Methods of determining susceptibility to or presence of schizophrenia, or a disorder related thereto

Abstract

Variations in the DNA sequence of the human proline dehydrogenase gene (PRODH) which correlate to an increased susceptibility to, or presence of schizophrenia or a disease or disorder related thereto, such as obsessive compulsive disorder (OCD), bipolar disorder (BP), or major depressive disorder (MDD) are provided, along with assays to diagnosing schizophrenia or a disease or disorder related thereto, and evaluating potential drugs or agents for using in treating such diseases or disorders.

What is claimed is:  
     
         1 . An isolated variant allele of a human proline dehydrogenase (PRODH) gene, wherein said PRODH gene comprises a DNA sequence of SEQ ID NO:1, and said variant allele comprises a DNA sequence having at least one variation in SEQ ID NO:1 wherein the at least one variation comprises: 
 a G to A transition in the third position of codon 83;    a C to T transition in the first position of codon 101;    a G to A transition in the second position of codon 101;    a C to T transition in the first position of codon 247;    a C to T transition in the third position of codon 342;    a C to T transition in the third position of codon 421;    an A to G transition in the second position of codon 437;    a T to C transition in the first position of codon 497; or    a combination thereof.    
     
     
         2 . An isolated nucleic acid molecule hybridizable to an isolated variant allele of a human PRODH gene of  claim 1  under standard hybridization conditions.  
     
     
         3 . The isolated variant allele of  claim 1 , detectably labeled.  
     
     
         4 . The isolated nucleic acid molecule of  claim 2 , detectably labeled.  
     
     
         5 . The isolated variant allele of  claim 3  wherein said detectable label comprises a radioactive element, a chemical which fluoresces, or an enzyme.  
     
     
         6 . The isolated nucleic acid molecule of  claim 4 , wherein said detectable label comprises a radioactive element, a chemical which fluoresces, or an enzyme.  
     
     
         7 . An isolated variant allele of a human PRODH gene, which encodes a variant proline dehydrogenase comprising an amino acid sequence comprising at least one variation in SEQ ID NO:2, wherein said at least one variation comprises: 
 Arg101Trp;    Arg101Glu;    Glu437Arg; or    a combination thereof.    
     
     
         8 . An isolated variant human proline dehydrogenase comprising an amino acid sequence comprising at least one variation in SEQ ID NO:2, wherein said at least one variation comprises: 
 Arg101Trp;    Arg101Glu;    Glu437Arg; or    a combination thereof.    
     
     
         9 . An antibody having the variant proline dehydrogenase of  claim 8  as an immunogen.  
     
     
         10 . The antibody of  claim 9 , which is a polyclonal antibody.  
     
     
         11 . The antibody of  claim 9 , which is a monoclonal antibody.  
     
     
         12 . The antibody of  claim 9 , which is a chimeric antibody.  
     
     
         13 . The antibody of  claim 9 , detectably labeled.  
     
     
         14 . The antibody of  claim 13 , wherein said detectable label comprises a radioactive element, a chemical which fluoresces, or an enzyme.  
     
     
         15 . A cloning vector comprising an origin of replication and an isolated variant allele of a human PRODH gene, wherein said human PRODH gene comprises a DNA sequence of SEQ ID NO:1, and said variant allele comprises a DNA sequence having at least one variation in SEQ ID NO:1, wherein said at least one variation comprises: 
 a G to A transition in the third position of codon 83;    a C to T transition in the first position of codon 101;    a G to A transition in the second position of codon 101;    a C to T transition in the first position of codon 247;    a C to T transition in the third position of codon 342;    a C to T transition in the third position of codon 421;    an A to G transition in the second position of codon 437;    a T to C transition in the first position of codon 497; or    a combination thereof.    
     
     
         16 . A cloning vector comprising an origin of replication and an isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated variant allele of a PRODH gene, wherein said PRODH gene comprises a DNA sequence of SEQ ID NO:1, and said isolated variant allele comprises a DNA sequence having at least one variation in SEQ ID NO:1, wherein said at least one variation comprises: 
 a G to A transition in the third position of codon 83;    a C to T transition in the first position of codon 101;    a G to A transition in the second position of codon 101;    a C to T transition in the first position of codon 247;    a C to T transition in the third position of codon 342;    a C to T transition in the third position of codon 421;    an A to G transition in the second position of codon 437;    a T to C transition in the first position of codon 497; or    a combination thereof.    
     
