Methods for detecting pre-diabetes and diabetes

• US 8,476,008 B2
• Filed: 07/23/2009
• Issued: 07/02/2013
• Est. Priority Date: 07/23/2008
• Status: Active Grant

• Current Assignee: DiabetOmics, Inc.
• Original Assignee: Diabetomics, LLC
• Inventors: Nagalla, Srinivasa R., Roberts, Charles T., Paturi, Vishnupriya Rao
• Primary Examiner(s): GITOMER, RALPH J
• Application Number:
  US13/055,605
• Publication Number:
  US 20110124022A1
• Time in Patent Office: 1,440 Days
• Field of Search: 435/4
• US Class Current: 435/4
• CPC Class Codes: G01N 2800/042 Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
• CPC Class Codes: G01N 2800/50 Determining the risk of developing a disease
• CPC Class Codes: G01N 2800/52 Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• CPC Class Codes: G01N 33/6893 related to diseases not provided for elsewhere

Abstract

Non-invasive methods are provided herein for the diagnosis of pre-diabetes and diabetes using biomarkers identified in a biological fluid, such as saliva. These biomarkers can be identified using proteomic methods, including but not limited to antibody based methods, such as an enzyme-linked immunosorbant assay (ELISA), a radioimmunoassay (RIA), or a lateral flow immunoassay.

Claims

1. A method for determining if a subject of interest has pre-diabetes or diabetes, comprising

(a) comparing a proteomic profile of a test sample of saliva from a subject of interest with a proteomic profile of at least one control sample, wherein the proteomic profile of the test sample and the at least one control sample comprise information on the expression of at least one of cystatin C, alpha 2-macroglobulin (A2MG), transthyretin (TTR), plasma retinol binding protein 4 (RBP4) and/or lipocalin 2, and wherein:

(i) if the at least one control sample is a non-diabetic reference value or a saliva sample from a non-diabetic subject, and the proteomic profile of the test sample does not show an elevated level of cystatin C, alpha 2-macroglobin (A2MG), transthyretin (TTR), plasma retinol binding protein 4 RBP4) and/or lipocalin 2 relative to the proteomic profile of the at least one control sample, the subject is determined not to have pre-diabetes or diabetes, and wherein if the proteomic profile of the test sample shows an elevated level of cystatin C, alpha 2-macroglobin (A2MG), transthyretin (TTR), plasma retinol binding protein 4 (RBP4) and/or lipocalin 2 relative to the proteomic profile of the at least one control-sample, the subject is determined to have prediabetes or diabetes;

(ii) if the at least one control sample is a diabetic reference value or a saliva sample from a subject with diabetes and the proteomic profile of the test sample does not show a decreased level of cystatin C, alpha 2-macroglobin (A2MG) transthyretin (TTR), plasma retinol binding protein 4 (RBP4) and/or lipocalin 2 relative to the proteomic profile of the at least one control sample, then the subject is determined to have diabetes, and wherein if the proteomic profile of the test sample shows a decreased level of cystatin C, alpha 2-macroglobin (A2MG), transthyretin (TTR), plasma retinol binding protein 4 (RBP4) and/or lipocalin 2 relative to the proteomic profile of the at least one control sample, the subject is determined not to have diabetes; and

(iii) if the at least one control sample comprises a negative control comprising a non-diabetic reference value or a saliva sample from a non-diabetic subject and a positive control comprising a diabetic reference value or a saliva sample from a subject with diabetes, and the proteomic profile of the test sample shows a level of cystatin C, alpha 2-macroglobin (A2MG), transthyretin (TTR), plasma retinol binding protein 4 (RBP4) and/or lipocalin 2 that is intermediate between the proteomic profiles of the negative control and the positive control, then the subject is determined to have pre-diabetes.

