Susceptibility gene for obesity and type II diabetes mellitus

• US 5,766,851 A
• Filed: 05/19/1995
• Issued: 06/16/1998
• Est. Priority Date: 05/19/1995
• Status: Expired due to Term

• Current Assignee: Johns Hopkins University
• Original Assignee: Johns Hopkins University
• Inventors: Shuldiner, Alan R., Roth, Jesse, Walston, Jeremy, Silver, Kristi
• Primary Examiner(s): Myers, Carla J.
• Application Number:
  US08/446,530
• Time in Patent Office: 1,124 Days
• Field of Search: 435/6, 435/91.2, 435/810, 436/63, 536/23.5, 536/24.31, 536/24.33, 935/8, 935/9, 935/17, 935/78
• US Class Current: 435/6.13
• 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 14/70571 for neuromediators, e.g. serotonin receptor, dopamine receptor
• CPC Class Codes: C12N 2799/027 where the vector is derived from a retrovirus
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 530/827 Proteins from mammals or birds

Abstract

The present invention provides a novel polypeptide characterized by a non-conservative missense mutation, Trp64Arg, in the β3-adrenergic receptor (β3AR) that increases susceptibility to obesity and non-insulin dependent diabetes mellitus (NIDDM; type II diabetes). Also provided are methods of diagnosis and methods of treatment of subjects having or at risk of having type II diabetes/obesity.

Claims

1. Isolated oligonucleotide primer(s) for diagnosis of a subject having or at risk of having an increased likelihood for developing type II diabetes mellitus and/or obesity, wherein the primer specifically hybridizes with a target polynucleotide sequence having a sequence selected from the group consisting of:

5'"'"'-GTGTTGGCCGTATTGGGCG-3'"'"' (SEQ ID NO;

1) and

5'"'"'-GGTGATGGGACTCCTGGTGG-3'"'"' (SEQ ID NO;

2).

2. The primer of claim 1, wherein the primer is

5'"'"'-CGCCCAATACCGCCAACAC-3'"'"' (SEQ ID NO:

3) or

5'"'"'-CCACCAGGAGTCCCATCACC-3'"'"' (SEQ ID NO;

4).

3. An isolated polynucleotide encoding a polypeptide having an amino acid sequence of a β

3-adrenergic receptor having a substitution at amino acid residue 64 of arginine for tryptophan and wherein the substituted receptor is associated with having or at risk of having an increased likelihood for developing Type II diabetes mellitus and/or obesity.

4. A method for diagnosis of a subject having or at risk of having an increased likelihood for developing type II diabetes mellitus and/or obesity comprising:

a) contacting a target nucleic acid of a sample from the subject with a reagent that detects a mutation in the β

3AR gene, wherein the mutation is a substitution at amino acid residue 64 of arginine for tryptophan; and

b) detecting the mutation, wherein detection of the mutation is indicative of a subject having or at risk of having an increased likelihood for developing type II diabetes mellitus and/or obesity.

5. The method of claim 4, wherein the target nucleic acid is DNA.

6. The method of claim 4, wherein the target nucleic acid is RNA.

7. The method of claim 4, wherein the reagent is a nucleic acid probe.

8. The method of claim 4, wherein the target nucleic acid is amplified prior to detection.

9. The method of claim 8, wherein the amplifying is by polymerase chain reaction (PCR).

10. The method of claim 7, wherein the probe comprises a detectable label.

11. The method of claim 10, wherein the label is selected from the group consisting of a radioisotope, a bioluminescent compound, a chemiluminescent compound, a fluorescent compound, a metal chelate, and an enzyme.

12. The method of claim 4, wherein the target nucleic acid has a nucleotide sequence selected from the group consisting of

5'"'"'-TCGGAGTCCAGGCGATG-3'"'"' (SEQ ID NO:

5) and

5'"'"'-TCGGAGTCCGGGCGATG-3'"'"' (SEQ ID NO;

6).

13. The method of claim 7, wherein the nucleic acid probe is

5'"'"'-CATCGCCTGGACTCCGA-3'"'"' (SEQ ID NO:

7) or

5'"'"'-CATCGCCCGGACTCCGA-3'"'"' (SEQ ID NO;

8).

14. The method of claim 4, wherein the reagent is a restriction endonuclease.

15. The method of claim 14, wherein the restriction endonuclease recognizes and cleaves a nucleic acid having a sequence CC↓

(A/T)GG.

16. The method of claim 15, wherein the restriction endonuclease is selected from the group consisting of BstNI, BstOI, ApyI, EcoRII, and MvaI.

17. The method of claim 4, wherein the sample comprises a blood cell.

18. The method of claim 4, wherein the mutation is a substitution of TGG at codon 64 with CGG.

19. A kit useful for the diagnosis of a subject having or at risk of having an increased likelihood for developing type II diabetes mellitus and/or obesity, the kit comprising means for amplifying a target β

3AR DNA, wherein the β

3AR DNA encodes a polypeptide having a substitution at amino acid residue 64 of arginine for tryptophan, said means comprising the necessary enzyme(s) and oligonucleotide primers for amplifying said target DNA of a sample from a subject, wherein said primers amplify a DNA fragment of about 100 to 350 nucleotides and wherein said DNA fragment contains DNA encoding amino acid residue 64.

20. The kit of claim 19, wherein the oligonucleotide primers include primers having a nucleotide sequence:

5'"'"'-CGCCCAATACCGCCAACAC-3'"'"' (SEQ ID NO;

3) or

5'"'"'-CCACCAGGAGTCCCATCACC-3'"'"' (SEQ ID NO;

4).

21. The kit of claim 19, wherein said primers specifically hybridize to a region of the target DNA having the sequence selected from the group consisting of:

5'"'"'-GTGTTGGCCGTATTGGGCG-3'"'"' (SEQ ID NO;

1)

and 5'"'"'-GGTGATGGGACTCCTGGTGG-3'"'"' (SEQ ID NO;

2).