MARKERS FOR DISEASE

Field of the Invention

The present invention relates to a method for diagnosing, or determining predisposition to disease.

Background to the Invention

The neurotransmitter, dopamine, orchestrates a wide-range of cognitive and motor functions associated with the brain, that affect memory, emotion and locomotion (Sibley and Monsama, 1992; Missale et al., 1998). Failure to regulate this process is implicated in a variety of neuropsychiatric disorders, including Parkinson's Disease, schizophrenia, Tourette syndrome, Attention Deficit Hyperactivity Disorder (ADHD) and alcohol and drug addiction, but dopamine signalling is poorly understood at the molecular level. It is important to gain a detailed insight into this process, because of its role in normal functioning of the brain, its relationship to other neurotransmitter systems and its role in major psychiatric disorders.

Dopamine signalling is coordinated through five dopamine receptors (Dl -D 5) that are subdivided into two sub-families, Dl like (including Dl and D5) and D2 like (including D2, D3 and D4), according to their genetic and pharmacological properties. Dopamine receptors belong to the family of G-protein (GTP -binding protein) coupled receptors (GPCRs).

hi WO 2006/043081, the contents of which are incorporated herein, we disclose 11 proteins that interact with one or more dopamine receptors. These proteins are referred to herein as dopamine receptor interacting proteins (DRIPs). All of the DRIP proteins are expressed in brain and involved in dopamine signalling. Their corresponding genes map to regions of the genome that have previously been linked to schizophrenia.

The genetic factors which predispose an individual to neuropsychiatric disorders such as schizophrenia and Parkinson's disease are thought to be variants of DNA structure

("polymorphisms") that alter the level of expression or the function of genes. Variants of DNA sequence at a particular site ("locus") are known as "alleles".

Close localisation of disease causing genes may be accomplished by the detection of associations between particular alleles and the disease phenotype. The detection of allelic association may therefore give information as to disease susceptibility in a particular individual. Furthermore, allelic association is indicative of a disease-causing gene being present within a limited distance (50- 500 Kb) of DNA in either direction from the allele. Such a region is likely to contain a gene or genetic element modifying disease susceptibility.

To date, no known disease associated genetic variants have been identified for schizophrenia. The identification of genetic polymorphisms in linkage disequilibrium with neuropsychiatric disorders such as described herein will allow the identification of children at risk of such diseases before the disease has developed (for example immediately after birth), with the potential for prevention of disease. The presence of particular polymorphisms or combinations of polymorphisms may predict the clinical course of disease (e. g. severe as opposed to mild) or the response to particular treatments. This diagnostic information will be of use to the health care, pharmaceutical and insurance industries.

The present inventors have identified a number of genetic polymorphisms in the genes encoding DRIPs that are associated with neuropsychiatric disorders and can be used as a diagnostic tool.

Statements of the Invention

According to a first aspect of the invention there is provided a method of diagnosing, or determining predisposition to, disease in a subject the method comprising determining the presence or absence of a variant form of one or more of the nucleic acid sequences shown in Figures 1 to 11 in an isolated sample wherein the presence of a variant is indicative of disease or susceptibility to disease. A variant includes any SNP from the wild-type (e. g. for humans Figures 1 to 11) or other mutation or alteration from the wild-type.

The nucleic acid sequences may be genomic DNA (gDNA) sequences or cDNA sequences. cDNA sequences encoding DRIPs 1 to 11 are shown in Figures l(b) to 11 (b) respectively. gDNA sequences encoding DRIPs 1 to 11 are shown in Figures l(a) to 11 (a) respectively. Preferably the nucleic acid sequences are gDNA sequences having the following accession numbers:

AA496212 (DRIPl)

ABO 14594 (DRIP2)

NM_024640 (DRIP3) BC020066 (DRIP4)

AK000669 (DRIP5)

X61118 (DRIP6)

M24194 (DRIP7)

Ll 3744 (DRIP8) U77735 (DRIP9)

NM_000917 (DRIPlO)

Q12756, X90840, NM_004321 (DRIPI l)

The method of the invention may comprise analysing the nucleic acid, for example gDNA or cDNA, in an isolated sample from a subject, preferably human, to determine the presence or absence of one or more polymorphisms within the nucleic acid sequences shown in Figures 1 to 11 ₅ wherein the presence of a risk allele is indicative of disease or predisposition to disease.