     
         17 . The cloning vector of either of  claim 15  or  16 , wherein said cloning vector is selected from the group consisting of  E. coli , bacteriophages, plasmids, and pUC plasmid derivatives.  
     
     
         18 . The cloning vector of  claim 17 , wherein bacteriophages further comprise lambda derivatives, plasmids further comprise pBR322 derivatives, and pUC plasmid derivatives further comprise pGEX vectors, or pmal-c, pFLAG.  
     
     
         19 . An expression vector comprising an isolated variant allele of a human PRODH gene operatively associated with a promoter, wherein said human PRODH gene comprises a DNA sequence of SEQ ID NO:1, and said isolated variant allele comprises a DNA sequence comprising at least one variation in SEQ ID NO:1, wherein said at least one variation and a variant allele of the present invention comprises a DNA sequence having at least one variation in SEQ ID NO:1 wherein the at least one variation comprises: 
 a G to A transition in the third position of codon 83;    a C to T transition in the first position of codon 101;    a G to A transition in the second position of codon 101;    a C to T transition in the first position of codon 247;    a C to T transition in the third position of codon 342;    a C to T transition in the third position of codon 421;    an A to G transition in the second position of codon 437;    a T to C transition in the first position of codon 497; or    a combination thereof.    
     
     
         20 . An expression vector comprising an isolated nucleic acid molecule operatively associated with a promoter, wherein the isolated nucleic acid molecule is hybridizable under standard hybridization conditions to an isolated variant allele of a human PRODH gene, wherein said human PRODH comprises a DNA sequence of SEQ ID NO:1, and said variant allele comprises a DNA sequence having at least one variation in SEQ ID NO:1, wherein said at least one variation comprises: 
 a G to A transition in the third position of codon 83;    a C to T transition in the first position of codon 101;    a G to A transition in the second position of codon 101;    a C to T transition in the first position of codon 247;    a C to T transition in the third position of codon 342;    a C to T transition in the third position of codon 421;    an A to G transition in the second position of codon 437;    a T to C transition in the first position of codon 497; or    a combination thereof.    
     
     
         21 . The expression vector of either of  claim 19  or  20 , wherein said promoter is selected from the group consisting of the immediate early promoters of hCMV, early promoters of SV40, early promoters of adenovirus, early promoters of vaccinia, early promoters of polyoma, late promoters of SV40, late promoters of adenovirus, late promoters of vaccinia, late promoters of polyoma, the lac the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage lambda, control regions of fd coat protein, 3-phosphoglycerate kinase promoter, acid phosphatase promoter, and promoters of yeast α mating factor.  
     
     
         22 . A unicellular host transformed or transfected with an expression vector comprising an isolated variant allele of a human PRODH gene operatively associated with a promoter, wherein a human PRODH gene comprises a DNA sequence of SEQ ID NO:1, and said variant allele comprises a DNA sequence having at least one variation in SEQ ID NO:1, wherein said at least one variation comprises: 
 a G to A transition in the third position of codon 83;    a C to T transition in the first position of codon 101;    a G to A transition in the second position of codon 101;    a C to T transition in the first position of codon 247;    a C to T transition in the third position of codon 342;    a C to T transition in the third position of codon 421;    an A to G transition in the second position of codon 437;    a T to C transition in the first position of codon 497; or    a combination thereof.    
     
     
         23 . A unicellular host transformed with an expression vector comprising an isolated nucleic acid molecule operatively associated with a promoter, wherein the isolated nucleic acid molecule is hybridizable under standard hybridization conditions to an isolated variant allele of a human PRODH gene, wherein said human PRODH gene comprises a DNA sequence of SEQ ID NO:1, and said variant allele comprises a DNA sequence having at least one variation in SEQ ID NO:1, wherein said at least one variation comprises: 
 a G to A transition in the third position of codon 83;    a C to T transition in the first position of codon 101;    a G to A transition in the second position of codon 101;    a C to T transition in the first position of codon 247;    a C to T transition in the third position of codon 342;    a C to T transition in the third position of codon 421;    an A to G transition in the second position of codon 437;    a T to C transition in the first position of codon 497; or    a combination thereof.    
     
