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).

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]     This application is a divisional application of pending U.S. Ser. No. 09/939,209, filed Aug. 24, 2001, which claims priority under 35 U.S.C. 119(e) to U.S. provisional application Ser. No. 60/288,021, filed Aug. 24, 1999. 
     
    
       [0002]     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  
       [0003]     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  
       [0004]     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.  
         [0005]     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.  
         [0006]     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.  
         [0007]     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.  
         [0008]     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.  
         [0009]     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 “atypical” 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.  
         [0010]     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.  
         [0011]     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.  
         [0012]     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).  
         [0013]     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.  
         [0014]     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.  
         [0015]     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.  
         [0016]     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  
       [0017]     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.  
         [0018]     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.  
         [0019]     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.  
         [0020]     A further aspect of the present invention is an expression system comprising a DNA sequence that corresponds to SEQ ID NO:3.  
         [0021]     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.  
         [0022]     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.  
         [0023]     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.  
         [0024]     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.  
         [0025]     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.  
         [0026]     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.  
         [0027]     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.  
         [0028]     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.  
         [0029]     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.  
         [0030]     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.  
         [0031]     A still additional aspect of the present invention is a transgenic mouse wherein a transgene comprises a nucleotide sequence that encodes a selectable marker.  
         [0032]     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  
       [0033]     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:  
         [0034]      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;  
         [0035]      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 (c-control subject) was 6.2-fold brighter than the cy5 signal intensity (s-schizophrenic subject);  
         [0036]      FIG. 1C  displays changes in RGS expression in the PFC of schizophrenic and control subjects reported by cDNA microarray analysis;  
         [0037]      FIG. 2A  shows in situ hybridization results for PFC RGS4 expression levels which are decreased in 9 of 10 schizophrenic subjects;  
         [0038]      FIG. 2B  shows the in situ hybridization data from 10 PFC pairwise comparisons which were quantified using film densitometry;  
         [0039]      FIG. 3A  shows that 632 G-protein signalling-related genes were detected out of 1644 possible detections (274 genes/microarray×six microarrays);  
         [0040]      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;  
         [0041]      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;  
         [0042]      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;  
         [0043]      FIG. 4C  shows high power photomicrographs of MC tissue sections from the same matched pair of schizophrenic and matched control subjects represented in  FIG. 2A , viewed under darkfield illumination;  
         [0044]      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;  
         [0045]      FIG. 5  shows a scatter plot of relative RGS4 expression changes across the experimental groups.  
         [0046]      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  
       [0047]     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.  
         [0000]     Expression Studies  
         [0048]     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.  
         [0000]     RGS4 Transcript Analysis  
         [0049]     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 ).  
         [0000]     Microarray Experiments  
         [0050]     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 10 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.  
         [0000]     Sample Labeling, Microarrays, Hybridization, and Data Analysis  
         [0051]     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 following NIH website: “http://www.ncbi.nlm.nih.gov/UniGene/index.html”. 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-cys 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.  
         [0000]     Gene Expression Criteria  
         [0052]     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 (“http://www.incyte.com/reagents/gem/products.shtml/GEM-reproducibility.pdf”) 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.        
 
         [0055]     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.  
         [0000]     Gene Group Analysis  
         [0056]     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.  
         [0057]     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.  
         [0000]     RGS4 Sequences  
         [0058]     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   60           tctttcctgc ttgcgaattc               caagctgtta aataagatgt gcaaagggct tgcaggtctg   120       ccggcttctt gcttgaggag               tgcaaaagat atgaaacatc ggctaggttt cctgctgcaa   180       aaatctgatt cctgtgaaca               caattcttcc cacaacaaga aggacaaagt ggttatttgc   240       cagagagtga gccaagagga               agtcaagaaa tgggctgaat cactggaaaa cctgattagt   300       catgaatgtg ggctggcagc               tttcaaagct ttcttgaa9t ctgaatatag tgaggagaat   360       attgacttct ggatcagctg               tgaagagtac aagaaaatca aatcaccatc taaactaagt   420       cccaaggcca aaaagatcta               taatgaattc atctcagtcc aggcaaccaa agaggtgaac   480       ctggattctt gcaccaggga               agagacaagc cggaacatgc tagagcctac aataacctgc   540       tttgatgagg cccagaagaa               gattttcaac ctgatggaga aggattccta ccgccgcttc   600       ctcaagtctc gattctatct               tgatttggtc aacccgtcca gctgtggggc agaaaagcag   660       aaaggagcca agagttcagc               agactgtgct tccctggtcc ctcagtgtgc ctaattctca   720       cctgaaggca gagggatgaa               atgccaagac tctatgctct ggaaaacctg aggccaaata   780       ttgatctgta ttaagctcca               gtgctttatc cacattgtag cctaatattc atgctgcctg   840       ccatgtgtga gtcacttcta               cgcataaact agatatagct tttggtgttt gagtgttcat   900       cagggtggga ccccattcca               gtccaatttt cctaagtttc tttgagggtt ccatgggagc   960       aaatatctaa ataatggcct               ggtaggtctg gattttcaaa gattgttggc agtttcctcc   1020       tcccaacagt tttacctcgg               gatggttggt tagtgcatgt cacatgacat ccacatgcac   1080       atgtattctg ttggccagca               cgttctccag actctagatg tttagatgag gttgagctat   1140       gatatgtgct tgtgtgtatg               tctatgtgta tatattatat atacattaga cacacatata   1200       cattatttct gtatatagat               gtctgtgtat acatatgtat gtgtgagtgt atgtatacac   1260       acacacacac acacacacac               acacttttgc aagagtgatg ggaaagaccc taggtgctca   1320       taactagagt atgtgtatgt               acttacatgg gtgttttgat ctctgttctt tcatactaca   1380       tttgaacagg gcaaaatgaa               ctaactgcca tgtaggctaa gaaagaaatg ctaacctgtg   1440       gaaagttggt tttgtaaaat               tccatggatc ttgctggaga agcatccaag gaacttcatg   1500       cttgatttga ccactgacag               cctccacctt gagcactatt ctaaggagca aataccttag   1560       ctcccttgag ctggttttct               ctgatggcac ttttgagctc ctaagctgcc agccttccct   1620       tcttttcctg ggtgctcagg               gcatgcttat tagcagctgg gttggtatgg agttggcaga   1680       caggatgttc aacttaatga               agaaatacag ctaaggcctt gccagcaaca cctgccgtaa   1740       gttactggct gagtgagggc               atagaagtta aaggttactg tttttatcct ctatcctttt   1800       ttcctttcct gatcaaggtg               ctcttctcat tttttcctga gaaccttagc catcagatga   1860       ggctccttag tttattgtgg               ttggttgttt tttctttata atggctctgg gctatatgcc   1920       tatatttata aaccagcagc               aggggaaaga ttatatttta taagagggaa caaattttca   1980       caatttgaaa agcccacata               agttttctct tttaaggtag aatcttgtta atttcattcc   2040       aaacatcggg gctaacagag               actggaggca tttcttttta ggctctgaga ctaaatgaga   2100       ggaaaagaaa agaaaaaaaa               aatgattgtc taaccaattg tgagaattac tgtttgaaac   2160       ttttcaaggc acattgaaat               acttgaaaac ttctcattta tgttatttat gatgttattt   2220       tgtacgtgtt attattatta               tattgtttta taaatggagg tacaggatat cacctgaatt   2280       attaatgaat gcccaggaag               taattttctt ctcattcttc taaaactact gcctttcaaa   2340       gtgcacacac acgcgtccac               atacactgca ttcgttgctc cagtataaat tacatgcatg   2400       agcacctttc tggcttttaa               gccaatataa tgggctgcaa aatgaagaca ccagagtgta   2460       tgcatacaaa tctcactgta               ttaaagatgc aggttttcta attgtaccct tcttgtctct   2520       ctggcaatct tgcccttaat               atccctggag ttcctcatca gtgtcatttt ctgttataca   2580       cagttccaca attttgtctc               tagttgactt caaatgtgta actttattgg tcttgcccta   2640       ttataattgt catgactttc               agattgtatc tgaactcaca gactgctgtc ttactaatag   2700       gtctggaagg tcacgctgaa               tgagaagtaa attattttat gtaatacatt tttgagtgtg   2760       tttttcagtt gtatttccct               gttatttcat cactatttcc aatggtgagc ttgcctgctc   2820       atgctccctg gacagaatac               tccttccttt tgcatgcctg tttctatcat gtgcttgata   2880       ggcctcaaag ctaatgcttc               cagtgaaaca cacgcatctt aataataagg gtaaataaac   2934       gctccatatg aaac          
 
