Patent Abstract:
A method of diagnosing, assessing susceptibility, and/or treating schizophrenia involving the observation of regulator of G-protein signaling 4 (RGS4) levels in a subject. Embodiments of the present invention include increasing RGS4 expression levels in the cortex, either by chemical means or by genetic complementation (e.g. gene therapy).

Full Description:
This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 60/228,021, filed Aug. 24, 2000. 
    
    
     This invention was made with United States Government support in the form of Grant Nos. MH45156, MH01489, MH56242, MH53459, and MH45156 from the National Institute of Mental Health. The United States Government may have certain rights in the invention. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates generally to the field of neurological and physiological dysfunctions associated with schizophrenia. The invention further relates to the identification, isolation, and cloning of genes which, when mutated or varied, are associated with schizophrenia. The present invention also relates to methods for diagnosing and detecting carriers of the genes and to diagnosis of schizophrenia. The present invention further relates to the construction of animal models of schizophrenia. 
     BACKGROUND OF THE INVENTION 
     Schizophrenia is a serious brain disorder that affects approximately 1% of the human population. The cause of this complex and devastating disease remains elusive, although genetic, nutritional, environmental, and developmental factors have been considered. A combination of clinical, neuroimaging, and postmortem studies have implicated the dorsal prefrontal cortex (PFC) as a prominent site of dysfunction in schizophrenia. 
     Schizophrenia is typically characterized as a disorder of thinking and cognition, as contrasted to other disorders of mental faculties, such as mood, social behavior, and those affecting learning, memory, and intelligence. Schizophrenia is characterized by psychotic episodes during which an individual may lose the ability to test reality or may have hallucinations, delusions, incoherent thinking, and even disordered memory. There are varying forms of schizophrenia differing in severity, from a schizotypal disorder to a catatonic state. A review of schizophrenia can be found in  Principles of Neural Science , 3 rd  ed., 1991, Kandel, Schwartz, and Jessel (Eds.), Connecticut: Appleton &amp; Lange, pp. 853–868; of which Chapter 55 is incorporated herein by reference. 
     Diseases of organ systems, such as those of the heart, lung, and kidney, are usually confirmed by tissue pathology. A demonstrable pathology includes identifying and defining a structural abnormality in the organ, along with an associated alteration in organ function. This type of diagnosis is also utilized in certain neurological diseases. However, there are few psychiatric disorders in which clinical manifestations and symptoms can be correlated with a demonstrable pathology. The majority of mental illnesses are evaluated by observing changes in key behaviors such as thinking, mood, or social behavior. These alterations are difficult to ascertain and nearly impossible to quantify. However, progress is being made in diagnosing mental illness and in determining the neuropathology of mental illnesses. 
     The Diagnostic and Statistical Manual of Mental Disorders, Third Edition (DSM-III-R) and the updated DSM-IV, published by the American Psychiatric Association, represent the progress made in providing a basis for objective and rigorous descriptive criteria for categories of psychiatric disorders. While the DSM-III-R is very thorough and detailed, it is also quite lengthy. Thus, the process of reviewing the categories and applying them to data from a patient is also very time-consuming and arduous. In addition, there is no mechanism by which a patient can be diagnosed either as having or being susceptible to schizophrenia prior to the expression of symptoms. Thus, there is a longstanding need for an easy and definitive method for diagnosing schizophrenia. A diagnostic tool that can be applied prior to the expression of symptoms would also have great utility, providing a basis for the development of therapeutic interventions. 
     There is strong evidence for a genetic linkage of schizophrenia. Historically, there have been a number of studies on monozygotic twins of schizophrenics that indicated that 30–50% of the twins also had schizophrenia. The fact that this number is not 100% indicates that there are other factors involved in this disease process that may protect some of these individuals from the disease. It is apparent from a number of studies that the patterns of inheritance in most forms of schizophrenia are more complex than the classical dominant or recessive Mendelian inheritance. Recently, locus 1q21-22, a chromosome region containing several hundred genes, has been strongly linked to schizophrenia as shown by Brzustowicz et al.,  Science  288, 678–82, 2000, which is hereby incorporated by reference. 
     Until the 1950&#39;s there were no specific, effective treatments for schizophrenia. Antipsychotic drugs were identified in the 1950&#39;s, and these drugs were found to produce a dramatic improvement in the psychotic phase of the illness. Reserpine was the first of these drugs to be used and was followed by typical antipsychotic drugs including phenothiazines, the butyrophenones, and the thioxanthenes. A new group of therapeutic drugs, typified by clozapine, has been developed and were referred to as “a typical” antipsychotics. Haloperidol has been employed extensively in the treatment of schizophrenia and is one of the currently preferred options for treatment. When these drugs are taken over the course of at least several weeks, they mitigate or eliminate delusions, hallucinations, and some types of disordered thinking. Maintenance of a patient on these drugs reduces the rate of relapse. Since there is no way of determining if an individual is susceptible to schizophrenia, it is currently unknown if these antipsychotic compounds are useful in the prophylactic treatment of schizophrenia. 
     Signal transduction is the general process by which cells respond to extracellular signals (e.g. neurotransmitters) through a cascade of biochemical reactions. The first step in this process is the binding of a signaling molecule to a cell membrane receptor that typically leads to the inhibition or activation of an intracellular enzyme. This type of process regulates many cell functions including cell proliferation, differentiation, and gene transcription. 
     One important mechanism by which signal transduction occurs is through G-proteins. Receptors on the cell surface are coupled intracellularly to a G-protein that becomes activated, when the receptor is occupied by an agonist, by binding to the molecule GTP. Activated G-proteins may influence a large number of cellular processes including voltage-activated calcium channels, adenylate cyclase, and phospholipase C. The G-protein itself is a critical regulator of the pathway by virtue of the fact that GTPase activity in the G-protein eventually hydrolyzes the bound GTP to GDP, restoring the protein to its inactive state. Thus, the G-protein contains a built-in deactivation mechanism for the signaling process. 
     Recently, an additional regulatory mechanism has been discovered for G-protein signaling that involves a family of mammalian gene products termed regulators of G-protein signaling, or RGS (Druey et al., 1996, Nature 379: 742–746 which is hereby incorporated by reference). RGS molecules play a crucial modulatory role in the G-protein signaling pathway. RGS proteins bind to the GTP-bound Gα subunits with a variable Gα specificity as a substrate. RGS molecules shorten the GTP binding of the activated Gα subunits by acting as GTPase activating proteins (GAPs), accelerating GTP hydrolysis by up to one hundred fold. By the virtue of this GAP action and by making available the GDP-bound Gα to re-attach to βγ dimers, RGS proteins shorten the duration of the intracellular signaling. RGS proteins are expressed in nearly every cell; however, they show a tissue-specific expression across the body and cell type-specific expression in the brain. For example, RGS4 is strongly expressed in the central nervous system, moderately expressed in the heart, and slightly expressed in skeletal muscle (Nomoto et al., 1997, Biochem. Biophys. Res. Commun. 241(2):281–287 which is herein incorporated by reference). 
     Several members of the G-protein signaling pathways, most located downstream of RGS4 modulation, have been implicated in schizophrenia. Gil, Gq and Golf messenger RNA (mRNA) and protein levels all have been reported to be altered in various brain regions of the schizophrenic subjects. Furthermore, changes in expression of adenylate cyclase, phospholipase C, and protein kinases, as well as DARPP (dopamine- and cAMP-regulated phosphoprotein) phosphorylation changes are expected to be influenced by RGS regulation of Gα signaling. In addition, RGS modulation changes are expected to have significant effects on the signal transduction effected by neurotransmitters including dopamine, serotonin, GABA, glutamate, and norepinephrine. 
     An additional genetic marker of schizophrenia has been identified by Meloni et al. (U.S. Pat. No. 6,210,879). These investigators found that an allele of the microsatellite HUNTH01 in the tyrosine hydroxylase gene correlated with the expression of schizophrenia. However, the allele only appears to be present in sporadic schizophrenias. 
     There has been a long-standing need for a definitive and easy method for diagnosing schizophrenia as well as for an effective treatment with minimal side effects. Further, a need has been recognized in connection with being able to detect schizophrenia prior to the expression of noticeable symptoms. 
     A need has been recognized in connection with overcoming the various limitations to the current implementation of a method for diagnosing and assessing the susceptibility to schizophrenia are addressed through the use of the current invention. 
     SUMMARY OF THE INVENTION 
     In accordance with at least one embodiment of the present invention, there is provided a system and method for diagnosing and determining the susceptibility to schizophrenia. 
     In summary, one aspect of the present invention provides an isolated and substantially purified DNA sequence corresponding to SEQ ID NOS: 3, 4, 5, 6, 7, 8, and contiguous portions thereof. 
     Another aspect of the present invention is a polynucleotide sequence that is complementary to a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and contiguous protions thereof. 
     A further aspect of the present invention is an expression system comprising a DNA sequence that corresponds to SEQ ID NO:3. 
     A yet further aspect of the present invention is a method for diagnosing schizophrenia in a human comprising obtaining a DNA sample comprising a RGS4 gene from a patient and detecting a variation in the RGS4 gene indicating schizophrenia. 
     A still further aspect of the present invention is a method for determining the susecptiblity to schizophrenia comprising obtaining from a patient a DNA sample comprising a RGS4 gene and detecting a variation in said RGS4 gene indicating susceptibility to schizophrenia. 
     An additional aspect of the present invention is a method for daignosing schizophrenia comprising obtaining from a patient to be tested for schizophrenia a sample of tissue, measuring RGS4 mRNA levels in said sample, and determing if there is a reduced level of RGS4 mRNA in the sample. 
     A still additional aspect of the present invention is a method of determing susceptibility to schizophrenia comprising obtaining from a patient to be tested for susceptibility to schizophrenia a sample of tissue, measuring RGS4 mRNA levels in said sample, and determing if there is a reduced level of RGS4 mRNA in the sample. 
     A yet further aspect of the present invention is A method of determining susceptibility to schizophrenia comprising obtaining from a patient to be tested for susceptibility to schizophrenia a sample of tissue, measuring RGS4 protein levels in said sample, and determining if there is a reduced level of RGS4 protein in the sample. 
     Yet another aspect of the present invention is A method of treating schizophrenia, said method comprising measuring RGS4 protein or mRNA levels in a patient, and altering said RGS4 protein levels to provide the patient with an improved psychiatric function. 
     Another aspect of the present invention is a kit for diagnosising schizophrenia in a patient, said kit comprising antibodies to RGS4, and a detector for ascertaining whether said antibodies bind to RGS4 in a sample. 
     Another aspect of the present invention is a kit for diagnosising schizophrenia in a patient, said kit comprising a detect of RGS4 transcript levels in a patient, and a standard to ascertain altered levels of RGS4 transcript in the patient. 
     A still further aspect of the present invention is the DNA sequence of SEQ ID NO: 3 containing variations as described in the text below. 
     A yet further aspect of the present invention is a transgenic mouse whose genome comprises a disruption of the endogenous RGS4 gene, wherein said disruption comprises the insertion of a transgene, and wherein said disruption results in said transgenic mouse not exhibiting normal expression of RGS4 protein. 
     A still additional aspect of the present invention is a transgenic mouse wherein a transgene comprises a nucleotide sequence that encodes a selectable marker. 
     These and other embodiments and advantages of the present invention will be better understood with reference to the following figures and detailed description. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The present invention and its presently preferred embodiments will be better understood by reference to the detailed disclosure hereinbelow and to the accompanying drawings, wherein: 
         FIG. 1A  displays the design of microarray immobilized probes and in situ probes for RGS4, wherein numbers on the RGS4 nucleic acid fragments denote nucleotide position in relationship to the RGS4 mRNA, as currently in the NCBI database; 
         FIG. 1B  is a pseudocolored intensity view of a single RGS4 feature from the 516 control/547 schizophrenic PFC comparison after a dual-fluorescent hybridization; both images represent the same spot under cy3 and cy5 excitation, respectively; the balanced cy3 signal intensity α-control subject) was 6.2-fold brighter than the cy5 signal intensity (s-schizophrenic subject); 
         FIG. 1C  displays changes in RGS expression in the PFC of schizophrenic and control subjects reported by cDNA microarray analysis; 
         FIG. 2A  shows in situ hybridization results for PFC RGS4 expression levels which are decreased in 9 of 10 schizophrenic subjects; 
         FIG. 2B  shows the in situ hybridization data from 10 PFC pairwise comparisons which were quantified using film densitometry; 
         FIG. 3A  shows that 632 G-protein signalling-related genes were detected out of 1644 possible detections (274 genes/microarray×six microarrays); 
         FIG. 3B  shows that 239 1q21-22 locus-related genes were detected out of 420 possible detections (70 genes/mircoarray×six microarrays); RGS4 contribution to the transcript distribution is denoted by a hatched bar; 
         FIG. 4A  shows high power photomicrographs of VC tissue sections from the same matched pair of schizophrenic and matched control subjects represented in  FIG. 2A , viewed under darkfield illumination; 
         FIG. 4B  shows a graph of 10 supragranular VC SCH pairwise comparisons, in which schizophrenic subjects showed a comparably significant RGS4 transcript reduction to the PFC comparisons; 
         FIG. 4C  shows high power photomicrographs of MC tissue sections from the same matched pair of schizophrenic and matched control subjects represented in FOG. 2A, viewed under darkfield illumination; 
         FIG. 4D  shows a graph in which schizophrenic subjects across the same 10 subject pairs across the MC had comparably decreased RGS4 expression levels (mean=−34.2%, F 1,15 =10.18; p=0.006) to VC and PFC; 
         FIG. 5  shows a scatter plot of relative RGS4 expression changes across the experimental groups. 
         FIG. 6  displays the genomic organization that is derived from available sequences for clone NT — 022030, as well as the sequence analyses presented here; five exons were identified from the coding sequence for RGS4 (approximately 8.5 kb); the critical RGS domain is encoded by exons 3 to 5; the SNPs that were analyzed are listed in the top panel; * (a small star) indicates SNPs identified by re-sequencing the RGS4 gene and * (a large star) indicates SNPs used for association analysis. 
     
