DETECTION OF VARIATIONS IN HUMAN H2 RECEPTORS

Abstract: A group of oligonucleotides for the identification of sequences in a sample comprising human histamine H2 receptor DNA, cDNA or RNA originating from a tissue sample or body fluid is employed in the diagnosis and/or treatment of human neurological and psychiatric disorders, particularly schizophrenia, and diseases of other systems or organs of the human body. The new oligonucleotides, suitable for use as primers for the amplification of DNA corresponding to a region of a human histamine H2 receptor, have nucleotide sequences selected from: 5' ACACCAGCCTGGATGTGA 3' (as listed in SEQ ID NO:12), 5' TCACATCCAGGCTGGTGT 3' (as listed in SEQ ID NO:13), 5' CAATCATACCACCTCTAA 3' (as listed in SEQ ID NO:14), 5' ACACAAACGCGGTGAAGT 3' (as listed in SEQ ID NO:15). Also described is a diagnostic kit comprising one or more of the above mentioned oligonucleotides.

Description: DETECTION OF VARIATIONS IN HUMAN H 2 RECEPTORS Teπhnical Field This invention relates to the detection of variations in human histamine H 2 receptors, and more particularly to the development of new compounds useful in the sequencing and identification of a human histamine H 2 receptor and their use in the diagnosis and treatment of certain human disorders, for example, brain disorders. The invention also relates to new compounds and a method for detecting an allelic polymorphic variation within the human population for the gene encoding the histamine H 2 receptor, and their use in the diagnosis and treatment of human disorders. Background Art The histamine H 2 receptor is one of many receptors in the body. Compounds used to treat many diseases work by activating a receptor or inhibiting the action of its natural ligand. Variations in some receptors amongst the population are known to be caused by allelic variation, and this variation can alter the response of a disease to a drug amongst patients. An example of this would be the response to clozapine, used to treat schizophrenia, associated with allelic variation in the 5-HT 2A receptor demonstrated by Arranz el al (1995) Lancet, 346(8970), 281-282. In co-pending UK Patent Applications Nos. GB9503866.7 and GB9522889.6, and in International Patent Application No. PCT/EP96/00397, it is reported that there is a hitherto unrecognised allele or subtype of the human histamine H 2 receptor gene which may be specific to the brain. In the above mentioned co-pending patent specifications there are described and claimed: 1) a sequence for a novel allele of a human histamine H 2 receptor gene comprising up to six single base substitutions compared with the cDNA sequence published by Gantz et al (1991) Biochem Biophys Res Comm 178,3,1386-1392 as follows: site Of Change base Change amino acid alteration 398 T - C Val - Gly 525 A - T Lys - Asn 620 A - G Lys - Asp 649 A - G Asn - Asp 692 A - G Lys - Arg 802 G - A Val - Met 2) a nucleotide sequence coding for a region of a human histamine H 2 receptor, comprising one or more of the following base substitutions compared with the published sequence in Gantz et al (1991) supra r and from which the positional notation is taken: site of change base. 398 C 525 T 620 G 649 G 692 G 802 A The nucleotide sequence can, for example, comprise the following sequence (as listed in SEQ ID NO: 1): 5' CAGCTCGGGTCGCCATCTCTCTGGTCTTAATTTGGGTCATCTCCATTACCCTGTC CTTTCTGTCTATCCACCTGGGGTGGAACAGCAGGAACGAGACCAGCAAGGGCAAT CATACCACCTCTAAGTGCAATGTCCAGGTCAATGAAGTGTACGGGCTGGTGGATG GGCTGGTCACCTTCTACCTCCCGCTACTGATCATGTGCATCACCTACTACCGCAT CTTCAGGGTCGCCCGGGATCAGGCCAAGAGGATCGATCACATTAGCTCCTGGAAG GCAGCCACCATCAGGGAGCACAGAGCCACAGTGACACTGGCCGCCGTCATGGGGG