Biomarkers for diagnosing schizophrenia and bipolar disorder

Abstract

The invention relates to the identification and selection of novel biomarkers and the identification and selection of novel biomarker combinations which are differentially expressed in blood and useful in diagnosing schizophrenia and/or bipolar disorder as well as monitoring therapeutic efficacy of treatment for schizophrenia or bipolar disorder. The measurement of expression levels of the products of the biomarkers and combinations of biomarkers of the invention can be used to diagnose schizophrenia and/or bipolar disorder. Measurement of the expression level of products of biomarkers of the invention using polynucleotides and proteins which specifically and/or selectively hybridize to the products of the biomarkers of the invention are also encompassed within the scope of the invention as are compositions and kits containing said polynucleotides and proteins. Further encompassed by the invention is the use of the polynucleotides and proteins to monitor the efficacy of therapeutic regimens. The invention also provides for the identification of methods of using the products of the biomarkers of the invention in the identification of novel therapeutic targets of schizophrenia and/or bipolar disorder and a method of screening the genes of said biomarkers for additional markers of disease.

1 . A composition comprising a collection of two or more isolated polynucleotides, said polynucleotides which selectively hybridize to at least two biomarkers of the invention, wherein the biomarkers are selected from the group consisting of the genes: adenylosuccinate synthase (ADSS); apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3B (APOBEC3B); ataxia telangiectasia mutated (includes complementation groups A, C and D) (ATM); Charcot-Leyden crystal protein (CLC); C-terminal binding protein 1 (CTBP1); chemokine (C—X—C motif) ligand 1 (melanoma growth stimulating activity, alpha) (CXCL1); death associated transcription factor 1 (DATF1); S100 calcium binding protein A9 (calgranulin B) (S100A9), and as set out in Table 1, and wherein the composition is used to measure the level of expression of said biomarker.  
     
     
         2 . A composition comprising a collection of two or more isolated polynucleotides which bind selectively to the RNA products of at least two biomarkers, wherein the biomarkers are selected from the group consisting of the genes: adenylosuccinate synthase (ADSS); apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3B (APOBEC3B); ataxia telangiectasia mutated (includes complementation groups A, C and D) (ATM); Charcot-Leyden crystal protein (CLC); C-terminal binding protein 1 (CTBP1); chemokine (C—X—C motif) ligand 1 (melanoma growth stimulating activity, alpha) (CXCL1); death associated transcription factor 1 (DATF1); S100 calcium binding protein A9 (calgranulin B) (S100A9), as set out in Table 1.  
     
     
         3 . The composition of  claim 2  wherein said polynucleotides are useful in quantitative RT-PCR (QRT-PCR).  
     
     
         4 . A composition comprising a collection of two or more isolated proteins which bind selectively to the protein products of at least two biomarkers, wherein the biomarkers are selected from the group consisting of the genes: adenylosuccinate synthase (ADSS); apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3B (APOBEC3B); ataxia telangiectasia mutated (includes complementation groups A, C and D) (ATM); Charcot-Leyden crystal protein (CLC); C-terminal binding protein 1 (CTBP1); chemokine (C—X—C motif) ligand 1 (melanoma growth stimulating activity, alpha) (CXCL1); death associated transcription factor 1 (DATF1); S100 calcium binding protein A9 (calgranulin B) (S100A9), as set out in Table 1  
     
     
         5 . A composition comprising a collection of two or more isolated polynucleotides which bind selectively to the RNA products of one or more biomarkers, wherein the biomarkers are selected from the group consisting of the genes: adenylosuccinate synthase (ADSS); death associated transcription factor 1 (DATF1); as listed in Table 3.  
     
     
         6 . A composition comprising a collection of one or more isolated proteins, which bind selectively to the protein products of one or more biomarkers, wherein the biomarkers are selected from the group consisting of the genes: adenylosuccinate synthase (ADSS); death associated transcription factor 1 (DATF1); as set out in Table 3.  
     
     
         7 . A composition comprising a collection of isolated polynucleotides which bind selectively to the RNA products of biomarkers, wherein the biomarkers are selected from the group of genes: adenylosuccinate synthase (ADSS); death associated transcription factor 1 (DATF1); as set out in Table 3.  
     