2. The method of claim 1, wherein the proteomic profile of the test sample and at least one control sample further comprise information on the expression of at least one additional protein, wherein the at least one additional protein comprises alpha-1-antitrypsin, alpha 1 acid glycoprotein, uteroglobin, carbonic anhydrase 6 pyruvate kinase isozymes MIIM2, neutrophil collagenase, purine nucleoside phosphorylase, aldehyde dehydrogenase, fatty acid biding protein (epidermal), peroxiredoxin-1 -2, +-6 lamin AIC apolipoprotein B-100, annexin A2, carbonic anhydrase 1, carbonic anhydrase 2,

protein plunc, pancreatic ribonuclease, inter-a-trypsin inhibitor heavy chain HI, inter-a-trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3- epsilon, cystatin A, annexin A3, Protein S100-A7, phosphoglycerate kinase 1, annexin Al , isoform2 of P67936 tropomyosin a-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and/or calnexin, and wherein:

(i) if the at least one control sample is a non-diabetic reference value or a saliva sample from a non-diabetic subject, and the proteomic profile of the test sample does not show an elevated level of the at least one additional protein relative to the proteomic profile of the at least one control sample, the subject is determined not to have pre-diabetes or diabetes, and wherein if the proteomic profile of the test sample shows an elevated level of the at least one additional protein relative to the proteomic profile of the at least one control sample, the subject is determined to have prediabetes or diabetes;

(ii) if the reference sample is a diabetic reference value or a saliva sample from a diabetic subject, and the proteomic profile of the test sample does not show a decreased level of the at least one additional protein relative to the proteomic profile of the at least one control sample, the subject is determined to have prediabetes or diabetes, and wherein if the proteomic profile of the test sample shows a decreased level of the at least one additional protein relative to the proteomic profile of the at least one control sample, the subject is determined not to have pre-diabetes or diabetes; and

(iii) if the at least one control sample comprises a negative control comprising a non-diabetic reference value or a saliva sample from a non-diabetic subject, and a positive control comprising a diabetic reference value or a saliva sample from a subject with diabetes, and the proteomic profile of the test sample shows a level of the at least one additional protein that is intermediate between the proteomic profiles of the negative control and the positive control, then the subject is determined to have pre-diabetes.

3. The method of claim 1, wherein an increased level of cystatin C, alpha 2-macroglobin (A2MG), transthyretin (TTR), plasma retinol binding protein 4 (RBP4) and/or lipocalin 2 comprises at least a 1.2-fold increase relative to the proteomic profile of the at least one control sample;

and wherein a decreased level of alpha 2-macroglobin (A2MG), transthyretin (TTR), plasma retinol binding protein 4 (RBP4) and/or lipocalin 2 comprises at least a 1.2-fold decrease relative to the proteomic profile of the at least one sample.

4. The method of claim 1, wherein the proteomic profile further comprises information on the expression of one or more of proteasome subunit, aldo-keto reductase family 1 member B 10, cathepsin Z, chitotriosidase isoform 2, 3, +4, transmembrane protease, serine 1 ID, transthyretin, glycogen phosphorylase, heterogeneous nuclear RNPs A21B 1, leukocyte elastase inhibitor, small proline-rich protein 2F, calmodulin-like protein 5, neuroblast differentiation AHNAK, histone cluster 1, Hle, kallikrein-13, chitinase-3-like protein 1, inter-alpha (Globulin) inhibitor H2, 14-3-3 protein eta, cofilin-1, retinol binding protein 4, plasma, basic proline-rich peptide IB-8a, isoform 2 of P60953 cdc 42 homolog, actin-related protein 213 complex subunit 5, ly61PLAUR domain-containing protein 3, actin-like protein 2, Rearranged VKA17V gene segment, brain acid soluble protein 1, golgi phosphoprotein 2, protein FAM49B (LI), and acidic leucine-rich nuclear phosphoprotein 32.

5. The method of claim 2, wherein the proteomic profile comprises information on the expression of five or more of protein plunc, pancreatic ribonuclease, inter-a-trypsin inhibitor heavy chain HI, inter-a-trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3-epsilon, cystatin A, annexin A3, Protein S100-A7, phosphoglycerate kinase 1, annexin Al, isotform2 of P67936 tropomyosin a-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and calnexin.

6. The method of claim 2, wherein the proteomic profile comprises information on the expression of ten or more of protein plunc, pancreatic ribonuclease, inter-a-trypsin inhibitor heavy chain HI, inter-a-trypsin heavy chain 5 H4, nucleobindin-2, moesin, 14-3-3-epsilon, cystatin A, annexin A3, Protein S100-A7, phosphoglycerate kinase 1, annexin Al, isotform2 of P67936 tropomyosin a-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and calnexin.