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The present invention focuses on the genetic underpinnings of schizophrenia. In the first phase of the research, cDNA microarrays were used to investigate potential alterations in transcript expression in six pairs of schizophrenic subjects. RGS4 was determined to be the most significantly and consistently changed transcript. In situ hybridization was also used to verify the microarray findings and to examine the regional and disease-related specificity of this change. Out of the several hundred genes on locus 1q21-22, the present studies indicate that RGS4 is a strong candidate for a major susceptibility gene on this locus. Genetic association and linkage studies were conducted using two samples independently in Pittsburgh and by the NIMH Collaborative Genetics Initiative. Using the Transmission Disequilibrium Test (TDT), significant transmission distortion was observed in both samples, albeit with different haplotypes. In support of the TDT results, increased sharing of alleles, identical by descent was observed for polymorphisms in this region among affected siblings of the NIMH cases, though associations were not observed when the cases were compared to a limited number of population-based controls. These analyses are consistent with the possibility that inheritable polymorphisms in the flanking untranslated regions (UTR) of the RGS4 gene confer susceptibility to schizophrenia. 
     Expression Studies 
     Two groups of human subjects, consisting of six and five pairs of schizophrenic and control subjects, were used in the present studies. Subject pairs were completely matched for sex (18 males and 4 females). The mean (±SD) difference within pairs was 4.6±3.5 years for age and 4.4±2.7 hours for post mortem interval (PMI). The entire group of schizophrenic and control subjects did not differ in mean (±SD) age at time of death (46.5±10.7 and 45.1±11.5 years, respectively), PMI (19.4±7.1 and 17.7±5.0 hours, respectively), brain pH (6.85±0.29 and 6.81±0.15, respectively), or tissue storage time at −80° C. (45.4±12.3 and 37.7±13.1 months, respectively) when the studies initiated. Nine of the schizophrenic subjects were receiving antipsychotic medications at the time of death, five had a history of alcohol abuse or dependence, and one died by suicide. Also studied were 10 subjects with major depressive disorder (MDD), each of whom were matched to one normal control subject. The MDD subject pairs were also completed matched for sex (18 males and 2 females). The mean (S.D.) difference within pairs was 1.2±1.4 years for age and 2.5±2.1 hours for PMI. The depressive and control subjects did not differ in mean(±S.D.) age at time of death (52.7±13.1 and 52.1±13.1 years, respectively), PMI (14.9±5.3 and 15.7±5.5 hours, respectively), brain pH (6.81±0.17 and 6.72±0.30), or tissue storage time at −80° C. (39.0±17.4 and 39.9±13.2 months, respectively). Two of the depressed subjects had a history of alcohol dependence, and six died by suicide. Two of the control subjects had also been matched to subjects with schizophrenia (685c, 604c). Consensus DSM-IIIR diagnoses were made for all subjects using data from clinical records, toxicology studies, and structured interviews with surviving relatives. 
     RCS4 Transcript Analysis 
     A Human Multiple Tissue Northern Blot (Clontech) and a  32 P-labeled cDNA probe were used to confirm the size of the RGS4 transcript reported previously (Druey, et al., 1996). However, our results reported the presence of single dark bands of ˜3 kB in lanes from multiple brain regions (whole cerebral cortex, frontal pole, occipital pole, temporal lobe), with much fainter or absent bands observed in lanes from other brain regions (cerebellum, medulla, spinal cord, putamen). Because the UniGene entry for the RGS4 cDNA (U27768) contained only the truncated transcript (800 bp), we designed custom PCR primers based on the BAC clone sequence containing the RGS4 gene (NT — 022030) to rapidly obtain the full-lenght RGS4 transcript sequence. For this analysis, mRNA from a control human brain was purified, DNased, and re-purified prior to first strand cDNA synthesis using Superscript II (Gibco) with an oligo dT primer. The resulting cDNA-mRNA mixture was diluted and used in a standard PCR reaction using AmpliTaq Gold (see above). All reaction products yielded single bright bands on 2% agarose/ethidium bromide-stained gels, and were subsequently purified and sequenced. Alignment of these sequences produced &gt;99% identity matches with the BAC clone sequence containing RGS4. The 3′ UTR for RGS4 obtained in this manner also aligned &gt;99% with a cDNA entry (AL137433.1) that contains both a poly A signal and a poly A attachment site, confirming that the human RGS4 transcript is 2949 bp without the poly A tail and includes a cDNA entry not previously associated with the human transcript in the NCBI database (see below;  FIG. 6 ). 
     Microarray Experiments 
     Fresh-frozen human tissue was obtained from the University of Pittsburgh&#39;s Center for the Neuroscience of Mental Disorders Brain Bank. Area 9 from the right hemisphere was identified and isolated and sectioned into tubes at −24° C. as described previously by Glantz, L. A. and Lewis, D. A. in Arch Gen Psychiatry 54: 943–952, 2000, which is herein incorporated by reference. Total RNA and mRNA were isolated according to manufacturer&#39;s instructions using Promega (Madison, Wis.) kit #Z5110, RNAgents® Total RNA Isolation System and Qiagen (Valencia, Calif.) kit #70022, Oligotex mRNA Kits, respectively. The volume was adjusted using Microcon columns YM-30 #42409 to 50 ng/μl. The quality and purity of the mRNA used in the reverse transcription labeling reactions was evaluated by size distribution on a 1% non-denaturing agarose gel (&gt;50% of mRNA smear over 1 kb; integrity of rRNA bands) and optical density (OD) measurements (260/280&gt;1.80), respectively. 
     Sample Labeling, Microarrays, Hybridization, and Data Analysis 
     Labeling was performed at Incyte Genomics, Inc. (Fremont, Calif.). Two hundred nanograms of mRNA was reverse transcribed using cy3- or cy5-labeled fluorescent primers; appropriate matched control and schizophrenic sample pairs were combined, and hybridized onto the same UniGEM-V cDNA microarray. Each UniGEM-V array contained over 7,000 unique and sequence-verified cDNA elements mapped to 6,794 UniGene  Homo sapiens  annotated clusters found at the NIH website. Hybridization and washing was performed using proprietary Incyte protocols. If a gene or expressed sequence tag (EST) was differentially expressed, the cDNA feature on the array bound more of the labeled probe from one sample than the other, producing either a greater cy3 or cy5 signal intensity. The microarrays were scanned under cy3-cy5 dual fluorescence, and the resulting images were analyzed for signal intensity. If the cy3 vs. cy5 signal intensity was within three fold, and the microarray detected spiked-in control standard less abundant than 1 copy in 50,000, the raw data were exported to a local SQL server database. On the server, the data were further analyzed using GemTools (Incyte&#39;s proprietary software) and MS-Excel 2000. Note that the operators performing the labeling, hybridization, scanning, and signal analysis were blind to the specific category to which each sample belonged. 
     A gene was considered to be expressed if the DNA sample was successfully amplified by PCR, produced signal from at least 40% of the spot surface, and had a signal/background ratio over 5-fold for either the cy3 or cy5 probe. Based on Incyte&#39;s control hybridization studies and control experiments, array data reliability and reproducibility cutoffs were established as follows:
         1. Genes were comparably expressed between the control and experimental samples if the cy3/cy5 ratio or cy5/cy3 ratio was &lt;1.6.   2. Gene expression was changed between the two samples at the 95% confidence level (95% CL) if the cy3/cy5 or cy5/cy3 signal was 1.6–1.89.   3. Gene expression was changed between the two samples at the 99% confidence level (99% CL) if the cy3/cy5 or cy5/cy3 signal was &gt;1.9. In the control experiments, &lt;0.5% of the observations fell into this category.       

     Gene Group Analysis 
     Of the genes represented on the array, a G-protein group was created for data analysis, and included transcripts on the microarray for G-protein-coupled receptors (GPCR), heterotrimeric G-protein subunits, Ras proteins, regulator of G-protein signaling (RGS) molecules, and G-protein-dependent inward rectifying potassium channels (GIRKs), totaling 274 genes. 
     At least two genes, RGS4 (Unigene cluster Hs 227571) and RGS5 (Unigene cluster Hs 24950) were mapped to the cytogenetic band 1q21-22. In order to determine whether there is altered expression of multiple genes mapped to this locus, a 1q21-22 group was created from genes represented on the microarray locus. The 1999 NCBI database human 1q21-22 map is represented by 70 genes on the microarray, although some of them are not expressed in the central nervous system. 
     RGS4 Sequences 
     The RGS4 microarray immobilized probes sequence matched the entry in the NCBI database (accession number U27768, UniGene cluster Hs.227571). Of the 800 bp full-length mRNA, the double-stranded DNA microarray immobilized probe was complementary to the 3′ region of 571 nucleotides, as shown in  FIG. 1A . The anti-sense, in situ hybridization probe was derived from the mRNA region spanning nucleotides 39–739, resulting in a 700 nucleotide long cRNA probe (see below). The RGS4 cDNA sequence, as determined from the complete mRNA coding sequence is listed as follows: 
     
       
         
               
               
               
             
           
               
                 gtacgctcaa agccgaagcc acagctcctc ctgccgcatt tctttcctgc ttgcgaattc 
                 60 
                   
               
               
                   
               
               
                 caagctgtta aataagatgt gcaaagggct tgcaggtctg ccggcttctt gcttgaggag 
                 120 
               
               
                   
               
               
                 tgcaaaagat atgaaacatc ggctaggttt cctgctgcaa aaatctgatt cctgtgaaca 
                 180 
               
               
                   
               
               
                 caattcttcc cacaacaaga aggacaaagt ggttatttgc cagagagtga gccaagagga 
                 240 
               
               
                   
               
               
                 agtcaagaaa tgggctgaat cactggaaaa cctgattagt catgaatgtg ggctggcagc 
                 300 
               
               
                   
               
               
                 tttcaaagct ttcttgaagt ctgaatatag tgaggagaat attgacttct ggatcagctg 
                 360 
               
               
                   
               
               
                 tgaagagtac aagaaaatca aatcaccatc taaactaagt cccaaggcca aaaagatcta 
                 420 
               
               
                   
               
               
                 taatgaattc atctcagtcc aggcaaccaa agaggtgaac ctggattctt gcaccaggga 
                 480 
               
               
                   
               
               
                 agagacaagc cggaacatgc tagagcctac aataacctgc tttgatgagg cccagaagaa 
                 540 
               
               
                   
               
               
                 gattttcaac ctgatggaga aggattccta ccgccgcttc ctcaagtctc gattctatct 
                 600 
               
               
                   
               
               
                 tgatttggtc aacccgtcca gctgtggggc agaaaagcag aaaggagcca agagttcagc 
                 660 
               
               
                   
               
               
                 agactgtgct tccctggtcc ctcagtgtgc ctaattctca cctgaaggca gagggatgaa 
                 720 
               
               
                   
               
               
                 atgccaagac tctatgctct ggaaaacctg aggccaaata ttgatctgta ttaagctcca 
                 780 
               
               
                   
               
               
                 gtgctttatc cacattgtag cctaatattc atgctgcctg ccatgtgtga gtcacttcta 
                 840 
               
               
                   
               
               
                 cgcataaact agatatagct tttggtgttt gagtgttcat cagggtggga ccccattcca 
                 900 
               
               
                   
               
               
                 gtccaatttt cctaagtttc tttgagggtt ccatgggagc aaatatctaa ataatggcct 
                 960 
               
               
                   
               
               
                 ggtaggtctg gattttcaaa gattgttggc agtttcctcc tcccaacagt tttacctcgg 
                 1020 
               
               
                   
               
               
                 gatggttggt tagtgcatgt cacatgacat ccacatgcac atgtattctg ttggccagca 
                 1080 
               
               
                   
               
               
                 cgttctccag actctagatg tttagatgag gttgagctat gatatgtgct tgtgtgtatg 
                 1140 
               
               
                   
               
               
                 tctatgtgta tatattatat atacattaga cacacatata cattatttct gtatatagat 
                 1200 
               
               
                   
               
               
                 gtctgtgtat acatatgtat gtgtgagtgt atgtatacac acacacacac acacacacac 
                 1260 
               
               
                   
               
               
                 acacttttgc aagagtgatg ggaaagaccc taggtgctca taactagagt atgtgtatgt 
                 1320 
               
               
                   
               
               
                 acttacatgg gtgttttgat ctctgttctt tcatactaca tttgaacagg gcaaaatgaa 
                 1380 
               
               
                   
               
               
                 ctaactgcca tgtaggctaa gaaagaaatg ctaacctgtg gaaagttggt tttgtaaaat 
                 1440 
               
               
                   
               
               
                 tccatggatc ttgctggaga agcatccaag gaacttcatg cttgatttga ccactgacag 
                 1500 
               
               
                   
               
               
                 cctccacctt gagcactatt ctaaggagca aataccttag ctcccttgag ctggttttct 
                 1560 
               
               
                   
               
               
                 ctgatggcac ttttgagctc ctaagctgcc agccttccct tcttttcctg ggtgctcagg 
                 1620 
               
               
                   
               
               
                 gcatgcttat tagcagctgg gttggtatgg agttggcaga caggatgttc aacttaatga 
                 1680 
               
               
                   
               
               
                 agaaatacag ctaaggcctt gccagcaaca cctgccgtaa gttactggct gagtgagggc 
                 1740 
               
               
                   
               
               
                 atagaagtta aaggttactg tttttatcct ctatcctttt ttcctttcct gatcaaggtg 
                 1800 
               
               
                   
               
               
                 ctcttctcat tttttcctga gaaccttagc catcagatga ggctccttag tttattgtgg 
                 1860 
               
               
                   
               
               
                 ttggttgttt tttctttata atggctctgg gctatatgcc tatatttata aaccagcagc 
                 1920 
               
               
                   
               
               
                 aggggaaaga ttatatttta taagagggaa caaattttca caatttgaaa agcccacata 
                 1980 
               
               
                   
               
               
                 agttttctct tttaaggtag aatcttgtta atttcattcc aaacatcggg gctaacagag 
                 2040 
               
               
                   
               
               
                 actggaggca tttcttttta ggctctgaga ctaaatgaga ggaaaagaaa agaaaaaaaa 
                 2100 
               
               
                   
               
               
                 aatgattgtc taaccaattg tgagaattac tgtttgaaac ttttcaaggc acattgaaat 
                 2160 
               
               
                   
               
               
                 acttgaaaac ttctcattta tgttatttat gatgttattt tgtacgtgtt attattatta 
                 2220 
               
               
                   
               
               
                 tattgtttta taaatggagg tacaggatat cacctgaatt attaatgaat gcccaggaag 
                 2280 
               
               
                   
               
               
                 taattttctt ctcattcttc taaaactact gcctttcaaa gtgcacacac acgcgtccac 
                 2340 
               
               
                   
               
               
                 atacactgca ttcgttgctc cagtataaat tacatgcatg agcacctttc tggcttttaa 
                 2400 
               
               
                   
               
               
                 gccaatataa tgggctgcaa aatgaagaca ccagagtgta tgcatacaaa tctcactgta 
                 2460 
               
               
                   
               
               
                 ttaaagatgc aggttttcta attgtaccct tcttgtctct ctggcaatct tgcccttaat 
                 2520 
               
               
                   
               
               
                 atccctggag ttcctcatca gtgtcatttt ctgttataca cagttccaca attttgtctc 
                 2580 
               
               
                   
               
               
                 tagttgactt caaatgtgta actttattgg tcttgcccta ttataattgt catgactttc 
                 2640 
               
               
                   
               
               
                 agattgtatc tgaactcaca gactgctgtc ttactaatag gtctggaagg tcacgctgaa 
                 2700 
               
               
                   
               
               
                 tgagaagtaa attattttat gtaatacatt tttgagtgtg tttttcagtt gtatttccct 
                 2760 
               
               
                   
               
               
                 gttatttcat cactatttcc aatggtgagc ttgcctgctc atgctccctg gacagaatac 
                 2820 
               
               
                   
               
               
                 tccttccttt tgcatgcctg tttctatcat gtgcttgata ggcctcaaag ctaatgcttc 
                 2880 
               
               
                   
               
               
                 cagtgaaaca cacgcatctt aataataagg gtaaataaac gctccatatg aaac 
                 2934 
               
             
          
         
       
     
     For purposes of the present invention, the RGS4 cDNA will be referred to as SEQ ID NO:1. 
     The 205 amino acid long sequence of RGS4, as determined and reported by Druey et al. in Nature, 379: 742–746 (1996) which is hereby incorporated by reference in its entirety, is listed as GenBank Accession number P49798 as follows: 
     
       
         
               
               
             
           
               
                   
                 MCKGLAGLPA SCLRSAKDMK HRLGFLLQKS DSCEHNSSHN 
               
               
                   
                   
               
               
                   
                 KKDKVVICQR VSQEEVKKWA ESLENLISHE CGLAAFKAFL 
               
               
                   
                   
               
               
                   
                 KSEYSEENID FWISCEEYKK IKSPSKLSPK AKKIYNEFIS 
               
               
                   
                   
               
               
                   
                 VQATKEVNLD SCTREETSRN MLEPTITCFD EAQKKIFNLM 
               
               
                   
                   
               
               
                   
                 EKDSYRRFLK SRFYLDLVNP SSCGAEKQKG AKSSADCASL 
               
               
                   
                   
               
               
                   
                 VPQCA 
               
             
          
         
       
     
     The above amino acid sequence of RGS4 is referred to as SEQ ID NO: 2 for purposes of the present invention. 
     Untranslated regions upstream and downstream from the RGS4 coding region are identified in the context of the present invention as being relevant components of the RGS4 gene. The RGS4 coding sequence along with these sequences are found on NT — 022030 as described in greater detail below. This sequence is 
     
       
         
               
               
               
             
           
               
                 agttcaagac cagcctgagc aacatggtga aaccccatct ctactaaaaa tacaaaatta 
                 60 
                   
               
               
                   
               
               
                 gacaggcatg gtgatacacg cctgtaatcc cagctacttc ggaggccgag gcaggagaat 
                 120 
               
               
                   
               
               
                 cacttgaacc tgctgggggt ggaggttgcg gggagcaaga tcatgccatt gcactccagc 
                 180 
               
               
                   
               
               
                 ccaggcaaca agagcgaaat gtcatctcag aaaaaaaaaa aggcatttta tatatatata 
                 240 
               
               
                   
               
               
                 tatatatata tacacacaca cacacatata tatatacaca tatatataca catatataca 
                 300 
               
               
                   
               
               
                 tatatacaca tatatacaca tatatataca catacatatg tacacatata tatacacata 
                 360 
               
               
                   
               
               
                 tgtatacaca tatatacaca tatatacaca catatataca catatataca cacatatata 
                 420 
               
               
                   
               
               
                 cacatatata cacatatata cacatataca catatataca catatataca tatatacaca 
                 480 
               
               
                   
               
               
                 tatatataat atacacacat atatatacac atatatacac acatatatac acatatatac 
                 540 
               
               
                   
               
               
                 acatatatat acacatatat acacatatat acatatatac acatatatat acatatatac 
                 600 
               
               
                   
               
               
                 acatatatac atatatacac atatatacat atatacacac atatatacac atacatatac 
                 660 
               
               
                   
               
               
                 acacacatag atatacatat atatacacat atatatacgt atatatatgt atatatatat 
                 720 
               
               
                   
               
               
                 gctccagagt tcataagagg tagcagttga ttaccactgg ggatagagga aaagagagtt 
                 780 
               
               
                   
               
               
                 tgacagcagt gtattgtgag aaggacattt caggttgatg gcaaatagta ggggaaatac 
                 840 
               
               
                   
               
               
                 ataaatgtgt aataaaacct atctgtaagg tagttaagaa ggtaacacta tatatatata 
                 900 
               
               
                   
               
               
                 tagtgaaagc agtgtaaacc taaaggatgg gccaaggatt taaatgttat agaagaatgg 
                 960 
               
               
                   
               
               
                 ctaagatgcc aaagctcagt gtatgtggca gaggcatggt gtagggtgtg tccaggttca 
                 1020 
               
               
                   
               
               
                 tatattgcat taagtgtgag aacaccctgg agtatgaacc aagaaaatgc aaaagccaga 
                 1080 
               