     
         8 . A composition comprising a collection of two or more isolated polynucleotides which bind selectively to the RNA products of at least one biomarker, wherein the biomarkers are selected from the group as set out in Table 4.  
     
     
         9 . A composition comprising a collection of two or more isolated polynucleotides which bind selectively to the RNA products of at least one biomarker, wherein the biomarkers are selected from the group as set out in Table 5.  
     
     
         10 . A composition comprising a collection of one or more isolated proteins which bind selectively to the protein products of at least one biomarker, wherein the biomarkers are selected from the group as set out in Table 4.  
     
     
         11 . A composition comprising a collection of one or more isolated proteins which bind selectively to the protein products of at least one biomarker, wherein the biomarkers are selected from the group as set out in Table 5.  
     
     
         12 . The composition of any one of claims  4 ,  6 ,  10  or  11 , wherein the isolated proteins are ligands.  
     
     
         13 . The composition of any one of claims  4 ,  6 ,  10  or  11 , wherein the ligands are antibodies.  
     
     
         14 . The composition of  claim 13 , wherein the antibodies are monoclonal antibodies.  
     
     
         15 . The composition of any one of claims  1 ,  2 ,  3 ,  5 ,  7 ,  8  or  9 , wherein the isolated oligonucleotides are single or double stranded RNA.  
     
     
         16 . The composition of any one of claims  1 ,  2 ,  3 ,  5 ,  7 ,  8  or  9  wherein the isolated polynucleotides are single or double stranded DNA.  
     
     
         17 . A method of diagnosing or prognosing schizophrenia in an individual, comprising the steps of: 
 a) determining the level of one or more RNA transcripts expressed in blood obtained from said individual, wherein said one or more RNA transcripts corresponds to said one or more biomarkers of Table 3, and    b) comparing the level of each of said one or more RNA transcripts in said blood according to step a) with the level of each of said one or more RNA transcripts in blood from one or more individuals not having schizophrenia,    wherein detecting differential expression of each of said one or more RNA transcripts in the comparison of step b) is indicative of schizophrenia in the individual of step a).    
     
     
         18 . A method of diagnosing or prognosing bipolar disorder in an individual, comprising the steps of: 
 a) determining the level of one or more RNA transcripts expressed in blood obtained from said individual, wherein said one or more RNA transcripts corresponds to said one or more biomarkers of Table 4, and    b) comparing the level of each of said one or more RNA transcripts in said blood according to step a) with the level of each of said one or more RNA transcripts in blood from one or more individuals not having bipolar disorder,    wherein detecting differential expression of each of said one or more RNA transcripts in the comparison of step b) is indicative of bipolar disorder in the individual of step a).    
     
     
         19 . A method of diagnosing or prognosing and individual as having either bipolar disorder or schizophrenia, comprising the steps of: 
 a) determining the level of one or more RNA transcripts expressed in blood obtained from said individual, wherein said one or more RNA transcripts corresponds to said one or more biomarkers of Table 5, and    b) comparing the level of each of said one or more RNA transcripts in said blood according to step a) with the level of each of said one or more RNA transcripts in blood from one or more individuals having schizophrenia,    wherein detecting differential expression of each of said one or more RNA transcripts in the comparison of step b) is indicative of bipolar disorder in the individual of step a).    
     
     
         20 . A method of diagnosing or prognosing and individual as having either bipolar disorder or schizophrenia, comprising the steps of: 
 a) determining the level of one or more RNA transcripts expressed in blood obtained from said individual, wherein said one or more RNA transcripts corresponds to said one or more biomarkers of Table 5, and    b) comparing the level of each of said one or more RNA transcripts in said blood according to step a) with the level of each of said one or more RNA transcripts in blood from one or more individuals having bipolar disorder,    wherein detecting differential expression of each of said one or more RNA transcripts in the comparison of step b) is indicative of schizophrenia in the individual of step a).    
     