         [0059]     For purposes of the present invention, the RGS4 cDNA will be referred to as SEQ ID NO:1.  
         [0060]     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          
 
         [0061]     The above amino acid sequence of RGS4 is referred to as SEQ ID NO: 2 for purposes of the present invention.  
         [0062]     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   60           ctactaaaaa tacaaaatta               gacaggcatg gtgatacacg cctgtaatcc cagctacttc   120       ggaggccgag gcaggagaat               cacttgaacc tgctgggggt ggaggttgcg gggagcaaga   180       tcatgccatt gcactccagc               ccaggcaaca agagcgaaat gtcatctcag aaaaaaaaaa   240       aggcatttta tatatatata               tatatatata tacacacaca cacacatata tatatacaca   300       tatatataca catatataca               tatatacaca tatatacaca tatatataca catacatatg   360       tacacatata tatacacata               tgtatacaca tatatacaca tatatacaca catatataca   420       catatataca cacatatata               cacatatata cacatatata cacatataca catatataca   480       catatataca tatatacaca               tatatataat atacacacat atatatacac atatatacac   540       acatatatac acatatatac               acatatatat acacatatat acacatatat acatatatac   600       acatatatat acatatatac               acatatatac atatatacac atatatacat atatacacac   660       atatatacac atacatatac               acacacatag atatacatat atatacacat atatatacgt   720       atatatatgt atatatatat               gctccagagt tcataagagg tagcagttga ttaccactgg   780       ggatagagga aaagagagtt               tgacagcagt gtattgtgag aaggacattt caggttgatg   840       gcaaatagta ggggaaatac               ataaatgtgt aataaaacct atctgtaagg tagttaagaa   900       ggtaacacta tatatatata               tagtgaaagc agtgtaaacc taaaggatgg gccaaggatt   960       taaatgttat agaagaatgg               ctaagatgcc aaagctcagt gtatgtggca gaggcatggt   1020       gtagggtgtg tccaggttca               tatattgcat taagtgtgag aacaccctgg agtatgaacc   1080       aagaaaatgc aaaagccaga               agtgatggag gaaatgagac acaataatga agatattgag   1140       aggagggtgt gggcctagag               tgaagctttt cgtgccagta cttcttttga aggcccagtt   1200       ctcttctctc tcgggggctc               cttcatctct catagagtcc acagctttta agggccaaca   1260       cttgaggtca gcctggctct               ctcatttgag ctggatagaa cattttagag caccatctat   1320       tcttcaagag gaagtttaaa               aataaaagaa ccttgaagag gaaaaaatgt agacattcaa   1380       tctaaccttt tcattttact               agccaaagct aaatagaatg caggttacct gtttttcagc   1440       caggcaccat catttcctaa               ttgttataaa atttattatt attgttgtta ttattattat   1500       ttgccataag aagtttccca               tatcctttta gtataacaaa aacacaattc acaagcatta   1560       taaaacccat ggtgtctaac               tattaaaaaa attaagtgga acacacttgt cccagctact   1620       ggggaggctg aggagggagg               atcacgtgat cccagggggt caaggttatg gagagctatg   1680       attgtgccac tgcactccag               cctgggtgac agggaaagac cctgtctcta aaattttttt   1740       taaaaaaact aaactggttt               tattacagag attctggaga cagctacaca taaaagggtg   1800       gtatgcctca tattagctac               ccagggaggt ggaatgccaa cttaggtggt gtcaccacta   1860       ttaaaaatgc cccaaagcaa               tcaaaactga gaacttcctg ggagcttagc attgtgcaaa   1920       agcagcacaa aacacttaaa               caattcacag ttgtgttgga atgggaaggc ctggaaatat   1980       aaaccaaaga gtatattgtc               taaattgata gagattacaa ttgcctgaaa gaaaaagttg   2040       acttttaact agaatgttca               gagtaggttt acagaagaag ctcttaaact gggctccagt   2100       ggatttgtca atgctttgga               agctggtggg gtgggagggt tggagggggc ataaaaagtc   2160       atgttggtat gctctgctca               agtctccatt ctgtttcctt ttcctctttt caatgtcatg   2220       tcccattatt tcattatggg               cttcccttta tccaggatca atatgccacc tcttggttgt   2280       cttttaccta cttctccacc               tcactatgga atcgtccttg ggtagctcct gtgcttggga   2340       acctgcacgg gcacttttct               gatgtcttga ttccagcttt actcctaaaa cttaaatgct   2400       gaggggccaa caccatggca               gtggtaggga tgggaatggg ggtcttgtaa cacactacat   2460       aaactacacg aaataaacta               catgaaactc aacatgtttg caagactcag ttcacatcca   2520       tgaggagctc atgcttctcc               ctcctgctcc cctagcacac atgattatct ctatttggaa   2580       atgtttggca tttttggtga               agtgaatggt tcaataactt tctccaccat cagaacaaaa   2640       gctctttaag gttagggatg               ggatcataca cacttccctt gtccaagtcc ccatcacccc   2700       ttatctagac aattgctaca               gtttcctaca cactcttcta acctcttgca gtctattttc   2760       ataaaacagc tagagaactt               tgagatgtaa gtcaaaaaat agaacatgtc gctctttccc   2820       attgtttttg aaataaagtt               caaccccctt accagggtca acaaggccct gcaatgattt   2880       ggtcctgtta aaaattcttt               agccttaact catgctgttc ttccttacac tcactgcatt   2940       ctagccattg aggtttctat               gcatcaaact ttttttggtc ccagcactgt gcacatcctt   3000       ctgggtagaa tgccccttga               tttgtataat tagcacctcc ttcatcattt aggtcttagt   3060       ataactacta ccttcttaga               gaagctctgc ttcttcatcc tataaaaaag taaaattcct   3120       taccctgtta ttttttaagt               catccgtgtt tcattctgtt aaagttctta tcacaattta   3180       tcattatttt atttacagtc               atgtgccaca taacaatgtt tcagtcaggg atagaacaca   3240       aatgtatctg gccccataat               