NOVEL DIAGNOSTIC MARKER FOR SPLICING VARIANTS OF GENES ASSOCIATED WITH NEUROLOGICAL FUNCTION

Abstract: Methods are described for detecting the presence or absence of a four amino acid motif (VRXQ) in expressed proteins that arise from aberrant alternative splicing of pre-mRNA in genes associated with normal neurological function. The presence of these variants suggest that mutational events in these genes have occurred. Nucleotide sequences and nitron-exon junctional sequences of examples of this splicing variant are provided.

Description: NOVEL DIAGNOSTIC MARKER FOR SPLICING VARIANTS OF GENES ASSOCIATED WITH NEUROLOGICAL FUNCTION BACKGROUND OF THE INVENTION In eukaryotes, the initial transcription of genomic DNA into RNA proceeds in the nucleus and yields a contiguous full-length reverse complementary heteronuclear RNA (hnRNA) primary transcript. The hnRNA contains regions or contiguous blocks of nucleotide sequence that end up in the final mRNA (exons) interspersed between "intervening" nucleotide sequences (introns) that do not. In addition to adenylyl methylation and polyadenylation, these hnRNAs are extensively modified in a process referred to as RNA "splicing" wherein discontiguous exons are joined and the intervening intron precisely deleted as an RNA "lariat" from the final mature mRNA transcript [B. Rushkin et al. Cell 1984, 38:317; R.A. Padgett et al. Science 1984, 225:898]. RNA splicing is a complex process involving large protein-RNA assemblies called spliceosomes that coordinate the concerted excision and ligation events to yield intron-free mRNAs [M.M. Konarska and P.A. Shaφ Cell 1987, 49:763; R. Reid et al. Cell 1988, 53:949; T.A. Steitz Sci. Am. 1988, 258:56]. In normal RNA processing, the resultant mRNA reflects the linear sequence orientation of the exons in the hnRNA; however all exons do not end up in the final transcripts. Rather, several of the resultant mRNAs have only certain exons that result from "alternatively spliced" hnRNA, wherein discontiguous intron-exon junctions are spliced to bring for instance exon 1 and exon 4 into juxtaposition rather than exon 1 and exon 2. Therefore, several mRNAs may arise from one gene sequence or hnRNA. Not all possible combinations of exons are normally represented in actual mRNA pools arising from one hnRNA as determined by mRNA, cDNA and protein analyses. As an example with three exons (Figure 1), while seven combinations are possible (exonl-exon2-exon3, exonl-exon2, exonl-exon3, exon2-exon3, exonl, exon2, or exon3) perhaps only two (exonl-exon2-exon3 and exonl-exon3) may actually result and be expressed at any appreciable level. These alternatively spliced transcripts are sometimes referred to as "variants". However, for purposes of this invention splice "variant" refers to heretofore unrepresented or expressed mRNAs arising from potential alternative splice sites that result from genomic mutation altering the structure of the hnRNA so that these splices now occur. The location of splice sites in an hnRNA primary transcript can be determined by comparing the sequences of the corresponding genomic DNA with that of cDNA prepared by copying the corresponding mature mRNA. Any discontinuities between the genomic DNA and cDNA sequences mark the exon-intron boundaries. Such analyses of a number of different RNAs have defined moderately short "consensus" sequences at the intron-exon boundaries in pre-mRNA and a tendency for a pyrimidine-rich region just upstream of the 3' splice junction (Figure 2). The only universally conserved nucleotides are the first two (GU) and last two (AG) in the intron (Figure 2), though there is a propensity for AG at the 5' exon termini and an initial G at the 3' exon. Only 