Novel allele of human histamine h2 receptor and methods of detection of h2 receptor variants

Description of equivalent WO 9623880 A2

NOVEL ALLELE OF HUMAN HISTAMINE Hj RECEPTOR AND METHODS OF DETECTION OF H₂ RECEPTOR VARIANTS

Technical Field

This invention relates to the detection of variations in human histamine H₂ receptors, and more particularly to the development of new compounds useful in the sequencing and identifica ion of a human histamine H₂ 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 H₂ receptor and their use in the diagnosis and treatment of human disorders.

Background Ar

The human H₂ receptor was first identified by Black et al Nature (1972), 236, 385 - 390. This was followed by the demonstration of the receptor in the mammalian brain by Baudry et al (1975) Nature 253, 362 - 363, and Haas and Bucher (1975) Nature 255, 634 - 635. Gantz et al (1991) Biochem. Biophys. Res. Comm. 178,3, 1386-1392 have recently identified the sequence of a human H₂ receptor cDNA from gastric parietal cells by using the polymerase chain reaction (PCR) and degenerated oligonucleotide primers whose sequence was obtained from the canine H₂ receptor previously cloned by this group, Gantz et al (1991) Proc. Nat. Acad. Sci. USA 88, 429 - 433. This sequence was characterised as an intronless gene encoding a typical seven transmembrane domain aminergic receptor protein.

The receptor is coupled to heterotrimeric GTPases (G proteins), but differs from other monoamine receptors in this G protein coupled superfamily in several respects.

The human gastric H₂ receptor is shorter than most other receptors in this class (359 amino acids) and lacks the two serine residues in the fifth transmembrane region (T 5). There exists instead an aspartate and a threonine residue, so far unique in this region. These two residues may be important for binding with the nitrogen atoms of the i idazole ring of histamine as suggested by

Birdsall (1991) Trends in Pharmacological Sci. Jan, 12, 9 - 10.

Histamine is a natural constituent of many organs and tissues including the gastrointestinal tract, the immune system and the brain, Green et al (1987) Agents and Actions 22, 1 - 15. It is a central neurotransmitter in the brain and is formed in the posterior hypothalamus from exogenous histidine by histidine decarboxylase (HDC). It is subsequently metabolised by histamine methyltransferase (HMT), Prell et al (1986) Ann. Rev. Neurosci. 9, 209 - 254. The cell bodies and neuronal pathways for histamine have been mapped in the human brain using immunocytochemistry by Panula et al (1990) Neuroscience 34, 127 - 132. Its cells project from the tuberomamillary nucleus of the posterior hypothalamus to almost every region of the brain. There are three known histamine receptors; H_lf H₂ and H₃, the latter functioning as an autoreceptor. The H₂ receptor specifically has been localised in the human brain by Traiffort et al (1992) J. of Neuroche . 59, 1, 290 - 299. using receptor autoradiography.

Histamine is known to have significant effects in the central nervous system (CNS). It has been implicated in the CNS mediated mechanisms of arousal ever since the sedating effect of H_λ receptor antagonists (eg, . chlorpheniramine, chloropromazine) had been noticed clinically. The use of H₂ receptor antagonists in the human brain however, has shown, that these compounds, unlike those acting on the H_x receptor, do not produce any effect on psycho otor functioning, or a subjective feeling of sedation or arousal in healthy subjects, White et al (1988) Psychopharmacology 95, 1 - 14. Some H₂ receptor antagonists (eg. cimetidine) are known to cause confusion in elderly or severely medically ill patients, perhaps in part due to a co-existing anti-cholinergic effect. H_: and H₂ receptor antagonists in large doses have been reported to cause hallucinations, Csillag et al (1973) Med. J. Aust. 1, 653 - 654, Argawal (1978) J. Am. Med. Assoc. 240, 214. Animal studies have shown that histamine applied directly to the hippocampus, where there is the highest level of activity of the H₂ receptor, will induce psychomotor withdrawal and decreased exploratory behaviour. The above evidence has led to the conclusion that H_: receptor systems are excitatory in the terms of arousal and motivated behaviour whilst H₂ receptor systems are inhibitory in this respect, Alvarez and Banzan (1985) Physiol. and Beh. 34, 661 - 664 and (1986) Physiol. and Beh. 37, 39 - 45, White et al (1988) supra.

