Diagnosing predisposition to fat deposition and therapeutic methods for reducing fat deposition and treatment of associated conditions

• US 20040018533A1
• Filed: 06/04/2003
• Published: 01/29/2004
• Est. Priority Date: 06/04/2002
• Status: Abandoned Application

• Current Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Langdown, Maria L., Smylie, Kevin J., Roth, Richard B., Denissenko, Mikhail F., Adam, Gail Isabel Reid
• Application Number:
  US10/455,552
• Publication Number:
  US 20040018533A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• CPC Class Codes: A61P 7/12 Antidiuretics, e.g. drugs for diabetes insipidus ADH A61P5/10
• 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

Provided herein are methods for prognosing and diagnosing fat deposition and related disorders (e.g., obesity and non-insulin diabetes dependent mellitus (NIDDM)) in a subject, reagents and kits for carrying out the methods, methods for identifying candidate therapeutics for reducing fat deposition and related disorders, and therapeutic methods for reducing fat deposition or treating fat deposition related disorders in a subject. These embodiments are based in part upon an analysis of polymorphic variations of the nucleic acid set forth in SEQ ID NO:1.

Claims

1. A method for diagnosing a predisposition to fat deposition in a subject, which comprises detecting the presence or absence of a polymorphic variation associated with fat deposition at a polymorphic site in a P2X4 nucleotide sequence in a nucleic acid sample from a subject, wherein the P2X4 nucleotide sequence comprises a polynucleotide sequence selected from the group consisting of:

(a) the nucleotide sequence of SEQ ID NO;

1;

(b) a nucleotide sequence which encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO;

2 or SEQ ID NO;

3;

(c) a nucleotide sequence which encodes a polypeptide that is 90% identical to the amino acid sequence of SEQ ID NO;

2 or SEQ ID NO;

3; and

(d) a fragment of a nucleotide sequence of (a), (b), or (c) comprising the polymorphic site;

whereby the presence of the polymorphic variation is indicative of a predisposition to fat deposition in the subject.

2. The method of claim 1, which further comprises obtaining the nucleic acid sample from the subject.

3. The method of claim 1, wherein the polymorphic variation is an adenine at position 11030 of SEQ ID NO:

1.

4. The method of claim 3, wherein the polymorphic variation is in linkage disequilibrium with the adenine at position 11030 of SEQ ID NO:

1.

5. The method of claim 1, wherein the polymorphic variation is a thymine at position 15847 of SEQ ID NO:

1.

6. The method of claim 5, wherein the polymorphic variation is in linkage disequilibrium with the thymine at position 15847 of SEQ ID NO:

1.

7. The method of claim 1, wherein the polymorphic variation is a cytosine at position 17338 of SEQ ID NO:

1.

8. The method of claim 7, wherein the polymorphic variation is in linkage disequilibrium with the cytosine at position 17338 of SEQ ID NO:

1.

9. The method of claim 1, wherein the polymorphic variation is a GGGCCCC nucleic acid insert between positions 2878 and 2891 in SEQ ID NO:

1.

10. The method of claim 9, wherein the polymorphic variation is in linkage disequilibrium with the GGGCCCC nucleic acid insert between positions 2878 and 2891 of SEQ ID NO:

1.

11. The method of claim 1, wherein detecting the presence or absence of a polymorphic variation comprises:

hybridizing an oligonucleotide to the nucleic acid sample, wherein the oligonucleotide is complementary to the P2X4 nucleotide sequence and hybridizes to a region of the P2X4 nucleotide sequence that is adjacent to the polymorphic variation;

extending the oligonucleotide in the presence of one or more nucleotides, yielding extension products; and

detecting the presence or absence of the polymorphic variation in the extension products.

12. The method of claim 11, wherein the oligonucleotide is selected from the group consisting of AAAAAAGACCGAACAC (SEQ ID NO:

5), CATTAGCTCGGTCTCC (SEQ ID NO;

     6), GCTTTCAACGGGTC (SEQ ID NO;

     7), TAGCTGGGATTACAGAC (SEQ ID NO;

     8), CATGTCCTGGAAAC (SEQ ID NO;

     9), GCCTTCTGCCTTGTA (SEQ ID NO;

     10), CCTCAAAAAAAGACCGAACAC (SEQ ID NO;

     11), GCGTAGCTTTCAACGGGTC (SEQ ID NO;

     12), ACGGCCATGTCCTGGAAAC (SEQ ID NO;

     13), GAAGAAAAGACACAGAGACAC (SEQ ID NO;

     14), TGGCAGGATGTTTCTCCTGT (SEQ ID NO;

     15), AGGATCCGGAAAGAGTTTCC (SEQ ID NO;

     16), CAGCTTTCTCTACTATATTCATGTAA (SEQ ID NO;

     17), GCTCATTAGCTCGGTCTCC (SEQ ID NO;

     18), GGAAAAACACGAAAGGCTTAGTT (SEQ ID NO;

     19), TCAGGCATGTGATCAGTTAGAGCC (SEQ ID NO;

     20), and CCTTCCCACTGAGCACATC (SEQ ID NO;

     21).