30-40 nucleotides in the center portion of introns are necessary for efficient splicing. There is also a conserved A within the context of the pyrimidine rich region of the intron (Figure 2) [..PyrPyrPurAPyrnAG; where Pyr is a pyrimidine and Pur is a purine nucleotide] which is the branch point where the cleaved 5' exon-intron junction loops back to form the "lariat" splicing intermediate [Padgett et al. Science 1984, 225:898]. Genetic point mutations that delete or alter these conserved intronic nucleotides (5' GU, 3' AG, or branch point A) would eliminate these splice junctions and prevent normal splicing yielding aberrantly truncated transcripts or transcripts where this exon is deleted and another downstream exon spliced in, that normally may not be spliced in. A final mechanism for splice variation occurs when several GU or AG dinucleotide motifs occur near consensus intron splice regions of 5' exon-intron or 3' intron-exon boundaries, respectively, such that the splicing system may sometimes not correctly distinguish the correct splice site resulting in alternate protein product some of which may be non-functional or aberrant. Multiple examples of splice variations exist, many of which are associated with diseases or related disorders. Previous genetic linkage studies have shown a G to A mutation at the 3' splice junction of exon 8 of the gene encoding lysosomal acid lipase. Defects in this gene are associated with cholesterol ester storage disease that result in premature artherosclerosis, hepatomegaly, and elevated LDL cholesterol [U. Seedorf et al. Arterioscler. Throb. Vase. Biol. 1995, 15: 773-778]. Two mutations at the exon 1 /intron 1 boundary altered the hepatic specific splicing of the human hydroxymethylbilane synthase gene (third enzyme in heme biosynthetic pathway) and resulted in an enzyme with half-normal activity [K.H. Astrin Human Mutat. 1994, 4:243-252]. Deficiency of this enzyme activity eventually results in acute intermittent poφhyria (AIP), an autosomal dominant inborn error of metabolism in which life-threatening attacks are precipitated by ecogenetic factors. Molecular cloning of cDNA and genomic DNA have provided probes allowing presymptomatic detection of these gene defects. In Menke's disease, a point mutation at the -2 exonic position of a splice donor site in the middle of the gene causes exon-skipping and activation of a cryptic splice acceptor site [S.G. Kaler et al. Nat. Genet. 1994, 8:195-202]. Exon skipping of the entire exon 19 results from a G to A point mutation at the 5' donor site of intron 19 in muscle phosphofructokinase deficiency [T. Hamaguchi Biochem. Biophys. Res. Comm. 1994, 202:444-449]. Aberrant RΝA splicing from a splice site mutant in the interleukin-2 receptor gamma (g_L2-R) gene results in the generation of an abundant non-functional gIL2-R containing a small intronic insertion and a second mutant form with 5-fold lower affinity [J.P. DiSanto et al. Proc. Natl. Acad. Sci. 1994, 91:9466-9470]. These isoforms produce an atypical form of an X chromosome-linked severe combined immunodeficiency disease. The presence of splice variants can be used as diagnostic markers of diseases associated with genetic mutations. For example, the expression of the exon 6 splice variant (v6) of the cell adhesion molecule CD44 is correlated with the expression of the tumor suppressor gene p53. Both have been shown to be markers of tumor progression in colorectal cancer [J.W. Mulder et al. Gut 1995, 36:76-80; Y. Matsumura Ixmcet 1992, 340:1053-1058]. Asymptomatic carriers of the acute intermittent poφhyria were identified by identification of a mutant allele containing a CG to CT transversion at the exonl/intron 1 