The present invention is advantageous in that it facilitates the accurate diagnosis of disease, or the determination of predisposition to disease. Thus, by genotyping, an individual may be identified as having or being predisposed to disease. This helps to identify those individuals who are likely to respond positively to particular treatments or preventative measures. Thus, more effective therapies or preventative measures can be administered.

In a preferred method the variant is of one or more of the sequences shown in Figure 2, 6 or 11.

In one embodiment , the method of the invention comprises analysing the nucleic acid in an isolated sample from a subject to determine the presence or absence of one or more polymorphisms within the nucleic acid sequence shown in Figure 2.

In one embodiment , the method of the invention comprises analysing the nucleic acid in an isolated sample from a subject to determine the presence or absence of one or more polymorphisms within the nucleic acid sequence shown in Figure 6.

In one embodiment , the method of the invention comprises analysing the nucleic acid in an isolated sample from a subject to determine the presence or absence of one or more polymorphisms within the nucleic acid sequence shown in Figure 11.

The term "polymorphism" refers to the coexistence of multiple forms of a sequence. Thus, a polymorphic site is the location at which sequence divergence occurs. The different forms of the sequence which exist as a result of the presence of a polymorphism are referred to as "alleles". The region comprising a polymorphic site may be referred to as a polymorphic region.

Examples of the ways in which polymorphisms are manifested include restriction fragment length polymorphisms (Bostein et al Am J Hum Genet 32 314-331 (1980)), variable number of tandem repeats, hypervariable regions, minisatellites, di-or multi- nucleotide repeats, insertion elements and nucleotide or amino acid deletions, additions or substitutions. A polymorphic site may be as small as one base pair, which may alter a codon thus resulting in a change in the encoded amino acid sequence.

Single nucleotide polymorphisms arise due to the substitution, deletion or insertion of a nucleotide residue at a polymorphic site. Such variations are referred to as SNPs. SNPs may occur in protein coding regions, in which case different polymorphic forms of the sequence may give rise to variant protein sequences. Other SNPs may occur in non-coding regions. In either case, SNPs may result in defective proteins or regulation of genes, thus resulting in disease. Other SNPs may have no direct phenotypic effects, but may show linkage to disease states, thus serving as markers for disease. SNPs typically occur more frequently throughout the genome than other forms of polymorphism discussed above, and there is therefore a greater probability of finding a SNP associated with a particular disease state.

In a preferred method of the invention, the method comprises determining the presence or absence of one or more of the polymorphisms listed in Tables 1 to 11. The method may involve determining the presence or absence or a risk allele of the one or more polymorphisms. In the context of the present invention, a risk allele is the allele of a polymorphism which is associated with disease or predisposition to disease. The risk allele may be the wild type or the variant allele. The method may also comprise genotyping one or more known polymorphisms. Any combination of such polymorphisms may be genotyped.

Polymorphisms according to the invention may be selected from those listed in Tables 1 to 11. The polymorphisms may be located at a chromosomal position shown Tables 1 to 11 where the chromosomal position corresponds to a position within a gene encoding DRIP 1 to 1 1 respectively or within a region (typically about 2kb) flanking said gene. Such flanking regions are often the site of regulatory sequences important in regulating gene expression. Preferably the polymorphism is located within a gene sequence encoding any of DRIP 1 to 11 as shown in Tables 1 to 11.

In one embodiment, the inventon comprises a method in which the polymorphism is selected from those listed in Tables 1, 2, 3, 5, 6, 7, 8 or 11.

In a preferred method of the invention, the polymorphism is a SNP in chromosome 2q36.3 as listed in Table 2.

In a preferred method of the invention, the polymorphism is a SNP in chromosome 1 Ip 13 as listed in Table 6.

In a preferred method of the invention, the polymorphism is a SNP in chromosome 2q37.3 as listed in Table 11.

Where two or more adjacent polymorphisms at a locus are genotyped, the method preferably defines determining the presence or absence of a haplotype which is indicative of disease or predisposition to disease. A haplotype is defined herein as a collection of polymorphic sites in a particular sequence that are inherited in a group, i. e. are in linkage disequilibrium with each other. The identification of haplotypes in the diagnosis of disease may help to reduce the possibility of false positives. The haplotype may be any particular combination of polymorphisms of any of Tables 1 to 11. The present inventors have, in Table 12, identified polymorphisms that are inherited in a group. Thus a preferred method of the invention provides for determining the presence or absence in an isolated sample/population of an SNP shown in Table 12, optionally in combination with one or more known polymorphisms. Preferred embodiments of the invention are as follows wherein the relationship between the DRIP, the gene encoding the DRIP and the chromosome number of the gene is shown in Table 13.