               
                   
               
               
                 agtgatggag gaaatgagac acaataatga agatattgag aggagggtgt gggcctagag 
                 1140 
               
               
                   
               
               
                 tgaagctttt cgtgccagta cttcttttga aggcccagtt ctcttctctc tcgggggctc 
                 1200 
               
               
                   
               
               
                 cttcatctct catagagtcc acagctttta agggccaaca cttgaggtca gcctggctct 
                 1260 
               
               
                   
               
               
                 ctcatttgag ctggatagaa cattttagag caccatctat tcttcaagag gaagtttaaa 
                 1320 
               
               
                   
               
               
                 aataaaagaa ccttgaagag gaaaaaatgt agacattcaa tctaaccttt tcattttact 
                 1380 
               
               
                   
               
               
                 agccaaagct aaatagaatg caggttacct gtttttcagc caggcaccat catttcctaa 
                 1440 
               
               
                   
               
               
                 ttgttataaa atttattatt attgttgtta ttattattat ttgccataag aagtttccca 
                 1500 
               
               
                   
               
               
                 tatcctttta gtataacaaa aacacaattc acaagcatta taaaacccat ggtgtctaac 
                 1560 
               
               
                   
               
               
                 tattaaaaaa attaagtgga acacacttgt cccagctact ggggaggctg aggagggagg 
                 1620 
               
               
                   
               
               
                 atcacgtgat cccagggggt caaggttatg gagagctatg attgtgccac tgcactccag 
                 1680 
               
               
                   
               
               
                 cctgggtgac agggaaagac cctgtctcta aaattttttt taaaaaaact aaactggttt 
                 1740 
               
               
                   
               
               
                 tattacagag attctggaga cagctacaca taaaagggtg gtatgcctca tattagctac 
                 1800 
               
               
                   
               
               
                 ccagggaggt ggaatgccaa cttaggtggt gtcaccacta ttaaaaatgc cccaaagcaa 
                 1860 
               
               
                   
               
               
                 tcaaaactga gaacttcctg ggagcttagc attgtgcaaa agcagcacaa aacacttaaa 
                 1920 
               
               
                   
               
               
                 caattcacag ttgtgttgga atgggaaggc ctggaaatat aaaccaaaga gtatattgtc 
                 1980 
               
               
                   
               
               
                 taaattgata gagattacaa ttgcctgaaa gaaaaagttg acttttaact agaatgttca 
                 2040 
               
               
                   
               
               
                 gagtaggttt acagaagaag ctcttaaact gggctccagt ggatttgtca atgctttgga 
                 2100 
               
               
                   
               
               
                 agctggtggg gtgggagggt tggagggggc ataaaaagtc atgttggtat gctctgctca 
                 2160 
               
               
                   
               
               
                 agtctccatt ctgtttcctt ttcctctttt caatgtcatg tcccattatt tcattatggg 
                 2220 
               
               
                   
               
               
                 cttcccttta tccaggatca atatgccacc tcttggttgt cttttaccta cttctccacc 
                 2280 
               
               
                   
               
               
                 tcactatgga atcgtccttg ggtagctcct gtgcttggga acctgcacgg gcacttttct 
                 2340 
               
               
                   
               
               
                 gatgtcttga ttccagcttt actcctaaaa cttaaatgct gaggggccaa caccatggca 
                 2400 
               
               
                   
               
               
                 gtggtaggga tgggaatggg ggtcttgtaa cacactacat aaactacacg aaataaacta 
                 2460 
               
               
                   
               
               
                 catgaaactc aacatgtttg caagactcag ttcacatcca tgaggagctc atgcttctcc 
                 2520 
               
               
                   
               
               
                 ctcctgctcc cctagcacac atgattatct ctatttggaa atgtttggca tttttggtga 
                 2580 
               
               
                   
               
               
                 agtgaatggt tcaataactt tctccaccat cagaacaaaa gctctttaag gttagggatg 
                 2640 
               
               
                   
               
               
                 ggatcataca cacttccctt gtccaagtcc ccatcacccc ttatctagac aattgctaca 
                 2700 
               
               
                   
               
               
                 gtttcctaca cactcttcta acctcttgca gtctattttc ataaaacagc tagagaactt 
                 2760 
               
               
                   
               
               
                 tgagatgtaa gtcaaaaaat agaacatgtc gctctttccc attgtttttg aaataaagtt 
                 2820 
               
               
                   
               
               
                 caaccccctt accagggtca acaaggccct gcaatgattt ggtcctgtta aaaattcttt 
                 2880 
               
               
                   
               
               
                 agccttaact catgctgttc ttccttacac tcactgcatt ctagccattg aggtttctat 
                 2940 
               
               
                   
               
               
                 gcatcaaact ttttttggtc ccagcactgt gcacatcctt ctgggtagaa tgccccttga 
                 3000 
               
               
                   
               
               
                 tttgtataat tagcacctcc ttcatcattt aggtcttagt ataactacta ccttcttaga 
                 3060 
               
               
                   
               
               
                 gaagctctgc ttcttcatcc tataaaaaag taaaattcct taccctgtta ttttttaagt 
                 3120 
               
               
                   
               
               
                 catccgtgtt tcattctgtt aaagttctta tcacaattta tcattatttt atttacagtc 
                 3180 
               
               
                   
               
               
                 atgtgccaca taacaatgtt tcagtcaggg atagaacaca aatgtatctg gccccataat 
                 3240 
               
               
                   
               
               
                 attataagct gagaaatttc tattaactag tgatatcgca gccatcataa gtgtaatgca 
                 3300 
               
               
                   
               
               
                 ggacattacc ttttctatgt ttagatatgt tagatacaca aatatatttc attgtgttat 
                 3360 
               
               
                   
               
               
                 aatttcctac agtattcagt acagtaacat gctgtacagg tttgtaacct aggagtaata 
                 3420 
               
               
                   
               
               
                 ggctatacca tacagcttag gtgtgtagta ggctataacc atctaggttt gtgtaagtac 
                 3480 
               
               
                   
               
               
                 attctatgat attcccacaa tgatgaaatc acctaactac acatttctca gaatgtttca 
                 3540 
               
               
                   
               
               
                 ctgttgtgaa gtgacccatg actatatttt cctatatact tgatattttt gtgcatctgc 
                 3600 
               
               
                   
               
               
                 ccatgagaat gtagtgtaag atcaaaggat gcaagaatgg gttctatcca gtatagtacc 
                 3660 
               
               
                   
               
               
                 cactacactg gtggatgtca atatgtattt gttagattaa tatctcaaga atgagcacct 
                 3720 
               
               
                   
               
               
                 ttctcagaca cataaaagat gctcaatata aaagtttgtt gaactgaacg ttattggcaa 
                 3780 
               
               
                   
               
               
                 atgtaacatg atcggattta aagaggagcg aaacagaggt ctggctcaaa caccatactt 
                 3840 
               
               
                   
               
               
                 ctagagtgca taagaggtag cagttgatta ccactggcga caggagaaaa aagagcttga 
                 3900 
               
               
                   
               
               
                 ccgcagggta ctgtgaagac atttcaggtt gatggcacag aacaggggaa atacataaat 
                 3960 
               
               
                   
               
               
                 gtgtgggaat attcagtggt ctgggatgac tacatagtag aatataatga agaaaagagt 
                 4020 
               
               
                   
               
               
                 ggaagggaaa gatgaaaagt tggaatgggg atgaattatg aaagtaccag aatgttatgc 
                 4080 
               
               
                   
               
               
                 taaggaatct agattttaaa atgtgagggc aaattgaagt  c c t gggcacg ttacaaaact 
                 4140 
               
               
                   
               
               
                 agaggtcata aagtttaccc taatttacca agatttccta gaggatctat aattggaatc 
                 4200 
               
               
                   
               
               
                 cagatctgcc tctctgtaaa gttcaagcac tttccatgac accatactgt ttctttccac 
                 4260 
               
               
                   
               
               
                 ctgcacaatg caaatgaact cttatgaaac tgctgtttct atcctgggct aaatgttgca 
                 4320 
               
               
                   
               
               
                 gaaaaaagat ttaatctttg ggataaggct attttgggtt ttctcct a ct tcttgggaaa 
                 4380 
               
               
                   
               
               
                 caaggttttc ttcccctggc taattaagtg tggtattgtt cttccaggga aatcagtgat 
                 4440 
               
               
                   
               
               
                 gcatcacctg ctgctatcaa atgtcagggt tggagttcct gatttattgc atgtgcccac 
                 4500 
               
               
                   
               
               
                 aaagcttggt gcaaagaatt ggacacattt cccaaaagta agacatactg ggaagtccct 
                 4560 
               
               
                   
               
               
                 gtttaccttc ctggtataca gcatcctcca gccccatatc tttgcttttt agtcctaaaa 
                 4620 
               
               
                   
               
               
                   a tcaataact gaactctcat tgatgtctag gccattgtag taaacaataa agaaggaggg 
                 4680 
               
               
                   
               
               
                 aggcttctga caactgagag gaaattgtca tctgaagtgg tgcaagcaca gcctggggct 
                 4740 
               
               
                   
               
               
                 gagccttggc ctacatcctg cccaagtgga ggatcagtg c cccatttaac atctggtaga   
                 4800 
               
               
                   
               
               
                 actaaagaac gcaac g cctg ccacaatgac ttatttccct gcatttgata ccgtcaatcc 
                 4860 
               
               
                   
               
               
                 ttgagaaatg ttttcttttg ttctccctga gcaaaggttg gaaaaatttg aaatttacct 
                 4920 
               
               
                   
               
               
                 agagaccaca catagttcac atcctgctgt gtggctgaat gtctgcccc c  cagtaggaaa 
                 4980 
               
               
                   
               
               
                 cagttcttct aaagcctatt gtcaacaata ccttccagat gttagcattt tacaatttaa 
                 5040 
               
               
                   
               
               
                 ggaacttaaa atag c cttca aactttttgc cagtttctct gatatccaat ctattctttt 
                 5100 
               
               
                   
               
               
                 actctgcctc ccaagctttc tttctagaat gctaacctga tcggcttaag tacttgaact 
                 5160 
               
               
                   
               
               
                 acctcttctc ctccattaac tacagagtaa attctggtct tcagagtaac aagaaacacc 
                 5220 
               
               
                   
               
               
                 ctttagttct cagcatattc gtgcaccttc atttatctct ccttctctct caaagctgca 
                 5280 
               
               
                   
               
               
                 gtaggggtga aaac g tgtga tacattttct cttccatcat aagggtcgca accaaaactc 
                 5340 
               
               
                   
               
               
                 ctatagtaaa agacaggtta ataagagcaa aacctaacaa atttatttaa tcaaagtttt 
                 5400 
               
               
                   
               
               
                 acatgacatg ggagtcttca gaaatgaaga cccaaagacc caggggaaac tgtctgtttt 
                 5460 
               
               
                   
               
               
                 ttttgctgag gttcgatgaa gaatggatag catgtagcca tgtagattag acaaaaggat 
                 5520 
               
               
                   
               
               
                 atgatctagt ggtaaaggac tcagggggaa acacagcaag gcctgtctat tcagattctt 
                 5580 
               
               
                   
               
               
                 cttgatctct ctctctctat gtatagcatt ctttcctcct gagtatgggg caggactctt 
                 5640 
               
               
                   
               
               
                 cttcaatgag ggtcttcaag ggagaaggga gaaagtggcc tttttagatt ttat gg cttg 
                 5700 
               
               
                   
               
               
                 cttcggggaa gaggagttct agtttctatg acccatcttg gggaagagga attctggttt 
                 5760 
               
               
                   
               
               
                 ctgtgacttg ctttcatgaa gaaagaggag taagaggcag gagggcagga gatggtcaga 
                 5820 
               
               
                   
               
               
                 aagagacttg gctgcttctg agggcttccg ctctccttta gttccaagta cttcttagca 
                 5880 
               
               
                   
               
               
                 taccaaagca ctatactttg gcatatggtt ttctgagctc taacactgca atcatgctaa 
                 5940 
               
               
                   
               
               
                 actcctctat gaccttcaaa cattccactt gcttttattc tttatggttg tgatggcata 
                 6000 
               
               
                   
               
               
                 gaggtcaata gcaaagaccc tggagtccca ctgtctgagc tggcataaca ttactaccac 
                 6060 
               
               
                   
               
               
                 ttaatcaatg tgtaagctca ggtaagtact taagtcctct atgcttcatc tgtaaaatga 
                 6120 
               
               
                   
               
               
                 gaatcattga agaacattct ctcaggatgg atcatgagga ataagtgaat taactggcat 
                 6180 
               
               
                   
               
               
                 atagtgctta aaccagtgcc ttgctcagtt agtgacagat aaaatcatct gttattactg 
                 6240 
               
               
                   
               
               
                 tgcccactat tgtgatgctc ttctcttctt tgtacaacga ctacatctct atttatcatt 
                 6300 
               
               
                   
               
               
                 ttagggtctc cttgtgaaaa accactccag attcaaaaga ttgagtttaa tctctatcct 
                 6360 
               
               
                   
               
               
                 ctgtgctttc ctggagtttt gtaaagtaaa tcttcacttg acatcatgga taggttcttg 
                 6420 
               
               
                   
               
               
                 gaaactacaa cttcaagtga aaggacataa ctaaaccaat ttttttctca tcaacgttat 
                 6480 
               
               
                   
               
               
                 aatgaaatgg cattgatgaa atgatggcat tcaaggacct gctgtacctt gtttcactta 
                 6540 
               
               
                   
               
               
                 aagtcactgt ttccaataat ctattgatga cattgaggac ttactatata ataataaata 
                 6600 
               
               
                   
               
               
                 tatatataat cgacgaaaca ggaatcaaac tgctaactct gctaactggt ctccctgctt 
                 6660 
               
               
                   
               
               
                 ccacactctg cccactcatc tcagtctttc tttcacaaga gtcagaatga tcagatgaga 
                 6720 
               
               
                   
               
               
                 cccctcctct gcttctgttt cttccatgga tttccactgc actctgataa agtccagcct 
                 6780 
               
               
                   
               
               
                 cttgaccaca gcctacaaat ccttgcacga tctatcgttt acttttccat ctccttttat 
                 6840 
               
               
                   
               
               
                 gctactttca tcttgttctc aattctctag ctatgctggc cccttcttgt tctttcccat 
                 6900 
               
               
                   
               
               
                 ttttttttaa tttttaaaat ttgtatatat ttatgggtta taagtgaaat ctttttagat 
                 6960 
               
               
                   
               
               
                 gcataggttg tatagtgata aaatcagggc ttttagggta ttcatcacct gaatgatgta 
                 7020 
               
               
                   
               
               
                 cattgtaccc cttaagtaat ttctcaccat ccgctgactt cttgccccct gggtattcat 
                 7080 
               
               
                   
               
               
                 cacctgaatg atgtgcattg taccccttaa gtaatttctc accatccgct gacttcttgc 
                 7140 
               
               
                   
               
               
                 cccctgggta ttcatcacct gaatgatgtg cattgtaccc cttaagtaat ttctcaccat 
                 7200 
               
               
                   
               
               
                 ccgctgactt cttgccccct catccttctg aggctccatt gtccatcatt ccacactcta 
                 7260 
               
               
                   
               
               
                 catctatgtg tacacattat ttagctccta cttataagtg ataacatgca atatttgtct 
                 7320 
               
               
                   
               
               
                 ttctgtgtct gtcttgtttt acttatgata atggccccca gttctatcta ggct g ctgca 
                 7380 
               
               
                   
               
               
                 aaaggcatga tttcattctt ttttatggct atgttctttc ccaatttaga taaagaacac 
                 7440 
               
               
                   
               
               
                 tcgcacttgc tcttacttct atttggaata ctaattccta ggcttcttgc attgctttct 
                 7500 
               
               
                   
               
               
                 ccttctcacc catcaaatct cattttagat accacctctt caaagagggc tttcctgacc 
                 7560 
               
               
                   
               
               
                 accttggctg aattagccct tcaccatctg attactctct agcacatcac ctgcccattt 
                 7620 
               
               
                   
               
               
                 tattcatggt acaggtcaaa atctggaatc acctgatttg tttattttct gactccttct 
                 7680 
               
               
                   
               
               
                 actgagatga aaactctact agagcggaga ttttatctgc ttgtatcagg tactgcttca 
                 7740 
               
               
                   
               
               
                 aacagcacct gatacaga g t aggtggtcaa aagatatttc ttaaacaaat gaacaaataa 
                 7800 
               
               
                   
               
               
                 aaagtagatc ttttgagagt aaagctcttc cacactacca gagtcattca ggaatgacaa 
                 7860 
               
               
                   
               
               
                 atcatagaat aacagaattt gatgctttgt gcatatcaga gaaagaaggt ggaaggttgt 
                 7920 
               
               
                   
               
               
                 caaggtatca tgatgtacca gtcctcgcct cctcaaacac aatctgcaag tcccacagtg 
                 7980 
               
               
                   