boundary via in vitro amplification of DNA followed by hybridization of the target sequence to allele-specific oligonucleotides . Accordingly, splicing variants have been observed in several gene loci and several diseases. Identification of these variants has proven to be especially useful in diagnosis and detection of asymptomatic carriers. SUMMARY OF THE INVENTION A novel insertional motif that arises from splice mutations or alternative utilization of cryptic or less preferred splice donor sites has now been identified. These splicing variations result in the in-frame insertion within a normal protein sequence of four amino acids, valine-arginine-X-glutamine (VRXQ), where X is a hydrophilic amino acid. This motif has been identified in splice variants of a receptor, an enzyme, and a putative channel protein, all of which are involved in normal neurological functioning. Identification of this motif allows for screening of genes and gene products for splice variations. A method for the detection of this motif in expressed proteins in vitro or in situ with the use of specific antisera, polyclonal or monoclonal antibodies is provided. A method for the detection of allele-specific genetic mutations using selected oligonucleotides with standard hybridization-based detection techniques is also provided. BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a schematic of potential alternative splicing with 3 exons and 4 introns. Figure 2 is a schematic of the consensus exon-intron-exon structure and sequence. Figure 3 provides the sequence of the VRSQ variant of the SI 82 gene. DETAILED DESCRIPTION OF THE INVENTION The SI 82 gene encodes a neuropeptide predicted to be a classical seven transmembrane protein (Sherrington et al. Nature 1995, 375:754-760). Missense mutations within this gene have been found in several families exhibiting early-onset Alzheimer's disease. Genomic analysis has revealed the intron-exon boundaries of the hnRNA. A common polymoφhism located within the intron 3' to exon 9 was identified in early onset AD patients. This polymoφhism also showed a strong association with the occurrence of typical late onset AD families. This particular mutation did not produce an alteration in the coding sequence but is typical of variations leading to alternatively spliced proteins. Other mutations within different introns of the SI 82 gene have been identified. These lead to alternatively spliced variants as well. One novel variant of the SI 82 protein isolated from a human cerebellar cDNA library contains a four amino acid insertion between codons 26 and 27 (VRSQ) (Figure 3). This variant arises from alternative use of a 5' exon donor site in the exon 3/intron 3 boundary and results in the loss of some potential phosphorylation sites. A similar motif (VRXQ- where X is a hydrophilic amino acid) arising from aberrant splicing has also arisen due to alternative splicing in several other neurological proteins as well. For example, the mRNA for tyrosine hydroxylase, the rate limiting enzyme in the synthesis of catecholomines, can undergo alternative splicing to produce several different isoforms (Kobayashi et al. J. Biochem. 1988, 103(6) 907-12; Lewis et al. Neuroscience 1993, 54(2) 477-92). The identified variants contain a 12 bp insertion encoding the sequence VRGQ. Isoforms containing the VRGQ insertion have also been found to exhibit alterations in phosphorylation by MAP kinase (Sutherland et al. Eur J Biochem. 1993, 217(2) 715-22). Furthermore, a tyrosine hydroxylase variant containing this insertion has been implicated in Parkinson's disease. Another neuropeptide, gamma-Aminobutyric acid A (GABAA) receptor, undergoes alternative splicing to yield a multiplicity of transcripts (Whiting et al. P.N.A.S. 1990, 87(24) 9966-70; Lasham et al. Biochem. Soc. Trans. 