CCTTCATCATCTGCTGGTTTCCCTACTTCACCGCGTTTGTGTACCGTGGGCTGAG AGGGGATGATGCCATCAATGAGATGTTA 3' As a specific exemplification, the nucleotide sequence can comprise the following sequence (as listed in SEQ ID NO: 2): 5 ' CCAATGGCACAGCCTCTTCCTTTTGCCTGGACTCTACCGCATGCAAGATCACCAT CACCGTGGTCCTTGCGGTCCTCATCCTCATCACCGTTGCTGGCAATGTGGTCGTC TGTCTGGCCGTGGGCTTGAACCGCCGGCTCCGCAACCTGACCAATTGTTTCATCG TGTCCTTGGCTATCACTGACCTGCTCCTCGGCCTCCTGGTGCTGCCCTTCTCTGC CATCTACCAGCTGTCCTGCAAGTGGAGCTTTGGCAAGGTCTTCTGCAATATCTAC ACCAGCCTGGATGTGATGCTCTGCACAGCCTCCATTCTTAACCTCTTCATGATCA GCCTCGACCGGTACTGCGCTGTCATGGACCCACTGCGGTACCCTGTGCTGGTCAC CCCAGCTCGGGTCGCCATCTCTCTGGTCTTAATTTGGGTCATCTCCATTACCCTG TCCTTTCTGTCTATCCACCTGGGGTGGAACAGCAGGAACGAGACCAGCAAGGGCA ATCATACCACCTCTAAGTGCAATGTCCAGGTCAATGAAGTGTACGGGCTGGTGGA TGGGCTGGTCACCTTCTACCTCCCGCTACTGATCATGTGCATCACCTACTACCGC ATCTTCAGGGTCGCCCGGGATCAGGCCAAGAGGATCGATCACATTAGCTCCTGGA AGGCAGCCACCATCAGGGAGCACAGAGCCACAGTGACACTGGCCGCCGTCATGGG GGCCTTCATCATCTGCTGGTTTCCCTACTTCACCGCGTTTGTGTACCGTGGGCTG AGAGGGGATGATGCCATCAATGAGATGTTAGAAGCCATCGTTCTGTGGCTGGGCT ATGCCAACTCAGCCCTGAACCCCATCCTGTATGCTGCGCTGAACAGAGACTTCCG CACCGGGTACCAACAGCTCTTCTGCTGCAGGCTGGCCAACCGCAACTCCCACAAA ACTTCTCTGAGGTCCAACGCCTCTCAGCTGTCCAGGACCCAAAGCCGAGAACCCA GGCAACAGGAAGAGAAACCCCTGAAGCTCCAGGTGTGGAGTGGGACAGAAGTCACG 3' 3) new oligonucleotides, suitable for use as primers for the amplification of DNA corresponding to a region of a human histamine H 2 receptor, having nucleotide sequences selected from: 1) 5' CCAATGGCACAGCCTCTT 3' (as listed in SEQ ID NO: 3) 2) 5' CGTGACTTCTGTCCCACT 3' (as listed in SEQ ID NO: 4) 3) 5' CCAGGCAACAGGAAGAGA 3' (as listed in SEQ ID NO: 5) 4) 5' TCTCTTCCTGTTGCCTGG 3' (as listed in SEQ ID NO: 6) 5) 5' GCAGCAGAAGAGCTGTTG 3' (as listed in SEQ ID NO: 7) 6) 5' TCCAGGTCAATGAAGTGT 3' (as listed in SEQ ID NO: 8) 7) 5' ACACTTCATTGACCTGGA 3' (as listed in SEQ ID NO: 9) 8) 5' CCAAGAGGATCAATCACA 3' (as listed in SEQ ID NO: 10) 9) 5' TGTGATTGATCCTCTTGG 3' (as listed in SEQ ID NO: 11) and 4) a diagnostic kit comprising one or more of the above new oligonucleotides. Disclosure of the Invention The present invention is concerned in one aspect with improvements in the diagnosis and/or treatment of human neurological and psychiatric disorders, and more particularly in the diagnosis and treatment of schizophrenia. In another aspect, the invention is concerned with improvements in the diagnosis and/or treatment of diseases of other systems or organs of the human body. According to the invention, a further group of oligonucleotides has now been developed for the identification of sequences in a sample comprising a human histamine H 2 receptor DNA, cDNA or RNA originating from a tissue sample or body fluid. In a first aspect, therefore, the invention provides new oligonucleotides, suitable for use as primers for the amplification of DNA corresponding to a region of a human histamine H 2 receptor, having nucleotide sequences selected from: 10) 5' ACACCAGCCTGGATGTGA 3' (as listed in SEQ ID NO:12) 11) 5' TCACATCCAGGCTGGTGT 3' (as listed in SEQ ID NO:13) 12) 5' CAATCATACCACCTCTAA 3' (as listed in SEQ ID NO:14) 13) 5' ACACAAACGCGGTGAAGT 3' (as listed in SEQ ID NO:15) and a diagnostic kit comprising one or more of the above mentioned oligonucleotides. The direction and base start numbers for the novel oligonucleotide primers are as follows: Primer Base Start NO. 10) Upstream 281 11) Downstream 298 12) Upstream 501 13) Downstream 766 Information on the human histamine H 2 receptor was obtained from the MRC Daresbury database accessing "Genem 61" File no. M64799 - Human histamine H 2 receptor gene. The above mentioned substitutions in the human histamine