     
         24 . The unicellular host of either of  claim 22  or  23 , wherein said host is selected from the group consisting of  E. coli , Pseudonomas, Bacillus, Strepomyces, yeast, CHO, R1.1, B-W, L-M, COS1, COS7, BSC1, BSC40, BMT10 and Sf9 cells.  
     
     
         25 . A method of producing an a variant human PRODH protein comprising an amino acid sequence comprising at least one variation in SEQ ID NO:1, wherein said at least one variation comprises: 
 Arg101Trp;    Arg101Glu;    Glu437Arg; or    a combination thereof,    wherein said method comprising the steps of:    (a) culturing a unicellular host of either of  claim 22  or  23  under conditions that provide for expression of said variant human PRODH protein; and    (b) recovering said variant human PRODH protein from said unicellular host, said culture, or both.    
     
     
         26 . A method for detecting a susceptibility to, or the presence of, schizophrenia or a disease or disorder related thereto, wherein the method comprises measurement of the levels of activity of an enzyme in a bodily sample, wherein said enzyme is involved in proline catabolism, and comparison of said levels to a standard, whereby modulated levels indicate the susceptibility to, or the presence of, schizophrenia or a disease or disorder related thereto.  
     
     
         27 . The method for detecting a susceptibility of  claim 26 , wherein a disease or disorder related to schizophrenia comprises obsessive compulsive disorder (OCD), bipolar disorder (BP) and major depressive disorder (MDD).  
     
     
         28 . The method for detecting a susceptibility as set forth in  claim 26 , wherein said enzyme is proline dehydrogenase (PRODH) comprising an amino acid sequence of SEQ ID NO:2, and reduced levels of PRODH in a bodily sample as compared to a standard indicates the susceptibility to, or the presence of schizophrenia or a disease or disorder related thereto.  
     
     
         29 . A method for determining a susceptibility in a subject to schizophrenia, or a disease or disorder related thereto, wherein the method comprises the steps of: 
 (a) removing a bodily sample from the subject, wherein the sample comprises a PRODH gene;    (b) determining whether the PRODH gene of the bodily sample comprises a DNA sequence comprising a variation in SEQ ID NO:1 comprising a T to C transition in the first position of codon 497, such that the presence of said at least one variation in said PRODH gene is indicative of the subject's susceptibility to schizophrenia or a disease or disorder related thereto, relative to the susecptibility of a standard.    
     
     
         30 . The method of  29 , wherein where the variation of PRODH gene is indicative of an increased susceptibility of the subject to schizophrenia or a disease or disorder related thereto relative to the susceptibility of a standard.  
     
     
         31 . An assay system for screening drugs and other agents for ability to treat schizophrenia or a disease or disorder related thereto, wherein the assay system comprises: 
 (a) culturing an observable cellular test colony inoculated with a drug or agent;    (b) harvesting a cellular extract from said cellular test colony; and    (c) examining said extract for the presence of PRODH; 
 wherein an increase or a decrease in a level of activity of said PRODH in said test colony compared to level of activity of PRODH in the control indicates the ability of the drug to modulate the production, stability, degradation or activity of said PRODH.  
   
     
     
         32 . The assay for screening drugs and other agents of  claim 31 , wherein an increase in the level or activity of said PRODH in said test colony compared to a control is indicative of the potential of the drug to treat schizophrenia or a disease or disorder related thereto.  
     
     
         33 . A test kit to facilitate diagnosis and treatment of schizophrenia or a disease or disorder related thereto, comprising: 
 (a) a predetermined amount of a detectably labeled specific binding partner of a PRODH;    (b) other reagents; and    (c) directions for use of said kit.    
     
     
         34 . The test kit of  claim 33 , wherein said labeled immunochemically reactive component is selected from the group consisting of polyclonal antibodies to the PRODH, monoclonal antibodies to the PRODH, fragments thereof, and mixtures thereof.  
     