7. The method of claim 2, wherein the proteomic profile comprises information on the expression of fifteen or more of protein plunc, pancreatic ribonuclease, inter-a-trypsin inhibitor heavy chain HI, inter-a-trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3-epsilon, cystatin A, annexin A3, Protein S100-A7, phosphoglycerate kinase 1, annexin Al, isotform2 of P67936 tropomyosin a-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and calnexin.

8. The method of claim 2, wherein the proteomic profile comprises all of protein plunc, pancreatic ribonuclease, inter-a-trypsin inhibitor heavy chain HI inter-a-trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3-epsilon, cystatin A, annexin A3, Protein S100-A7, phosphoglycerate kinase 1, annexin Al, isotform2 of P67936 tropomyosin a-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and calnexin.

9. The method of claim 2, wherein the proteomic profile further comprises information on the expression of alpha-1-antitrypsin, alpha 2-macroglobulin, cystatin C, plasma retinol binding protein 4 (RBP4), and transthyretin.

10. The method of claim 1, wherein the diabetes is type 2 diabetes.

11. The method of claim 1, wherein the diabetes is type 1 diabetes.

12. The method of claim 1, further comprising determining a level of hemoglobin A1C for the subject, wherein an elevated level of hemoglobin A1C relative to a non-diabetic A1C control value confirms a diagnosis of pre-diabetes or diabetes.

13. The method of claim 1, wherein the subject is obese.

14. The method of claim 1, wherein the proteomic profile is determined using a lateral flow device.

15. A method for monitoring the efficacy of an anti-diabetes therapy in a subject comprising:

obtaining a first saliva sample from the subject at a first time point;

administering the anti-diabetes therapy to the subject;

obtaining a second saliva sample from the subject at a second time point;

determining a proteomic profile for each of the first and second saliva samples, wherein the proteomic profiles of the first and second saliva samples comprise information on the expression of at least one of cystatin C,alpha 2-macroglobulin (A2MG), transthyretin (TTR), plasma retinol binding protein 4 (RBP4) and lipocalin 2, and wherein;

(i) if the proteomic profile of the second saliva sample shows a decrease in a level of cystatin C, alpha 2-macroglobin (A2MG), transthyretin (TTR), plasma retinol binding protein 4 (RBP4) and/or lipocalin 2 relative to the proteomic profile of the first saliva sample, the anti-diabetes therapy is determined to be effective; and

(ii) if the proteomic profile of the second saliva sample does not show a decrease in a level of cystatin C, alpha 2-macroglobin (A2MG), transthyretin (TTR), plasma retinol binding protein 4 (RBP4) and/or lipocalin 2 relative to the proteomic profile of the first saliva sample, the anti-diabetes therapy is determined to be ineffective.

16. The method of claim 15, wherein the proteomic profiles of the first and second saliva samples further comprise information on the expression of at least one additional protein, wherein the at least one additional protein comprises alpha-1-antitrypsin, alpha 1 acid glycoprotein, uteroglobin, carbonic anhydrase 6, pyruvate kinase isozymes MIIM2, neutrophil collagenase, purine nucleoside phosphorylase, aldehyde dehydrogenase, fatty acid biding protein (epidermal), peroxiredoxin-1, -2, +-6, lamin AIC, apolipoprotein B-100, annexin A2, carbonic anhydrase 1, carbonic anhydrase 2,protein plunc, pancreatic ribonuclease, inter-a-trypsin inhibitor heavy chain HI, inter-a-trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3- epsilon, cystatin A, annexin A3, Protein S100-A7, phosphoglycerate kinase 1, annexin Al, isoform2 of P67936 tropomyosin a-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and/or calnexin, and wherein:

(i) if the proteomic profile of the second saliva sample shows a decrease in a level of the at least one additional protein relative to the proteomic profile of the first saliva sample, the anti-diabetes therapy is determined to be effective; and

(ii) if the proteomic profile of the second saliva sample does not show a decrease in a level of the at least one additional protein relative to the proteomic profile of the first saliva sample, the anti-diabetes therapy is determined to be ineffective.