H 2 receptor gene alter and in some instances introduce or remove new sites for cleavage by specific restriction endonucleases as follows: base change _si±e alters restriction map of: 398 Alul, Aval, BspWI, Bsrl, C\ 525 620 Eco57 649 Clal, Sau3A, Taql 692 802 Mnll In another aspect the invention provides a diagnostic kit comprising one or more of the new oligonucleotide primers and, preferably, one or more of the above mentioned endonucleases, optionally with one or more buffers. In a still further aspect, the invention provides a diagnostic kit comprising one or more of the new oligonucleotide primers whereby detection of human histamine subtypes can be achieved using single-stranded conformational polymorphism (SSCP). The kit can, for example, comprise one or more of the above mentioned oligonucleotide primers, and, optionally, formamide. A kit may be used to establish genotype or base variations. This information may be used in predicting an individual's disease susceptibility, disease course, prognosis and/or response to treatment, as would be understood by those skilled in the art from the disclosure contained herein. The treatment response or efficacy which may be predicted may include drug treatment such as for example, use of H 2 receptor antagonists, for example, famotidine, or other forms of treatment such as social or psychological intervention. Eucaryotic expression vectors and host cells transformed thereby can be prepared as described in the above mentioned co-pending patent specifications and the encoded protein analysed. The invention is illustrated by the following Examples: EXAMPLE 1 This example describes the identification and sequencing of an allelic human H 2 receptor gene using certain novel oligonucleotide primers according to the invention. A polymerase chain reaction (PCR) product is prepared from human DNA. DNA was extracted from human brain tissue by first pulverizing approximately lg of tissue in liquid nitrogen then adding to 10ml lysis buffer (0.32M sucrose, lOmM Tris, 5mM magnesium chloride 1% Triton X-100 pH8.0). This solution was centrifuged (9,000 rpm 15 mins) to pellet the tissue, the lysis buffer was drawn off and the pellet resuspended in 4.5ml 75 mM sodium chloride, 24mM EDTA. This solution was then incubated for 3 hours with 250 μl 10% SDS and 2mg proteinase K at 56°C. This aqueous phase was then extracted twice with 5ml of phenol:chloroform:isoamyl alcohol (25:24:1). Then sodium acetate to 0.3M pH7.5 and 2 volumes of ethanol (at -20°C) were added to the aqueous phase and the DNA hooked out into TE buffer. The concentration of the DNA was determined by measuring the optical density of the sample, at a wavelength of 260nm. The DNA was then amplified by the polymerase chain reaction using the oligonucleotide primers 1) and 2) (as hereinbefore described) for 36 cycles. The timing for each cycle was as follows; 1 min at 94°C, 1.5 min at 56°C and 2 mins at 72°C, this was then followed by a 10 min extension at 72°C (Amplitaq DNA polymerase Perkin-Elmer Cetus). This reaction produced a DNA fragment of 1047 base pair when analyzed by gel electrophoresis. Following PCR amplification of the DNA, the PCR products were immediately ligated and cloned into the TA cloning system (InvitroGen). The transformed cells were plated onto Luria-Bertani plates containing 50μl/ml amplicillin and 1.6mg X-Gal. Plates were then incubated overnight at 37°C, then moved to 4°C for 4 hours to allow for colour development. Positive (white colonies) were then analyzed by growing a 5ml culture overnight at 37°C extracting the plasmids (Qiaspin minipreps (Qiagen)) and performing an EcoRI digest