In a preferred method of the invention, the polymorphism is a SNP in chromosome 2q36.3 as listed in Table 12.

In a preferred method of the invention, the polymorphism is a SNP is chromosome 1 Ipl3 as listed in Table 12.

In a preferred method of the invention, the polymorphism is a SNP in chromosome 2q37.3 as listed in Table 12.

In a further preferred method, the invention comprises determining the presence or absence of one or more of the polymorphisms listed in any of Tables 14 to 24.

Methods for use in the first aspect of the invention may include those known to persons skilled in the art for identifying differences between nucleic acid sequences (in diseased and non-diseased individuals), for example direct probing, allele specific hybridisation, polymerase chain reaction (PCR) methodology including Pyrosequencing (Ahmadian A, Gharizadeh B, Gustafsson AC, Sterky F, Nyren P, Uhlen M, Lundeberg J. Single- nucleotide polymorphism analysis by pyrosequencing, Anal Biochem. 2000 Apr 10; 280 (1) : 103-10; Nordstrom T, Ronaghi M, Forsberg L, de Faire U, Morgenstern R, Nyren P. Direct analysis of single-nucleotide polymorphism on double-stranded DNA by pyrosequencing. Biotechnol Appl Biochem. 2000 Apr; 31 (Pt 2): 107-12) Allele Specific Amplification (ASA) (W093/22456), Allele Specific Hybridisation, single base extension (US patent No. 4,656,127), ARMS-PCR, Taqman (US 4683202; 4683195 ; and 4965188), oligo ligation assays, single-strand conformational analysis ( (SSCP) Orita et al PNAS 86 2766-2770 (1989)), Genetic Bit Analysis (WO 92/15712) and RFLP direct sequencing, mass-spectrometry (MALDI- TOF) and DNA arrays. The appropriate restriction enzyme, will, of course, be dependent upon the polymorphism and restriction site, and will include those known to persons skilled in the art. Analysis of the digested fragments may be performed using any method in the art, for example gel analysis, or southern blots.

Preferably the method for use in the invention comprises the steps of:

i) isolating genomic DNA from a subject; ii) amplifying said isolated DNA by PCR with primers that flank a polymorphism in a gene encoding a DRIP to produce amplified DNA regions; and iii) sequencing said amplified regions to determine the presence or absence of said polymorphism wherein the presence of said polymorphism is indicative of disease or susceptibility to disease.

In an alternative preferred method of the invention the variant is a polymorphism resulting from trinucleotide (or triplet base) repeat instability. The polymorphism is an AGC repeat sequence or complement thereof.

Thus the method may comprise the steps of amplifying by PCR said AGC repeat sequence present in a test genomic DNA using at least one oligonucleotide primer capable of amplifying a AGC nucleotide repeat sequence present in the gene encoding a DRIP 8, thereby producing amplified genomic DNA fragments. In a preferred embodiment the method comprises the steps of: i) isolating DNA from a sample from a subject; ii) amplifying said isolated DNA with primers that flank an AGC repeat sequence in a gene encoding DRIP 8 (Figure 8) to produce amplified AGC repeat regions; and iii) determining a number of AGC repeat in said AGC repeat region in said sample.

Depending on the disease, the number of AGC repeats in a gene encoding a DRIP in a diseased subject may be higher or lower than the number in a non-diseased subject. Typically, however, the method of the invention involves determining whether AGC repeats are observed at a relatively high frequency in a DNA sample from a diseased subject compared to a control DNA sample, wherein if AGC repeats are observed at a relatively high frequency in a DNA sample from a diseased subject but occurs at a lower frequency in a control sample, said subject has, or is at risk of developing, said disease. As used herein a control sample is a wild type DNA sample from a non-diseased subject.

The AGC repeat sequence is preferably of formula (AGC) _n wherein n is an integer greater than 40, for example greater than 42 such as greater than 45. Thus in a preferred method of the invention, a subject has, or is at risk of developing, a disease if said number of AGC repeats in a gene encoding a DRIP is greater than 40, for example greater than 42, 45 or 50.

As used herein a "gene encoding a DRIP" is generally one or more of the sequences shown in Figures 1 to 11 but also may include other known gene sequences encoding a DRIP. Preferably the gene encodes DRIP 8 (Figure 8).

