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WO1997015688A9 - Nouveau marqueur diagnostique pour l'epissage de genes associes a la fonction neurologique - Google Patents

Nouveau marqueur diagnostique pour l'epissage de genes associes a la fonction neurologique

Info

Publication number
WO1997015688A9
WO1997015688A9 PCT/US1996/017128 US9617128W WO9715688A9 WO 1997015688 A9 WO1997015688 A9 WO 1997015688A9 US 9617128 W US9617128 W US 9617128W WO 9715688 A9 WO9715688 A9 WO 9715688A9
Authority
WO
WIPO (PCT)
Prior art keywords
motif
vrxq
leu
val
exon
Prior art date
Application number
PCT/US1996/017128
Other languages
English (en)
Other versions
WO1997015688A1 (fr
Filing date
Publication date
Application filed filed Critical
Priority to JP9516125A priority Critical patent/JPH11513570A/ja
Priority to EP96937747A priority patent/EP0859862A4/fr
Priority to AU75216/96A priority patent/AU7521696A/en
Priority to CA 2235593 priority patent/CA2235593A1/fr
Publication of WO1997015688A1 publication Critical patent/WO1997015688A1/fr
Publication of WO1997015688A9 publication Critical patent/WO1997015688A9/fr

Links

Definitions

  • hnRNA reverse complementary heteronuclear RNA
  • 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. Sha ⁇ Cell 1987, 49:763; R. Reid et al. Cell 1988, 53:949; T.A. Steitz Sci. Am. 1988, 258:56].
  • 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 "altematively 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.
  • 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).
  • 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.
  • RNA splicing from a splice site mutant in the interleukin-2 receptor gamma (gIL2-R) gene results in the generation of an abundant non-functional gLL2-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].
  • gIL2-R interleukin-2 receptor gamma
  • splice variants can be used as diagnostic markers of diseases associated with genetic mutations.
  • 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 Lancet 1992, 340: 1053-1058].
  • Asymptomatic carriers of the acute intermittent porphyria 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.
  • 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.
  • 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.
  • Figure 3 provides the sequence of the VRSQ variant of the S 182 gene.
  • 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 polymorphism located within the intron 3' to exon 9 was identified in early onset AD patients. This polymorphism 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 altematively spliced proteins.
  • the mRNA for tyrosine hydroxylase can undergo altemative 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).
  • GABAA gamma-Aminobutyric acid A
  • GAB A 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 altemative 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).
  • the alternative splice site generates variants containing a specific motif (VRXQ) which appears to be intracellularly located and alters phosphorylation by various kinases.
  • 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.
  • codon and anticodon oligonucleotides for VRNQ comprise GU(N) AG(A/G) AA(CAJ) 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.
  • 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.
  • 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).
  • An ELISA assay initially comprises preparing an antibody specific to a VRXQ motif, preferably a monoclonal antibody.
  • a reporter antibody is prepared against the monoclonal antibody.
  • 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.
  • 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.
  • detectable reagents 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.
  • 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.
  • beta-4 subunit gene 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).
  • AACACATGAA AGAAAGAACC TCAAGAGGCT TTGTTTTCTG TGAAACAGTA 200

Abstract

L'invention concerne des méthodes de détection de la présence ou l'absence d'un motif à quatre acides aminés (VRXQ) dans des protéines exprimées issues d'un épissage alterne aberrant de pré-ARNm dans des gènes associés à la fonction neurologique normale. La présence de ces variantes d'épissage suggère que des mutations se sont produites dans ces gènes. Des séquences nucléotidiques et des séquence de jonction intron-exon des exemples de variantes d'épissage sont également décrites.
PCT/US1996/017128 1995-10-25 1996-10-25 Nouveau marqueur diagnostique pour l'epissage de genes associes a la fonction neurologique WO1997015688A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP9516125A JPH11513570A (ja) 1995-10-25 1996-10-25 神経学的機能に関連した遺伝子のスプライシング変種についての新規診断マーカー
EP96937747A EP0859862A4 (fr) 1995-10-25 1996-10-25 Nouveau marqueur diagnostique pour l'epissage de genes associes a la fonction neurologique
AU75216/96A AU7521696A (en) 1995-10-25 1996-10-25 Novel diagnostic marker for splicing variants of genes associated with neurological function
CA 2235593 CA2235593A1 (fr) 1995-10-25 1996-10-25 Nouveau marqueur diagnostique pour l'epissage de genes associes a la fonction neurologique

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US704595P 1995-10-25 1995-10-25
US60/007,045 1995-10-25

Publications (2)

Publication Number Publication Date
WO1997015688A1 WO1997015688A1 (fr) 1997-05-01
WO1997015688A9 true WO1997015688A9 (fr) 1997-06-26

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/017128 WO1997015688A1 (fr) 1995-10-25 1996-10-25 Nouveau marqueur diagnostique pour l'epissage de genes associes a la fonction neurologique

Country Status (5)

Country Link
EP (1) EP0859862A4 (fr)
JP (1) JPH11513570A (fr)
AU (1) AU7521696A (fr)
WO (1) WO1997015688A1 (fr)
ZA (1) ZA968899B (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0814157A3 (fr) * 1996-06-18 1998-01-07 Smithkline Beecham Corporation Marqueur diagnostique pour variations des gènes codant presenilin en association avec la maladie d'Alzheimer et familial adulte apparition de la maladie d'Alzheimer
GB9902776D0 (en) * 1999-02-08 1999-03-31 Marie Curie Cancer Care Materials and methods relating to a cancer cell marker

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0791660A1 (fr) * 1996-02-22 1997-08-27 Smithkline Beecham Corporation Nouveau marqueur diagnostique pour variations d'épissage de gènes en association avec fonctions neurologiques
EP0814157A3 (fr) * 1996-06-18 1998-01-07 Smithkline Beecham Corporation Marqueur diagnostique pour variations des gènes codant presenilin en association avec la maladie d'Alzheimer et familial adulte apparition de la maladie d'Alzheimer

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