               
               
                 aaaaagtaag ttaactcatg tgaagcgttt tacaaacact tttttaaaag tcttaaaact 
                 8040 
               
               
                   
               
               
                 cctaagaaag caagatttaa tagtcaaaga agtgagtaaa catgaaatgc ctgaacagag 
                 8100 
               
               
                   
               
               
                 taatgagcta agcacaaagt tagagacatg ttagttaata tgtcttgaaa gcagcagctc 
                 8160 
               
               
                   
               
               
                 ctgctttcaa ggagcaagaa caaattgggc aagtgaacac tccttgaata aaatgtgtaa 
                 8220 
               
               
                   
               
               
                 aattaatttt gggttatgtt ctatactgtg tataatagaa tgataaaaat tatttgacta 
                 8280 
               
               
                   
               
               
                 gcactttgta gtttagaaat atctctattt acacagttta ccttatttga taagactgtt 
                 8340 
               
               
                   
               
               
                 gagtgatggg atagcatggt ggacaatcca cataactgag tatcgagaca cctgtatctg 
                 8400 
               
               
                   
               
               
                 gacccagctc tgttagtaag aagctgtaac ctcagcaagt cactttctct ttctgggtct 
                 8460 
               
               
                   
               
               
                 ctatttcctt tttggtgaaa tgagagtgtt aggctagatt gcctttgaag tcccattttg 
                 8520 
               
               
                   
               
               
                 tctttaaagt cccatctatt gcagtgattt atatttaact catgacaaat caggcttctc 
                 8580 
               
               
                   
               
               
                 ttattctaag tgcaa g acat aaaactttta ttgtggaatt tcaggcatca gtaaatcttt 
                 8640 
               
               
                   
               
               
                 ttgggtactc acttatgttc ctgaaatcaa tctatttgag tgatcactct tttaggtgcc 
                 8700 
               
               
                   
               
               
                 caggtaaaca aagaaggcca tggtctttct ttgagtgacc ttctttccct tttaattagt 
                 8760 
               
               
                   
               
               
                 ctgacctctt taatgtcagt tctgactgat tcatttccct ggtccatctt ccttggtctg 
                 8820 
               
               
                   
               
               
                 agggccttcc tagtttcata ttgcacttca gttccttcca caccaccatc aaggatggct 
                 8880 
               
               
                   
               
               
                 gtcaacattc atttgttcta tgttataatt caaggaaaag ttgcccagta gctaatccaa 
                 8940 
               
               
                   
               
               
                 taaatgccct cttatgggcg gctagagact ttttcctata atttaaatgc atcttctgta 
                 9000 
               
               
                   
               
               
                 gattatggtc cctccaccac tttacatttg tctgctgtct ccttgctctg ctagtcatgg 
                 9060 
               
               
                   
               
               
                 aacgtgttgg tagtgggggc agtgtgggat gttcaagggc acgtattggg tagggccaca 
                 9120 
               
               
                   
               
               
                 tatgggcatt gctttgtgcc attctttcta tatttttggt attttgcatc tcactggaac 
                 9180 
               
               
                   
               
               
                 ccaactattt ttcatctctt ccacctaaac tatttgatgc ctctgtttct tatatataaa 
                 9240 
               
               
                   
               
               
                 gtatagctca ctgtagccta tgatcaggaa cctatctgct ttctaaatga aagctgtttt 
                 9300 
               
               
                   
               
               
                 ggtcagatct agcaattaat tcccttcttc cacttatagc tttcctctgt aactctggtg 
                 9360 
               
               
                   
               
               
                 taggtatttg gtttatggct ataagatgtg aaacacctga atgattctgt ccatgcaggc 
                 9420 
               
               
                   
               
               
                 atttcagttc atgatattgt atgtaaaaga tactgattgt ctaggtgttc agaaacacct 
                 9480 
               
               
                   
               
               
                 atagggctta atattcttac aatcagtttg aaggctggtg atacgcaaag caaactacat 
                 9540 
               
               
                   
               
               
                 atttttctgc ctgctctctc tctttctctc tacatctctc tttctttatc ttttgaaata 
                 9600 
               
               
                   
               
               
                 tcagtttgga gacttagaat tacataagac ataaacccat ttgatataag aattgctgtg 
                 9660 
               
               
                   
               
               
                 tatatttgct catctactcc ctcctttggt cctcgagctg ccggtttaga ctttttacag 
                 9720 
               
               
                   
               
               
                 gacgcaggca tgtgaaggag aaactgtcag tgctaggctg aattctgttg ttaccaagat 
                 9780 
               
               
                   
               
               
                 ttctagaaaa gtattcctca gtcaggttga ttacagatat agcaaatcta tttttcctag 
                 9840 
               
               
                   
               
               
                 ggtagtttct gtatgctgcc gggcttataa ctgtctgtca tccagctatt t c tctccacc 
                 9900 
               
               
                   
               
               
                 ttcttgtttg cataacaacc aaggcaactt ccgcaaatca ctgcgtggag acgatgatcc 
                 9960 
               
               
                   
               
               
                 tg c cagctcc cttttggaaa tcgtgaggat cagatcttgg accatgtata atatgatgct 
                 10020 
               
               
                   
               
               
                 tctaatccaa aagaggaaag gcattgggag tcagctccta agtaagctcc agaattcctg 
                 10080 
               
               
                   
               
               
                 ctggtacttt tccttccagg aagcaacttc cttgatattt tttttttaca g g catatgaa 
                 10140 
               
               
                   
               
               
                 taaaaactat attttgcagc attgtacact ttttttcctt ttctagaaat tctaaacctc 
                 10200 
               
               
                   
               
               
                 tgacattggt ggagacattg agtacatttt ttcccatatc cctacttttc agaaggattt 
                 10260 
               
               
                   
               
               
                 tctctgctcg ttcacttaac attgctgatg cgtcagtctt ttcttcctca tctctttcag 
                 10320 
               
               
                   
               
               
                 gggctggaga ggcagaggga gacagaggag ctggtactgc agagcggtcg tctgattggc 
                 10380 
               
               
                   
               
               
                 tggacggtcg tagctgggct ataaaagaga cccctacagg cttagcagga agacgctcag 
                 10440 
               
               
                   
               
               
                 aggattctga caatatcttt accggagaag aggcaaagta cgctcaaagc cgaagccaca 
                 10500 
               
               
                   
               
               
                 gctcctcctg ccgcatttct ttcctgcttg cgaattccaa gctgttaaat aagatgtgca 
                 10560 
               
               
                   
               
               
                 aagggcttgc aggtctgccg gcttcttgct tgaggaggta agattgcttt cagccattaa 
                 10620 
               
               
                   
               
               
                 ccatattaaa cttttggcta gactttctca gttatttaca tgttgtactt actaacctag 
                 10680 
               
               
                   
               
               
                 ttctgtgcaa ttagaaacag tgtggtcagg agagcacgac tttctaactt tcctccaaga 
                 10740 
               
               
                   
               
               
                 ctagctagat attgtgactt aagacatgtg ctccccaaat ttcagccctt atgtgttgtt 
                 10800 
               
               
                   
               
               
                 ttgtgtgacc tcagttttga gaactgttct attctttaag ccaggtctaa gaaagctagt 
                 10860 
               
               
                   
               
               
                 tttaattaag aagcgagatg aggtttgagg ctatgtacag tgatctgtaa tatctccatc 
                 10920 
               
               
                   
               
               
                 tgtgattact actgctattt gagcatccct ggagtacata gaagcctggc tctgggcttt 
                 10980 
               
               
                   
               
               
                 ctgattgtat gctacaactt gtttcaggaa aggtacccca gaatgaggtt tggctccatc 
                 11040 
               
               
                   
               
               
                 atcagaaagg cacta t gctt tccgtgtggt ggtgcagtaa ctttcactct  c tatgttctt 
                 11100 
               
               
                   
               
               
                 ataag c aaat gttacaatga gatatgagtt ttaaagccag atcttcctta tctctctgcc 
                 11160 
               
               
                   
               
               
                 ccatctctag ttcttgaagt gtctcatatg agtttggttg agaaatattg atcattacaa 
                 11220 
               
               
                   
               
               
                 atcagttaat agttttgtag aagatctcat cttaaagaca ttgttttgtt aatatactcc 
                 11280 
               
               
                   
               
               
                 cttgattttt ttaaaagacc ttacagacat acagctattc atttgttttt ggtttgttca 
                 11340 
               
               
                   
               
               
                 aaaaaggtat aaagaaatgc attcagagaa agatcatata ttagccagtt gaaaattaaa 
                 11400 
               
               
                   
               
               
                 cacaaaatga gtgcatatta cattacttaa tcttgcagtc aaaggtaaaa agtcaaccta 
                 11460 
               
               
                   
               
               
                 aaggtatact acctgctttc ttatcgcact gcaaatagaa attaccacaa attttatttt 
                 11520 
               
               
                   
               
               
                 ggaaataatc tcagaaaaca taatttttta tgtactatta aaacatttac tttccaaata 
                 11580 
               
               
                   
               
               
                 ttctgtcatt caggagtatg gaagtatcga tggcttcttt aaaatgaagc aggagggtct 
                 11640 
               
               
                   
               
               
                 ggcagagagt atctatgaaa taagttcctc tgaccttcac gcttaatttt ctgaatggag 
                 11700 
               
               
                   
               
               
                 tggagcaaat tacttcaagc ttcacttaac ttgcatatga aatgaaccgt acaaaaatac 
                 11760 
               
               
                   
               
               
                 aagagtgtca gga g aaagtt atgctctggt aatatttttg caaaacagat aaaagataat 
                 11820 
               
               
                   
               
               
                 actagagctc tgtcctcaaa gagttaagca gctaatctaa ggaggtaaac tctatgtcag 
                 11880 
               
               
                   
               
               
                 caggatgaac tgctcttccc tttcctcctc aataaattgc aaatcatcta gtccaacatc 
                 11940 
               
               
                   
               
               
                 tttaccacca gtgcctgagg ctccagagga gccattgcct tctcaaggtc acataggtgg 
                 12000 
               
               
                   
               
               
                 tgggtgagtt aggaccaaat ctagaattcc tgactccagt aacttctgaa gtcattttgt 
                 12060 
               
               
                   
               
               
                 tttttatttt tatggtttta ttataagaat acttgctaag cacacttacc ccctgcattg 
                 12120 
               
               
                   
               
               
                 attaataact ctaggatctc ag g t g gatcc agcacataga aatatgaatt cgtttctatt 
                 12180 
               
               
                   
               
               
                 tggacttcat gatatattta cattatcacc ttggaatcac cctaacattc aggattgtat 
                 12240 
               
               
                   
               
               
                 cttgttataa tcaaaaagga tgttgcatcc cctgaacagt catcagtcag ggaagcagag 
                 12300 
               
               
                   
               
               
                 gagggaaagt aatcttgcga ggaagagaaa atactattta agggacagtc agagaacata 
                 12360 
               
               
                   
               
               
                 atggaattca aactttctgg gaaaacctac atacataaat gtattagtgg ccatcctaaa 
                 12420 
               
               
                   
               
               
                 tgtctttata tctttgaggc tttattttcc ctactccaaa tagacacatt tagttattca 
                 12480 
               
               
                   
               
               
                 tttcttttaa aatggtattt ctctttttaa actatttctt gactttttta ataaaaagag 
                 12540 
               
               
                   
               
               
                 atgcaagcaa gaggatattt aataaaaagt aagagagttg agcttaaggc ttattaaaag 
                 12600 
               
               
                   
               
               
                 accccctttt tctagttagt caggagctct aatgtgccct ggctacctat taaatggtgg 
                 12660 
               
               
                   
               
               
                 caataaactg gaagctcagt gatgactcta gcctgcttct cctaatagct gttaagcctc 
                 12720 
               
               
                   
               
               
                 aaatgccctt tagagtgtgt atgtccttta aagtagctat taagaaggaa agcagcagca 
                 12780 
               
               
                   
               
               
                 gcagatattg tctagaaaga agccccaaga agctgaggtt tcagcttggg catttgtttt 
                 12840 
               
               
                   
               
               
                 cgccatccca tgctccattt ccctctgctg gaactgtgca cctcagtgta ttctccctct 
                 12900 
               
               
                   
               
               
                 atacctcaca gcaggaactg cttgcccccc cccccccccc ccaacataca tggctggaac 
                 12960 
               
               
                   
               
               
                 tgaatagact tttactttcc cgaggtgctt ctacagttcc ctctgccagc aggggaacag 
                 13020 
               
               
                   
               
               
                 atggaaatag caatcacctg ccagaaggtg gcgtgcagca aggatgtgca tcttttgccg 
                 13080 
               
               
                   
               
               
                 ctactgcttt ctgattccta aaaattactc agagatcact catgtgttca gtgattcagg 
                 13140 
               
               
                   
               
               
                 ttctgttgaa gataccaaag atattcggtt ggtcaaaatg acgggcatat aaaggcttct 
                 13200 
               
               
                   
               
               
                 caggtttctg aggtaaactg aagggtcaga attccagttg tggatgaagg aaatggtgtt 
                 13260 
               
               
                   
               
               
                 atgactgcct caaggttttg tagcaagtca tagggaacca agaggaatct tgttttcctc 
                 13320 
               
               
                   
               
               
                 agaggtcatg ccaactccaa ctcccgttcc ctaaactgtc tctgagccat agactagtaa 
                 13380 
               
               
                   
               
               
                 tggactcttc aagctctacc attaggtatc ttttaaagaa agctggttat tactatttat 
                 13440 
               
               
                   
               
               
                 tcattttttt ctcttctgtg cagtgcaaaa gatatgaaac atcggctagg tttcctgctg 
                 13500 
               
               
                   
               
               
                 caaaaatctg attcctgtga acacaattct tcccacaaca agaaggacaa agtggttatt 
                 13560 
               
               
                   
               
               
                 tgccagaggt aagagaaaag gccttggtga agatgtactt agtattaact atctgatgat 
                 13620 
               
               
                   
               
               
                 ggggatgttc tgtgagaagg aacttgtgct cctagttaag ccagatttgg atcaagatag 
                 13680 
               
               
                   
               
               
                 cctccatttt catggagatc ataactacat ttgaaatttc tatacattta gtgaaaaact 
                 13740 
               
               
                   
               
               
                 gccctcatca ataacatatt ttgtcataac gatggaaaat aaaatctttg ccttcattca 
                 13800 
               
               
                   
               
               
                 ggatcttaga tttcttgccc caattttttt accatggcat tccaattatt ctgtttctct 
                 13860 
               
               
                   
               
               
                 ctattttttc tagagtgagc caagaggaag tcaagaaatg ggctgaatca ctggaaaacc 
                 13920 
               
               
                   
               
               
                 tgattagtca tgaatgtaag tctgacagca acctgggatg aggtactctg gataagacaa 
                 13980 
               
               
                   
               
               
                 gttatattat gctggtctaa tagaaactgc agcaaggcct ggcttctttc tgatgttcag 
                 14040 
               
               
                   
               
               
                 actcaggaga ctctttaggt cttaaattca gtctgtttaa aattttaata tgccctagag 
                 14100 
               
               
                   
               
               
                 ctttgtgata tacaatgaaa agtttatgca ggaaccatgt ggaaaaccat ctctctcatc 
                 14160 
               
               
                   
               
               
                 acaaggaaaa acggaagaga gaaaaaaaat gataaatatc aataccttct tgcaaaatca 
                 14220 
               
               
                   
               
               
                 atctcagttt ctctttccca aattgacctt ggtaattgat agctgcatag gcatttcaga 
                 14280 
               
               
                   
               
               
                 agcaaaatac ttccttgaaa gaggcttcca acttgagtaa gaatcattag gtagaactgg 
                 14340 
               
               
                   
               
               
                 gaaccactgg atatcaaaca cagatt a ggg ttacctgact ccaggtgact tgaaaaaagc 
                 14400 
               
               
                   
               
               
                 aggggaaaaa gggattgctt gaatccatgc tttatccccc aagtacctca gctttatgtg 
                 14460 
               
               
                   
               
               
                 aaatagcata tccaagaggc caaccagtgt gatgacaact gtggtccttt ctcctgtatc 
                 14520 
               
               
                   
               
               
                 ataggtgggc tggcagcttt caaagctttc ttgaagtctg aatatagtga ggagaatatt 
                 14580 
               
               
                   
               
               
                 gacttctgga tcagctgtga agagtacaag aaaatcaaat caccatctaa actaagtccc 
                 14640 
               
               
                   
               
               
                 aaggccaaaa agatctataa tgaattcatc tcagtccagg caaccaaaga ggtaggtttt 
                 14700 
               
               
                   
               
               
                 ttatggatac ataaaaattg tacgtattta tggagtatgt gtgatatttt gatacatgca 
                 14760 
               
               
                   
               
               
                 tacaatgtga taacaatcaa atcagggcaa ttgctatata catatctcaa acatttatta 
                 14820 
               
               
                   
               
               
                 tttctacgtg ttgagaacat tccaaatctc ctcttctagc tatcttaaaa tatacaataa 
                 14880 
               
               
                   