13. The method of claim 1, wherein the fat deposition is central fat deposition in the subject.

14. The method of claim 1, wherein the subject is a human.

15. A method for diagnosing a predisposition to leanness in a subject, which comprises detecting the presence or absence of a polymorphic variation associated with leanness at a polymorphic site in a P2X4 nucleotide sequence in a nucleic acid sample from a subject, wherein the P2X4 nucleotide sequence is selected from the group consisting of:

(a) the nucleotide sequence of SEQ ID NO;

1;

(b) a nucleotide sequence which encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO;

2 or SEQ ID NO;

3;

(c) a nucleotide sequence which encodes a polypeptide that is 90% identical to the amino acid sequence of SEQ ID NO;

2 or SEQ ID NO;

3; and

(d) a fragment of a nucleotide sequence of (i), (ii), or (iii) comprising the polymorphic site;

whereby the presence of the polymorphic variation is indicative of leanness in the subject.

16. The method of claim 15, wherein the polymorphic variation is selected from the group consisting of a thymine at position 11030, a thymine at position 15847, a thymine at position 17338, and the sequence GGGCCCCGGGCCCC between positions 2878 and 2891 in SEQ ID NO:

1.

17. The method of claim 15, wherein the polymorphic variation is a thymine at position 11030, a thymine at position 15847, a thymine at position 17338, an adenine at position 21708, and a thymine at position 22713 of SEQ ID NO:

1.

18. A method for identifying a polymorphic variation associated with fat deposition proximal to an incident polymorphic variation associated with fat deposition, which comprises:

identifying a polymorphic variant proximal to the incident polymorphic variant associated with fat deposition, wherein the incident polymorphic variant is in a P2X4 nucleotide sequence and the P2X4 nucleotide sequence comprises a polynucleotide sequence selected from the group consisting of;

(a) a polynucleotide sequence set forth in SEQ ID NO;

1;

(b) a polynucleotide sequence that encodes a polypeptide having an amino acid sequence encoded by a nucleotide sequence set forth as SEQ ID NO;

1;

or

(c) a polynucleotide sequence that encodes a polypeptide having an amino acid sequence that is 90% identical to an amino acid sequence encoded by a nucleotide sequence set forth in SEQ ID NO;

1; and

determining the presence or absence of an association of the proximal polymorphic variant with fat deposition.

19. The method of claim 18, wherein the first polymorphic variant is located at position 2878, 11030, 15847, or 17338 of SEQ ID NO:

1.

20. The method of claim 18, wherein the proximal polymorphic variant is within a region spanning about 5 kb 5′

of the incident polymorphic variant and about 5 kb 3′

of the incident polymorphic variant.

21. The method of claim 18, which further comprises determining if the proximal polymorphic variant is in linkage disequilibrium with the incident polymorphic variant.

22. The method of claim 18, which further comprises identifying a second polymorphic variant proximal to a proximal polymorphic variant of claim 18 associated with fat deposition and determining if the second polymorphic variant is associated with fat deposition.

23. The method of claim 22, wherein the second proximal polymorphic variant is within a region spanning about 5 kb 5′

of the incident polymorphic variant and about 5 kb 3′

of the proximal polymorphic variant associated with fat deposition.

25. The method of claim 18, wherein the polymorphic variation is a thymine at position 15847 of SEQ ID NO:

1.

26. The method of claim 19, wherein the polymorphic variation is in linkage disequilibrium with the thymine at position 15847 of SEQ ID NO:

1.

24. A method for diagnosing a predisposition to non-insulin dependent diabetes mellitus (NIDDM) in a subject, which comprises detecting the presence or absence of a polymorphic variation associated with NIDDM at a polymorphic site in a P2X4 nucleotide sequence in a nucleic acid sample from a subject, wherein the P2X4 nucleotide sequence comprises a polynucleotide sequence selected from the group consisting of:

(a) the nucleotide sequence of SEQ ID NO;

1;

(b) a nucleotide sequence which encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO;

2 or SEQ ID NO;

3;

(c) a nucleotide sequence which encodes a polypeptide that is 90% identical to the amino acid sequence of SEQ ID NO;

2 or SEQ ID NO;

3; and

(d) a fragment of a nucleotide sequence of (i), (ii), or (iii) comprising the polymorphic site;

whereby the presence of the polymorphic variation is indicative of a predisposition to NIDDM in the subject.