     
         35 . A test kit to facilitate diagnosis and treatment of schizophrenia or a disease or disorder related thereto in a eukaryotic cellular sample, comprising: 
 (a) PCR oligonucleotide primers suitable PRODH detection;    (b) other reagents; and    (c) directions for use of said kit.    
     
     
         36 . A test kit to facilitate diagnosis and treatment of schizophrenia or a disease or disorder related thereto in a subject, wherein the test kit comprises: 
 (a) PCR oligonucleotide primers suitable for detection of an isolated variant allele of a PRODH gene in a bodily sample of the subject comprising a DNA sequence having a variation in SEQ ID NO:1 comprising a T to C transition in the first position of codon 497;    (b) other reagents; and    (c) directions for using said kit.    
     
     
         37 . A method of treating schizophrenia or a disease or disorder related thereto, comprising administering to a mammal a therapeutically effective amount of a PRODH comprising an amino acid sequence of SEQ ID NO:2, a conservative variant thereof, a fragment thereof, or an analog or derivative thereof.  
     
     
         38 . The method of  claim 37  wherein said PRODH comprising an amino acid sequence of SEQ ID NO:2 is administered to modulate the course of therapy where said PRODH is being co-administered with one or more additional therapeutic agents.  
     
     
         39 . A method of determining the schizophrenic-related pharmacological activity of a compound comprising: 
 administering the compound to a mammal;    determining the level of activity of PRODH comprising an amino acid sequence of SEQ ID NO:2 in the mammal; and    comparing the level of activity of PRODH in the mammal to the level of activity of PRODH in a standard to which the compound was not administered,    wherein an increase in the level of activity of PRODH in the mammal relative the level of activity of PRODH in the standard is indicative of the ability of the compound to treat schizophrenia or a disease or disorder related thereto.    
     
     
         40 . An isolated nucleic acid molecule comprising the DNA sequence of SEQ ID NO:1, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof.  
     
     
         41 . The isolated nucleic acid molecule of  claim 40  detectably labeled.  
     
     
         42 . The isolated nucleic acid molecule of  claim 41 , wherein the detectable label comprises a radioactive element, a chemical which fluoresces, or an enzyme.  
     
     
         43 . An isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:1, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof.  
     
     
         44 . The isolated nucleic acid molecule of  claim 43 , detectably labeled.  
     
     
         45 . The isolated nucleic acid molecule of  claim 49 , wherein the detectable label comprises a radioactive element, a chemical which fluoresces, or an enzyme.  
     
     
         46 . An isolated nucleic acid molecule which encodes human proline dehydrogenase protein, wherein the protein comprises an amino acid sequence of SEQ ID NO:2.  
     
     
         47 . The isolated nucleic acid molecule of  claim 46 , comprising a DNA sequence of SEQ ID NO:1.  
     
     
         48 . An isolated protein comprising an amino acid sequence of SEQ ID NO:2, conservative variants thereof, fragments thereof, or analogs or derivatives thereof.  
     
     
         49 . An isolated nucleic acid molecule comprising the DNA sequence of SEQ ID NO:3, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof.  
     
     
         50 . The isolated nucleic acid molecule of  claim 49  detectably labeled.  
     
     
         51 . The isolated nucleic acid molecule of  claim 50 , wherein the detectable label comprises a radioactive element, a chemical which fluoresces, or an enzyme.  
     
     
         52 . An isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:3, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof.  
     
     
         53 . The isolated nucleic acid molecule of  claim 52 , detectably labeled.  
     
     
         54 . The isolated nucleic acid molecule of  claim 53 , wherein the detectable label comprises a radioactive element, a chemical which fluoresces, or an enzyme.  
     
     
         55 . An isolated nucleic acid molecule which encodes human proline dehydrogenase protein, wherein the protein comprises an amino acid sequence of SEQ ID NO:4.  
     
     
         56 . The isolated nucleic acid molecule of  claim 55 , comprising a DNA sequence of SEQ ID NO:3.  
     
     
         57 . An isolated protein comprising an amino acid sequence of SEQ ID NO:4, conservative variants thereof, fragments thereof, or analogs or derivatives thereof.  
     
