primer comprising at least 15 contiguous nucleotides of any one of SEQ ID NOs:

1 to 4 or any one of the complements of SEQ ID NOs;

1 to 4, wherein detection of the mutation indicates that the individual has Peutz-Jeghers syndrome.

2. The method of claim 1, wherein the step of detecting one or more mutations in the LKB1 gene comprises the steps of:

(a) providing a DNA sample from the individual;

(b) amplifying the DNA using a primer DNA comprising at least 15 contiguous nucleotides of any one of SEQ ID NOs;

1 to 4 or any one of the complements of SEQ ID NOs;

1 to 4;

(c) cleaving the amplified DNA into first DNA fragments;

(d) fractionating the first DNA fragments according to their size;

(e) hybridizing a DNA probe comprising at least 15 contiguous nucleotides of any one of SEQ ID NO;

1 to 4 or any one of the complements of SEQ ID NOs;

1 to 4 with the fractionated first DNA fragments; and

(f) comparing the size of the first DNA fragments to the size of second DNA fragments from a healthy control, wherein a difference in size between the first fragments and the second fragments indicates the presence of one or more mutations in the individual'"'"'s LKB1 gene.

3. The method of claim 1, wherein the step of detecting one or more mutations in the LKB1 gene comprises the steps of:

(a) providing a RNA sample from the individual;

(b) fractionating the RNA in the sample by size;

(c) hybridizing a DNA probe comprising at least 15 contiguous nucleotides of any one of SEQ ID NOs;

1 to 4 or any one of the complements of SEQ ID NOs;

1 to 4 with the fractionated RNA; and

(d) comparing the size of the hybridized RNA thus detected to that from a healthy control, wherein a difference in size between the RNA from the control and the individual indicates the presence of one or more mutations in the individual'"'"'s LKB1 gene.

4. The method of claim 1, wherein the step of detecting one or more mutations in the LKB1 gene comprises the steps of:

(a) providing a DNA sample from the individual;

(b) amplifying the DNA using a primer DNA comprising at least 15 contiguous nucleotides of any one of SEQ ID NOs;

1 to 4 or any one of the complements of SEQ ID NOs;

1 to 4;

(c) separating the amplified DNA into single stranded DNA;

(d) fractionating the single stranded DNA on a non-denaturing gel; and

(e) comparing the mobility of the fractionated single stranded DNA to that from a healthy control, wherein a difference in mobility between the single stranded DNA from the control and the individual indicates the presence of one or more mutations in the individual'"'"'s LKB1 gene.

5. The method of claim 1, wherein the step of detecting one or more mutations in the LKB1 gene comprises the steps of:

(a) providing a DNA sample from the individual;

(b) amplifying the DNA using a primer DNA comprising at least 15 contiguous nucleotides of any one of SEQ ID NOs;

1 to 4 or any one of the complements of SEQ ID NOs;

1 to 4;

(c) fractionating the amplified DNA on a DNA denaturing gradient gel; and

(d) comparing the mobility of the fractionated DNA on the gel to that from a healthy control, wherein a difference in mobility between the DNA from the control and the individual indicates the presence of one or more mutations in the patient'"'"'s LKB1 gene.

6. The method according to claim 1, wherein the individual has Peutz-Jeghers syndrome.

7. The method of claim 1, wherein the one or more mutations result in a disruption of the kinase domain of the LKB1 protein.

8. The method of claim 1, wherein at least one of the one or more mutations is an inversion.

9. The method of claim 1, wherein at least one of the one or more mutations is a deletion.

10. The method of claim 1, wherein at least one of the one or more mutations is aberrant splicing.

11. The method of claim 1, wherein at least one of the one or more mutations is a point mutation.

12. The method of claim 1, wherein at least one of the one or more mutations is premature termination.

13. The method of claim 1, wherein at least one of the one or more mutations is a frameshift mutation.