1991, 19(1) 9S). GABA receptors are multisubunit ligand gated ion channels which mediate neuronal inhibition by GABAA and are composed of at least four subunit types (alpha, beta, gamma, and delta). The beta 4 subunit can undergo alternative splicing at two 5 '-donor splice sites separated by 12 bp in the region that encodes the presumed intracellular loop between transmembrane domains M3 and M4. The insertion of the 12 bp sequence results in the addition of a VREQ motif (Bateson et al. J. Neurochem 1991, 56(4) 1437-40). In all three neurological proteins, the alternative splice site generates variants containing a specific motif (VRXQ) which appears to be intracellularly located and alters phosphorylation by various kinases. In the present invention, a method for detecting the presence of the VRXQ motif in polyadenylated messenger RNA transcripts (polyA mRNA) and resultant expressed proteins, (where V is valine, R is arginine, X is any hydrophilic amino acid residue, and Q is glutamine) or in cDNA resulting from these RNAs is provided. Oligonucleotides having the anticodon sequences associated with the VRXQ motif having degenerate positions at the third base position of each codon can be used for the detection of mRNA. Additionally, these oligonucleotides can be associated with codon sequences and used for the detection of cDNAs. For example, codon and anticodon oligonucleotides for VRNQ comprise GU(N) AG(A/G) AA(C/U) CA(A/G) and the reverse complement. Hybridization of appropriate oligonucleotides can be detected directly by procedures well known to those of skill in the art using radioactively or fluorescently labeled oligonucleotides. Indirect detection procedures such as, but not limited to, biotinylated oligonucleotides/strepavidin-horseradish peroxidase, enhanced chemiluminescent detection, or fluorescently tagged strepavidins can also be performed. Specific antibodies against the VRXQ motif can also be used for detection. Various procedures known in the art may be used for the production of such antibodies. For example, these antibodies can be obtained by direct injection of a polypeptide containing a VRXQ motif into an animal, preferably a nonhuman. The antibody so obtained will then bind to polypeptides containing this motif. Such antibodies can then be used to isolate polypeptides containing this motif from tissues. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, Nature 1975, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today 1983, 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al. in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., 1985, pp. 77-96). Techniques described for the production of single chain antibodies (U.S. Patent 4,946,778) can be adapted to produce single chain antibodies to the immunogenic motif of this invention. Also, transgenic mice may be used to express humanized antibodies to polypeptides containing this motif. Primary antibody-antigen reactions can be visualized secondarily by standard enzyme-linked immunosorbent assay (ELISA) procedures. An ELISA assay initially comprises preparing an antibody specific to a VRXQ motif, preferably a monoclonal antibody. In addition a reporter antibody is prepared against the monoclonal antibody. To the reporter antibody is attached a detectable reagent such as horse radish peroxidase. A sample is then removed from a host and incubated on a solid support, e.g., a polystyrene dish, that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non¬ specific protein like BSA. Next, the monoclonal antibody is incubated in the dish during which time the monoclonal antibodies