attataagct gagaaatttc tattaactag tgatatcgca   3300       gccatcataa gtgtaatgca               ggacattacc ttttctatgt ttagatatgt tagatacaca   3360       aatatatttc attgtgttat               aatttcctac agtattcagt acagtaacat gctgtacagg   3420       tttgtaacct aggagtaata               ggctatacca tacagcttag gtgtgtagta ggctataacc   3480       atctaggttt gtgtaagtac               attctatgat attcccacaa tgatgaaatc acctaactac   3540       acatttctca gaatgtttca               ctgttgtgaa gtgacccatg actatatttt cctatatact   3600       tgatattttt gtgcatctgc               ccatgagaat gtagtgtaag atcaaaggat gcaagaatgg   3660       gttctatcca gtatagtacc               cactacactg gtggatgtca atatgtattt gttagattaa   3720       tatctcaaga atgagcacct               ttctcagaca cataaaagat gctcaatata aaagtttgtt   3780       gaactgaacg ttattggcaa               atgtaacatg atcggattta aagaggagcg aaacagaggt   3840       ctggctcaaa caccatactt               ctagagtgca taagaggtag cagttgatta ccactggcga   3900       caggagaaaa aagagcttga               ccgcagggta ctgtgaagac atttcaggtt gatggcacag   3960       aacaggggaa atacataaat               gtgtgggaat attcagtggt ctgggatgac tacatagtag   4020       aatataatga agaaaagagt               ggaagggaaa gatgaaaagt tggaatgggg atgaattatg   4080       aaagtaccag aatgttatgc               taaggaatct agattttaaa atgtgagggc aaattgaagt   4140                 c         gggcacg ttacaaaact               agaggtcata aagtttaccc taatttacca agatttccta   4200       gaggatctat aattggaatc               cagatctgcc tctctgtaaa gttcaagcac tttccatgac   4260       accatactgt ttctttccac               ctgcacaatg caaatgaact cttatgaaac tgctgtttct   4320       atcctgggct aaatgttgca               gaaaaaagat ttaatctttg ggataaggct attttgggtt   4380       ttctcct         ct tcttgggaaa               caaggttttc ttcccctggc taattaagtg tggtattgtt   4440       cttccaggga aatcagtgat               gcatcacctg ctgctatcaa atgtcagggt tggagttcct   4500       gatttattgc atgtgcccac               aaagcttggt gcaaagaatt ggacacattt cccaaaagta   4560       agacatactg ggaagtccct               gtttaccttc ctggtataca gcatcctcca gccccatatc   4620       tttgcttttt agtcctaaaa                         tcaataact gaactctcat tgatgtctag gccattgtag   4680       taaacaataa agaaggaggg               aggcttctga caactgagag gaaattgtca tctgaagtgg   4740       tgcaagcaca gcctggggct               gagccttggc ctacatcctg cccaagtgga ggatcagtg             4800       cccatttaac atctggtaga               actaaagaac gcaac         cctg ccacaatgac ttatttccct   4860       gcatttgata ccgtcaatcc               ttgagaaatg ttttcttttg ttctccctga gcaaaggttg   4920       gaaaaatttg aaatttacct               agagaccaca catagttcac atcctgctgt gtggctgaat   4980       gtctgcccc          cagtaggaaa               cagttcttct aaagcctatt gtcaacaata ccttccagat   5040       gttagcattt tacaatttaa               ggaacttaaa atag         cttca aactttttgc cagtttctct   5100       gatatccaat ctattctttt               actctgcctc ccaagctttc tttctagaat gctaacctga   5160       tcggcttaag tacttgaact               acctcttctc ctccattaac tacagagtaa attctggtct   5220       tcagagtaac aagaaacacc               ctttagttct cagcatattc gtgcaccttc atttatctct   5280       ccttctctct caaagctgca               gtaggggtga aaac         tgtga tacattttct cttccatcat   5340       aagggtcgca accaaaactc               ctatagtaaa agacaggtta ataagagcaa aacctaacaa   5400       atttatttaa tcaaagtttt               acatgacatg ggagtcttca gaaatgaaga cccaaagacc   5460       caggggaaac tgtctgtttt               ttttgctgag gttcgatgaa gaatggatag catgtagcca   5520       tgtagattag acaaaaggat               atgatctagt ggtaaaggac tcagggggaa acacagcaag   5580       gcctgtctat tcagattctt               cttgatctct ctctctctat gtatagcatt ctttcctcct   5640       gagtatgggg caggactctt               cttcaatgag ggtcttcaag ggagaaggga gaaagtggcc   5700       tttttagatt ttat                   cttg               cttcggggaa gaggagttct agtttctatg acccatcttg   5760       gggaagagga attctggttt               ctgtgacttg ctttcatgaa gaaagaggag taagaggcag   5820       gagggcagga gatggtcaga               aagagacttg gctgcttctg agggcttccg ctctccttta   5880       gttccaagta cttcttagca               taccaaagca ctatactttg gcatatggtt ttctgagctc   5940       taacactgca atcatgctaa               actcctctat gaccttcaaa cattccactt gcttttattc   6000       tttatggttg tgatggcata               gaggtcaata gcaaagaccc tggagtccca ctgtctgagc   6060       tggcataaca ttactaccac               ttaatcaatg tgtaagctca ggtaagtact taagtcctct   6120       atgcttcatc tgtaaaatga               gaatcattga agaacattct ctcaggatgg atcatgagga   6180       ataagtgaat taactggcat               atagtgctta aaccagtgcc ttgctcagtt agtgacagat   6240       aaaatcatct gttattactg               tgcccactat tgtgatgctc ttctcttctt tgtacaacga   6300       ctacatctct atttatcatt               ttagggtctc cttgtgaaaa accactccag attcaaaaga   6360       ttgagtttaa tctctatcct               ctgtgctttc ctggagtttt gtaaagtaaa tcttcacttg   6420       acatcatgga taggttcttg               gaaactacaa cttcaagtga aaggacataa ctaaaccaat   6480       ttttttctca tcaacgttat               aatgaaatgg cattgatgaa atgatggcat tcaaggacct   6540       gctgtacctt gtttcactta               aagtcactgt ttccaataat ctattgatga cattgaggac   6600       ttactatata ataataaata               tatatataat cgacgaaaca ggaatcaaac tgctaactct   6660       gctaactggt