     
         21 . A method of diagnosing or prognosing schizophrenia in an individual, comprising the steps of: 
 a) determining the level of two or more RNA transcripts expressed in blood obtained from said individual, wherein said two or more RNA transcripts corresponds to said one or more biomarkers of Table 1 and    b) comparing the level of each of said two or more RNA transcripts in said blood according to step a) with the level of each of said two or more RNA transcripts in blood from one or more individuals having schizophrenia,    c) comparing the level of each of said two or more RNA transcripts in said blood according to step a) with the level of each of said two or more RNA transcripts in blood from one or more individuals not having schizophrenia,    d) determining whether the level of said two or more RNA transcripts of step a) classify with the levels of said transcripts in step b) as compared with levels of said transcripts in step c),    wherein said determination is indicative of said individual of step a) having schizophrenia.    
     
     
         22 . A method of diagnosing or prognosing bipolar disorder in an individual, comprising the steps of: 
 a) determining the level of two or more RNA transcripts expressed in blood obtained from said individual, wherein said two or more RNA transcripts corresponds to said two or more biomarkers of Table 1 and    b) comparing the level of each of said two or more RNA transcripts in said blood according to step a) with the level of each of said two or more RNA transcripts in blood from one or more individuals having bipolar disorder,    c) comparing the level of each of said two or more RNA transcripts in said blood according to step a) with the level of each of said two or more RNA transcripts in blood from one or more individuals not having bipolar disorder,    d) determining whether the level of said two or more RNA transcripts of step a) classify with the levels of said transcripts in step b) as compared with levels of said transcripts in step c),    wherein said determination is indicative of said individual of step a) having bipolar disorder.    
     
     
         23 . A method of diagnosing or prognosing an individual as having bipolar disorder or schizophrenia, comprising the steps of: 
 a) determining the level of two or more RNA transcripts expressed in blood obtained from said individual, wherein said two or more RNA transcripts corresponds to said two or more biomarkers of Table 1 and    b) comparing the level of each of said two or more RNA transcripts in said blood according to step a) with the level of each of said two or more RNA transcripts in blood from one or more individuals having bipolar disorder,    c) comparing the level of each of said two or more RNA transcripts in said blood according to step a) with the level of each of said two or more RNA transcripts in blood from one or more individuals having schizophrenia,    d) determining whether the level of said two or more RNA transcripts of step a) classify with the levels of said transcripts in step b) as compared with levels of said transcripts in step c),    wherein said determination is indicative of said individual of step a) having bipolar disorder.    
     
     
         24 . A method of diagnosing or prognosing schizophrenia in an individual, comprising the steps of: 
 a) determining the level of one or more RNA transcripts expressed in blood obtained from said individual, wherein said one or more RNA transcripts corresponds to said one or more biomarkers of Table 1 and    b) using the results from step (a) in combination with a classifier so as to determine a diagnosis with respect to schizophrenia.    
     
     
         25 . A method of diagnosing or prognosing bipolar disorder in an individual, comprising the steps of: 
 a) determining the level of one or more RNA transcripts expressed in blood obtained from said individual, wherein said one or more RNA transcripts corresponds to said one or more biomarkers of Table 1 and    b) using the results from step (a) in combination with a classifier so as to determine a diagnosis with respect to bipolar disorder.    
     
     
         26 . The method of any one of claims  17 - 25 , wherein said blood sample consists of whole blood.  
     
     
         27 . The method of any one of claims  17 - 25 , wherein said blood sample consists of a drop of blood.  
     
     
         28 . The method of any one of claims  17 - 25 , wherein said blood sample consists of blood that has been lysed.  
     
     
         29 . The method of any one of claims  17 - 25 , further comprising the step of isolating RNA from said blood samples.  
     
     
         30 . The method of any one of claims  17 - 25 , wherein the step of determining the level of each of said one or more RNA transcripts comprises quantitative RT-PCR (QRT-PCR), wherein said one or more transcripts are from step a) and/or step b) of claims  17 - 25 .  
     
     
         31 . The method of  claim 30 , wherein said QRT-PCR utilizes primers which hybridize to said one or more transcripts or the complement thereof, wherein said one or more transcripts are from step a) and/or step b) of claims  17 - 25 .  
     