In an alternative method, the invention provides a method of diagnosing, or determining predisposition to, disease caused by AGC repeat instability in a subject the method comprising the steps of:

i) isolating genomic DNA from a sample from a subject; ii) amplifying by PCR said isolated DNA with primers that flank an AGC repeat sequence in a gene encoding a DRIP to produce amplified AGC repeat regions; and iii) electrophoresing said amplified sample genomic DNA fragments to produce a sample electrophoresis pattern.

Depending on the disease, the labelled fragments in the electrophoresis pattern may be larger or smaller in a diseased subject compared to a non-diseased subject. Typically, however, the method of the invention involves comparing said sample electrophoresis pattern to a control electrophoresis pattern; and determining whether the subject is at risk of developing a disease caused by AGC repeat instability, wherein if said sample genomic DNA electrophoresis pattern contains labelled fragments larger than labelled fragments from said control genomic DNA electrophoresis pattern, said subject may be at risk for developing diseases caused by AGC repeat instability. As used herein a "control" electrophoresis pattern refers to that obtained from wild type DNA.

The methods of the invention are preferably carried out on a sample removed from a subject. Any biological sample comprising cells containing nucleic acid is suitable for this purpose. Examples of suitable samples include whole blood, leukocytes, semen, saliva, tears, buccal, skin or hair. For analysis of cDNA, mRNA or protein, the sample must come from a tissue in which the sequence of interest is expressed. Blood is a readily accessible sample. Thus, the methods of the invention typically include the steps of obtaining a sample from a subject, and preparing nucleic acid from the sample.

The subject is preferably a mammal, and more preferably a human. The subject may be an infant, a child or an adult. Alternatively, the sample may be obtained from the subject prepartum e. g. by amniocentesis, or post mortum brain tissue.

As used herein the term "disease" refers to dopamine mediated disorders for example neuropsychiatric disorders, including Parkinson's Disease, Attention Deficit Hyperactivity Disorder (ADHD), depression (bipolar disorder) and schizophrenia and addiction (e.g drug addiction such as alcohol, nicotine or cocaine addiction). Other "dopamine-mediated disorders" may include neurodegenerative disorders (e.g Alzheimer's disease, Tourette Syndrome, Parkinson's disease, Huntington's disease, Amyotrophic Lateral Sclerosis, senile chorea, Sydenham's chorea, autism, head and spinal cord trauma, acute and chromic pain, epilepsy and seizures, dementia, distonia, tremor, autism, cerebral ischemia and neuronal cell death) and disorders linked to apoptosis (particularly neuronal apoptosis). Dopamine related disorders may include cardiovascular, pulmonary or renal conditions.

Generally the disease is a neuropsychiatric disorder such as Parkinson's disease, schizophrenia or depression.

The above described methods may require amplification of the DNA sample from the subject, and this can be done by techniques known in the art, such as PCR (see PCR

Technology : Principles and Applications for DNA Amplification (ed. H. A. Erlich, Freeman Press, NY 1992; PCR Protocols : A Guide to methods and Applications (eds.

Innis et al., Academic press, San Diego, CA 1990); Mattila et al., Nucleic Acids Res.

19 4967 (1991) ; Eckert et al. _s PCR Methods and Applications 117 (1991) and US Patent No 4,683,202. Other suitable amplification methods include ligase chain reaction (LCR) (Wu et al., Genomics 4 560 (1989); Landegran et al., Science 241 1077 (1988)), transcription amplification (Kwoh et al., Proc Natl Acad Sci USA 86 1173 (1989)), self sustained sequence replication (Guatelli et al., Proc Natl Acad Sci USA 87 1874 (1990)) and nucleic acid based sequence amplification (NASBA). The latter two methods both involve isothermal reactions based on isothermal transcription which produce both single stranded RNA and double stranded DNA as the amplification products, in a ratio of 30 or 100 to 1, respectively.

Where it is desirable to analyse multiple samples simultaneously, it may be preferable to use arrays as described in W095/11995. The array may contain a number of probes, each designed to identify variants of the gene sequence shown in Figures 1 to 11 from a sample. Where a restriction enzyme is required, it can be selected according to the nature of the polymorphism and restriction site. Suitable enzymes will be known to persons skilled in the art. Analysis of the digested fragments may be performed using any method in the art, for example gel analysis, or southern blots.

Determination of an allele of a polymorphism using the above methods typically involves the use of anti-sense sequences i. e. sequences which are complementary to the nucleic acid sequences of interest, which may include part of the sequence of Figures 1 to 11.