ctccctgctt               ccacactctg cccactcatc tcagtctttc tttcacaaga   6720       gtcagaatga tcagatgaga               cccctcctct gcttctgttt cttccatgga tttccactgc   6780       actctgataa agtccagcct               cttgaccaca gcctacaaat ccttgcacga tctatcgttt   6840       acttttccat ctccttttat               gctactttca tcttgttctc aattctctag ctatgctggc   6900       cccttcttgt tctttcccat               ttttttttaa tttttaaaat ttgtatatat ttatgggtta   6960       taagtgaaat ctttttagat               gcataggttg tatagtgata aaatcagggc ttttagggta   7020       ttcatcacct gaatgatgta               cattgtaccc cttaagtaat ttctcaccat ccgctgactt   7080       cttgccccct gggtattcat               cacctgaatg atgtgcattg taccccttaa gtaatttctc   7140       accatccgct gacttcttgc               cccctgggta ttcatcacct gaatgatgtg cattgtaccc   7200       cttaagtaat ttctcaccat               ccgctgactt cttgccccct catccttctg aggctccatt   7260       gtccatcatt ccacactcta               catctatgtg tacacattat ttagctccta cttataagtg   7320       ataacatgca atatttgtct               ttctgtgtct gtcttgtttt acttatgata atggccccca   7380       gttctatcta ggct         ctgca               aaaggcatga tttcattctt ttttatggct atgttctttc   7440       ccaatttaga taaagaacac               tcgcacttgc tcttacttct atttggaata ctaattccta   7500       ggcttcttgc attgctttct               ccttctcacc catcaaatct cattttagat accacctctt   7560       caaagagggc tttcctgacc               accttggctg aattagccct tcaccatctg attactctct   7620       agcacatcac ctgcccattt               tattcatggt acaggtcaaa atctggaatc acctgatttg   7680       tttattttct gactccttct               actgagatga aaactctact agagcggaga ttttatctgc   7740       ttgtatcagg tactgcttca               aacagcacct gatacaga         t aggtggtcaa aagatatttc   7800       ttaaacaaat gaacaaataa               aaagtagatc ttttgagagt aaagctcttc cacactacca   7860       gagtcattca ggaatgacaa               atcatagaat aacagaattt gatgctttgt gcatatcaga   7920       gaaagaaggt ggaaggttgt               caaggtatca tgatgtacca gtcctcgcct cctcaaacac   7980       aatctgcaag tcccacagtg               aaaaagtaag ttaactcatg tgaagcgttt tacaaacact   8040       tttttaaaag tcttaaaact               cctaagaaag caagatttaa tagtcaaaga agtgagtaaa   8100       catgaaatgc ctgaacagag               taatgagcta agcacaaagt tagagacatg ttagttaata   8160       tgtcttgaaa gcagcagctc               ctgctttcaa ggagcaagaa caaattgggc aagtgaacac   8220       tccttgaata aaatgtgtaa               aattaatttt gggttatgtt ctatactgtg tataatagaa   8280       tgataaaaat tatttgacta               gcactttgta gtttagaaat atctctattt acacagttta   8340       ccttatttga taagactgtt               gagtgatggg atagcatggt ggacaatcca cataactgag   8400       tatcgagaca cctgtatctg               gacccagctc tgttagtaag aagctgtaac ctcagcaagt   8460       cactttctct ttctgggtct               ctatttcctt tttggtgaaa tgagagtgtt aggctagatt   8520       gcctttgaag tcccattttg               tctttaaagt cccatctatt gcagtgattt atatttaact   8580       catgacaaat caggcttctc               ttattctaag tgcaa         acat aaaactttta ttgtggaatt   8640       tcaggcatca gtaaatcttt               ttgggtactc acttatgttc ctgaaatcaa tctatttgag   8700       tgatcactct tttaggtgcc               caggtaaaca aagaaggcca tggtctttct ttgagtgacc   8760       ttctttccct tttaattagt               ctgacctctt taatgtcagt tctgactgat tcatttccct   8820       ggtccatctt ccttggtctg               agggccttcc tagtttcata ttgcacttca gttccttcca   8880       caccaccatc aaggatggct               gtcaacattc atttgttcta tgttataatt caaggaaaag   8940       ttgcccagta gctaatccaa               taaatgccct cttatgggcg gctagagact ttttcctata   9000       atttaaatgc atcttctgta               gattatggtc cctccaccac tttacatttg tctgctgtct   9060       ccttgctctg ctagtcatgg               aacgtgttgg tagtgggggc agtgtgggat gttcaagggc   9120       acgtattggg tagggccaca               tatgggcatt gctttgtgcc attctttcta tatttttggt   9180       attttgcatc tcactggaac               ccaactattt ttcatctctt ccacctaaac tatttgatgc   9240       ctctgtttct tatatataaa               gtatagctca ctgtagccta tgatcaggaa cctatctgct   9300       ttctaaatga aagctgtttt               ggtcagatct agcaattaat tcccttcttc cacttatagc   9360       tttcctctgt aactctggtg               taggtatttg gtttatggct ataagatgtg aaacacctga   9420       atgattctgt ccatgcaggc               atttcagttc atgatattgt atgtaaaaga tactgattgt   9480       ctaggtgttc agaaacacct               atagggctta atattcttac aatcagtttg aaggctggtg   9540       atacgcaaag caaactacat               atttttctgc ctgctctctc tctttctctc tacatctctc   9600       tttctttatc ttttgaaata               tcagtttgga gacttagaat tacataagac ataaacccat   9660       ttgatataag aattgctgtg               tatatttgct catctactcc ctcctttggt cctcgagctg   9720       ccggtttaga ctttttacag               gacgcaggca tgtgaaggag aaactgtcag tgctaggctg   9780       aattctgttg ttaccaagat               ttctagaaaa gtattcctca gtcaggttga ttacagatat   9840       agcaaatcta tttttcctag               ggtagtttct gtatgctgcc gggcttataa ctgtctgtca   9900       tccagctatt t         tctccacc               ttcttgtttg cataacaacc aaggcaactt ccgcaaatca   9960       ctgcgtggag acgatgatcc               tg         cagctcc cttttggaaa tcgtgaggat cagatcttgg   10020       accatgtata atatgatgct               tctaatccaa aagaggaaag gcattgggag tcagctccta   10080       agtaagctcc agaattcctg               