The H₂ receptor is a site of action of various compounds used in the treatment of psychiatric disorders eg. amitriptyline and mianserin, Traiffort et al (1992) supra. Kaminsky et al (1990) The Lancet 335, 1351 - 1352 and (1991) Schizophrenia Bull. 4, 318 - 319 have reported the successful response of patients with chronic, predominantly negative type schizophrenia, to the highly specific H₂ receptor antagonist famotidine. For example in one patient there was a substantial amelioration of the deficit symptoms of schizophrenia (eg apathy, social withdrawal, and blunted affect) while on famotidine, relapse in these symptoms on withdrawal, and improvement on re-institution of this drug, Kaminski, US Patents Nos. 5070101 and 5177081. Prell et al (1992) Abstract, part 1, 199.6 Soc. for Neurosci. Annual Meeting, Anaheim Cal. have shown substantially raised levels of N-tele-methyl histamine, a metabolite of histamine in the cerebrospinal fluid of patients with schizophrenia which correlates with those patients with the occurrence of negative symptoms of this disorder assessed using the Psychiatric Symptoms Assessment Scale. These levels were not significantly different between patients free from medication and those on neuroleptic therapy. It is therefore postulated that there is an increase in histaminergic activity in patients with chronic schizophrenia.

The disclosures of all the above mentioned publications are incorporated herein by reference for all purposes.

Additionally, histamine, acting via its receptors, including the H₂ receptor, is believed to be critically involved in a number of diseases of organs other than the brain; these include peptic ulceration, allergic reactions, including asthma, immune-mediated disorders, and possibly some tumours.

The histamine H₂ receptor is one of many receptors in the body. Compounds used to treat many diseases work by activating a receptor or inhibiting the action if its natural ligand. Variations in 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.

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.

As a first step to the present invention, the Applicants devised a new oligonucleotide probe to the human H₂ receptor mRNA in accordance with the published cDNA sequence available for the gastric parietal cell. Surprisingly, studies using this probe with in-situ hybridization histochemistry on human post-mortem brain tissue produced evidence of a mismatch in the nucleotide sequence for the brain H₂ receptor and the sequence for the gastric parietal cell H₂ receptor. This discovery was made by recording melt-curve estimations for the optimum hybridization incubation temperature using the method of Davis et al (1986) "Basic Methods in Molecular Biology" page 77 Elsevier Science Publishing Co. It was found that the sequence mismatch is of the order of 10%.

It was apparent, therefore, that there is a hitherto unrecognised allele or subtype of the human histamine H₂ receptor gene, which may be specific to the brain.

In a first aspect, the invention provides a sequence for a novel allele of a human histamine H₂ 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

In another aspect, the invention provides a nucleotide sequence coding for a region of a human histamine H₂ receptor, comprising one or more of the following base substitutions compared with the published sequence in Gantz et al (1991) supra, 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 of the invention can, for example, comprise the following sequence (as also listed in SEQ ID NO: 1):

5' CAGCTCGGGTCGCCATCTCTCTGGTCTTAATTTGGGTCATCTCCATTACCCTGTC

CTTTCTGTCTATCCACCTGGGGTGGAACAGCAGGAACGAGACCAGCAAGGGCAAT

CATACCACCTCTAAGTGCAATGTCCAGGTCAATGAAGTGTACGGGCTGGTGGATG

GGCTGGTCACCTTCTACCTCCCGCTACTGATCATGTGCATCACCTACTACCGCAT

CTTCAGGGTCGCCCGGGATCAGGCCAAGAGGATCGATCACATTAGCTCCTGGAAG GCAGCCACCATCAGGGAGCACAGAGCCACAGTGACACTGGCCGCCGTCATGGGGG

CCTTCATCATCTGCTGGTTTCCCTACTTCACCGCGTTTGTGTACCGTGGGCTGAG

AGGGGATGATGCCATCAATGAGATGTTA 3 '

As a specific exemplification, the nucleotide sequence of the invention can comprise the following sequence (as also 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*

In another aspect of the invention, a series of new oligonucleotide primers have been developed for the identification of sequences in a sample comprising a human histamine H₂ receptor DNA, cDNA or RNA originating from a tissue sample or body fluid. In this aspect, the invention provides new oligonucleotides, suitable for use as primers for the amplification of DNA corresponding to a region of a human histamine H₂ 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 a diagnostic kit comprising one or more of the new oligonucleotides.

The direction and base start numbers for the novel oligonucleotide primers are as follows:

Primer Base Start No.

1) Upstream 8 2) Downstream 1036 and 1095

3) Upstream 995

4) Downstream 1012 (no. 3) in reverse)

5) Downstream 898 and 1171 6) Upstream 527

7) Downstream 544 ( no . 6 ) in reverse )

8) Upstream 638

9) Downstream 655 (no. 8) in reverse)

Information on the human histamine H₂ receptor was obtained from the MRC Daresbury database accessing "Genem 61" File no. M64799 - Human histamine H₂ receptor gene.