               
               
                 actattgata actatatcac cctaatgtgc tatcaaacac tagaacctat tccctctacc 
                 14940 
               
               
                   
               
               
                 caactttcta tctattcctt ctacccatta gccaacctga ccaaaaaggt aagcttttat 
                 15000 
               
               
                   
               
               
                 ggcagagaac tctctggatc ttagtgaagg ttcctagaat agtggagctg actatcataa 
                 15060 
               
               
                   
               
               
                 tcttgacaac cccaaataaa tcagtttttt aaaaaatctc ttttatccat gtggcttacc 
                 15120 
               
               
                   
               
               
                 ataacctccc tgcatgaatt tttctgatga atctccccaa tttgttagac agaacagaag 
                 15180 
               
               
                   
               
               
                 atcttgccct gctctctcta aagcagaaag gttcattctg aacctttcat actctctcac 
                 15240 
               
               
                   
               
               
                 atgtgccaag gaggacccca atgtcacttt tgttttttgc ttctgaaata cagagggtgc 
                 15300 
               
               
                   
               
               
                 actgccactt acaagtcact acaaagcata caggcttgca tcctcaacag ggatataggt 
                 15360 
               
               
                   
               
               
                 ctaatgaagc cttggccttt gcccctcagg tgaacctgga ttcttgcacc agggaagaga 
                 15420 
               
               
                   
               
               
                 caagccggaa catgctagag cctacaataa cctgctttga tgaggcccag aagaagattt 
                 15480 
               
               
                   
               
               
                 tcaacctgat ggagaaggat tcctaccgcc gcttcctcaa gtctcgattc tatcttgatt 
                 15540 
               
               
                   
               
               
                 tggtcaaccc gtccagctgt ggggcagaaa agcagaaagg agccaagagt tcagcagact 
                 15600 
               
               
                   
               
               
                 gtgcttccct ggtccctcag tgtgcctaat tctcacctga aggcagaggg atgaaatgcc 
                 15660 
               
               
                   
               
               
                 aagactctat gctctggaaa acctgaggcc aaatattgat ctgtattaag ctccagtgct 
                 15720 
               
               
                   
               
               
                 ttatccacat tgtagcctaa tattcatgct gcctgccatg tgtgagtcac ttctacgcat 
                 15780 
               
               
                   
               
               
                 aaactagata tagcttttgg tgtttgagtg ttcatcaggg tgggacccca ttccagtcca 
                 15840 
               
               
                   
               
               
                 attttcctaa gtttctttga gggttccatg ggagcaaata tctaaataat ggcctggtag 
                 15900 
               
               
                   
               
               
                 gtctggattt tcaaagattg ttggcagttt cctcctccca acagttttac ctcgggatgg 
                 15960 
               
               
                   
               
               
                 ttggttagtg catgtcacat gacatccaca tgcacatgta ttctgttggc cagcacgttc 
                 16020 
               
               
                   
               
               
                 tccagactct agatgtttag atgaggttga gctatgatat gtgcttgtgt gtatgtctat 
                 16080 
               
               
                   
               
               
                 gtgtatatat tatatataca ttagacacac atatacatta tttctgtata tagatgtctg 
                 16140 
               
               
                   
               
               
                 tgtatacata tgtatgtgtg agtgtatgta tacacacaca cacacacaca cacacacact 
                 16200 
               
               
                   
               
               
                 tttgcaagag tgatgggaaa gaccctaggt gctcataact agagtatgtg tatgtactta 
                 16260 
               
               
                   
               
               
                 catgggtgtt ttgatctctg ttctttcata ctacatttga acagggcaaa atgaactaac 
                 16320 
               
               
                   
               
               
                 tgccatgtag gctaagaaag aaatgctaac ctgtggaaag ttggttttgt aaaattccat 
                 16380 
               
               
                   
               
               
                 ggatcttgct ggagaagcat ccaaggaact tcatgcttga tttgaccact gacagcctcc 
                 16440 
               
               
                   
               
               
                 accttgagca ctattctaag gagcaaatac cttagctccc ttgagctggt tttctctgat 
                 16500 
               
               
                   
               
               
                 ggcacttttg agctcctaag ctgccagcct tcccttcttt tcctgggtgc tcagggcatg 
                 16560 
               
               
                   
               
               
                 cttattagca gctgggttgg tatggagttg gcagacagga tgttcaactt aatgaagaaa 
                 16620 
               
               
                   
               
               
                 tacagctaag gccttgccag caacacctgc cgtaagttac tggctgagtg agggcataga 
                 16680 
               
               
                   
               
               
                 agttaaaggt tactgttttt atcctctatc cttttttcct ttcctgatca aggtgctctt 
                 16740 
               
               
                   
               
               
                 ctcatttttt cctgagaacc ttagccatca gatgaggctc cttagtttat tgtggttggt 
                 16800 
               
               
                   
               
               
                 tgttttttct ttataatggc tctgggctat atgcctatat ttataaacca gcagcagggg 
                 16860 
               
               
                   
               
               
                 aaagattata ttttataaga gggaacaaat tttcacaatt tgaaaagccc acataagttt 
                 16920 
               
               
                   
               
               
                 tctcttttaa ggtagaatct tgttaatttc attccaaaca tcggggctaa cagagactgg 
                 16980 
               
               
                   
               
               
                 aggcatttct ttttaggctc tgagactaaa tgagaggaaa agaaaag a aa aaaaaaatga 
                 17040 
               
               
                   
               
               
                 ttgtctaacc aattgtgaga attactgttt gaaacttttc aaggcacatt gaaatacttg 
                 17100 
               
               
                   
               
               
                 aaaacttctc atttatgtta tttatgatgt tattttgtac gtgttattat tattatattg 
                 17160 
               
               
                   
               
               
                 ttttataaat ggaggtacag gatatcacct gaattattaa tgaatgccca ggaagtaatt 
                 17220 
               
               
                   
               
               
                 ttcttctcat tcttctaaaa ctactgcctt tcaaagtgca cacacacgcg tccacataca 
                 17280 
               
               
                   
               
               
                 ctgcattcgt tgctccagta taaattacat gcatgagcac ctttctggct tttaagccaa 
                 17340 
               
               
                   
               
               
                 tataatgggc tgcaaaatga agacaccaga gtgtatgcat acaaatctca ctgtattaaa 
                 17400 
               
               
                   
               
               
                 gatgcaggtt ttctaattgt acccttcttg tctctctggc aatcttgccc ttaatatccc 
                 17460 
               
               
                   
               
               
                 tggagttcct catcagtgtc attttctgtt atacacagtt ccacaatttt gtctctagtt 
                 17520 
               
               
                   
               
               
                 gacttcaaat gtgtaacttt attggtcttg ccctattata attgtcatga ctttcagatt 
                 17580 
               
               
                   
               
               
                 gtatctgaac tcacagactg ctgtcttact aataggtctg gaaggtcac g ctgaatgaga   
                 17640 
               
               
                   
               
               
                 agtaaattat tttatgtaat acatttttga gtgtgttttt cagttgtatt tccctgttat 
                 17700 
               
               
                   
               
               
                 ttcatcacta tttccaatgg tgagcttgcc tgctcatgct ccctggacag aatactcctt 
                 17760 
               
               
                   
               
               
                 ccttttgcat gcctgtttct atcatgtgct tgataggcct caaagctaat gcttccagtg 
                 17820 
               
               
                   
               
               
                 aaacacacgc atcttaataa taagggtaaa taaacgctcc atatgaaact atttgcttgg 
                 17880 
               
               
                   
               
               
                 aaacacatta atgatccaga gacatgctat gagaaacatc agggtgtagg gtgactttag 
                 17940 
               
               
                   
               
               
                 aaaaatactc atactgagtc tttaatccct cctgtgccag tgaactctgg gaaagaaagt 
                 18000 
               
               
                   
               
               
                 acaaactgaa tattgtttat tctttagttc atgccactgc tctgcttggc tctactcata 
                 18060 
               
               
                   
               
               
                 gaaccaaggc aatcttagct tcagagactg caaaacagat taagtgattt gcttgcagat 
                 18120 
               
               
                   
               
               
                 tctcaatcaa ttttcaaggg atagagttca ccttccagag ccattctttt atttccagtt 
                 18180 
               
               
                   
               
               
                 acccgcctgt ttgagagatg atagagcagt gggaaattga gagagttgaa aggagatata 
                 18240 
               
               
                   
               
               
                 gattcttacc caaacttcaa aaatccttcc ctcccttttg ttaattctct ttcctggaaa 
                 18300 
               
               
                   
               
               
                 agaggtcata aaatgttcac atcctcagta ataggccctg tgctgtgtct attatgtcat 
                 18360 
               
               
                   
               
               
                 gagactccca tttcctgacc cttctttccc attgtaagag tagtagttac aaggtgttaa 
                 18420 
               
               
                   
               
               
                 ggatagatga tcttcaacac ttttgagaaa tagatccatt tacggatctg gtaaaaacta 
                 18480 
               
               
                   
               
               
                 tggaccgaac catcttttaa gaaaaaaatt cagagaggaa tctaaatttt gtgtgctttg 
                 18540 
               
               
                   
               
               
                 aggggaaact ctcagaatct cccctcaaaa ctatcattct tctcttatac tatagatgtg 
                 18600 
               
               
                   
               
               
                 tcagactctc actgggactg tatagttgct gctccctgta tttgataata tctatcaaga 
                 18660 
               
               
                   
               
               
                 actgcagggt aattcaaagt cacgctatta gcagcaagtg tgagcagtgt tggtttcccc 
                 18720 
               
               
                   
               
               
                 agtctctaca tccctcatcc tttctttctt ctttatggtt gtctattaaa gaaataaaaa 
                 18780 
               
               
                   
               
               
                 aaaatattgg ctgaccgttt ttctgaagat aatgtatatc aaggaccacc ttttgaaaaa 
                 18840 
               
               
                   
               
               
                 cactcattat tcgagaacaa agacacaaca tacgagaatc tctgggatac attcaaagca 
                 18900 
               
               
                   
               
               
                 gtgtgtagag ggaaatttat agcactaaat gcccacaaga gaaagcagga aagatctaaa 
                 18960 
               
               
                   
               
               
                 attgataccc taacatcaca attaaaagaa ctagaaaagc aagagcaaac acattcaaaa 
                 19020 
               
               
                   
               
               
                 gctagcagaa gacaagaaat aactaagatc agagcagaac tgaaggaaat agagacacaa 
                 19080 
               
               
                   
               
               
                 aaaacccttc aaaaaattaa tgaatccagg agctggtttt ttgaaaagat taacaaaatt 
                 19140 
               
               
                   
               
               
                 gatagactgc tagcaagact aataaagaag aaaagagaga agaatcaaat agacacaata 
                 19200 
               
               
                   
               
               
                 aaaaatgata aaggggatat caccaccgat cccacagaaa tacaaactac catcagagaa 
                 19260 
               
               
                   
               
               
                 tactataaac acctctacgc aaataaacta gaaaatctag aagaaatgga taaattcctc 
                 19320 
               
               
                   
               
               
                 gatacataca ccctcccaag accaaaccag gaagaagttg aatctctgaa tagaccaata 
                 19380 
               
               
                   
               
               
                 acaggctctg aaattgaggc aataatcaat agcttaccaa ccaaaaaaag tccaggacca 
                 19440 
               
               
                   
               
               
                 gatggattca cagctgaatt ctaccagacg tacaaagagg agctggtacc attccttctg 
                 19500 
               
               
                   
               
               
                 aaactattcc aatcaataga aaaagaggga atcctcccta actcatttta tgaggccagc 
                 19560 
               
               
                   
               
               
                 atcatcctga taccaaagcc tggcagagac acaaccaaaa aagagaattt tagaccaata 
                 19620 
               
               
                   
               
               
                 tccttgatga acattgatgc aaaaatcctc aataaaatac tggcaaaccg aatccagcag 
                 19680 
               
               
                   
               
               
                 cacatcaaaa agcttatcca ccatgatcaa gtgggtttca tccctgggat gcaaggctgg 
                 19740 
               
               
                   
               
               
                 ttcaacatac gcaaatcaat aaatgtaatc cagcatataa acagaaacaa agacaaaaac 
                 19800 
               
               
                   
               
               
                 cacatgatta tctcaataga tgcagaaaag gcatttgaca aaatttaaca actcttcatg 
                 19860 
               
               
                   
               
               
                 ctaaaaactc tcaatcaatt aggtattgat gggacgtatc tcaaaataat aagcactatc 
                 19920 
               
               
                   
               
               
                 tatgacaaac tcacagccaa tatcatactg aatgggcaaa aactggaagc attccctttg 
                 19980 
               
               
                   
               
               
                 aaaacgggca caagacaggg atgccctctc tcaccactcc tattcaacat agtgttggaa 
                 20040 
               
               
                   
               
               
                 gctctggcca gggcaattag gcaggagaag gaaataaagg gtattcaatt aggagaagag 
                 20100 
               
               
                   
               
               
                 gaagtcaaat tgtccctgtt tgcagatgac atgattgtat atctagaaaa ccccatcgtc 
                 20160 
               
               
                   
               
               
                 tcagcccaaa atctccttaa gctgataagc aacttcagca aagtctcagg atacaaaatc 
                 20220 
               
               
                   
               
               
                 aatgtacaaa aatcacaagc actcttatac atcaataaca gacaaacaga gagccaaatc 
                 20280 
               
               
                   
               
               
                 atgagtgaac tcccattcac 20300 
               
             
          
         
       
     
     For purposes of the present invention, this DNA sequence will be referred to as SEQ ID NO:3. The location of the SNPs discussed further below is indicated by bold and larger font letters. Several additional sequences of DNA that are upstream from SEQ ID NO:3 are identified as relevant to the present invention. These DNA sequences are also found on NT — 022030 and are 
     
       
         
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
           
               
                 ggattaatca tgacaaaagt aatctaaatc tcgttaagac 
                 60 
                   
               
             
          
           
               
                 tacttaatga tcaatctttc 
                   
                   
               
               
                   
               
             
          
           
               
                 cctctgtttt ccctgactat agggaagtga attgccccaa 
                 120 
                   
               
             
          
           
               
                 tccttctcta tcacccccct 
                   
                   
               
               
                   
               
             
          
           
               
                 gcagccatgc caatgcctta cctctgttat attcagccat 
                 180 
                   
               
             
          
           
               
                 aggggaagct tattctcata 
                   
                   
               
               
                   
               
             
          
           
               
                 gaatcagggg ttggcatg t a gtcactagct attcttggtg 
                 240 
                   
               
             
          
           
               
                 agactagtga agatgagtga 
                   
                   
               
               
                   
               
             
          
           
               
                 aggaaaatat tgcataggtg aaatctcata ggcacaaata 
                 300 
                   
               
             
          
           
               
                 ggtgtttgtg agagtaacaa 
                   
                   
               
               
                   
               
             
          
           
               
                 taaaagaaag tcattcccat actctagtag atgactcatt 
                 360 
                   
               
             
          
           
               
                 ttctcctcat tttttttttt 
                   
                   
               
               
                   
               
             
          
           
               
                 tcaaggcgtt ctctacaacg gttaacctag taccaaaaat 
                 420 
                   
               
             
          
           
               
                 ccttctcttt tttcttggac 
                   
                   
               
               
                   
               
             
          
           
               
                 aaatcctgtt caagttagca tggcatttac tacgtccaag 
                 480 
                   
               
             
          
           
               
                 acattgtcca gatgctgtgg 
                   
                   
               
             
          
         
       
     
     For purposes of the present invention, this DNA sequence will be referred to as SEQ ID NO:4. 
     
       
         
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
           
               
                 agagaaagaa aggcaggcag caaggagaaa aaacattttt 
                 60 
                   
               
             
          
           
               
                 taaaaaaaga aaattaaaat 
                   
                   
               
               
                   
               
             
          
           
               
                 ccatgtaatg tctgatatct gttctgctgt atgtgtagat 
                 120 
                   
               
             
          
           
               
                 ctttccatat accaactcat 
                   
                   
               
               
                   
               
             
          
           
               
                 tagccttatt ttacaggtga ggaaaatgag ac c gagagtc 
                 180 
                   
               
             
          
           
               
                 cttcttactt gaccaagttc 
                   
                   
               
               
                   
               
             
          
           
               
                 acacagcaag atcacacatg gtagaaccaa tgttagaacc 
                 240 
                   
               
             
          
           
               
                 taggtgtata cttgctcatt 
                   
                   
               
               
                   
               
             
          
           
               
                 caatatgtac aataattgca aaagtttcca taggtcttat 
                 300 
                   
               
             
          
           
               
                 tatatatcag gcactataaa 
                   
                   
               
               
                   
               
             
          
           
               
                 tgctatgcat gtgtcaacta atttaaacct aagcaatatt 
                 360 
                   
               
             
          
           
               
                 ataaggaagg tactattata 
                   
                   
               
               
                   
               
             
          
           
               
                 gaaatctcag ccttacaggt aagggaacag gaataaagag 
                 420 
                   
               
             
          
           
               
                 atgtgaggta atggcccaag 
                   
                   
               
             
          
         
       
     
     For purposes of the present invention, this DNA sequence will be referred to as SEQ ID NO:5. 
     