attach to any proteins containing the VRXQ motif attached to the polystyrene dish. All unbound monoclonal antibody is washed out with buffer. The reporter antibody linked to horseradish peroxidase is then placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to proteins containing the VRXQ motif. Unattached reporter antibody is then washed out. Peroxidase substrates are then added to the dish and the amount of color developed in a given time period is a measurement of the amount of protein containing the VRXQ motif present in a given volume of patient sample when compared against a standard curve. Examples of other detectable reagents which can be used include, but are not limited to, luciferase and fluorescently or radioactively tagged secondary antibodies. Specific populations of immune cells or chimeric cells (e.g., hybridomas) that express antibodies to VRXQ epitopes on their cell surfaces and respond by degranulation or release of cellular contents such as histamines that can be detected functionally or preloaded radiolabeled metals such as chromium are also useful. The following examples are provided for illustrative purposes only and are not intended to limit the invention. EXAMPLES Example 1 A novel splice variant of the SI 82 gene described by Sherrington et al. Nature 1995, 375:754-760, was isolated from a human cerebellar and a human fibroblast library. In this novel splice variant there is a deletion of four amino acids at codons 26-27 (VRSQ). This arises from alternative use of a 5' exon donor site in the exon3/intron 3 (-52 to 75 nt) boundary. The ...CAG/gta... boundary of the final Gin codon of exon 3 of the VRSQ motif provides a 5' exon AG donor site and GT intron consensus 5' boundary and use of this splice site results in the insertion of the 12-nts encoding the VRSQ motif. The upstream ...ACT/GTA... boundary of the Thr- Val codons provides the less preferred CT (AG preferred) 5' exonic boundary to the consensus GT 5' intronic boundary and splicing at this site would remove the VRSQ motif. Interestingly, in the SI 82 protein of Sherrington et al. Nature 1995, 375:754-760, this is the sole observed product and point mutations are interspersed elsewhere. Example 2 In the GABA receptor 4 subunit alternative splicing adds a VREQ motif (Bateson et al. J. Neurochem 1991, 56(4) 1437-40). A chicken genomic cDNA library was screened with chicken beta-4' subunit cDNA at high stringency. Southern blot analysis, using cDNA sequence specific oligonucleotides as probes and subsequent restriction mapping allowed the identification of overlapping DNA fragments containing the coding regions of the beta-4 subunit gene. These fragments were subcloned into pBluescript and sequenced. Complete sequencing of one of the clones revealed the presence of 12 bp in the part encoding the intracellular loop (amino acid residues 335-338). Analysis of the beta-4 subunit gene reveals that the different transcripts encoding the two variants (absence or presence of 12bp loop) arise by the use of one of two 5'-donor splice sites (located in the intron immediately 3' of the 12 bp sequence). SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Washington University School of Medicine and University of South Florida (ii) TITLE OF INVENTION: A Novel Diagnostic Marker for Splicing Variants of Genes Associated with Neurological Function (iii) NUMBER OF SEQUENCES: 7 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: SmithKline Beecham Corporation (B) STREET: 709 Swedeland Road, P.O. Box 1539 (C) CITY: King of Prussia (D) STATE: PA (E) COUNTRY: USA (F) ZIP: 19406-0939 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: DISKETTE, 3.5 INCH, 1.44 Mb STORAGE (B) COMPUTER: IBM 486 (C) OPERATING SYSTEM: WINDOWS FOR WORKGROUPS (D) SOFTWARE: WORDPERFECT 5.1 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: not yet assigned (B) FILING DATE: Herewith (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 60/007,045 (B) FILING DATE: October 25, 1995 (viii) ATTORNE /AGENT INFORMATION: (A) NAME: William T. Han (B) REGISTRATION NUMBER: 34,344 (C) REFERENCE/DOCKET NUMBER: P50389 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 610-270-5219 (B) TELEFAX: 610-270-5090 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1914 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: CCGTACGTAG CCGCGGCGGC AGCGGGGCGG CGGGGAAGCG TATGCATACA 50 AATTTATTAG CATGCAGACT GGGAGAACCA CAAGACCTAA TCTGGGAGCC 100 TGCAAGTGAC AACAGCCTTT GCGGTCCTTA GACAGCTTGG CCTGGAGGAG 150 AACACATGAA AGAAAGAACC TCAAGAGGCT TTGTTTTCTG TGAAACAGTA 200 TTTCTATACA GTTGCTCCAA TGACAGAGTT ACCTGCACCG TTGTCCTACT 250 TCCAGAATGC ACAGATGTCT GAGGACAACC ACCTGAGCAA TACTAATGAC 300 AATAGAGAAC GGCAGGAGCA CAACGACAGA CGGAGCCTTG GCCACCCTGA 350 GCCATTATCT AATGGACGAC CCCAGGGTAA CTCCCGGCAG GTGGTGGAGC 400 AAGATGAGGA AGAAGATGAG GAGCTGACAT TGAAATATGG CGCCAAGCAT 450 GTGATCATGC TCTTTGTCCC TGTGACTCTC TGCATGGTGG TGGTCGTGGC 500 TACCATTAAG TCAGTCAGCT TTTATACCCG GAAGGATGGG CAGCTAATCT 550 ATACCCCATT CACAGAAGAT ACCGAGACTG TGGGCCAGAG AGCCCTGCAC 600 TCAATTCTGA ATGCTGCCAT CATGATCAGT GTCATTGTTG TCATGACTAT 650 CCTCCTGGTG GTTCTGTATA AATACAGGTG CTATAAGGTC ATCCATGCCT 700 GGCTTATTAT ATCATCTCTA TTGTTGCTGT TCTTTTTTTC ATTCATTTAC 750 TTGGGGGAAG TGTTTAAAAC CTATAACGTT GCTGTGGACT ACATTACTGT 800 TGCACTCCTG ATCTGGAATT TTGGTGTGGT GGGAATGATT TCCATTCACT 850 GGAAAGGTCC ACTTCGACTC CAGCAGGCAT ATCTCATTAT GATTAGTGCC 900 CTCATGGCCC TGGTGTTTAT CAAGTACCTC CCTGAATGGA CTGCGTGGCT 950 CATCTTGGCT GTGATTTCAG TATATGATTT AGTGGCTGTT TTGTGTCCGA 1000 AAGGTCCACT TCGTATGCTG GTTGAAACAG CTCAGGAGAG AGATGAAACG 1050 CTTTTTCCAG CTCTCATTTA CTCCTCAACA ATGGTGTGGT TGGTGAATAT 1100 GGCAGAAGGA GACCCGGAAG CTCAAAGGAG AGTATCCAAA AATTCCAAGT 1150 A AATGCAGA AAGCACAGAA AGGGAGTCAC AAGACACTGT TGCAGAGAAT 1200 GATGATGGCG GGTTCAGTGA GGAATGGGAA GCCCAGAGGG ACAGTCATCT 1250 AGGGCCTCAT CGCTCTACAC CTGAGTCACG AGCTGCTGTC CAGGAACTTT 1300 CCAGCAGTAT CCTCGCTGGT GAAGACCCAG AGGAAAGGGG AGTAAAACTT 1350 GGATTGGGAG ATTTCATTTT CTACAGTGTT CTGGTTGGTA AAGCCTCAGC 1400 AACAGCCAGT GGAGACTGGA ACACAACCAT AGCCTGTTTC GTAGCCATAT 1450 TAATTGGTTT GTGCCTTACA TTATTACTCC TTGCCATTTT CAAGAAAGCA 1500 TTGCCAGCTC TTCCAATCTC CATCACCTTT GGGCTTGTTT TCTACTTTGC 1550 CACAGATTAT CTTGTACAGC CTTTTATGGA CCAATTAGCA TTCCATCAAT 1600 TTTATATCTA GCATATTTGC GGTTAGAATC CCATGGATGT TTCTTCTTTG 1650 ACTATAACAA AATCTGGGGA GGACAAAGGT GRTTTTCCTG TGTCCCACAT 1700 CTAACAAAGT CAAGATTCCC GKCTGGACTT TTGCAGCTTC CTKCCAAGTC 1750 TTCCTGACCA CCTTGCACTW TTGGACTTTG GARGGAGGTG CCTAKAGAAA 1800 ACGRTTTTGA MCATACTTCA TCGCAGTGGA CTGTGTCCCT CGGTGCAGAA 1850 ACTACCAGAT TTGAGGGACG AGGTCAAGGA GATATGATAG GCCCGGAAGT 1900 TGCTGTGCCC ATCA 1914 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: VAL ARG XAA GLN 1 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: VAL ARG SER GLN 1 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: V R G Q 1 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: VAL ARG GLU GLN 1 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: VAL ARG ASN GLN 1 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 463 (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: MET THR GLU LEU PRO ALA PRO LEU SER TYR PHE GLN ASN ALA GLN 1 5 10 15 MET SER GLU ASP ASN HIS LEU SER ASN THR ASN ASP ASN ARG GLU 20 25 30 ARG GLN GLU HIS ASN ASP ARG ARG SER LEU GLY HIS PRO GLU PRO 35 40 45 LEU SER ASN GLY ARG PRO GLN GLY ASN SER ARG GLN VAL VAL GLU 50 55 60 GLN ASP GLU GLU GLU ASP GLU GLU LEU THR LEU LYS TYR GLY ALA 65 70 75 LYS HIS VAL