to ensure the correct size product was contained in the plasmid. The plasmid used to clone the PCR product is the pCR™II Vector, which is transformed into One Shot™ INVαF' Competent cells. Both strands of the cloned PCR product were sequenced using the dideoxynucleotide chain-terminated method, carried out with Sequenase version 2.0 (Amersham/USB). Partial sequencing of short stretches of the cloned DNA utilised all the oligonucleotide primers 1) to 11) hereinbefore described. The cloned PCR product was shown to be identical to the gastric cDNA of Gantz et al except for the previously mentioned six single base changes. Results and discussions The sequence derived from the above described method is listed below and in SEQ ID NO: 2. The novel form of the human Histamine H 2 receptor gene is designated the H 2 R 6490 allele (SEQ ID N0:2), and the original form as described by Gantz et al is designated the H 2 R 649A allele, as described by Orange et al (1996) Neuro Report 7(7) (to be published) . 5' CCAATGGCACAGCCTCTTCCTTTTGCCTGGACTCTACCGCATGCAAGATCACCAT CACCGTGGTCCTTGCGGTCCTCATCCTCATCACCGTTGCTGGCAATGTGGTCGTC TGTCTGGCCGTGGGCTTGAACCGCCGGCTCCGCAACCTGACCAATTGTTTCATCG TGTCCTTGGCTATCACTGACCTGCTCCTCGGCCTCCTGGTGCTGCCCTTCTCTGC CATCTACCAGCTGTCCTGCAAGTGGAGCTTTGGCAAGGTCTTCTGCAATATCTAC ACCAGCCTGGATGTGATGCTCTGCACAGCCTCCATTCTTAACCTCTTCATGATCA GCCTCGACCGGTACTGCGCTGTCATGGACCCACTGCGGTACCCTGTGCTGGTCAC CCCAGCTCGGGTCGCCATCTCTCTGGTCTTAATTTGGGTCATCTCCATTACCCTG TCCTTTCTGTCTATCCACCTGGGGTGGAACAGCAGGAACGAGACCAGCAAGGGCA ATCATACCACCTCTAAGTGCAATGTCCAGGTCAATGAAGTGTACGGGCTGGTGGA TGGGCTGGTCACCTTCTACCTCCCGCTACTGATCATGTGCATCACCTACTACCGC ATCTTCAGGGTCGCCCGGGATCAGGCCAAGAGGATCGATCACATTAGCTCCTGGA AGGCAGCCACCATCAGGGAGCACAGAGCCACAGTGACACTGGCCGCCGTCATGGG GGCCTTCATCATCTGCTGGTTTCCCTACTTCACCGCGTTTGTGTACCGTGGGCTG AGAGGGGATGATGCCATCAATGAGATGTTAGAAGCCATCGTTCTGTGGCTGGGCT ATGCCAACTCAGCCCTGAACCCCATCCTGTATGCTGCGCTGAACAGAGACTTCCG CACCGGGTACCAACAGCTCTTCTGCTGCAGGCTGGCCAACCGCAACTCCCACAAA ACTTCTCTGAGGTCCAACGCCTCTCAGCTGTCCAGGACCCAAAGCCGAGAACCCA GGCAACAGGAAGAGAAACCCCTGAAGCTCCAGGTGTGGAGTGGGACAGAAGTCACG 3' EXAMPLE 2 This example describes the identification and sequencing of an allelic human H 2 receptor gene using certain novel oligonucleotide primers according to the invention. The example describes confirmation of the existence of polymorphisms in the human histamine H 2 receptor gene utilising single-stranded conformational polymorphism (SSCP). This is made possible by an assay based upon amplification of a 266 base pair PCR fragment of the human histamine H 2 receptor gene, followed by specific acrylamide gel electrophoresis. (It will be apparent to those skilled in the art that single base changes may also be detected by other techniques known to those in the art, including, chemical cleavage, PCR thermoligase reactions, restriction endonuclease digestion of human histamine H 2 receptor gene PCR fragments etc. ) Samples of blood are collected from human volunteers into EDTA coated tubes. 1ml of this blood is heated to 100"C for 15 minutes and then spun in a microcentrifuge at 13,000Xg for 15 minutes. The supernatant is collected and the cell debris is discarded, lμl of the supernatant is utilised as template DNA for a PCR reaction to amplify a portion of the human histamine H 2 receptor gene between bases 501 and 766. The conditions for this PCR reaction are 3mM MgCl 2 (Gibco BRL), 1XPCR buffer (Gibco BRL), 0.2mM of each dATP, dCTP, dTTP, dGTP (Gibco BRL) 10 pmoles of each of oligonucleotide primers 12) and 13) (hereinbefore described) and 1 unit Taq DNA polymerase, in a total volume adjusted to 50μl by sterile DNAse free water. This mix is then subjected to the following conditions in a Progene thermal cycler (Techne); 96°C, 5 minutes, then 35 cycles of 96°C, 45 seconds, 56°C, 45 seconds, 72 β C, 45 seconds. lOμl of the resultant product is analysed on a 1% agarose gel to ensure that the above reaction is correctly amplifying the target DNA fragement. Then 7.5μl of the PCR mix is added to 7.5μl of 95% formamide, lOmM EDTA pH 8.0, heated to 100 β C for 5-10 minute, and then quenched immediately in slushy ice for 5 minutes. 