       
         
               
               
               
             
               
               
               
             
               
               
               
             
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
           
               
                 ataatctcct ttcaagtttt tatcctgtca cttgctagtt 
                 60 
                   
               
             
          
           
               
                 gtgtgatttg ggacaaatca 
                   
                   
               
               
                   
               
             
          
           
               
                 tttaactcct tgtaaaggga gagaag g aag gctgtaaaaa 
                 120 
                   
               
             
          
           
               
                 aattaagtaa taaaaagata 
                   
                   
               
               
                   
               
             
          
           
               
                 aactccttgt ggtatatttt gttattgttc aaaaatattt 
                 180 
                   
               
             
          
           
               
                 attgcccctc ttaggatgtc 
                   
                   
               
               
                   
               
             
          
           
               
                 ttaggtcatt cttgcattgc tataaagaaa tacccaagtc 
                 240 
                   
               
             
          
           
               
                 tgggtaattt ataaagaata 
                   
                   
               
               
                   
               
             
          
           
               
                 gaggttaaat tggctcacag ttctgcaggc tgcacaggaa 
                 300 
                   
               
             
          
           
               
                 gcatcccact ggcgtctact 
                   
                   
               
               
                   
               
             
          
           
               
                 cacttctggt gaggactcag aaagcttttg cttatgacag 
                 360 
                   
               
             
          
           
               
                 caggctaagt gagagcaggt 
                   
                   
               
             
          
         
       
     
     For purposes of the present invention, this DNA sequence will be referred to as SEQ ID NO:6. 
     Several additional sequences of DNA that are downstream from SEQ ID NO:3 are identified as relevant to the present invention. These DNA sequences are also found on NT — 022030 and are 
     
       
         
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
           
               
                 catggtattt ttactaccca ttgccttcta ggaaagggta 
                 60 
                   
               
             
          
           
               
                 taacaaatag gaaatattaa 
                   
                   
               
               
                   
               
             
          
           
               
                 tatttttaat gcctttgagg gtgttaaaaa gcacaactct 
                 120 
                   
               
             
          
           
               
                 aaggactgtt tgtaaattc c   
                   
                   
               
               
                   
               
             
          
           
               
                 aggtcaaatg ttgtttctcc ttctctattt cctaccttgg 
                 180 
                   
               
             
          
           
               
                 tgatggcctg atcttatatg 
                   
                   
               
               
                   
               
             
          
           
               
                 gagtcactcc aactagaaac cacagaatca tccctagttc 
                 240 
                   
               
             
          
           
               
                 ctacttctga ctcactccat 
                   
                   
               
               
                   
               
             
          
           
               
                 acactcaaaa gtcacctgac tctgcagaat ttctctagaa 
                 300 
                   
               
             
          
           
               
                 aaactctatg aaaacctatt 
                   
                   
               
               
                   
               
             
          
           
               
                 cctgcctctc cacctgcata gatgtagctt catccaggct 
                 360 
                   
               
             
          
           
               
                 cttatggtgc atggcctcgg 
                   
                   
               
               
                   
               
             
          
           
               
                 ttactgcctt atcctttcta ctggcctctc aatctcccat 
                 420 
                   
               
             
          
           
               
                 ctgataccca ttaatgtact 
                   
                   
               
             
          
         
       
     
     For purposes of the present invention, this DNA sequence will be referred to as SEQ ID NO:7. 
     
       
         
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
           
               
                 ccaaatactt tttaggcaca ctgggaagtt acattgtttc 
                 60 
                   
               
             
          
           
               
                 ttgcaagtga caggttgtcc 
                   
                   
               
               
                   
               
             
          
           
               
                 tttaattagt tctttctctc aaaaagagac tgctgactcc 
                 120 
                   
               
             
          
           
               
                 aaactgggaa gaaacccact 
                   
                   
               
               
                   
               
             
          
           
               
                 caccagcaaa atgctgctga attcactctg atagttttct 
                 180 
                   
               
             
          
           
               
                 aatctctcat cagtagatga 
                   
                   
               
               
                   
               
             
          
           
               
                 caataatgaa gccagtattg ttaccacaag actcagatat 
                 240 
                   
               
             
          
           
               
                   g tctatcacc caagatgatt 
                   
                   
               
               
                   
               
             
          
           
               
                 tctctttaag acgcaataaa agggaacttt tctccccatt 
                 300 
                   
               
             
          
           
               
                 tattagcaac taagatgaaa 
                   
                   
               
               
                   
               
             
          
           
               
                 tgagagccag agaaataaag tgaggaagga aagagaattt 
                 360 
                   
               
             
          
           
               
                 actaccttta caagctgaaa 
                   
                   
               
             
          
         
       
