Citrullinated brain and neurological proteins as biomarkers of brain injury or neurodegeneration

Patent metadata

Field	Value
Publication number	US-2017370943-A1
Application number	US-201715636076-A
Country	US
Kind code	A1
Filing date	Jun 28, 2017
Priority date	Mar 13, 2012
Publication date	Dec 28, 2017
Grant date	—

Abstract

The present invention relates to the field of biomarkers. More specifically, the present invention relates to biomarkers useful in diagnosing brain injury or neurodegeneration. In one embodiment, a method for diagnosing brain injury in a patient comprises the steps of (a) obtaining a sample from the patient; (b) determining the ratio of citrullinated to unmodified arginine residues at one or more arginine residues of one or more brain injury biomarker proteins; and (c) correlating the ratio to a patient having brain injury or to a patient not having brain injury, thereby providing the diagnosis.

First claim

1 - 60 . (canceled)

61 . A method for detecting citrullinated brain injury biomarker proteins in a sample from a subject, the method comprising:
detecting in a subject's sample citrullinated arginine residues in citrullinated brain injury biomarker protein glial fibrillary acidic protein (GFAP) and/or citrullinated brain injury biomarker protein myelin basic protein (MBP) by contacting the citrullinated arginine residues in the citrullinated brain injury biomarker proteins with an antibody that specifically binds to said citrullinated arginine residues or by detecting the citrullinated arginine residues using mass spectrometry; thereby detecting citrullinated brain injury biomarker proteins in the subject's sample.

62 . The method of claim 61 , wherein the citrullinated GFAP is citrullinated at one or more arginine (R) amino acid residues selected from R88, R105, R124, R126, R136, R173, R217, R258, R270, R286, R287, R367, and R406, and the citrullinated MBP is citrullinated at one or more arginine (R) amino acid residues selected from R31, R43, R49, R65, R107, R113, R162, and R169.

63 . The method of claim 61 , further comprising detecting citrullinated brain injury biomarker protein neurogranin (NRGN).

64 . The method of claim 63 , wherein the citrullinated NRGN is citrullinated at one or more arginine (R) amino acid residues selected from R38, R43, R51, R53, and R68.

65 . The method of claim 61 , further comprising detecting in the subject's sample one or more citrullinated brain injury biomarker proteins selected from the group consisting of citrullinated tubulin beta-4B chain, tubulin alpha-1B chain, CNPase, PPIA, Septin-7, Elongation factorl-alpha2, TPPP, TPPP3, Ermin Isoform 2, NDRG2 Isoform 2, astrotactin 1 (ASTN1), brain angiogenesis inhibitor 3 (BAI3), carnosine dipeptidase 1 (CNDP1), ERMIN, glutamate receptor metabotropic 3 (GRM3), kelch-like protein 32 (KLH32), melanoma antigen family E,2 (MAGE2), neuregulin 3 (NRG3), oligodendrocyte myelin glycoprotein (OMG), solute carrier family 39 (zinc transporter), reticulon 1 (RTN1), MT3, and peptidylarginine deiminase types 1-4 and 6 (PAD1-4 and PAD6).

66 . The method of claim 61 , wherein the subject's sample is selected from the group consisting of blood, peripheral blood, serum, plasma, cerebrospinal fluid (CSF), urine, saliva, stool and synovial fluid.

67 . The method of claim 61 , wherein the detecting of antibody binding is accomplished using an immunoassay, and wherein the detecting by mass spectrometry is accomplished using multiple reaction monitoring mass spectrometry (MRM-MS).

68 . The method of claim 61 , wherein the subject has or is suspected of having a brain injury or a neurodegenerative disease.

69 . The method of claim 68 , wherein the neurodegenerative disease is selected from hemorrhagic stroke, ischemic stroke, chronic traumatic encephalopathy, Alzheimer's disease, multiple sclerosis, or Parkinson's Disease.

70 . A method for detecting autoantibodies against a citrullinated brain injury biomarker protein in a sample from a subject, the method comprising the steps of:
(a) contacting a biological sample obtained from the subject with citrullinated brain injury biomarker protein glial fibrillary acidic protein (GFAP) and/or citrullinated brain injury biomarker protein myelin basic protein (MBP); and (b) detecting the binding of the citrullinated GFAP and/or citrullinated MBP brain injury biomarker proteins to autoantibodies in the sample which are specific for the citrullinated brain injury biomarker proteins, said detection of binding being indicative of the presence of citrullinated brain injury biomarker protein autoantibodies.