     
         32 . The method of  claim 31 , wherein said primers are 15-25 nucleotides in length.  
     
     
         33 . The method of any one of claims  17 - 25 , wherein the step of determining the level of each of said one or more RNA transcripts comprises hybridizing a first plurality of isolated nucleic acid molecules that correspond to said one or more transcripts, to an array comprising a second plurality of isolated nucleic acid molecules.  
     
     
         34 . The method of  claim 33 , wherein said first plurality of isolated nucleic acid molecules comprises RNA, DNA, cDNA, PCR products or ESTs.  
     
     
         35 . The method of  claim 33 , wherein said array comprises a plurality of isolated nucleic acid molecules comprising RNA, DNA, cDNA, PCR products or ESTs.  
     
     
         36 . The method of  claim 33 , wherein said second plurality of isolated nucleic acid molecules on said array comprises polynucleotides corresponding to one or more of the biomarkers of Table 1.  
     
     
         37 . A kit for diagnosing or prognosing schizophrenia comprising: 
 a) at least two sets of biomarker specific priming means wherein each set of biomarker specific priming means produces double stranded DNA complementary to a unique biomarker selected from Table 1; wherein each first priming means of said sets contains a sequence which can selectively hybridize to RNA, cDNA or an EST complementary to one of said biomarkers to create an extension product and each said second priming means of said sets is capable of selectively hybridizing to said extension product;    b) an enzyme with reverse transcriptase activity;    c) an enzyme with thermostable DNA polymerase activity, and    d) a labeling means;    wherein each of said primer sets is used to detect the quantitative expression levels of said biomarker in a test subject.    
     
     
         38 . A kit for diagnosing or prognosing bipolar disorder comprising: 
 a) at least two sets of biomarker specific priming means wherein each set of biomarker specific priming means produces double stranded DNA complementary to a unique biomarker selected from Table 1; wherein each first priming means of said sets contains a sequence which can selectively hybridize to RNA, cDNA or an EST complementary to one of said biomarkers to create an extension product and each said second priming means of said sets is capable of selectively hybridizing to said extension product;    b) an enzyme with reverse transcriptase activity;    c) an enzyme with thermostable DNA polymerase activity, and    d) a labeling means;    wherein each said primer set is used to detect the quantitative expression levels of a biomarker in a test subject.    
     
     
         39 . A method of diagnosing or prognosing schizophrenia in an individual, comprising the steps of: 
 a) determining the level of two or more proteins expressed in blood obtained from said individual, wherein said two or more proteins are encoded by two or more biomarkers of Table 1, and    b) comparing the level of each of said two or more proteins in said blood according to step a) with the level of each of said two or more proteins in blood from one or more individuals having schizophrenia,    c) comparing the level of each of said two or more proteins in said blood according to step a) with the level of each of said two or more proteins in blood from one or more individuals not having schizophrenia,    d) determining whether the level of said two or more proteins of step a) classify with the levels of said proteins in step b) as compared with levels of said proteins in step c),    wherein said determination is indicative of said individual of step a) having schizophrenia.    
     
     
         40 . A method of diagnosing or prognosing bipolar disorder in an individual, comprising the steps of: 
 a) determining the level of two or more proteins expressed in blood obtained from said individual, wherein said two or more proteins are encoded by two or more biomarkers of Table 1, and    b) comparing the level of each of said two or more proteins in said blood according to step a) with the level of each of said two or more proteins in blood from one or more individuals having bipolar disorder    c) comparing the level of each of said two or more proteins in said blood according to step a) with the level of each of said two or more proteins in blood from one or more individuals not having bipolar disorder,    d) determining whether the level of said two or more proteins of step a) classify with the levels of said proteins in step b) as compared with levels of said proteins in step c),    wherein said determination is indicative of said individual of step a) having bipolar disorder.    
     