ctggtacttt tccttccagg aagcaacttc cttgatattt   10140       tttttttaca g         catatgaa               taaaaactat attttgcagc attgtacact ttttttcctt   10200       ttctagaaat tctaaacctc               tgacattggt ggagacattg agtacatttt ttcccatatc   10260       cctacttttc agaaggattt               tctctgctcg ttcacttaac attgctgatg cgtcagtctt   10320       ttcttcctca tctctttcag               gggctggaga ggcagaggga gacagaggag ctggtactgc   10380       agagcggtcg tctgattggc               tggacggtcg tagctgggct ataaaagaga cccctacagg   10440       cttagcagga agacgctcag               aggattctga caatatcttt accggagaag aggcaaagta   10500       cgctcaaagc cgaagccaca               gctcctcctg ccgcatttct ttcctgcttg cgaattccaa   10560       gctgttaaat aagatgtgca               aagggcttgc aggtctgccg gcttcttgct tgaggaggta   10620       agattgcttt cagccattaa               ccatattaaa cttttggcta gactttctca gttatttaca   10680       tgttgtactt actaacctag               ttctgtgcaa ttagaaacag tgtggtcagg agagcacgac   10740       tttctaactt tcctccaaga               ctagctagat attgtgactt aagacatgtg ctccccaaat   10800       ttcagccctt atgtgttgtt               ttgtgtgacc tcagttttga gaactgttct attctttaag   10860       ccaggtctaa gaaagctagt               tttaattaag aagcgagatg aggtttgagg ctatgtacag   10920       tgatctgtaa tatctccatc               tgtgattact actgctattt gagcatccct ggagtacata   10980       gaagcctggc tctgggcttt               ctgattgtat gctacaactt gtttcaggaa aggtacccca   11040       gaatgaggtt tggctccatc               atcagaaagg cacta         gctt tccgtgtggt ggtgcagtaa   11100       ctttcactct          tatgttctt               ataag         aaat gttacaatga gatatgagtt ttaaagccag   11160       atcttcctta tctctctgcc               ccatctctag ttcttgaagt gtctcatatg agtttggttg   11220       agaaatattg atcattacaa               atcagttaat agttttgtag aagatctcat cttaaagaca   11280       ttgttttgtt aatatactcc               cttgattttt ttaaaagacc ttacagacat acagctattc   11340       atttgttttt ggtttgttca               aaaaaggtat aaagaaatgc attcagagaa agatcatata   11400       ttagccagtt gaaaattaaa               cacaaaatga gtgcatatta cattacttaa tcttgcagtc   11460       aaaggtaaaa agtcaaccta               aaggtatact acctgctttc ttatcgcact gcaaatagaa   11520       attaccacaa attttatttt               ggaaataatc tcagaaaaca taatttttta tgtactatta   11580       aaacatttac tttccaaata               ttctgtcatt caggagtatg gaagtatcga tggcttcttt   11640       aaaatgaagc aggagggtct               ggcagagagt atctatgaaa taagttcctc tgaccttcac   11700       gcttaatttt ctgaatggag               tggagcaaat tacttcaagc ttcacttaac ttgcatatga   11760       aatgaaccgt acaaaaatac               aagagtgtca gga         aaagtt atgctctggt aaatattttg   11820       caaaacagat aaaagataat               actagagcta tgtcctcaaa gagttaagca gctaatctaa   11880       ggaggtaaac tctatgtcag               caggatgaac tgctcttccc tttcctcctc aataaattgc   11940       aaatcatcta gtccaacatc               tttaccacca gtgcctgagg ctccagagga gccattgcct   12000       tctcaaggtc acataggtgg               tgggtgagtt aggaccaaat ctagaattcc tgactccagt   12060       aacttctgaa gtcattttgt               tttttatttt tatggtttta ttataagaat acttgctaag   12120       cacacttacc ccctgcattg               attaataact ctaggatctc ag         t         gatcc agcacataga   12180       aatatgaatt cgtttctatt               tggacttcat gatatattta cattatcacc ttggaatcac   12240       cctaacattc aggattgtat               cttgttataa tcaaaaagga tgttgcatcc cctgaacagt   12300       catcagtcag ggaagcagag               gagggaaagt aatcttgcga ggaagagaaa atactattta   12360       agggacagtc agagaacata               atggaattca aactttctgg gaaaacctac atacataaat   12420       gtattagtgg ccatcctaaa               tgtctttata tctttgaggc tttattttcc ctactccaaa   12480       tagacacatt tagttattca               tttcttttaa aatggtattt ctctttttaa actatttctt   12540       gactttttta ataaaaagag               atgcaagcaa gaggatattt aataaaaagt aagagagttg   12600       agcttaaggc ttattaaaag               accccctttt tctagttagt caggagctct aatgtgccct   12660       ggctacctat taaatggtgg               caataaactg gaagctcagt gatgactcta gcctgcttct   12720       cctaatagct gttaagcctc               aaatgccctt tagagtgtgt atgtccttta aagtagctat   12780       taagaaggaa agcagcagca               gcagatattg tctagaaaga agccccaaga agctgaggtt   12840       tcagcttggg catttgtttt               cgccatccca tgctccattt ccctctgctg gaactgtgca   12900       cctcagtgta ttctccctct               atacctcaca gcaggaactg cttgcccccc cccccccccc   12960       ccaacataca tggctggaac               tgaatagact tttactttcc cgaggtgctt ctacagttcc   13020       ctctgccagc aggggaacag               atggaaatag caatcacctg ccagaaggtg gcgtgcagca   13080       aggatgtgca tcttttgccg               ctactgcttt ctgattccta aaaattactc agagatcact   13140       catgtgttca gtgattcagg               ttctgttgaa gataccaaag atattcggtt ggtcaaaatg   13200       acgggcatat aaaggcttct               caggtttctg aggtaaactg aagggtcaga attccagttg   13260       tggatgaagg aaatggtgtt               atgactgcct caaggttttg tagcaagtca tagggaacca   13320       agaggaatct tgttttcctc               agaggtcatg ccaactccaa ctcccgttcc ctaaactgtc   13380       tctgagccat agactagtaa               tggactcttc aagctctacc attaggtatc ttttaaagaa   13440       agctggttat tactatttat               tcattttttt ctcttctgtg cagtgcaaaa gatatgaaac   