ILE MET LEU PHE VAL PRO VAL THR LEU CYS MET VAL 80 85 90 VAL VAL VAL ALA THR ILE LYS SER VAL SER PHE TYR THR ARG LYS 95 100 105 ASP GLY GLN LEU ILE TYR THR PRO PHE THR GLU ASP THR GLU THR 110 115 120 VAL GLY GLN ARG ALA LEU HIS SER ILE LEU ASN ALA ALA ILE MET 125 130 135 ILE SER VAL ILE VAL VAL MET THR ILE LEU LEU VAL VAL LEU TYR 140 145 150 LYS TYR ARG CYS TYR LYS VAL ILE HIS ALA TRP LEU ILE ILE SER 155 160 165 SER LEU LEU LEU LEU PHE GLU GLU SER PHE ILE TYR LEU GLY GLU 170 175 180 VAL PHE LYS THR TYR ASN VAL ALA VAL ASP TYR ILE THR VAL ALA 185 190 195 LEU LEU ILE TRP ASN PHE GLY VAL VAL GLY MET ILE SER ILE HIS 200 205 210 TRP LYS GLY PRO LEU ARG LEU GLN GLN ALA TYR LEU ILE MET ILE 215 220 225 SER ALA LEU MET ALA LEU VAL PHE ILE LYS TYR LEU PRO GLU TRP 230 235 240 THR ALA TRP LEU ILE LEU ALA VAL ILE SER VAL TYR ASP LEU VAL 245 250 255 ALA VAL LEU CYS PRO LYS GLY PRO LEU ARG MET LEU VAL GLU THR 260 265 270 ALA GLN GLU ARG ASP GLU THR LEU PHE PRO ALA LEU ILE TYR SER 275 280 285 SER THR MET VAL TRP LEU VAL ASN MET ALA GLU GLY ASP PRO GLU 290 295 300 ALA GLN ARG ARG VAL SER LYS ASN SER LYS TYR ASN ALA GLU SER 305 310 315 THR GLU ARG GLU SER GLN ASP THR VAL ALA GLU ASN ASP ASP GLY 320 325 330 GLY PHE SER GLU GLU TRP GLU ALA GLN ARG ASP SER HIS LEU GLY 335 340 345 PRO HIS ARG SER THR PRO GLU SER ARG ALA ALA VAL GLN GLU LEU 350 355 360 SER SER SER ILE LEU ALA GLY GLU ASP PRO GLU GLU ARG GLY VAL 365 370 375 LYS LEU GLY LEU GLY ASP PHE ILE PHE TYR SER VAL LEU VAL GLY 380 385 390 LYS ALA SER ALA THR ALA SER GLY ASP TRP ASN THR THR ILE ALA 395 400 405 CYS PHE VAL ALA ILE LEU ILE GLY LEU CYS LEU THR LEU LEU LEU 410 415 420 LEU ALA ILE PHE LYS LYS ALA LEU PRO ALA LEU PRO ILE SER ILE 425 430 435 THR PHE GLY LEU VAL PHE TYR PHE ALA THR ASP TYR LEU VAL GLN 440 445 450 PRO PHE MET ASP GLN LEU ALA PHE HIS GLN PHE TYR ILE 455 460

Claims: What is claimed is: 1. A method for diagnosing a neurological disease or condition associated with a genetic mutation comprising: providing a sample of genetic material from an individual suspected of having a neurological disease or condition associated with a genetic mutation; and detecting the presence of the VRXQ motif in selected polyadenylated messenger RNA transcripts in said sample. 2. The method of claim 1 wherein the selected messenger RNA is derived from the SI 82 gene. 3. The method of claim 1 wherein the selected messenger RNA is derived from the gamma-aminobutyric acid A receptor gene. 4. The method of claim 1 wherein the selected messenger RNA is derived from the tyrosine hydroxylase gene. 5. The method of claim 1 wherein the presence of the VRXQ motif is detected using oligonucleotides having the anticodon sequences associated with the VRXQ motif having degenerate positions at the third base position of each codon. 6. The method of claim 1 wherein the presence of the VRXQ motif is detected in cDNA resulting from selected polyadenylated messenger RNA transcripts using oligonucleotides having the anticodon sequences associated with the VRXQ motif having degenerate positions at the third base position of each codon; associating said oligonucleotides with codon sequences; and detecting the presence of the VRXQ motif in cDNA resulting from the selected polyadenylated messenger RNA. 7. The method of claim 1 wherein the presence of the VRXQ motif is detected using antibodies against the VRXQ motif.

Inventors: GOATE ALISON M, HARDY JOHN A, GOATE, ALISON, M., HARDY, JOHN, A.
Applicants: GOATE ALISON M, HARDY JOHN A, UNIV SOUTH FLORIDA, UNIV WASHINGTON, GOATE, ALISON, M., HARDY, JOHN, A., UNIVERSITY OF SOUTH FLORIDA, WASHINGTON UNIVERSITY SCHOOL OF MEDICINE
Application year: 1996
Main classification: C12Q1
IPC codes: C12N15, C12Q1