     
         41 . A method of diagnosing or prognosing an individual with bipolar disorder as compared with schizophrenia, comprising the steps of: 
 a) determining the level of two or more proteins expressed in blood obtained from said individual, wherein said two or more proteins are encoded by two or more biomarkers of Table 1, and    b) comparing the level of each of said two or more proteins in said blood according to step a) with the level of each of said two or more proteins in blood from one or more individuals having bipolar disorder    c) comparing the level of each of said two or more proteins in said blood according to step a) with the level of each of said two or more proteins in blood from one or more individuals having schizophrenia,    d) determining whether the level of said two or more proteins of step a) classify with the levels of said proteins in step b) as compared with levels of said proteins in step c),    wherein said determination is indicative of said individual of step a) having bipolar disorder.    
     
     
         42 . A method of diagnosing or prognosing schizophrenia in an individual, comprising the steps of: 
 a) determining the level of two or more protein products expressed in blood obtained from said individual, wherein said two or more protein products corresponds to two or more biomarkers of Table 1 and    b) using the results from step (a) in combination with a classifier designed to differentiate schizophrenia from non schizophrenia so as to determine a diagnosis with respect to schizophrenia.    
     
     
         43 . A method of diagnosing or prognosing bipolar disorder in an individual, comprising the steps of: 
 a) determining the level of two or more protein products expressed in blood obtained from said individual, wherein said two or more protein products corresponds to two or more biomarkers of Table 1 and    b) using the results from step (a) in combination with a classifier designed to differentiate bipolar disorder from non bipolar disorder so as to determine a diagnosis with respect to bipolar disorder.    
     
     
         44 . A method of diagnosing or prognosing and individual as having bipolar disorder or schizophrenial, comprising the steps of: 
 a) determining the level of two or more protein products expressed in blood obtained from said individual, wherein said two or more protein products corresponds to two or more biomarkers of Table 1 and    b) using the results from step (a) in combination with a classifier designed to differentiate bipolar disorder from schizophrenia so as to determine a diagnosis with respect to bipolar disorder or schizophrenia.    
     
     
         45 . The method of any one of claims  39 - 44 , wherein the step of determining the level of each of said one or more proteins comprises the use of two or more antibodies, wherein each of said two or more antibodies is specific for a protein product of a biomarker listed in Table 1.  
     
     
         46 . The method of  claim 45 , wherein said one or more antibodies is selected from the group consisting of a monoclonal antibody, fv. scfv, dab, fd, fab, and fab′2.  
     
     
         47 . A method of developing a classifier useful for diagnosing schizophrenia, said method comprising: 
 (a) measuring the level of expression of the products of the biomarkers identified in Table 1 in a training population wherein said training population is comprised of two subgroups, a first subgroup diagnosed as having schizophrenia and said second subgroup diagnosed as not having schizophrenia.    (b) apply one or more mathematical models to the levels of expression of step (a) to develop one or more classifiers which differentiate between said first subgroup and said second subgroup.    
     
     
         48 . A method of developing a classifier useful for diagnosing bipolar disorder, said method comprising: 
 (a) measuring the level of expression of the products of the biomarkers identified in Table 1 in a training population wherein said training population is comprised of two subgroups, a first subgroup diagnosed as having bipolar disorder and said second subgroup diagnosed as not having bipolar disorder.    (b) apply one or more mathematical models to the levels of expression of step (a) to develop one or more classifiers which differentiate between said first subgroup and said second subgroup.    
     
     
         49 . A method of developing a classifier useful for diagnosing bipolar disorder or schizophrenia, said method comprising: 
 (a) measuring the level of expression of the products of the biomarkers identified in Table 1 in a training population wherein said training population is comprised of two subgroups, a first subgroup diagnosed as having bipolar disorder and said second subgroup diagnosed as having schizophrenia.    (b) apply one or more mathematical models to the levels of expression of step (a) to develop one or more classifiers which differentiate between said first subgroup and said second subgroup.    
     
     
         50 . The method of any one of claims  47 - 49 , further comprising the step of evaluating one or more of said classifiers of step (b) for the classifier's ability to properly characterize each individual of the training population.  
     
     
         51 . The method of any one of claims  47 - 49 , further comprising the step of evaluating one or more of said classifiers of step (b) for the classifier's ability to properly characterize one or more individuals of a population which is not the training population.