27. A method for identifying a polymorphic variation associated with NIDDM proximal to an incident polymorphic variation associated with NIDDM, which comprises:

identifying a polymorphic variant proximal to the incident polymorphic variant associated with NIDDM, wherein the incident polymorphic variant is in a P2X4 nucleotide sequence and the P2X4 nucleotide sequence comprises a polynucleotide sequence selected from the group consisting of;

(a) a polynucleotide sequence set forth in SEQ ID NO;

1;

(b) a polynucleotide sequence that encodes a polypeptide having an amino acid sequence encoded by a nucleotide sequence set forth as SEQ ID NO;

1;

or

(c) a polynucleotide sequence that encodes a polypeptide having an amino acid sequence that is 90% identical to an amino acid sequence encoded by a nucleotide sequence set forth in SEQ ID NO;

1; and

determining the presence or absence of an association of the proximal polymorphic variant with NIDDM.

28. The method of claim 27, wherein the first polymorphic variant is a thymine at position 15847 of SEQ ID NO:

1.

29. The method of claim 27, wherein the proximal polymorphic variant is within a region spanning about 5 kb 5′

of the incident polymorphic variant and about 5 kb 3′

of the incident polymorphic variant.

30. The method of claim 27, which further comprises determining if the proximal polymorphic variant is in linkage disequilibrium with the incident polymorphic variant.

31. The method of claim 27, which further comprises identifying a second polymorphic variant proximal to a proximal polymorphic variant of claim 27 associated with NIDDM and determining if the second polymorphic variant is associated with NIDDM.

32. The method of claim 31, wherein the second proximal polymorphic variant is within a region spanning about 5 kb 5′

of the incident polymorphic variant and about 5 kb 3′

of the proximal polymorphic variant associated with NIDDM.

33. An isolated nucleic acid which comprises a P2X4 nucleotide sequence selected from the group consisting of:

(a) the nucleotide sequence of SEQ ID NO;

1;

(b) a nucleotide sequence which encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO;

2 or SEQ ID NO;

3;

(c) a nucleotide sequence which encodes a polypeptide that is 90% identical to the amino acid sequence of SEQ ID NO;

2 or SEQ ID NO;

3;

(d) a fragment of a nucleotide sequence of (a), (b), or (c) comprising a polymorphic variation; and

(e) a nucleotide sequence complementary to the nucleotide sequences of (a), (b), (c), or (d);

wherein the P2X4 nucleotide sequence comprises one or more polymorphic variations selected from the group consisting of thymine at position 14744, an adenine at position 14977, a cytosine at position 15046, a thymine at position 21538, a guanine at position 21630, and a single nucleotide deletion at position 25264 of SEQ ID NO;

1.

36. A microarray comprising an isolated nucleic acid of claim 33 linked to a solid support.

37. An isolated polypeptide encoded by the isolated nucleic acid sequence of claim 33.

34. An oligonucleotide which comprises a nucleotide sequence complementary to a portion of a P2X4 nucleotide sequence selected from the group consisting of:

(a) the nucleotide sequence of SEQ ID NO;

1;

(b) a nucleotide sequence which encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO;

2 or SEQ ID NO;

3; and

(c) a nucleotide sequence which encodes a polypeptide that is 90% identical to the amino acid sequence of SEQ ID NO;

2 or SEQ ID NO;

3;

wherein the 3′

end of the oligonucleotide is adjacent to a polymorphic variation in the P2X4 nucleotide sequence.

35. The oligonucleotide of claim 34, which consists of a nucleotide sequence selected from the group consisting of AAAAAAAGACCGAACAC (SEQ ID NO:

5), CATTAGCTCGGTCTCC (SEQ ID NO;

     6), GCTTTCAACGGGTC (SEQ ID NO;

     7), TAGCTGGGATTACAGAC (SEQ ID NO;

     8), CATGTCCTGGAAAC (SEQ ID NO;

     9), GCCTTCTGCCTTGTA (SEQ ID NO;

     10), CCTCAAAAAAAGACCGAACAC (SEQ ID NO;

     11), GCGTAGCTTTCAACGGGTC (SEQ ID NO;

     12), ACGGCCATGTCCTGGAAAC (SEQ ID NO;

     13), GAAGAAAAGACACAGAGACAC (SEQ ID NO;

     14), TGGCAGGATGTTTCTCCTGT (SEQ ID NO;

     15), AGGATCCGGAAAGAGTTTCC (SEQ ID NO;

     16), CAGCTTTCTACTATATTCATGTAA (SEQ ID NO;

     17), GCTCATTAGCTCGGTCTCC (SEQ ID NO;

     18), GGAAAAACACGAAAGGCTTAGTT (SEQ ID NO;

     19), TCAGGCATGTGATCAGTTAGAGCC (SEQ ID NO;

     20), and CCTTCCCACTGAGCACATC (SEQ ID NO;

     21).