3μl of 6X blue/orange loading dye is then added (Promega) and the mix is loaded into a 1mm thick 14% 59:1 (acrylamide:bis-acrylamide) electrophoresis gel containing 2% glycerol, prepared in 0.5X TBE buffer. The gel is run for 18-24 hours at a constant voltage of 260V, at room temperature. The gel is then fixed in 10% methanol, 10% ethanoic acid for 10 minutes. The gel is next placed into lmg/ml silver nitrate for 15 minutes, then rinsed briefly with distilled water. The gel is then placed into 0.26% formaldehyde, 0.38M sodium hydroxide for 15 minutes. The gel is finally placed into 0.75% sodium carbonate for 10 minutes. Typical results are illustrated diagrammatically in Figure 1 which shows a SSCP gel, prepared as above described, on which different samples have been loaded. The following patterns of banding are seen: Lane 1 shows a pattern corresponding to a homozygons H 2 R 6 4 9o genotype. Lane 2 shows patterns corresponding to an individual heterozygons H 2 R 649A /H 2 R 649G genotype. Lane 3 shows a pattern corresponding to a homozygous H 2 R 6 * 9A genotype. Alternative embodiments of the invention can be envisaged by those skilled in the art from the information contained herein. All such alternative embodiments are intended to lie within the scope of this application. The reader's attention is directed to all papers and documents which are filed concurrently with this specification and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference. All the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps or any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features. The invention is not restricted to the details of the foregoing embodiments. This invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: The University of Sheffield (B) STREET: Western Bank (C) CITY: Sheffield (D) STATE OR PROVINCE: South Yorkshire (E) COUNTRY: United Kingdom (F) POSTAL CODE: S10 2TN INVENTORS (A) NAME: Heath, Paul Roy (B) STREET: 91 Abbey Lane (C) CITY: Sheffield (D) STATE OR PROVINCE: South Yorkshire (E) COUNTRY: United Kingdom (F) POSTAL CODE: S8 OBR (A) NAME: Orange, Paul Richard (B) STREET: 56 Salisbury Road, Crookes, (C) CITY: Sheffield (D) STATE OR PROVINCE: South Yorkshire (E) COUNTRY: United Kingdom (F) POSTAL CODE: S10 1WB (A NAME: Pearson, Ronald Carl Alan (B STREET: 12 Kingswood Road, Jesmond, (C CITY: Newcastle-upon-Tyne (D STATE OR PROVINCE: Tyne and Wear (E COUNTRY: United Kingdom (F POSTAL CODE: NE2 3NS (A NAME: Wright, Simon Ralph (B STREET: 56 Hunter Hill Road, Hunter's Bar (C CITY: Sheffield (D STATE OR PROVINCE: South Yorkshire (E COUNTRY: United Kingdom (F POSTAL CODE: Sll 8UE (ϋ) TITLE OF INVENTION: Improvements in or Relating to the Detection of Variations in Human Histamine H 2 Receptors (iii) NUMBER OF SEQUENCES: 15 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Dibb Lupton Broomhead (B) STREET: Fountain Precinct, Balm Green (C) CITY: Sheffield (D) STATE OR PROVINCE: South Yorkshire (E) COUNTRY: United Kingdom (F) POSTAL CODE: SI 1RZ (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy Disc 1.44 MB DSHD (B) COMPUTER: IBM PC Compatible (C) OPERATING SYSTEM: MS-DOS 6.22 (D) SOFTWARE: Word Perfect 5.1 