13500       atcggctagg tttcctgctg               caaaaatctg attcctgtga acacaattct tcccacaaca   13560       agaaggacaa agtggttatt               tgccagaggt aagagaaaag gccttggtga agatgtactt   13620       agtattaact atctgatgat               ggggatgttc tgtgagaagg aacttgtgct cctagttaag   13680       ccagatttgg atcaagatag               cctccatttt catggagatc ataactacat ttgaaatttc   13740       tatacattta gtgaaaaact               gccctcatca ataacatatt ttgtcataac gatggaaaat   13800       aaaatctttg ccttcattca               ggatcttaga tttcttgccc caattttttt accatggcat   13860       tccaattatt ctgtttctct               ctattttttc tagagtgagc caagaggaag tcaagaaatg   13920       ggctgaatca ctggaaaacc               tgattagtca tgaatgtaag tctgacagca acctgggatg   13980       aggtactctg gataagacaa               gttatattat gctggtctaa tagaaactgc agcaaggcct   14040       ggcttctttc tgatgttcag               actcaggaga ctctttaggt cttaaattca gtctgtttaa   14100       aattttaata tgccctagag               ctttgtgata tacaatgaaa agtttatgca ggaaccatgt   14160       ggaaaaccat ctctctcatc               acaaggaaaa acggaagaga gaaaaaaaat gataaatatc   14220       aataccttct tgcaaaatca               atctcagttt ctctttccca aattgacctt ggtaattgat   14280       agctgcatag gcatttcaga               agcaaaatac ttccttgaaa gaggcttcca acttgagtaa   14340       gaatcattag gtagaactgg               gaaccactgg atatcaaaca cagatt         ggg ttacctgact   14400       ccaggtgact tgaaaaaagc               aggggaaaaa gggattgctt gaatccatgc tttatccccc   14460       aagtacctca gctttatgtg               aaatagcata tccaagaggc caaccagtgt gatgacaact   14520       gtggtccttt ctcctgtatc               ataggtgggc tggcagcttt caaagctttc ttgaagtctg   14580       aatatagtga ggagaatatt               gacttctgga tcagctgtga agagtacaag aaaatcaaat   14640       caccatctaa actaagtccc               aaggccaaaa agatctataa tgaattcatc tcagtccagg   14700       caaccaaaga ggtaggtttt               ttatggatac ataaaaattg tacgtattta tggagtatgt   14760       gtgatatttt gatacatgca               tacaatgtga taacaatcaa atcagggcaa ttgctatata   14820       catatctcaa acatttatta               tttctacgtg ttgagaacat tccaaatctc ctcttctagc   14880       tatcttaaaa tatacaataa               actattgata actatatcac cctaatgtgc tatcaaacac   14940       tagaacctat tccctctacc               caactttcta tctattcctt ctacccatta gccaacctga   15000       ccaaaaaggt aagcttttat               ggcagagaac tctctggatc ttagtgaagg ttcctagaat   15060       agtggagctg actatcataa               tcttgacaac cccaaataaa tcagtttttt aaaaaatctc   15120       ttttatccat gtggcttacc               ataacctccc tgcatgaatt tttctgatga atctccccaa   15180       tttgttagac agaacagaag               atcttgccct gctctctcta aagcagaaag gttcattctg   15240       aacctttcat actctctcac               atgtgccaag gaggacccca atgtcacttt tgttttttgc   15300       ttctgaaata cagagggtgc               actgccactt acaagtcact acaaagcata caggcttgca   15360       tcctcaacag ggatataggt               ctaatgaagc cttggccttt gcccctcagg tgaacctgga   15420       ttcttgcacc agggaagaga               caagccggaa catgctagag cctacaataa cctgctttga   15480       tgaggcccag aagaagattt               tcaacctgat ggagaaggat tcctaccgcc gcttcctcaa   15540       gtctcgattc tatcttgatt               tggtcaaccc gtccagctgt ggggcagaaa agcagaaagg   15600       agccaagagt tcagcagact               gtgcttccct ggtccctcag tgtgcctaat tctcacctga   15660       aggcagaggg atgaaatgcc               aagactctat gctctggaaa acctgaggcc aaatattgat   15720       ctgtattaag ctccagtgct               ttatccacat tgtagcctaa tattcatgct gcctgccatg   15780       tgtgagtcac ttctacgcat               aaactagata tagcttttgg tgtttgagtg ttcatcaggg   15840       tgggacccca ttccagtcca               attttcctaa gtttctttga gggttccatg ggagcaaata   15900       tctaaataat ggcctggtag               gtctggattt tcaaagattg ttggcagttt cctcctccca   15960       acagttttac ctcgggatgg               ttggttagtg catgtcacat gacatccaca tgcacatgta   16020       ttctgttggc cagcacgttc               tccagactct agatgtttag atgaggttga gctatgatat   16080       gtgcttgtgt gtatgtctat               gtgtatatat tatatataca ttagacacac atatacatta   16140       tttctgtata tagatgtctg               tgtatacata tgtatgtgtg agtgtatgta tacacacaca   16200       cacacacaca cacacacact               tttgcaagag tgatgggaaa gaccctaggt gctcataact   16260       agagtatgtg tatgtactta               catgggtgtt ttgatctctg ttctttcata ctacatttga   16320       acagggcaaa atgaactaac               tgccatgtag gctaagaaag aaatgctaac ctgtggaaag   16380       ttggttttgt aaaattccat               ggatcttgct ggagaagcat ccaaggaact tcatgcttga   16440       tttgaccact gacagcctcc               accttgagca ctattctaag gagcaaatac cttagctccc   16500       ttgagctggt tttctctgat               ggcacttttg agctcctaag ctgccagcct tcccttcttt   16560       tcctgggtgc tcagggcatg               cttattagca gctgggttgg tatggagttg gcagacagga   16620       tgttcaactt aatgaagaaa               tacagctaag gccttgccag caacacctgc cgtaagttac   16680       tggctgagtg agggcataga               agttaaaggt tactgttttt atcctctatc cttttttcct   16740       ttcctgatca aggtgctctt               ctcatttttt cctgagaacc ttagccatca gatgaggctc   16800       cttagtttat tgtggttggt               tgttttttct ttataatggc tctgggctat atgcctatat   16860       ttataaacca gcagcagggg               aaagattata ttttataaga gggaacaaat tttcacaatt   