     
     For purposes of the present invention, this DNA sequence will be referred to as SEQ ID NO:8. In all upstream and downstream sequences (i.e. SEQ ID NOS: 4, 5, 6, 7, and 8), the location of SNPs are indicated by bold and larger font letters. 
     In Situ Hybridization 
     Double-stranded cDNA containing the RGS4 sequence was first amplified from normal human brain cDNA using custom designed primers (Forward primer sequence: CCGAAGCCACAGCTCCTC (corresponding to SEQ ID NO: 3); Reverse primer sequence: CATCCCTCTCCCTTCAGGTG (corresponding to SEQ ID NO: 4), and “touchdown” PCR with AmpliTaq Gold (PE Biosystems): (94° C. for 10 minutes (min), followed by 10 PCR cycles with a high annealing temperature 94° C. for 30 seconds (sec), 62° C. for 30 sec, and 72° C. for 60 sec), 10 cycles with a medium annealing temperature (94° C. for 30 sec, 60° C. for 30 sec, 72° C. for 60 sec), and 20 cycles at a low annealing temperature (94° C. for 30 sec, 58° C. for 30 sec, 72° C. for 60 sec). The product of this touchdown PCR reaction produced a single bright band on a 2% agarose gel and was purified and ligated into a T/A plasmid cloning vector (AdvanTAge, Clontech) and transformed into competent  Escherichia coli  cells and plated overnight at 37° C. Colony PCR was performed on selected colonies containing the insert, and the products of these reactions were restriction digested and sequenced to verify orientation and insert identity. 
     [ 35 S]-labeled riboprobes were synthesized using the T7 Riboprobe In Vitro Transcription System (Promega kit #P1460) and purified using RNeasy kit (Qiagen #74104). A scintillation counter was used to verify the specific radioactivity and yield of the probe. During hybridization, approximately 3 nanograms (ng) of probe was used per slide in a total volume of 90 μl. All other methods used were those described previously in Campbell et al., in Exp. Neurol. 160: 268–278, 1999, which is hereby incorporated by reference. 
     Tissue blocks containing the regions of interest (PFC area 9, motor cortex [MC] and visual cortex [VC]) were identified using surface landmarks and sulci (the superior frontal gyrus, the central sulcus and precentral gyrus, and the calcarine sulcus, respectively). After histological verification of the regions, 20 μm sections containing these regions were cut with a cryostat at −20° C., mounted onto gelatin-coated glass slides, and stored at −80° C. until use. The slides were coded so that the investigator performing the analysis was blind to the diagnosis of the subjects. 
     Following hybridization and washing, slides were air dried and exposed to BioMax MR film (Kodak) for 8–22 hours and then dipped in emulsion (NTB-2, Kodak), and exposed for 3–5 days at 4° C. High resolution scans of each film image were used for quantification of signal with Image (Scion Corporation, Fredrick, Maryland), version 4.0b), and darkfield images were captured from the developed slides. Throughout all steps and procedures, subject pairs were processed in parallel. Hybridization of sections with sense RGS4 riboprobe, used as a specificity control, did not result in detectable signal. 
     Quantification was performed by subtracting the background white matter OD from the average signal OD measured in five non-overlapping rectangular regions on each section (3 sections per tissue block). In PFC and MC, these rectangular regions spanned cortical layers II–VI. Due to the lack of RGS4 signal in layer IV throughout the neocortex, and the great expansion of this layer in VC, the supragranular and infranular signal intensities were analyzed separately in VC. However, there were no significant differences in the levels of signal contained in the supra- and infragranular layers, so they were combined as a measure of overall VC signal intensity. 
     Each in situ hybridization was repeated three times in separate hybridization reactions. The resulting ODs were background-corrected and averaged. Visual cortex (V1) OD quantification, due to a bi-laminar transcript distribution, was performed separately for the supragranular and infragranular layers. 
     In order to search for novel candidate genes whose expression is consistently altered in schizophrenia, high-density cDNA microarrays (UniGEM-V, Incyte Genomics) were used to examine the expression patterns of over 7,800 genes and ESTs in post mortem samples of prefrontal cortex area 9 from six matched pairs of schizophrenic and control subjects. 
     Comparison and Statistical Analyses 
     As illustrated in  FIG. 1B , a gene was determined to be expressed if the arrayed immobilized probe or target (the design of which is shown in  FIG. 1A ) was successfully amplified by PCR, produced a signal from at least 40% of the spot surface and had a signal/background ratio over 5-fold for either the cy3 or cy5 probe. Both images represent the same spot under cy3 and cy5 excitation, respectively. In this experiment, the balanced cy3 signal intensity (control or c-subject) was 6.2-fold brighter than the cy5 signal intensity (schizophrenic or s-subject). 
     Genes were comparably expressed between the control and experimental samples if the cy3/cy5 ratio or cy5/cy3 ratio was &lt;1.6. Over 80% of observations fell into this class. Gene expression was changed between the two samples at the 95% confidence level (95% CL) if the cy3/cy5 or cy5/cy3 signal was 1.6–1.89. Gene expression was changed between the two samples at the 99% confidence level (99% CL) if the cy3/cy5 or cy5/cy3 signal was 1.9. 
     In the microarray analyses, data from experimental subjects were compared to data from matched control subjects in a pairwise design to control for the effects of age, race, sex and PMI on gene expression. To evaluate potential changes in gene group expression on the microarrays, two types of statistical measures were employed: 1) χ-square analysis was performed on the distribution of genes in a group versus the distribution of all genes called present on each individual microarray. The distribution of gene expression ratios was divided into five different bins based on confidence levels for individual gene comparisons: &lt;−1.9, −1.89 to −1.6, −1.59 to 1.59, 1.6 to 1.89 and &gt;1.9. 2) A paired t-test (degrees of freedom=5) was used to compare mean expression ratios for a given gene group to the mean expression ratios for all expressed genes across all six subject pairs. A gene group was considered to be changed only if it reported differential expression by both the χ-square and t-test compared to the mean and distribution of all expressed genes. Microarray changes were also analyzed by descriptive statistics and correlation. 
     To mimic the microarray comparisons, the in situ hybridization data were analyzed using ANCOVA with diagnosis as the main effect, subject pair as a blocking factor, and brain pH and tissue storage time as covariates. Furthermore, to verify that the pairing of subjects adequately controlled for sex, age, and PMI, we also conducted an ANCOVA with diagnosis as a main effect, and sex, age, PMI brain pH, and tissue storage time as covariates. Since both models produced similar results, the values from the ANCOVA with subject pair as a blocking factor are reported. Changes between groups were also analyzed by descriptive statistics, Pearson correlation, and Factor analysis. 
     Pittsburgh Cases and Parents for Genotyping Analysis 
     Inpatients and outpatients were recruited at Western Psychiatric Institute and Clinic, a University of Pittsburgh-affiliated tertiary care center and 35 other treatment facilities within a 500 mile radius of Pittsburgh. The Diagnostic Interview for Genetic Studies (DIGS) was the primary source for clinical information for probands (Nurnberger, et al.  Archives of General Psych . 51, 849–59; discussion 863–4, 1994). Additional information was obtained from available medical records and appropriate relatives, who also provided written informed consent. Consensus diagnoses were established by board certified psychiatrists. There were 93 Caucasian and 70 African-American cases. Genomic DNA, but not clinical information was available from all parents of the Caucasian cases. Cord blood samples were obtained from live births at Pittsburgh and served as unscreened, population-based controls. There were 169 individuals. They included 76 Caucasians and 93 African-Americans. 
     National Institute of Mental Health Collaborative Genetics Initiative (NIMH CGI) Sample 
     From 1991–98, pedigrees having probands with schizophrenia or schizoaffective disorder, depressed (DSM IV criteria) were ascertained at Columbia University, Harvard University, and Washington University. The DIGS was the primary interview schedule. The families were ascertained if they included two or more affected first degree relatives (Cloninger et al.  Am. J. Med. Gen . 81, 275–81, 1998, which is hereby incorporated by reference). We selected case-parent trios and available affected siblings from this cohort. Thus, 39 cases, their parents and 30 affected sibling-pairs were obtained. They comprised 25 Caucasian families, 10 who reported African-American ethnicity and 4 from other ethnic groups. Transmission disequilibrium test (TDT) analysis utilized only one case/family. 
     Written, informed consent was obtained from all participants. Ethnicity was based on self-report (maternal report for neonatal samples). 
     DNA Sequencing and Polymorphism Detection 
     The genomic sequence for RGS4 was obtained from NT — 022030 (390242 bp), a currently unfinished clone from Human Genome Project, Chromosome 1 database. The annotated data revealed three identified genes, namely, RGS4, MSTPO 32  and RGS5. The genomic organization of RGS4 and RGS5 includes 5 exons which is typical for the RGS family gene. 
     A panel of 10 African-American cases and 6 Caucasian controls was initially used to screen for polymorphisms in the exonic, intronic, and flanking genomic sequences of the RGS4 gene. The re-sequenced region included 6.8 kb upstream and 2.9 kb downstream of the coding sequence. The genomic sequence was used to design primers and amplicons ˜500 bp were generated, with overlapping sequences. The amplified fragments were sequenced using an ABI 3700 DNA sequencer. The sequencing panel that was used (n=16) has over 80% power to detect SNPs with minor allele frequency over 5% (Kruglyak et al.  Nature Gen . 27, 234–236, 2001, which is hereby incorporated by reference). We also sequenced cDNA sequences from the post-mortem samples reported on earlier (Mirnics et al.  Mol. Psychiatry  6, 293–301, 2001). The sequences were aligned using Sequencher (version 4.5) and polymorphisms were numbered consecutively. Additional SNPs localized to NT — 022030 were obtained from the NCBI SNP database. We also obtained genotype data from a prior study of the NIMH sample. 
     Polymorphism Analysis 
     PCR based assays included primers (5 pmol) with 200 μM dNTP, 1.5 mM MgCl2, 0.5 U of AmpliTaq Polymerase (PE Biosystems), 1× buffer and 60 ng of genomic DNA in 10 or 20 μl reactions. The PCR conditions were 95° C. for 10 min followed by 35 cycles (94° C. for 45 sec, 60° C. 45 sec and 72° C. for 1 min). The final extension at 72° C. for 7 min. The amplified products were digested with restriction endonucleases, electrophoresed on agarose gels, and visualized using ethidium stain. SNPs 4 and 18 were identified as single strand conformational polymorphisms (SSCP) (Orita et al.  DNAS  86, 2766–70, 1989). All genotypes were read independently by two investigators. 
     Polymorphisms were detected only in the intronic and flanking sequences of RGS4 ( FIG. 6 ). Among 34 identified SNPs, one was selected from each of six sets which appeared to be in complete linkage disequilibirum in the re-sequenced panel. SNPs were further evaluated for informativeness (minor allele frequency &gt;0.1) and availability of reliable genotyping assays. Among the Caucasian cases from Pittsburgh, deviations from Hardy Weinberg equilibrium (HWE) were noted for SNP 7 (p&lt;0.03) and SNP 13 (p&lt;0.01). Though all maternal genotypes conformed to HWE, deviations were noted at SNPs for the fathers of Pittsburgh cases at SNPs 4 and 18 (p&lt;0.05). For the analysis of IBD sharing among affected sibling-pairs from the NIMH samples, we also used genotypes for markers D1S1595, D1S484, D1S1677, D1S431 and D1S1589 (Faraone et al.  Am. J. of Med. Gen . 81, 290–5, 1998). 
     Statistical Analysis 
     PEDCHECK software was used to check for Mendelian inconsistencies (O&#39;Connell et al.  Am. J. of Hum. Gen . 63, 259–266, 1998, which is hereby incorporated by reference). χ 2  tests were employed for comparisons between cases and unrelated controls. We also used SNPEM software based on the EM algorithm to estimate and compare haplotype frequencies (Fallin, 2001, which is hereby incorporated by reference). We utilized GENEHUNTER software for TDT analysis of individual SNPs and haplotypes, as well as analysis of identity by descent among affected sibling-pairs (Kruglyak et al.  Am. J. of Hum. Gen . 58, 1347–63, 1996; Spielman et al.  Am. J. of Hum. Gen . 54, 559–60, 1994, both of which are hereby incorporated by reference). We also used TRANSMIT for global tests of association involving multiple haplotypes (Clayton et al.  Am. J. of Med. Gen . 65, 1161–1169, 1999a; Clayton et al.  Am. J. of Hum. Gen . 65, 1170–1177, 1999b, both of which are hereby incorporated by reference). 
     Microarray Results 
     Single gene transcripts were analyzed across all cDNA microarray comparisons. Across the six microarray comparisons over 90,000 data points were collected, and from these 44,000 were expression-positive observations, resulting in an average of 3,735 expressed genes/microarray. Of the expressed transcripts, 4.8% were judged to be differentially expressed (99% CL) between the schizophrenic and control subjects. The observed differences for any subject pair, in general, were comparably distributed in both directions: 2.6% of the genes were expressed at higher levels in schizophrenic subjects than in the matched controls, whereas 2.2% were expressed at lower levels in the schizophrenic subject. 
     Of all the expressed genes, RGS4 transcript reported the most significant decrease across all schizophrenic subjects. In fact, it was the only gene decreased at the 99% CL in all microarray comparisons. The microarray-bound, 571 base pair long, double-stranded cDNA immobilized probe corresponded to the 3′ end of RGS4 and had a less than 50% sequence homology to any other known transcript, including RGS family members. This high binding specificity, coupled with strong cy3 and cy5 hybridization signal intensities, as shown in  FIG. 1B , showed that RGS4 was robustly expressed in the human prefrontal cortex. Across the six microarray comparisons, RGS4 mRNA levels were decreased 50–84% in the PFC of schizophrenic subjects, as illustrated in  FIG. 1C , while the expression of the ten other RGS family members represented on the microarray were unchanged in the schizophrenic subjects. In the scatter plot shown in  FIG. 1C , the X-axis reports subject pairs, the Y-axis reports percent change between schizophrenic and control subjects. Individual symbols represent a gene expression difference between a schizophrenic and control subject in a single pairwise comparison. The black dashed line denotes equal cy3 and cy5 signal intensity (similar expression) between schizophrenic and control subjects (0% change), green dashed line denotes the 95% confidence interval (37.5% change), red dashed line represents 99% confidence interval (47.5% change). Missing symbols in some pairwise comparisons indicate that the corresponding genes&#39; microarray hybridization did not meet expression criteria. Across all the RGS members represented on the microarray, only RGS4 showed a consistent expression change over the 99% CL in schizophrenic subjects. 
     To confirm the microarray findings for the RGS4 expression changes, in situ hybridization was performed on the PFC from the same five subject pairs used for the microarray experiments (for pair 794c/665s, no sections were available from the same block of tissue used in the microarray experiment). As a further test of the robustness of the microarray data, five additional subject pairs were added to the in situ hybridization analysis. Radiolabeled cRNA probes designed against RGS4 mRNA were used to localize and quantify relative transcript levels. In the control subjects, RGS4 labeling was heavy in the prefrontal cortex, as shown in  FIG. 2A , mimicking previously described labeling in the rat. In the gray matter of prefrontal cortex, the RGS4 riboprobe heavily labeled various size and shape cell profiles, including both projection neurons and interneurons. This labeling was the most prominent in layers III and V, with sparse labeling in the intervening granular layer IV, and appeared to be present over both large pyramidal neurons and smaller cells that could represent interneurons. High power photomicrographs of PFC tissue sections from a schizophrenic (622s) and matched control (685c) subjects were viewed under darkfield illumination. Micrographs for each subject were taken under identical conditions. Roman numbers denote cortical layers. Pial surface is to the left. Strong labeling across all cortical layers except lamina IV was observed, and diminished labeling in the matched schizophrenic subject across all the layers was noted (scale bar=400 μm). White matter labeling was absent. 
     Based on optical density analysis, 9/10 subject pairs exhibited a 10.2% to 74.3% decrease in PFC RGS4 expression, as shown in  FIG. 2B . The in situ hybridization data from 10 PFC pairwise comparisons were quantified using film densitometry. The X-axis represents subject classes, the Y-axis reports average film OD from 3 repeated hybridizations, measured across all layers. Lines connecting symbols indicate a matched subject pair. Note that in 10 PFC pairwise comparisons, 9 schizophrenic subjects showed RGS4 transcript reduction (mean=−34.5%; F 1,15 =6.95; p=0.019). 
     Specificity of RGS4 Expression Changes 
     To investigate whether RGS4 transcript decrease is a specific alteration in schizophrenia, the same microarray data was analyzed for consistent gene expression changes across other RGS-family members ( FIG. 1C ). Nine of the eleven RGS family members represented with immobilized probes on the microarrays reported expression in four or more microarray comparisons. RGS13, primarily lung-specific family member, was not expressed in any of the comparisons, while p115-RhoGEF reported expression in only one comparison. RGS4 was the only family member (and the only gene on the microarray) to report a consistent change in expression over the 99% CL in every schizophrenic subject. RGS5 mRNA (a gene also localized to cytogenetic position 1q21-22) was decreased at the 99% CL in one subject pair, at the 95% CL in another subject pair, and unchanged in the remaining 2 pairs that showed detectable RGS5 expression by microarrays. Expression of the other RGS family members did not display any consistent differences across the schizophrenic subjects. The mRNA from pair 567c/537s was analyzed a second time on the newest Incyte microarray, UniGEM-V2, which includes five additional RGS family members (RGSZ, RGS1, RGS7, RGS11, and RGS14). This analysis confirmed that, in the comparisons, RGS4 was the only significantly changed RGS family member. 
     Heterotrimeric G-proteins, the main substrates for RGS family members, were assessed for expression patterns. Several reports suggest Gα changes associated with schizophrenia. Thus, it was desirable to assess whether the decrease in RGS4 expression correlated with changes in Gα expression levels. Of the eight Gα RGS substrates represented on the microarrays, only G o  expression was changed beyond the 95% CL in three or more pairwise comparisons. These three subjects with increased Go levels (317s, 547s, and 622s) showed the most robust decrease in RGS4 expression both in the PFC microarray and in situ hybridization assays. 
     Expression of 274 genes known to be involved in the G-protein signaling cascades (GPCR, heterotrimeric G-proteins, RGS, GIRKS, G-protein receptor kinases, and mitogen-activated protein kinases) were analyzed in a gene group comparison. An average of 105 genes belonging to this group were expressed in each comparison. The results of microarray analyses showing G-protein and 1q21-22 locus-related expression differences in the PFC of six pairs of schizophrenic and control subjects are shown in  FIGS. 3A and 3B . For both gene groups, all expressed genes were classified into signal intensity difference intervals (0.1 bins) according to their cy5/cy3 signal ratio. Transcripts in a “1” bin had identical cy5 vs. cy3 signal intensities. Positive values (to the right) on the X-axis denote higher cy5 signal in schizophrenic subjects (S&gt;C), negative values (to the left) correspond to higher cy3 signal intensity in the control subjects (C&gt;S). The Y-axis reports percentage of expressed genes across the six subject pairs per bin for each gene group. In both panels, the white bars (All genes) denote distribution of all expressed genes across the six PFC pairwise comparisons (n=22,408). Additionally, in both panels, RGS4 contribution to the transcript distribution is denoted by a hatched bar. Note that in both  FIG. 3A  and  FIG. 3B , the cy3/cy5 signal distribution of G-protein and 1q21-22 gene groups was comparable to the distribution of all expressed genes across the six microarray comparisons. 
     At the 99% confidence level, 5.6% of G-proteins showed a different distribution between schizophrenic and control subjects, as shown in  FIG. 3A : 2.8% of G-proteins were decreased, while 2.8% were increased in the PFC of schizophrenic subjects. Of the 2.8% decrease in schizophrenic subjects, RGS4 observations alone accounted for nearly half of the decrease. When RGS4 was removed from the G-protein group, a gene group analysis by χ 2  test and t-test closely matched the distribution of all expressed genes, suggesting that the majority of different expression levels can be attributed to normal human variability. Except RGS4, no other member of the G-protein gene group was consistently changed across the subject pairs over the 95% or 99% confidence levels. 
     The RGS4 gene has been mapped to locus 1q21-22, a novel schizophrenia locus recently implicated by pedigree studies with a linkage of disease score (LOD) of 6.5 as described by Brzustowicz et al. supra. To address if any other genes at this locus displayed altered expression in the PFC of schizophrenic subjects, 70 additional transcripts originating from this cytogenetic region were analyzed. At the 99% CL, 0.4% of 1q21-22 genes were increased, and 5.9% were decreased in the schizophrenic subjects. Of the transcripts decreased in schizophrenic subjects, RGS4 observations alone accounted for nearly half of the decreases, as shown in  FIG. 3B . Furthermore, of all the genes on the 1q21-22 locus, only RGS4 showed a consistent expression change across all the pairwise comparisons over the 95% or 99% confidence levels. Of the remaining genes on this locus, only the ALL1-FUSED gene (AF1q GenBank Accesion #U16954) reported consistent expression change over the 95% CL in the schizophrenic subjects in three or more pairwise comparisons. Furthermore, as a gene group, the expression of the remaining genes on locus 1q21-22 showed the same overall pattern as genes located on non-schizophrenia loci or the overall average gene expression which is shown in  FIG. 3B . 
     Regional RGS4 Gene Expression Changes 
     To test whether RGS4 transcript decrease is specific to the prefrontal cortex or includes a more widespread cortical deficiency, RGS4 expression was assessed by in situ hybridization in the visual cortex (VC) and motor cortex (MC) from the same 10 pairs of control and schizophrenic subjects (for pair 558c/317s MC material was not available, and this pair was substituted with pair 794c/665s). The figure layout for  FIGS. 4A–D  is similar to that of  FIGS. 2A–B . In VC, RGS4 in situ hybridization showed heavy labeling under darkfield illumination of diverse cell population in the gray matter, with a very prominent bi-laminar labeling pattern in the supragranular and infragranular layers, as shown in  FIG. 4A . Roman numbers denote cortical layers, scale bar=400 μm. There was very sparse labeling in the well-developed layer IV, with very few cellular elements exhibiting detectable levels of RGS4 mRNA. These high power photomicrographs show that RGS4 levels are significantly decreased in the VC region of the schizophrenic subjects. The OD measurements on these two layers were performed separately. 
     Across the same ten pairwise comparisons that were examined in the PFC hybridizations, combined RGS4 expression in supragranular and infragranular layers of VC was decreased by 32.8% (F 1,15 =8.24; p=0.012) as shown in  FIG. 4B . 
     In MC, RGS4 expression was concentrated over the cell-rich layers I–III and V–VI of both control and schizophrenic subjects, as shown in  FIG. 4C . High power photomicrographs of MC tissue sections from the same matched pair of schizophrenic and control subject are represented in  FIG. 2A  and  FIG. 4A , viewed under darkfield illumination. Roman numbers denote cortical layers, scale bar=400 μm. Because of the attenuated layer IV in motor cortex, the RGS4 labeling is almost uniform across all layers. 
     Similar to the RGS4 transcript decrease observed in supragranular VC, schizophrenic subjects across the same 10 subject pairs were analyzed in MC. The mean RGS4 expression in MC shown in  FIG. 4D , measured across all the layers, was decreased by 34.2% across the 10 schizophrenic subjects (F 1,15 =10.18; p=0.006) 
     In the PFC, VC, and MC of subjects with schizophrenia, RGS4 expression was consistently decreased compared to the PFC of subjects with the diagnosis of MDD, as shown in the schematic of  FIG. 5 . In contrast, factor analysis of the pairwise differences in RGS4 gene expression across 3 different cortical areas for all 9 common schizophrenic and control subject pairs revealed that over 84% of the total variance in expression was accounted for by diagnosis (variance proportion=0.848, eigenvalue=2.544, p=0.001. The X-axis represents experimental groups, the Y-axis reports percent RGS4 expression change in PFC, VC, MC, in schizophrenic subjects (SCH) and PFC of subjects with MDD viewed by in situ hybridization. Each symbol represents percent of change between a single pairwise comparison; same symbols represent the same subject pairs. Arrows represent mean expression difference for each group. The same schizophrenic subjects showed a comparable and highly correlated decrease in RGS4 expression across all three cortical regions (PFC-VC: r=0.88, p=0.0003; PFC-MC: r=0.69, p=0.0384; VC-MC: r=0.76, p=0.0144). In contrast, subjects with MDD reported variable RGS4 expression changes when compared to their matched controls. 
     The combined data indicate that RGS4 transcript changes are a result of the pathophysiological changes related to schizophrenia and not due to confounds. Furthermore, the RGS4 expression decrease appears to be specific and unique to schizophrenia, and not a hallmark of the major depressive disorder. 
     RGS4 labeling in the white matter was comparable to background labeling across all brain regions, suggesting that RGS4 is primarily expressed in neuronal cells. The labeling was abundant in the majority of interneurons and projection neurons. However, in some pyramidal cells and interneurons RGS4 labeling could not be detected. RGS4 labeling was heavy in all cortical layers, except layer IV, where RGS4 expression was both sparse and light. This overall pattern of labeling was comparable across all three cortical regions (PFC, VC, MC). As the granular layer IV is the widest in the primary visual cortex, in this region RGS4 labeling was prominent in supragranular and infragranular layers, separated by a wide zone of mostly unlabeled granular cells. The overall distribution pattern of the RGS4 message does not mimic the known expression patterns of neurotransmitter systems, suggesting that RGS4 regulates many functionally distinct neuronal populations. 
     Together, the microarray and in situ hybridization methods suggest decreased RGS4 expression is a consistent characteristic of schizophrenic subjects. Several causes of the reduced RGS4 expression may be offered, including adaptive and genetic changes in schizophrenic patients. It was hypothesized that reduction in RGS4 expression was generated by alterations in the RGS4 gene. In addition, it was contemplated that variations in the DNA upstream and downstream from the coding region of the RGS4 gene may also impact the expression of the RGS4 transcript. These possibilities were investigated by searching for SNPs in the RGS4 gene. 
     The specificity of the reduced expression of RGS4 message for schizophrenic patients was confirmed in a series of control experiments. The same reduced level of RGS4 message was not observed in patients suffering from major depressive disorder. In addition, prolonged treatment of non-human primates with the anti-psychotic haloperidol did not result in decreased levels of RGS message in the cerebral cortex. This result indicates that chronic exposure to anti-psychotic drugs are unlikely to be responsible for the depressed levels of RGS4 message observed in schizophrenic patients. 
     Genotyping Results 
     34 single nucleotide polymorphisms (SNPs) were identified after re-sequencing all exons, introns and flanking 5′ and 3′ UTRs of the RGS4 coding region ( FIG. 6 ). Thirteen SNPs were chosen for analysis using the TDT. SNPs are explicitly defined in Table 1. When the SNPs were tested individually, significantly increased transmission at SNP4 was observed in the Pittsburgh sample. ‘Moving window’ haplotype analyses using two to four contiguous SNPs, revealed significant association for several haplotypes; all but one included SNPs 1, 4, 7, or 18 (Table 2). A global test of association for haplotypes encompassing these SNPs was significant (TRANSMIT software, χ 2 =16.6, 8 df, p=0.035). There were 39 cases with schizoaffective disorder in the sample; these trends remained significant when the sample was restricted to individuals with schizophrenia (χ 2 =13.0, 6 df, p=0.043). 
     TDT analysis was conducted next in the ethnically diverse NIMH sample using the same set of SNPs. Significant transmission distortion was observed individually at SNPs 1, 4 and 18 (Table 2). Exclusion of African-American families from the sample also 
     
       
         
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                   
                   
                 Nu- 
                   
                   
               
               
                   
                   
                 cleotide 
                   
               
               
                   
                   
                 identity 
                   
               
               
                   
                   
                 in 
                 Observed 
               
               
                   
                 Location of the SNP 
                 SEQ ID 
                 Nucleotide 
               
               
                 SNIP # 
                 within the SEQ 
                 NO:3 
                 variation 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 27,859 
                 199  
                 {SEQ ID NO:4} 
                 T 
                 C 
                   
               
               
                   
               
               
                 34,653 
                 153 
                 {SEQ ID NO:5} 
                 C 
                 T 
               
               
                   
               
               
                 90,387 
                 87 
                 {SEQ ID NO:6} 
                 G 
                 A 
               
               
                   
               
               
                 SNP1 
                 4121 
                 {SEQ ID NO:3} 
                 C 
                 T 
               
               
                   
               
               
                 SNP2 
                 4123 
                 {SEQ ID NO:3} 
                 T 
                 A 
               
               
                   
               
               
                 SNP3 
                 4368 
                 {SEQ ID NO:3} 
                 A 
                 C 
               
               
                   
               
               
                 SNP4 
                 4621 
                 {SEQ ID NO:3} 
                 A 
                 C 
               
               
                   
               
               
                 SNP5 
                 4790 
                 {SEQ ID NO:3} 
                 C 
                 T 
               
               
                   
               
               
                 SNP6 
                 4816 
                 {SEQ ID NO:3} 
                 G 
                 T 
               
               
                   
               
               
                 SNP7 
                 4970 
                 {SEQ ID NO:3} 
                 C 
                 T 
               
               
                   
               
               
                 SNP8 
                 5055 
                 {SEQ ID NO:3} 
                 C 
                 G 
               
               
                   
               
               
                 SNP9 
                 5295 
                 {SEQ ID NO:3} 
                 G 
                 A 
               
               
                   