for DOS (vi) CURRENT APPLICATION DATA: (A) APPLICATION NO: Not known (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Hall, Robert Leonard (B) REGISTRATION NO: (C) REFERENCE/DOCKET NUMBER: P31533GB (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 0114 283 3253 (B) TELEFAX: 0114 273 0312 (C) TELEX: (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 413 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double stranded (D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE: (A) ORGANISM: homo sapiens (C) INDIVIDUAL/ISOLATE: human tissue sample no:Sl 13 (D) DEVELOPMENT STAGE: fully mature (F) TISSUE TYPE: brain (vii) IMMEDIATE SOURCE: (B) CLONES: ONESHOT™ INVα F' clone PO1 (ix) FEATURES: (A) NAME/KEY: Taq I restriction endonuclease site (B) LOCATION: bases 253-257 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: CA GCT CGG GTC GCC ATC TCT CTG GTC TTA ATT TGG GTC ATC TCC ATT 47 Ala Arg Val Ala lie Ser Leu Val Leu lie Trp Val lie Ser lie 135 140 145 ACC CTG TCC TTT CTG TCT ATC CAC CTG GGG TGG AAC AGC AGG AAC GAG 95 Thr Leu Ser Phe Leu Ser lie His Leu Gly Trp Asn Ser Arg Asn Glu 150 155 160 ACC AGC AAG GGC AAT CAT ACC ACC TCT AAG TGC AAT GTC CAG GTC AAT 143 Thr Ser Lys Gly Asn His Thr Thr Ser Lys Cys Asn Val Gin Val Asn 165 170 175 GAA GTG TAC GGG CTG GTG GAT GGG CTG GTC ACC TTC TAC CTC CCG CTA 191 Glu Val Tyr Gly Leu Val Asp Gly Leu Val Thr Phe Tyr Leu Pro Leu 180 185 190 195 CTG ATC ATG TGC ATC ACC TAC TAC CGC ATC TTC AGG GTC GCC CGG GAT 239 Leu He Met Cys He Thr Tyr Tyr Arg He Phe Arg Val Ala Arg Asp 200 205 210 CAG GCC AAG AGG ATC GAT CAC ATT AGC TCC TGG AAG GCA GCC ACC ATC 287 Gin Ala Lys Arg He Asp His He Ser Ser Trp Lys Ala Ala Thr He 215 220 225 AGG GAG CAC AGA GCC ACA GTG ACA CTG GCC GCC GTC ATG GGG GCC TTC 335 Arg Glu His Arg Ala Thr Val Thr Leu Ala Ala Val Met Gly Ala Phe 230 235 240 ATC ATC TGC TGG TTT CCC TAC TTC ACC GCG TTT GTG TAC CGT GGG CTG 383 He He Cys Trp Phe Pro Tyr Phe Thr Ala Phe Val Tyr Arg Gly Leu 245 250 255 AGAGGGGATGATGCCATCAATGAGATGTTA 413 Arg Gly Asp Asp Ala He Asn Glu Met Leu 260 265 (3) INFORMATION FOR SEQ ID NO: 2: (I) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1046 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double stranded (D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic dna (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE: (A) ORGANISM: homo sapiens (C) INDIVIDUA1/ISOLATE: human tissue sample no: S 113 (D) DEVELOPMENT STAGE: fully mature (F) TISSUE TYPE: brain (vii) IMMEDIATE SOURCE: (B) CLONES. ONESHOT™ INV a F' clone PO1 (ix) FEATURES: (A) NAME/KEY: Taq I restriction endonuclease sites (B) LOCATION, bases 640-644 and 334-337 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: CC AAT GGC ACA GCC TCT TCC TTT TGC CTG GAC TCT ACC GCA TGC AAG 47 Asn Gly Thr Ala Ser Ser Phe Cys Leu Asp Ser Thr Ala Cys Lys 5 10 15 ATC ACC ATC ACC GTG GTC CTT GCG GTC CTC ATC CTC ATC ACC GTT GCT 95 He Thr He Thr Val Val Leu Ala Val Leu He Leu He Thr Val Ala 20 25 30 GGC AAT GTG GTC GTC TGT CTG GCC GTG GGC TTG AAC CGC CGG CTC CGC 143 Gly Asn Val Val Val Cys Leu Ala Val Gly Leu Asn Arg Arg Leu Arg 35 40 45 50 AAC CTG ACC AAT TGT TTC ATC GTG TCC TTG GCT ATC ACT GAC CTG CTC 191 Asn Leu Thr Asn Cys Phe He Val Ser Leu Ala He Thr Asp Leu Leu 55 60 65 CTC GGC CTC CTG GTG CTG CCC TTC TCT GCC ATC TAC CAG CTG TCC TGC 239 Leu Gly Leu Leu Val Leu Pro Phe Ser Ala He Tyr Gin Leu Ser Cys 70 75 80 AAG TGG AGC TTT GGC AAG GTC TTC TGC AAT ATC TAC ACC AGC CTG GAT 287 Lys Trp Ser Phe Gly Lys Val Phe Cys Asn He Tyr Thr Ser Leu Asp 85 90 95 GTG ATG CTC TGC ACA GCC TCC ATT CTT AAC CTC TTC ATG ATC AGC CTC 335 Val Met Leu Cys Thr Ala Ser He Leu Asn Leu Phe Met