38. A method for identifying a candidate therapeutic for fat reduction, which comprises:

(a) introducing a test molecule to a system which comprises a nucleic acid comprising a P2X4 nucleotide sequence selected from the group consisting of;

(i) the nucleotide sequence of SEQ ID NO;

1;

(ii) a nucleotide sequence which encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO;

2 or SEQ ID NO;

3;

(iii) a nucleotide sequence which encodes a polypeptide that is 90% identical to the amino acid sequence of SEQ ID NO;

2 or SEQ ID NO;

3; and

(iv) a fragment of a nucleotide sequence of (i), (ii), or (iii);

or

introducing a test molecule to a system which comprises a protein encoded by a nucleotide sequence of (i), (ii), (iii), or (iv); and

(b) determining the presence or absence of an interaction between the test molecule and the nucleic acid or protein,

whereby the presence of an interaction between the test molecule and the nucleic acid or protein identifies the test molecule as a candidate therapeutic for fat reduction.

39. The method of claim 38, wherein the system is an animal.

40. The method of claim 38, wherein the system is a cell.

41. The method of claim 38, wherein the P2X4 nucleotide sequence comprises one or more polymorphic variations selected from the group consisting of a an adenine at position 11030, a thymine at position 11030, a thymine at position 15847, a cytosine at position 15847, a cytosine at position 17338, and a thymine at position 17338.

42. A method for reducing fat deposition in a subject, which comprises administering a candidate therapeutic of claim 38 to a subject in need thereof, whereby the candidate therapeutic reduces fat deposition in the subject.

43. A method for reducing fat deposition in a subject, which comprises contacting a P2X4 nucleic acid with one or more cells of a subject in need thereof, wherein the P2X4 nucleic acid comprises a nucleotide sequence selected from the group consisting of:

(a) the nucleotide sequence of SEQ ID NO;

1;

(b) a nucleotide sequence which encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO;

2 or SEQ ID NO;

3;

(c) a nucleotide sequence which encodes a polypeptide that is 90% identical to the amino acid sequence of SEQ ID NO;

2 or SEQ ID NO;

3; and

(d) a fragment of a nucleotide sequence of (i), (ii), or (iii);

whereby contacting the one or more cells of the subject with the P2X4 nucleic acid reduces fat deposition.

44. A method for reducing fat deposition in a subject, which comprises contacting a P2X4 protein with one or more cells of a subject in need thereof, wherein the P2X4 protein is encoded by a P2X4 nucleotide sequence which comprises a polynucleotide sequence selected from the group consisting of:

(a) the polynucleotide sequence of SEQ ID NO;

1;

(b) a polynucleotide sequence which encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO;

2 or SEQ ID NO;

3;

(c) a polynucleotide sequence which encodes a polypeptide that is 90% identical to the amino acid sequence of SEQ ID NO;

2 or SEQ ID NO;

3; and

(d) a fragment of a polynucleotide sequence of (i), (ii), or (iii);

whereby contacting the one or more cells of the subject with the P2X4 protein reduces fat deposition.

45. A method for reducing fat deposition in a subject, which comprises:

detecting the presence or absence of a polymorphic variant associated with fat deposition in a P2X4 nucleotide sequence in a nucleic acid sample from a subject, wherein the P2X4 nucleotide sequence comprises a polynucleotide sequence selected from the group consisting of;

(a) the polynucleotide sequence of SEQ ID NO;

1;

(b) a polynucleotide sequence which encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO;

2 or SEQ ID NO;

3;

(c) a polynucleotide sequence which encodes a polypeptide that is 90% identical to the amino acid sequence of SEQ ID NO;

2 or SEQ ID NO;

3; and

(d) a fragment of a polynucleotide sequence of (i), (ii), or (iii); and

administering a treatment that reduces fat deposition to a subject from whom the sample originated where the presence of a polymorphic variation associated with fat reduction is detected in the P2X4 nucleotide sequence.