71 . The method of claim 70 , wherein the citrullinated GFAP is citrullinated at one or more arginine (R) amino acid residues selected from R88, R105, R124, R126, R136, R173, R217, R258, R270, R286, R287, R367, and R406, and the citrullinated MBP is citrullinated at one or more arginine (R) amino acid residues selected from R31, R43, R49, R65, R107, R113, R162, and R169.

72 . The method of claim 70 , further comprising contacting the sample with one or more citrullinated brain injury biomarker proteins selected from the group consisting of tubulin beta-4B chain, tubulin alpha-1B chain, CNPase, PPIA, Septin-7, Elongation factorl-alpha2, TPPP, TPPP3, Ermin Isoform 2, NDRG2 Isoform 2, astrotactin 1 (ASTN1), brain angiogenesis inhibitor 3 (BAI3); carnosine dipeptidase 1 (CNDP1); ERMIN; glutamate receptor metabotropic 3 (GRM3); kelch-like protein 32 (KLH32); melanoma antigen family E,2 (MAGE2); neuregulin 3 (NRG3); oligodendrocyte myelin glycoprotein (OMG); solute carrier family 39 (zinc transporter); reticulon 1 (RTN1); MT3, and peptidylarginine deiminase, types 1-4 and 6 (PAD1-4 or PAD6)

73 . The method of claim 70 , wherein the binding is detected by immunosorbent assay, by immunoprecipitation, or by immunoblotting.

74 . The method of claim 70 , further comprising contacting the biological sample obtained from the subject with citrullinated brain injury biomarker protein neurogranin (NRGN).

75 . The method of claim 74 , wherein citrullinated NRGN is citrullinated at one or more arginine (R) amino acid residues selected from R38, R43, R51, R53, or R68.

76 . The method of claim 70 , wherein the biological sample is blood, serum, plasma, cerebrospinal fluid (CSF), urine, saliva, stool and synovial fluid.

77 . A method of detecting efficacy of treatment in a subject who is being treated for brain injury, the method comprising:
a. measuring at a first time point the level of autoantibodies to at least two citrullinated brain injury biomarker proteins selected from the group consisting of neurogranin (cit-NRGN), glial fibrillary acidic protein (cit-GFAP), and/or citrullinated myelin basic protein (cit-MBP), in a sample obtained from the subject prior to initiating brain injury treatment in the subject; b. measuring the levels of autoantibodies to cit-NRGN, cit-GFAP and/or cit-MBP biomarker proteins using a cit-NRGN-specific peptide, a cit-GFAP-specific peptide and/or a cit-MBP-specific peptide that specifically binds the autoantibodies in a sample obtained from the subject at one or more time points after initiating brain injury treatment in the subject; and c. detecting that the subject's brain injury treatment is efficacious if the measured levels of autoantibodies that bind to the cit-NRGN, cit-GFAP and/or cit-MBP peptide in step (b) are decreased relative to the levels of autoantibodies to cit-NRGN, cit-GFAP and/or cit-MBP measured at the first time point in step (a).

78 . The method of claim 77 , wherein the cit-NRGN is citrullinated at one or more arginine (R) amino acid residues selected from R38, R43, R51, R53, or R68; the cit-GFAP is citrullinated at one or more arginine (R) amino acid residues selected from R88, R105, R124, R126, R136, R173, R217, R258, R270, R286, R287, R367, and R406; and the cit-MBP is citrullinated at one or more arginine (R) amino acid residues selected from R31, R43, R49, R65, R107, R113, R162, and R169.

79 . The method of claim 77 , further comprising contacting the sample with one or more citrullinated brain injury biomarker proteins selected from the group consisting of tubulin beta-4B chain, tubulin alpha-1B chain, CNPase, PPIA, Septin-7, Elongation factorl-alpha2, TPPP, TPPP3, Ermin Isoform 2, NDRG2 Isoform 2, astrotactin 1 (ASTN1), brain angiogenesis inhibitor 3 (BAI3); carnosine dipeptidase 1 (CNDP1); ERMIN; glutamate receptor metabotropic 3 (GRM3); kelch-like protein 32 (KLH32);

Assignees

• Univ Johns Hopkins

Inventors

• Van Eyk Jennifer E
• Everett Allen Dale
• Jin Zhicheng

Classifications

• G01N2800/28

  Neurological disorders · CPC title

• G01N33/5306

  Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding · CPC title

• G01N2440/18

  citrullination · CPC title

• G01N33/6848 Primary

  Methods of protein analysis involving mass spectrometry · CPC title

• G01N33/6842 Primary

  Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins · CPC title