The above mentioned substitutions alter and in some instances introduce or remove new sites for cleavage by specific restriction endonucleases as follows: base change site alters restriction map of:

398 Alul, Aval, BspWI, BsrI, CviJI 525

620 Eco57

649 Clal, Sau3A, TaqI

692

802 Mnll

The invention further 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.

A kit may be used to establish genotype or base variations. This information may be used in predicting an individuals 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 H₂ receptor antagonist like famotidine or other forms of treatment such as social or psychological intervention.

Eucaryotic expression vectors comprising a DNA sequence coding for a protein and.or a peptide according to the invention are new materials and are also included in the invention. Host cells, for example, cloned human cell lines, such as NTera 2 c.dl, can be transformed using the new expression vectors and are also included in the invention.

Expression vectors and host cells transformed thereby, in accordance with the invention, can be prepared, for example, as detailed below, and the encoded protein studied, by one or more of the following exemplary methods:

1. Total RNA is extracted from homogenised human tissue, eg. brain, by the acid guanidine thiocyanate method (Chomczynski & Saach (1987), anal. Biochem. 161, 156-159). Messenger RNA (mRNA) is purified from this by hybridisation of oligo(d)T to the polyadenylated tails present on the majority of mRNA's, for example, using the Promega PolyAttract ® system. Reverse transcription of the mRNA using specific reverse transcriptase enzyme, eg. Superscipt II, Gibco BRL, is followed by PCR amplification of the resultant product using specific oligonucleotide primers, for example, those previously described. The resulting amplified cDNA is ligated into an expression vector, eg. pGEMEX®-l vector available from Promega. Competent cells, eg. bacterial strain JM109(DE3), also available from Promega, are transformed using this vector, effective transforms selected and cultured. Expression of the encoded protein is then induced with a suitable promotor, eg. IPTG, and the expressed protein purified from the cell culture using standard biochemical procedures, eg. cell lysis and polyacrylamide gel electrophoresis.

2. An alternative method for examining the functional protein encoded by the cDNA described above, is to induce transcription of the cloned cDNA, as above, and to purify the specific mRNA from the cell culture as described. The purified mRNA is introduced into competent cells, eg. frog oocytes or Chinese hamster ovary cells, and the function of the encoded protein studied by standard pharmacological and physiological techniques, eg. microelectrode recording and receptor binding techniques. 3. As 1 above, but introducing the cDNA into a coupled transcription-translation system, eg. TNT, Promega with subsequent purification and analysis of the encoded protein as described.

The invention is illustrated by the following Examples:

EXAMPLE 1

This example describes the identification and sequencing of an allelic human H₂ 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 M 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/ιnl 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 9) 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.

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 confirmation of the presence of the base changes in a larger population. This is made possible by an assay based upon PCR amplification of a 909 base pair fragment of the H₂ receptor gene from human DNA, followed by cleavage utilising specific restriction endonucleases. It will apparent to those skilled in the art that single base changes could be detected using other techniques known to those in the art which include single stranded confirmational polymorphisms (sscp), chemical cleavage, PCR thermoligase reactions etc.

Samples of blood are collected from human volunteers into EDTA coated tube, 1ml of this blood is heated to 100°C for 15 minutes then spun in a microcentrifuge at 13,000Xg for 15 minutes. This supernatant is collected, and the cell debris is discarded. Then 0.5-3μl of this supernatant is utilised as template DNA for a PCR reaction to amplify a portion of the receptor gene between bases 8 and 915. The conditions for this PCR reaction are 3mM MgCl₂ (Gibco BRL),1X PCR buffer (Gibco BRL) lmM of each dATP, dGTP, dGTP and dTTP (Promega) 10 pmoles of each of oligonucleotide primers 1) and 5) (hereinbefore described) and 1 unit Taq DNA polymerase (Gibco BRL), in a total volume adjusted to 50μl by sterile DNAse free water. This mix is then subjected to the following conditions; 96°C 5 minutes, then 35 cycles of 96°C for 1 minute, 56°C for 1 minute, 72°C for 1 minute and 20 seconds.

lOμl of the resultant products are then analysed on a 1% agarose gel to ensure that the above reaction is correctly amplifying the target DNA fragment. Then 11.5μl of the PCR mix is added to 2 units Taql restriction endonuclease (Fermentas) and 1.5μl of 10X buffer and incubated at 65°c for 3-24 hours. The products of this reaction are then analysed on a 2.5% agarose gel. If the original sequence described by Gantz (nominated H₂A) has been amplified, then bands of 574 and 335 base pairs are seen which indicates that the individual is an A/A homozygote. If the sequence described in Example 1 (nominated H₂B) has been amplified, then following the TaqI cleavage of the PCR product , bands of 335, 306 and 268 base pairs can be seen, indicating that that individual is a B/B homozygote. If bands of 574, 335, 306 and 268 base pairs can be seen, then that individual is an A/B heterozygote.