46. The method of claim 45, wherein the polymorphic variation is selected from the group consisting of an adenine at position 11030, a thymine at position 15847, and a cytosine at position 17338.

47. The method of claim 45, wherein the treatment is one or more selected from the group consisting of an appetite suppressant, a lipase inhibitor, a phospholipase inhibitor, an exercise regimen, a dietary regimen, psychological consoling, psychotherapy, and a psychotherapeutic.

48. A method for identifying a candidate therapeutic for alleviating NIDDM, which comprises:

(a) introducing a test molecule to a system which comprises a nucleic acid comprising a P2X4 nucleotide sequence selected from the group consisting of;

(i) the nucleotide sequence of SEQ ID NO;

1;

(ii) a nucleotide sequence which encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO;

2 or SEQ ID NO;

3;

(iii) a nucleotide sequence which encodes a polypeptide that is 90% identical to the amino acid sequence of SEQ ID NO;

2 or SEQ ID NO;

3; and

(iv) a fragment of a nucleotide sequence of (i), (ii), or (iii);

or

introducing a test molecule to a system which comprises a protein encoded by a nucleotide sequence of (i), (ii), (iii), or (iv); and

(b) determining the presence or absence of an interaction between the test molecule and the nucleic acid or protein,

whereby the presence of an interaction between the test molecule and the nucleic acid or protein identifies the test molecule as a candidate therapeutic for treating NIDDM.

49. The method of claim 48, wherein the system is an animal.

50. The method of claim 48, wherein the system is a cell.

51. The method of claim 48, wherein the P2X4 nucleotide sequence comprises a thymine at position 15847 of SEQ ID NO:

1.

52. A method for treating NIDDM in a subject, which comprises administering a candidate therapeutic of claim 48 to the subject in need thereof, whereby the candidate therapeutic treats NIDDM in the subject.

53. A method for alleviating NIDDM in a subject, which comprises contacting a P2X4 nucleic acid with one or more cells of a subject in need thereof, wherein the P2X4 nucleic acid comprises a nucleotide sequence selected from the group consisting of:

(a) the nucleotide sequence of SEQ ID NO;

1;

(b) a nucleotide sequence which encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO;

2 or SEQ ID NO;

3;

(c) a nucleotide sequence which encodes a polypeptide that is 90% identical to the amino acid sequence of SEQ ID NO;

2 or SEQ ID NO;

3; and

(d) a fragment of a nucleotide sequence of (i), (ii), or (iii);

whereby contacting the one or more cells of the subject with the P2X4 nucleic acid alleviates NIDDM.

54. A method for alleviating NIDDM in a subject, which comprises contacting a P2X4 protein with one or more cells of a subject in need thereof, wherein the P2X4 protein is encoded by a P2X4 nucleotide sequence which comprises a polynucleotide sequence selected from the group consisting of:

(a) the polynucleotide sequence of SEQ ID NO;

1;

(b) a polynucleotide sequence which encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO;

2 or SEQ ID NO;

3;

(c) a polynucleotide sequence which encodes a polypeptide that is 90% identical to the amino acid sequence of SEQ ID NO;

2 or SEQ ID NO;

3; and

(d) a fragment of a polynucleotide sequence of (i), (ii), or (iii);

whereby contacting the one or more cells of the subject with the P2X4 protein alleviates NIDDM.

55. A method for alleviating NIDDM in a subject, which comprises:

detecting the presence or absence of a polymorphic variant associated with NIDDM in a P2X4 nucleotide sequence in a nucleic acid sample from a subject, wherein the P2X4 nucleotide sequence comprises a polynucleotide sequence selected from the group consisting of;

(a) the polynucleotide sequence of SEQ ID NO;

1;

(b) a polynucleotide sequence which encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO;

2 or SEQ ID NO;

3;

(c) a polynucleotide sequence which encodes a polypeptide that is 90% identical to the amino acid sequence of SEQ ID NO;

2 or SEQ ID NO;

3; and

(d) a fragment of a polynucleotide sequence of (i), (ii), or (iii); and

administering a treatment that alleviates NIDDM to a subject from whom the sample originated where the presence of a polymorphic variation associated with NIDDM is detected in the P2X4 nucleotide sequence.

56. The method of claim 55, wherein the polymorphic variation is a thymine at position 15847 of SEQ ID NO:

1.

57. The method of claim 55, wherein the treatment is one or more selected from the group consisting of insulin, a hypoglycemic, a starch blocker, a liver glucose regulating agent, an insulin sensitizer, a glucose level monitoring regimen, dietary counseling, and a dietary regimen for managing blood glucose levels.