     
         58 . A method of identifying drugs or agents useful in treating schizophrenia or a disease, comprising the steps of: 
 performing an first pre-pulse inhibition test (PPI) test on an F3 generation mouse from a cross Pro/Re X C57B1/6J wild-type, wherein the F3 generation mouse has two copies within its genome of an isolated variant allele of a Prodh gene comprising a DNA sequence of SEQ ID NO:7 which are capable of expressing a mutant Prodh comprising an amino acid sequence of SEQ ID NO:8, to obtain a first percentage of inhibition of startle response;    administering the potential drug or agent to the F3 generation mouse from a cross of Pro/Re X C57B1/6J wild-type;    performing a second PPI test on the F3 generation mouse from a cross of Pro/Re X C57B1/6J wild-type to obtain a second percentage of inhibition of startle response; and    comparing the first percentage to the inhibition of startle response with the second percentage of startle response,    wherein an increase in percentage of inhibition in the second percentage of inhibition relative to the first percentage of inhibition is indicative of the ability of the drug or agent to treat schizophrenia or a disease or disorder related thereto.    
     
     
         59 . A method of identifying drugs or agents useful in treating schizophrenia or a disease, comprising the steps of: 
 a) administering the drug or agent to an F3 generation mouse from a cross of Pro/Re X C57B1/6J wild-type, wherein the F3 generation mouse has two copies within its genome of an isolated variant allele of a Prodh gene comprising a DNA sequence of SEQ ID NO:7 which are capable of expressing a mutant Prodh comprising an amino acid sequence of SEQ ID NO:8;    b) performing a PPI test on the F3 generation mouse from a cross of Pro/Re X C57B1/6J wild-type administered the drug or agent to obtain a percentage of inhibition of the startle response in the mouse; and    c) comparing the percentage of inhibition of the startle response in F3 generation mouse from a cross of Pro/Re X C57B1/6J wild-type administered the drug with the percentage of inhibition of the startle response in an F3 generation mouse from a cross of Pro/Re X C57B1/6J wild-type, wherein the F3 generation mouse has two copies within its genome of an isolated Prodh gene comprising a DNA sequence of SEQ ID NO:3 which are capable of expressing a Prodh comprising an amino acid sequence of SEQ ID NO:4,    such that the percentage of inhibition of the startle response in the medicated mouse is statistically equivalent to the percentage of inhibition in the mouse capable of expressing Prodh comprising a DNA sequence of SEQ ID NO:4, then the drug or agent has the ability to treat schizophrenia or a disease or disorder related thereto.    
     
     
         60 . A method for identifying a drug or agent for use in treating schizophrenia or a disease or disorder related thereto, comprising the steps of: 
 a) administering the drug or agent to an F3 generation mouse from a cross of Pro/Re X C57B1/6J wild-type, wherein the F3 generation mouse has two copies within its genome of an isolated variant allele of a Prodh gene comprising a DNA sequence of SEQ ID NO:7 which are capable of expressing a mutant Prodh comprising an amino acid sequence of SEQ ID NO:8;    b) performing a PPI test on the F3 generation mouse from a cross of Pro/Re X C57B1/6J wild-type to obtain a percentage of inhibition of the startle response in the F3 generation mouse from a cross of Pro/Re X C57B1/6J wild-type which was administered the drug or agent; and    c) comparing the percentage of inhibition of the startle response in the F3 generation mouse from a cross of Pro/Re X C57B1/6J wild-type with the percentage of inhibition of the startle response in an unmedicated F3 generation mouse from a cross of Pro/Re X C57B1/6J wild-type, wherein the F3 generation mouse has two copies within its genome of an isolated variant allele of a Prodh gene comprising a DNA sequence of SEQ ID NO:7 which are capable of expressing a mutant Prodh comprising an amino acid sequence of SEQ ID NO:8,    wherein an increase in percentage of inhibition in the percentage of inhibition in the medicated F3 generation mouse relative to the percentage of inhibition in the unmedicated F3 generation mouse is indicative of the ability of the drug or agent to treat schizophrenia or a disease or disorder related thereto.