Typical results are illustrated in Figure 1, which shows a 2.5% TBE Agarose gel stained with ethidium bromide, showing T ql digestion patterns of a 909 base pair PCR fragment, from 4 separate individuals.

Lanes A + G - 100 base pair DNA marker (Gibco BRL)

Lanes B + F - Band pattern indicative of an A/B heterozygote

Lanes C + E - Band pattern indicative of an B/B homozygote

Lane D - Blank

Arrows indicated the sizes of the DNA fragments in lanes

B to F.

Primers: Oswell DNA Services)

1) upstream: 5' CCAATGGCACAGCCTCTT 3' (as in SEQ ID N0:1)

2) upstream: 5' CCAGGCAACAGGAAGAGA 3' (as in SEQ ID NO:5) 5)downstream: 5' GCAGCAGAAGAGCTGTTG 3' (as in SEQ ID NO:7)

Example 3 A method as described in example 2 is applied to a series of DNA samples extracted from schizophrenic individuals, their first degree relatives, and normal controls. There is observed a statistically significant difference of P less than 0.01 the pattern seen in the genotype of these individuals, as described in the table below:

Diagnosis -H. . Genotypιe

A/A A/B B/B

Controls 12.1% 48.5% 39.4%

Schizophrenia 9.8% 26.8% 63.4%

1st degree 6.1% 12.1% 81.8% relatives

Discussion

The variable sequence is explained by a polymorphic allelic variation within the human population for the gene encoding the H₂ receptor protein. This allelic polymorphism may lead to substantial variation in the effect of activation of the encoded receptor by histamine, either in the efficacy of histamine binding, the duration of activation, or the intracellular effects of such activation. It is envisaged that such variation resulting from allelic polymorphism may underline susceptibility to specific disorders, both affecting the brain and/or involving other systems or organs. In summary, this variation in the human H₂ receptor gene and its products, including, for example, mRNA and proteins, could be used as a method of establishing individual risk to a particular psychiatric or neurological or other illness eg. schizophrenia.

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

(A NAME: Heath, Paul Roy (US only) (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 (US only) (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 (US only) (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 (US only) (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 H₂ Receptors

(iii) NUMBER OF SEQUENCES: 11

(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: P31409PC

(ix) TELECOMMUNCATION 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:S113

(D) DEVELOPMENT STAGE: fully mature (F) TISSUE TYPE: brain

(vii) IMMEDIATE SOURCE:

(B) CLONES: ONESHOT™ INVαF' clone POl

(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 lie Met Cys lie Thr Tyr Tyr Arg lie 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 lie Asp His lie Ser Ser Trp Lys Ala Ala Thr lie 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 lie lie Cys Trp Phe Pro Tyr Phe Thr Ala Phe Val Tyr Arg Gly Leu 245 250 255

AGA GGG GAT GAT GCC ATC AAT GAG ATG TTA 413

Arg Gly Asp Asp Ala lie Asn Glu Met Leu 260 265 28 (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) INDIVIDUAL/ISOLATE: human tissue sample no:S113

(D) DEVELOPMENT STAGE: fully mature (F) TISSUE TYPE: brain

(vii) IMMEDIATE SOURCE:

(B) CLONES: ONESHOT™ INVαF' clone POl

(i ) 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 lie Thr lie Thr Val Val Leu Ala Val Leu lie Leu lie 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 lie Val Ser Leu Ala lie 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 lie 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 lie 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 lie Leu Asn Leu Phe Met lie 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 lie Ser Leu Val Leu lie Trp Val lie Ser

135 140 145

ATT ACC CTG TCC TTT CTG TCT ATC CAC CTG GGG. TGG AAC AGC AGG AAC 479 lie Thr Leu Ser Phe Leu Ser lie 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 lie Met Cys lie Thr Tyr Tyr Arg lie 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 lie Asp His lie 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 lie 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 lie lie 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 lie Asn Glu Met Leu Glu Ala lie 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 lie 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) MOLECULE 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 IB

(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