16920       tgaaaagccc acataagttt               tctcttttaa ggtagaatct tgttaatttc attccaaaca   16980       tcggggctaa cagagactgg               aggcatttct ttttaggctc tgagactaaa tgagaggaaa   17040       agaaaag         aa aaaaaaatga               ttgtctaacc aattgtgaga attactgttt gaaacttttc   17100       aaggcacatt gaaatacttg               aaaacttctc atttatgtta tttatgatgt tattttgtac   17160       gtgttattat tattatattg               ttttataaat ggaggtacag gatatcacct gaattattaa   17220       tgaatgccca ggaagtaatt               ttcttctcat tcttctaaaa ctactgcctt tcaaagtgca   17280       cacacacgcg tccacataca               ctgcattcgt tgctccagta taaattacat gcatgagcac   17340       ctttctggct tttaagccaa               tataatgggc tgcaaaatga agacaccaga gtgtatgcat   17400       acaaatctca ctgtattaaa               gatgcaggtt ttctaattgt acccttcttg tctctctggc   17460       aatcttgccc ttaatatccc               tggagttcct catcagtgtc attttctgtt atacacagtt   17520       ccacaatttt gtctctagtt               gacttcaaat gtgtaacttt attggtcttg ccctattata   17580       attgtcatga ctttcagatt               gtatctgaac tcacagactg ctgtcttact aataggtctg   17640       gaaggtcac          ctgaatgaga               agtaaattat tttatgtaat acatttttga gtgtgttttt   17700       cagttgtatt tccctgttat               ttcatcacta tttccaatgg tgagcttgcc tgctcatgct   17760       ccctggacag aatactcctt               ccttttgcat gcctgtttct atcatgtgct tgataggcct   17820       caaagctaat gcttccagtg               aaacacacgc atcttaataa taagggtaaa taaacgctcc   17880       atatgaaact atttgcttgg               aaacacatta atgatccaga gacatgctat gagaaacatc   17940       agggtgtagg gtgactttag               aaaaatactc atactgagtc tttaatccct cctgtgccag   18000       tgaactctgg gaaagaaagt               acaaactgaa tattgtttat tctttagttc atgccactgc   18060       tctgcttggc tctactcata               gaaccaaggc aatcttagct tcagagactg caaaacagat   18120       taagtgattt gcttgcagat               tctcaatcaa ttttcaaggg atagagttca ccttccagag   18180       ccattctttt atttccagtt               acccgcctgt ttgagagatg atagagcagt gggaaattga   18240       gagagttgaa aggagctata               gattcttacc caaacttcaa aaatccttcc ctcccttttg   18300       ttaattctct ttcctggaaa               agaggtcata aaatgttcac atcctcagta ataggccctg   18360       tgctgtgtct attatgtcat               gagactccca tttcctgacc cttctttccc attgtaagag   18420       tagtagttac aaggtgttaa               ggatagatga tcttcaacac ttttgagaaa tagatccatt   18480       tacggatctg gtaaaaacta               tggaccgaac catcttttaa gaaaaaaatt cagagaggaa   18540       tctaaatttt gtgtgctttg               aggggaaact ctcagaatct cccctcaaaa ctatcattct   18600       tctcttatac tatagatgtg               tcagactctc actgggactg tatagttgct gctccctgta   18660       tttgataata tctatcaaga               actgcagggt aattcaaagt cacgctatta gcagcaagtg   18720       tgagcagtgt tggtttcccc               agtctctaca tccctcatcc tttctttctt ctttatggtt   18780       gtctattaaa gaaataaaaa               aaaatattgg ctgaccgttt ttctgaagat aatgtatatc   18840       aaggaccacc ttttgaaaaa               cactcattat tcgagaacaa agacacaaca tacgagaatc   18900       tctgggatac attcaaagca               gtgtgtagag ggaaatttat agcactaaat gcccacaaga   18960       gaaagcagga aagatctaaa               attgataccc taacatcaca attaaaagaa ctagaaaagc   19020       aagagcaaac acattcaaaa               gctagcagaa gacaagaaat aactaagatc agagcagaac   19080       tgaaggaaat agagacacaa               aaaacccttc aaaaaattaa tgaatccagg agctggtttt   19140       ttgaaaagat taacaaaatt               gatagactgc tagcaagact aataaagaag aaaagagaga   19200       agaatcaaat agacacaata               aaaaatgata aaggggatat caccaccgat cccacagaaa   19260       tacaaactac catcagagaa               tactataaac acctctacgc aaataaacta gaaaatctag   19320       aagaaatgga taaattcctc               gatacataca ccctcccaag accaaaccag gaagaagttg   19380       aatctctgaa tagaccaata               acaggctctg aaattgaggc aataatcaat agcttaccaa   19440       ccaaaaaaag tccaggacca               gatggattca cagctgaatt ctaccagacg tacaaagagg   19500       agctggtacc attccttctg               aaactattcc aatcaataga aaaagaggga atcctcccta   19560       actcatttta tgaggccagc               atcatcctga taccaaagcc tggcagagac acaaccaaaa   19620       aagagaattt tagaccaata               tccttgatga acattgatgc aaaaatcctc aataaaatac   19680       tggcaaaccg aatccagcag               cacatcaaaa agcttatcca ccatgatcaa gtgggtttca   19740       tccctgggat gcaaggctgg               ttcaacatac gcaaatcaat aaatgtaatc cagcatataa   19800       acagaaacaa agacaaaaac               cacatgatta tctcaataga tgcagaaaag gcatttgaca   19860       aaatttaaca actcttcatg               ctaaaaactc tcaatcaatt aggtattgat gggacgtatc   19920       tcaaaataat aagcactatc               tatgacaaac tcacagccaa tatcatactg aatgggcaaa   19980       aactggaagc attccctttg               aaaacgggca caagacaggg atgccctctc tcaccactcc   20040       tattcaacat agtgttggaa               gctctggcca gggcaattag gcaggagaag gaaataaagg   20100       gtattcaatt aggagaagag               gaagtcaaat tgtccctgtt tgcagatgac atgattgtat   20160       atctagaaaa ccccatcgtc               tcagcccaaa atctccttaa gctgataagc aacttcagca   20220       aagtctcagg atacaaaatc               aatgtacaaa aatcacaagc actcttatac atcaataaca   20280       gacaaacaga gagccaaatc               atgagtgaac tcccattcac   20300          
 