               
               
                 SNP10 
                 5695 
                 {SEQ ID NO:3} 
                 G 
                 A 
               
               
                   
               
               
                 SNP11 
                 7375 
                 {SEQ ID NO:3} 
                 G 
                 T 
               
               
                   
               
               
                 SNP12 
                 7759 
                 {SEQ ID NO:3} 
                 G 
                 A 
               
               
                   
               
               
                 SNP13 
                 8596 
                 {SEQ ID NO:3} 
                 G 
                 A 
               
               
                   
               
               
                 SNP14 
                 9603–9609 
                 {SEQ ID NO:3} 
                 AGTTTGG 
                 7 bases 
               
               
                   
                   
                   
                   
                 Absent 
               
               
                   
               
               
                 SNP15 
                 9892 
                 {SEQ ID NO:3} 
                 C 
                 A 
               
               
                   
               
               
                 SNP16 
                 9963 
                 {SEQ ID NO:3} 
                 C 
                 A 
               
               
                   
               
               
                 SNP17 
                 10132 
                 {SEQ ID NO:3} 
                 G 
                 A 
               
               
                   
               
               
                 SNP18 
                 11056 
                 {SEQ ID NO:3} 
                 T 
                 C 
               
               
                   
               
               
                 SNP19 
                 11091 
                 {SEQ ID NO:3} 
                 C 
                 T 
               
               
                   
               
               
                 SNP20 
                 11106 
                 {SEQ ID NO:3} 
                 C 
                 A 
               
               
                   
               
               
                 SNP21 
                 11774 
                 {SEQ ID NO:3} 
                 G 
                 T 
               
               
                   
               
               
                 SNP22 
                 12143 
                 {SEQ ID NO:3} 
                 G 
                 A 
               
               
                   
               
               
                 SNP23 
                 12145 
                 {SEQ ID NO:3} 
                 G 
                 T 
               
               
                   
               
               
                 SNP24 
                 14367 
                 {SEQ ID NO:3} 
                 A 
                 G 
               
               
                   
               
               
                 SNP25 
                 17028 
                 {SEQ ID NO:3} 
                 A 
                 Base absent 
               
               
                   
               
               
                 SNP26 
                 17630 
                 {SEQ ID NO:3} 
                 G 
                 T 
               
               
                   
               
               
                 118740 
                 120 
                 {SEQ ID NO:7} 
                 C 
                 G 
               
               
                   
               
               
                 130121 
                 221 
                 {SEQ ID NO:8} 
                 G 
                 C 
               
               
                   
               
               
                 Location of single nucleotide polymorphisms relevant to the present invention. The location of the SNIP within the sequence is listed as is the variation observed in the collected samples. SNP 14 is the absence of the listed 7 bases at the indicated location. 
               
             
          
         
       
     
     revealed significant results for these SNPs (p=0.023, 0.011 and 0.033 respectively). However, the transmitted alleles differed from the Pittsburgh sample. Moving window haplotype analyses revealed preferential transmission for more extensive chromosomal segments than the Pittsburgh sample. Like the Pittsburgh sample, all but one of haplotypes with significant increased transmission included SNPs 1, 4, 7 or 18. A global test for association was also significant for haplotypes encompassing these SNPs (TRANSMIT analysis; χ 2 =18.8, p=0.016, 8 df). 
     If the significant TDT results were due to linkage, it was reasoned that the affected sibships in the NIMH sample should yield evidence for increased haplotype sharing. For 30 available affected sib-pairs, the proportion of 0, 1, or 2 haplotypes identical by descent (IBD) were elevated over expectations of 0.25, 0.50, 0.25; namely 0.11, 0.44, 0.45 respectively (for SNPs 1, 4, 7 and 18 analyzed in conjunction with 5 flanking short tandem repeat polymorphisms genotyped previously). Increased IBD sharing was also observed when these sets of SNPs or STRPs were analyzed separately. 
     Association at the population level was assessed by comparing Caucasian cases from each sample separately with two independent groups of Caucasian community-based controls. Since SNPs 1, 4, 7 and 18 appeared to be critical for transmission distortion in both samples, genotypes and allele frequencies for these SNPs were analyzed. Haplotypes frequencies were estimated using an expectation-maximization algorithm (EM), paying particular attention to haplotypes VI and XI, the haplotypes with excess transmission in the NIMH and Pittsburgh samples, respectively (Table 3). SNP 14 was informative only among African-Americans and so was analyzed separately using 70 African-American cases and 93 control individuals from Pittsburgh. Significant case-control differences were not noted for any of the comparisons. The failure to detect association may reflect superior power for the TDT in the context of population sub-structure. 
     
       
         
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 Haplotype based comparisons among cases and unrelated controls. The 
               
               
                 Caucasian cases from Pittsburgh (n = 93) and NIMH (n = 25) were 
               
               
                 compared separately with unscreened Caucasian controls from Pittsburgh 
               
               
                 (n = 76). Bonferoni corrections have been applied for the Pittsburgh case- 
               
               
                 control comparisons, but not for comparisons involving the NIMH cases. 
               
               
                 An omnibus test based on likelihood ratios was used to estimate overall 
               
               
                 differences in haplotype frequencies (Fallin et al., Gen. Res. 11, 143–51, 
               
               
                 2001) and was significant for both comparisons (χ 2  = 88.7, p &lt; 0.0001 
               
               
                 and χ 2  = 30.1, p &lt; 0.0003 respectively for Pittsburgh and NIMH cases). 
               
               
                 Similar significant differences based on 3 SNP haplotypes were 
               
               
                 present, but are not shown. For each SNP, ‘o’ represents allele 1 
               
               
                 and ‘•’ represents allele 2. OR—Odds ratio; NS—Not significant. 
               
               
                   
               
             
             
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Pair-wise linkage disequlibrium between SNPs at RGS4. Population based control 
               
               
                 individuals (n = 76) were used to estimate linkage disequilibrium. The figures above the 
               
               
                 diagonal represent D′ and estimates for statistical significance (p values) are below the diagonal. 
               
             
          
           
               
                 SNP 
                 27859 
                 90387 
                 snp1 
                 snp4 
                 snp7 
                 snp18 
                 snp23 
                 118740 
                 130121 
               
               
                   
               
             
          
           
               
                 27859 
                   
                 0.096 
                 0.064 
                 0.076 
                 0.287 
                 0.009 
                 0.000 
                 0.000 
                 0.000 
               
               
                 90387 
                 0.132 
                   
                 0.000 
                 0.000 
                 0.000 
                 0.000 
                 0.000 
                 0.001 
                 0.627 
               
               
                 snp1 
                 −0.123 
                 −0.501 
                   
                 0.000 
                 0.000 
                 0.000 
                 0.000 
                 0.450 
                 0.477 
               
               
                 snp4 
                 0.101 
                 −0.501 
                 −1.000 
                   
                 0.000 
                 0.000 
                 0.000 
                 0.128 
                 0.515 
               
               
                 snp7 
                 −0.075 
                 0.783 
                 0.970 
                 −0.961 
                   
                 0.000 
                 0.000 
                 0.012 
                 0.068 
               
               
                 snp18 
                 0.177 
                 0.377 
                 −0.677 
                 0.989 
                 −0.961 
                   
                 0.000 
                 0.000 
                 0.041 
               
               
                 snp23 
                 0.527 
                 −0.302 
                 −1.000 
                 1.000 
                 −0.847 
                 0.674 
                   
                 0.499 
                 0.002 
               
               
                 118740 
                 0.385 
                 0.163 
                 0.048 
                 −0.083 
                 0.172 
                 −0.233 
                 0.042 
                   
                 0.000 
               
               
                 130121 
                 −0.505 
                 0.049 
                 −0.059 
                 0.046 
                 −0.163 
                 0.174 
                 −0.154 
                 −0.956 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
           
               
                 TABLE 4 
               
               
                   
               
               
                 SNPs and Haplotypes at RGS4 with increased transmission distortion. TDT analysis of case-parent trios 
               
               
                 included 93 families from Pittsburgh and 39 families from the NIMH cohort. Only statistically significant 
               
               
                 increased transmissions are shown. The shaded haplotypes correspond to haplotypes VII and X, 
               
               
                 respectively from Table 2. T/NT-Transmitted/not transmitted; o-Allele 1, ●-Allele 2 at each SNP; 
               
               
                 /-Allele not specified at this locus; *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.005 
               
               
                   
               
             
             
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
             
          
         
       
     
     The demonstration of the association between these SNPs and schizophrenia offers a large number of applications in the diagnostic and therapeutic fields. Thus, embodiments of the present invention offer the possibility of diagnosing schizophrenia by means of a biological test and no longer exclusively by means of clinical evaluations. Embodiments of the present invention can also be applied to diagnosing pathologies of the schizophrenia spectrum, such as, in particular, schizotypy, schizoid individuals, etc. Embodiments of the present invention make it possible to refine the criteria for diagnosing these pathologies, which is currently entirely established clinically. Furthermore, embodiments of the invention also makes it possible to demonstrate susceptibility to schizophrenia by means of identifying a genetic vulnerability in the families of patients who posses the identified SNPs in the RGS4 coding region and flanking regions. Once individuals have been identified as being susceptible to schizophrenia, the utility of prophylactic treatment may be investigated. 
     The DNA sample to be tested can be obtained from cells that have been withdrawn from the patient. These cells are preferably blood cells (e.g. mononucleated cells), that are easily obtained by the simple withdrawal of blood from the patient. Other cell types, such as fibroblasts, epithelial cells, keratinocytes, etc., may also be employed. The DNA may then extracted from the cells and used to detect the presence of SNPs in the RGS4 coding region and flanking regions. 
     Most preferably, the DNA extract is initially subjected to one or more amplification reactions in order to obtain a substantial quantity of material corresponding to the region carrying the RGS4 coding region and flanking regions. The amplification can be achieved by any technique known to the skilled person, and in particular by means of the so-called PCR technique as described above. To this end, embodiments of the present invention also relate to specific primers which make it possible to amplify DNA fragments that are of small size and which carry the RGS4 gene, flanking regions thereof, or portions thereof generated from SEQ ID NOS. 3, 4, 5, 6, 7, or 8. Portion of a polynucleotide sequence is specifically intended to refer to any section of SEQ ID NOS. 3, 4, 5, 6, 7, or 8 that can be used in the practice of this invention, such as use as a primer to identify the presence of SEQ ID NOS. 3, 4, 5, 6, 7, or 8 or variations thereof in a patient or a section of SEQ ID NOS. 3, 4, 5, 6, 7, or 8 that can be used to amplify the entire sequence. The phrase contiguous portion is meant to refer to a series of bases that are adjacent to one another within a polynucleotide sequence. In the context of the present invention, the word gene is intended to mean the protein coding region, the proximal 5′ and 3′ untranslated regions, as well as any distal and proximal regulatory domains. The phrase gene-coding region is meant to refer to the stretch of DNA that begins at the transcription initiation site and includes all exionic and intrionic sequences that encode a protein. 
     Embodiments of the present invention may also involve isolating DNA sequences and ligating the isolated sequence into a replicative cloning vector which comprises the isolated DNA of the RGS4 gene, based upon or derived from the cDNA of SEQ ID NOS. 3, 4, 5, 6, 7, or 8 and a replicon operative in a host cell. Additional embodiments include an expression system which comprises isolating DNA of the RGS4 gene, based upon complimentarity to SEQ ID NOS. 3, 4, 5, 6, 7, or 8 and operably linking this DNA to suitable control sequences. Recombinant host cells can be transformed with any of these replicative cloning vectors and may be used to overproduce the RGS4 protein. 
     Embodiments of the present invention also include kits that will facilitate the diagnosis of schizophrenia through the amplification of segments of the 1q21-22 locus. Several methods providing for this amplification are described including: at least a pair of single-stranded DNA primers wherein use of said primers in a polymerase chain reaction results in amplification of a portion of the RGS4 gene fragment, wherein the sequence of said primers is derived from the regions of the cDNA defined by or complementary to SEQ ID NOS: 1, 3, 4, 5, 6, 7, or 8. Similarly, embodiments of the invention also provide for a pair of single-stranded DNA primers wherein use of said primers in a polymerase chain reaction results in amplification of an RGS4 gene fragment, wherein the sequence of said primers is based on the exon regions of chromosomal DNA derived from SEQ ID NOS:1 or 3. 
     Various nucleic acid probes and primers specific for RGS4 (derived from or complementary to SEQ ID NOS. 3, 4, 5, 6, 7, or 8) may also be useful in diagnostic and therapeutic techniques and are included within the present invention. Among these are a nucleic acid probe complementary to portions or the entirety of human RGS4 gene as well as a nucleic acid probe complementary to human altered RGS4 gene sequences wherein said nucleic acid probe hybridizes to a variant of the RGS4 gene under hybridization conditions which prevent hybridizing of said nucleic acid probe to a wild-type RGS4 gene. Probes that are complementary to portions or the entirety of the RGS4 coding region and flanking regions that contain SNPs may also be used in these diagnostic tests. Any primer which makes it possible to amplify a fragment of the RGS4 coding region or flanking regions also forms part of the present invention. The primers that are used within the context of the invention can be synthesized by any technique known to the skilled person. The primers can also be labeled by any technique known to the skilled person. 
     The invention may also be practiced through detection of SNPs in the RGS4 coding region or flanking regions by a variety of techniques. The techniques which may preferably be employed are DNA sequencing and gel separation. 
     Any sequencing method known to the skilled person may be employed. In particular, it is advantageous to use an automated DNA sequencer. The sequencing is preferably carried out on double-stranded templates by means of the chain-termination method using fluorescent primers. An appropriate kit for this purpose is the Taq Dye Primer sequencing kit from Applied Biosystem (Applied Biosystem, Foster City, Calif.). Sequencing the SNPs in the RGS4 coding region and the flanking regions makes it possible to identify directly the SNPs that are present in the patient. 
     An additional preferred technique for demonstrating the SNPs in the RGS4 coding region and flanking regions is that of separation on a gel. This technique is based on the migration, under denaturing conditions, of the denatured DNA fragments in a polyacrylamide gel. The bands of DNA can be visualized by any technique known to the skilled person, with the technique being based, such as by using labeled probes that are complementary to the entirety or portions of the RGS4 coding region and flanking regions. Alternatively, the bands may be visualized by using ethidium bromide or else by means of hybridization with a radiolabeled probe. 
     In addition, measuring the expression of RGS4 message in peripheral tissue allows the diagnosis and determination of the susceptibility to schizophrenia in humans. As a matter of convenience, the reagents employed in the present invention can be provided in a kit packaged in combination with predetermined amounts of reagents for use in determining and/or quantifying the level of RGS4 expression. For example, a kit can comprise in packaged combination with other reagents any or all of the following components: appropriate detectors, buffers, deoxynucleotide triphosphates, ions provided by MgCl 2  or MnCl 2 , and polymerase(s). The diagnostic kits of the invention may further comprise a positive control and/or a negative control as well as instructions for quantitating RGS4 expression. 
     Additionally, an embodiment of the present invention relates to ascertaining levels of the RGS4 protein. The level of RGS4 protein can be detected by analyzing binding of a sample from a subject with an antibody capable of binding to RGS4. An embodiment of this detection method utilizes an immunoassay. The sample from a subject may preferably be a biopsy of skeletal muscle, though any tissue accessible to biopsy may be used. 
     In addition to providing generally useful diagnostic kits and methods, embodiments of the present invention may provide a method for augmenting traditional treatments by supplying the RGS4 protein to a subject and/or augmenting the subject&#39;s medication, such as antipsychotic drugs, and providing an improved therapeutic outcome. 
     Further embodiments of the present invention may relate to the construction of an animal model of schizophrenia. Transgenic mice technology involves the introduction of new or altered genetic material into the mouse germ line by microinjection, retroviral infection or embryonic stem cell transfer. This results in lineages that carry the new integrated genetic material. Insertional mutagenesis occurs when integration of the microinjected genetic material into the host genome alters an endogenous gene resulting in a mutation. Methods of transferring genes into the germline, the expression of natural and hybrid genes and phenotypic changes that have occurred in transgenic mice are described by Palmiter and Brinster in Ann. Rev. Genet. 20 (1986) 465–499. Methods of foreign gene insertion, applications to foreign gene expression, and the use of transgenic mice to study immunological processes, neoplastic disease and other proliferative disorders are described by Gordon in Intl. Rev. Cytol. 115, 1989, 171–299 both of which are hereby incorporated by reference. A further example of genetic ‘knock-in’ technology may be found in Nebert, et al., Ann. N.Y. Acad. Sci. 919, 2000, 148–170 which is hereby incorporated by reference. The insertion of SEQ ID NO:3 containing some or all of the described SNPs into a mouse germ line may be expected to result in adult mice that may be used as an experimental model of schizophrenia. The insertion of SEQ ID NO:3 containing one or more of the variations listed in Table 1 with standard on:off regulatory domains will allow for the creation of mice deficient in RGS4 expression at specified times, and may be used as an experimental model of schizophrenia. 
     Having now fully described embodiments of the present invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the invention.

Technology Classification (CPC): 2