He Ser Leu 100 105 110 GAC CGG TAC TGC GCT GTC ATG GAC CCA CTG CGG TAC CCT GTG CTG GTC 383 Asp Arg Tyr Cys Ala Val Met Asp Pro Leu Arg Tyr Pro Val Leu Val 115 120 125 130 ACC CCA GCT CGG GTC GCC ATC TCT CTG GTC TTA ATT TGG GTC ATC TCC 431 Thr Pro Ala Arg Val Ala He Ser Leu Val Leu He Trp Val He Ser 135 140 145 ATT ACC CTG TCC TTT CTG TCT ATC CAC CTG GGG TGG AAC AGC AGG AAC 479 He Thr Leu Ser Phe Leu Ser He His Leu Gly Trp Asn Ser Arg Asn 150 155 160 GAG ACC AGC AAG GGC AAT CAT ACC ACC TCT AAG TGC AAT GTC CAG GTC 527 Glu Thr Ser Lys Gly Asn His Thr Thr Ser Lys Cys Asn Val Gin Val 165 170 175 AAT GAA GTG TAC GGG CTG GTG GAT GGG CTG GTC ACC TTC TAC CTC CCG 575 Asn Glu Val Tyr Gly Leu Val Asp Gly Leu Val Thr Phe Tyr Leu Pro 180 185 190 CTA CTG ATC ATG TGC ATC ACC TAC TAC CGC ATC TTC AGG GTC GCC CGG 623 Leu Leu He Met Cys He Thr Tyr Tyr Arg He Phe Arg Val Ala Arg 195 200 205 210 GAT CAG GCC AAG AGG ATC GAT CAC ATT AGC TCC TGG AAG GCA GCC ACC 671 Asp Gin Ala Lys Arg He Asp His He Ser Ser Trp Lys Ala Ala Thr 215 220 225 ATC AGG GAG CAC AGA GCC ACA GTG ACA CTG GCC GCC GTC ATG GGG GCC 719 He Arg Glu His Arg Ala Thr Val Thr Leu Ala Ala Val Met Gly Ala 230 235 240 TTC ATC ATC TGC TGG TTT CCC TAC TTC ACC GCG TTT GTG TAC CGT GGG 767 Phe He He Cys Trp Phe Pro Tyr Phe Thr Ala Phe Val Tyr Arg Gly 245 250 255 CTG AGA GGG GAT GAT GCC ATC AAT GAG ATG TTA GAA GCC ATC GTT CTG 815 Leu Arg Gly Asp Asp Ala He Asn Glu Met Leu Glu Ala He Val Leu 260 265 270 TGG CTG GGC TAT GCC AAC TCA GCC CTG AAC CCC ATC CTG TAT GCT GCG 863 Trp Leu Gly Tyr Ala Asn Ser Ala Leu Asn Pro He Leu Tyr Ala Ala 275 280 285 290 CTG AAC AGA GAC TTC CGC ACC GGG TAC CAA CAG CTC TTC TGC TGC AGG 911 Leu Asn Arg Asp Phe Arg Thr Gly Tyr Gin Gin Leu Phe Cys Cys Arg 295 300 305 CTG GCC AAC CGC AAC TCC CAC AAA ACT TCT CTG AGG TCC AAC GCC TCT 959 Leu Ala Asn Arg Asn Ser His Lys Thr Ser Leu Arg Ser Asn Ala Ser 310 315 320 CAG CTG TCC AGG ACC CAA AGC CGA GAA CCC AGG CAA CAG GAA GAG AAA 1007 Gin Leu Ser Arg Thr Gin Ser Arg Glu Pro Arg Gin Gin Glu Glu Lys 325 330 335 CCC CTG AAG CTC CAG GTG TGG AGT GGG ACA GAA GTC ACG 1046 Pro Leu Lys Leu Gin Val Trp Ser Gly Thr Glu Val Thr 340 345 350 (4) INFORMATION FOR SEQ ID NO: 3 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single stranded (D) TOPOLOGY, linear (ii) MOLECULAR TYPE: synthetic oligonucleotide (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 : CCAATGGCAC AGCCTCTT 18 (5) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic oligonucleotide (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: CGTGACTTCT GTCCCACT 18 (6) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic oligonucleotide (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: CCAGGCAACA GGAAGAGA 18 (7) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic oligonucleotide (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: TCTCTTCCTG TTGCCTGG 18 (8) INFORMATION FOR SEQ ID NO:, 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic oligonucleotide (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GCAGCAGAAG AGCTGTTG 18 (9) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic oligonucleotide (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: TCCAGGTCAA TGAAGTGT 18 (10) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic oligonucleotide (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: ACACTTCATT GACCTGGA 18 (11) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic oligonucleotide (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: CCAAGAGGAT CAATCACA 18 (12) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic oligonucleotide (iii) HYPOTHETICAL: no ( iv ) ANTI -SENSE : no (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: TGTGATTGAT CCTCTTGG 18 (13) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic oligonucleotide (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: ACACCAGCCT GGATGTGA 18 (14) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic oligonucleotide (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: TCACATCCAG GCTGGTGT 18 (15) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic oligonucleotide (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: CAATCATACC ACCTCTAA 18 (16) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single stranded (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic oligonucleotide (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: ACACAAACGC GGTGAAGT 18

Claims: CLAIUS. 1. Oligonucleotides, suitable for use as primers for the amplification or sequencing of DNA corresponding to a region of a human histamine H 2 receptor, selected from: 10) 5' ACACCAGCCTGGATGTGA 3' (also listed in SEQ ID NO: 12) 11) 5' TCACATCCAGGCTGGTGT 3* (also listed in SEQ ID NO: 13) 12) 5' CAATCATACCACCTCTAA 3' (also listed in SEQ ID NO: 14) 13) 5' ACACAAACGCGGTGAAGT 3' (also listed in SEQ ID NO: 15) 2. The use, as a primer for the amplification or sequencing of DNA corresponding to a region of a human histamine H 2 receptor, of an oligonucleotide selected from: 10) 5' ACACCAGCCTGGATGTGA 3' (also listed in SEQ ID NO: 12) 11) 5' TCACATCCAGGCTGGTGT 3' (also listed in SEQ ID NO: 13) 12) 5' CAATCATACCACCTCTAA 3' (also listed in SEQ ID NO: 14) 13) 5' ACACAAACGCGGTGAAGT 3' (also listed in SEQ ID NO: 15) 3. A process for the detection of a nucleotide sequence coding for a region of a human H 2 receptor, or of cDNA corresponding to transcription products thereof, in a biological sample thereof, which comprises: a) the amplification of the DNA with at least a pair of primers selected from the oligonucleotides of Claim 1 b) the detection of the amplified sequences on an electrophoretic gel. 4. A diagnostic kit comprising at least one oligonucleotide according to Claim 1. 5. A diagnostic kit according to Claim 4, which also comprises one or more specific restriction endonucleases capable of demonstrating one or more of the single base substitutions hereinbefore described. 6. A method for the detection of one or more inherited or acquired allelic polymorphic variations in a sample of a DNA encoding the human H 2 receptor comprising one or more of the six base substitutions hereinbefore defined, which comprises the use of single-stranded conformational polymorphism (SSCP). 7. A method according to Claim 6, wherein the step of PCR Amplification is followed by the step of detecting one or more single base variations in the human H 2 receptor using SSCP. 8. The use of at least one oligonucleotide according to Claim 1, for determining the base variations in the human H 2 receptor of an individual for providing information relating to disease susceptibility, disease course, prognosis and/or response to treatment. 9. An oligonucleotide suitable for use as a primer for the amplification of DNA corresponding to a region of a human H 2 receptor substantially as hereinbefore described. 10. A process according to Claim 3, substantially as described in the Examples.

Inventors: HEATH PAUL, ORANGE PAUL, PEARSON RONALD CARL ALAN, WRIGHT SIMON, HEATH, PAUL, ORANGE, PAUL, PEARSON, RONALD, CARL, ALAN, WRIGHT, SIMON
Applicants: HEATH PAUL, ORANGE PAUL, PEARSON RONALD CARL ALAN, UNIV SHEFFIELD, WRIGHT SIMON, HEATH, PAUL, ORANGE, PAUL, PEARSON, RONALD, CARL, ALAN, THE UNIVERSITY OF SHEFFIELD, WRIGHT, SIMON
Application year: 1997
Main classification: C12Q1
IPC codes: C07K14, C12N15, C12Q1