         [0063]     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         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          
 
         [0064]     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         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          
 
         [0065]     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         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          
 
         [0066]     For purposes of the present invention, this DNA sequence will be referred to as SEQ ID NO:6.  
         [0067]     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                         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          
 
         [0068]     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                 tctatcacc caagatgatt               tctctttaag acgcaataaa agggaacttt tctccccatt   300       tattagcaac taagatgaaa               tgagagccag agaaataaag tgaggaagga aagagaattt   360       actaccttta caagctgaaa          
 
         [0069]     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.  
         [0000]     In Situ Hybridization  
         [0070]     Double-stranded cDNA containing the RGS4 sequence was first amplified from normal human brain cDNA using custom designed primers (Forward primer sequence: CCGAAGCCACAGCTCCTC (SEQ ID NO: 3); Reverse primer sequence: CATCCCTCTCCCTTCAGGTG (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.  
         [0071]     [ 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.  
         [0072]     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.  
         [0073]     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, Md.), 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.  
         [0074]     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.  
         [0075]     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.  
         [0076]     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.  
         [0000]     Comparison and Statistical Analyses  
         [0077]     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).  
         [0078]     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.  
         [0079]     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.  
         [0080]     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.  
         [0000]     Pittsburgh Cases and Parents for Genotyping Analysis  
         [0081]     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.  
         [0000]     National Institute of Mental Health Collaborative Genetics Initiative (NIMH CGI) Sample  
         [0082]     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.  
         [0083]     Written, informed consent was obtained from all participants. Ethnicity was based on self-report (maternal report for neonatal samples).  
         [0000]     DNA Sequencing and Polymorphism Detection  
         [0084]     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, MSTP032 and RGS5. The genomic organization of RGS4 and RGS5 includes 5 exons which is typical for the RGS family gene.  
         [0085]     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 (“http://www.ncbi.nlm.nih.gov/SNP”). We also obtained genotype data from a prior study of the NIMH sample (“http://zork.wustl.edu/nimh”).  
         [0000]     Polymorphism Analysis  
         [0086]     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.  
         [0087]     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).  
         [0000]     Statistical Analysis  
         [0088]     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).  
         [0000]     Microarray Results  
         [0089]     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.  
         [0090]     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.  
         [0091]     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.  
         [0092]     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 1,1   5 =6.95; p=0.019).  
         [0000]     Specificity of RGS4 Expression Changes  
         [0093]     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.  
         [0094]     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.  
         [0095]     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.  
         [0096]     At the 99% confidence level, 5.6W 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.  
         [0097]     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 .  
         [0000]     Regional RGS4 Gene Expression Changes  
         [0098]     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  FIG. 4A -D is similar to that of  FIG. 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.  
         [0099]     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 .  
         [0100]     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 darkf ield 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.  
         [0101]     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).  
         [0102]     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.  
         [0103]     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.  
         [0104]     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.  
         [0105]     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.  
         [0106]     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.  
         [0000]     Genotyping Results  
         [0107]     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).  
         [0108]     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                           Location of single nucleotide polymorphisms           relevant to the present invention. The location       of the SNP within the sequence is listed as is       the variation observed in the collected sam-       ples. SNP 14 is the absence of the listed 7       bases at the indicated location.                        Observed                   Nucleotide   Nucleo-           Location of the SNP   identity in   tide       SNP #   within the SEQ   SEQ ID 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   AGTTTGG   7 bases           {SEQ ID NO: 3}       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                  
 
 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). 
 
         [0109]     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.  
         [0110]     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 tsburgh samples, respectively (Table 3). SNP 14 was ormative only among African-Americans and so was lyzed separately using 70 African-American cases and control individuals from Pittsburgh. Significant e-control differences were not noted for any of the parisons. The failure to detect association may lect superior power for the TDT in the context of ulation sub-structure.  
                                         TABLE 2                               Neonatal   Adult   Pittsburgh           No.   Haplotype   Controls   controls   Cases   NIMH Cases                       SNP                           1-4-7-18       I                                 0.096   0.066   0.078   0.067               II                                 0.004   0.021   0.022   0.083               III                                 0.006   0.006   0.000   0.000               IV                                 0.000   0.000   0.000   0.000               V                                 0.000   0.000   0.006   0.000                                                                                                                                                                                                               VII                                 0.000   0.006   0.000   0.000               VIII                                 0.000   0.000   0.006   0.000               IX                                 0.000   0.004   0.000   0.017               X                                 0.000   0.006   0.000   0.000                                                                                                                                                                                                               XII                                 0.008   0.013   0.000   0.000               XIII                                 0.000   0.000   0.000   0.000               XIV                                 0.053   0.013   0.017   0.025               XV                                 0.006   0.000   0.000   0.000               XVI                                 0.000   0.000   0.000   0.000                    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                                         represents allele 1 and                                         represents allele       2. OR—Odds ratio; NS—Not significant.          
 
         [0111]    
       
         
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 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 
               
               
                   
               
             
          
         
       
     
         [0112]    
       
         
               
               
               
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
             
               
             
               
               
             
               
               
               
             
               
             
           
               
                   
                 TABLE 4 
               
               
                   
                   
               
               
                   
                   
               
             
             
               
                   
                 Pittsburgh 
                   
                 NIMH 
               
             
          
           
               
                   
                 SNP 
                 Transmitted allele 
                 T/NT 
                 SNP 
                 Transmitted allele 
                 T/NT 
               
               
                   
                   
               
               
                   
                 SNP 1 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 53/35 (0.055) 
                 SNP 1 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 30/13 (0.01) 
               
               
                   
                   
               
               
                   
                 SNP 4 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 51/33 (&lt;0.05) 
                 SNP 4 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 22/6 (0.003) 
               
               
                   
                   
               
               
                   
                   
                   
                   
                 SNP 18 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 24/8 (0.005) 
               
               
                   
                   
               
             
          
           
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
             
          
           
               
                   
                   
                   
               
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 34/18(0.03) 
               
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 33/14(0.006) 
               
               
                   
                   
               
               
                   
                   
               
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 38/17(0.005) 
               
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 37/13(0.0007) 
               
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 39/19(0.009) 
               
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 39/19(0.009) 
               
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 35/19(0.03) 
               
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 40/22(0.022) 
               
               
                   
                   
               
               
                   
                   
               
               
                   
                   
               
               
                   
                   
               
               
                   
                   
               
               
                   
                   
               
               
                   
                   
               
               
                   
                   
               
               
                   
                   
               
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 6/0(0.01) 
               
               
                   
                   
               
             
          
           
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
             
          
           
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
             
          
           
               
                   
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 9/1 (0.02) 
               
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 8/0 (0.005) 
               
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 11/3 (0.04) 
               
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 23/3 (0.0001) 
               
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 19/3 (0.001) 
               
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 20/7 (0.02) 
               
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 20/7 (0.02) 
               
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 11/3 (0.04) 
               
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 20/7 (0.02) 
               
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 11/3 (0.04) 
               
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 20/6 (0.006) 
               
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 20/4 (0.001) 
               
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 11/3 (0.04) 
               
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 11/3 (0.04) 
               
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 4/0 (0.05) 
               
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 4/0 (0.05) 
               
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 7/1 (0.04) 
               
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 5/0 (0.03) 
               
               
                   
                   
               
             
          
           
               
                 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; 
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                 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. 
               
             
          
         
       
     
         [0113]     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.  
         [0114]     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.  
         [0115]     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.  
         [0116]     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.  
         [0117]     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.  
         [0118]     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.  
         [0119]     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.  
         [0120]     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.  
         [0121]     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.  
         [0122]     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.  
         [0123]     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.  
         [0124]     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.  
         [0125]     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.  
         [0126]     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.