Where it is desirable to identify the presence of multiple single nucleotide polymorphisms, or haplotypes, in a sample from a subject, it may be preferable to use arrays as described in W095/11995. The array may contain a number of probes, each

designed to identify one or more of the above single nucleotide polymorphisms of the invention.

In a further aspect of the invention there is provided isolated nucleic acid molecules comprising all or part of the sequence of Figures 1 to 11 wherein the all or part of the sequence comprises one or more of the variants described herein. The variant may be a polymorphism, preferably SNP ₃ selected from those listed in any one of Tables 1 to 12 respectively. Alternatively the variant may be a polymorphism selected from those listed in any one of Tables 14 to 24. Alternatively the variant may be an AGC repeat sequence as described herein.

The nucleic acid molecules of the invention may be DNA, RNA, and single or double stranded sequences. All the molecules of the present invention are isolated, or alternatively may be recombinant. By isolated is meant a nucleic acid molecule which has been purified, and is substantially free of protein and other nucleic acid. Such molecules may be obtained by PCR amplification, cloning techniques, or synthesis on a synthesiser. By recombinant is meant nucleic acid molecules which have been recombined by the hand of man.

The isolated nucleic acid molecules of the present invention are different to the "wild type" or "reference" sequence of Figures 1 to 11. The nucleic acid sequences of the invention which differ from the sequence of Figures 1 to 11 at any one or more of the positions detailed in Tables 1 to 11 or 12 to 24, or include one or more polymorphsims as listed in Table 12, or with respect to the number of AGC repeat sequences therein, are referred to as polymorphic variants of the sequence of Figures 1 to 11 and form part of the invention.

The invention also provides antisense sequences. Such sequences are typically single stranded and are capable of hybridizing to the above mentioned nucleic acid sequences of

the invention under stringent conditions. Preferred antisense sequences are those which are capable of hybridising to an allele of a polymorphism of the invention, and most preferably is capable of distinguishing between alleles of a polymorphism.

Stringent conditions are defined below. The antisense sequences may be prepared synthetically or by nick translation, and are preferably isolated or recombinant. The antisense sequences include primers and probes, for example for use in the methods of the present invention. Primer sequences are capable of acting as an initiation site for template directed nucleic acid synthesis, under appropriate conditions which will be known to skilled persons. Probes are useful in the detection, identification and isolation of particular nucleic acid sequences. Probes and primers are preferably 15 to 30 nucleotides in length.

For amplification purposes, pairs and primers are provided. These include a 5 'primer which hybridizes to the 5'end of the nucleic acid sequence to be amplified, and a 3' primer which hybridizes to the complementary strand of the 3'end of the nucleic acid to be amplified. Probes and primers may be labelled, for example to enable their detection.

Suitable labels include for example, a radiolabel, enzyme label, fluoro-label, biotin-avidin label for subsequent visualization in, for example, a southern blot procedure or high throughput genotyping methods such as TaqMan (ABI). A labelled probe or primer may be reacted with a sample DNA or RNA, and the areas of the DNA or RNA which carry complimentary sequences will hybridise to the probe, and become labelled themselves. The labelled areas may be visualized, for example by autoradiography.

Preferably, the probes and/or primers hybridise under, "stringent conditions", which refers to the washing conditions used in a hybridisation protocol. The hybridisation conditions for probes are preferably sufficiently stringent to allow distinction between different alleles of a polymorphism upon binding of the probes. In general, the washing conditions should be combination of temperature and salt concentration so that the denaturation temperature is approximately 5 to 20°C below the calculated Tm of the nucleic acid under study. The Tm of a nucleic acid probe of 20 bases or less is calculated under standard conditions (IM NaCl) as [4°C x (G+C) + 2°C x (A+T)], according to Wallace rules for

short oligonucleotides. For longer DNA fragments, the nearest neighbor method, which combines solid thermodynamics and experimental data may be used, according to the principles set out in Breslauer et al., PNAS 83: 3746-3750 (1986). The optimum salt and temperature conditions for hybridisation may be readily determined in preliminary experiments in which DNA samples immobilised on filters are hybridised to the probe of interest and then washed under conditions of different stringencies. While the conditions for PCR may differ from the standard conditions, theTm may be used as a guide for the expected relative stability of the primers. For short primers of approximately 14 nucleotides, low annealing temperatures of around 44°C to 50°C are used. The temperature may be higher depending upon the base composition of the primer sequence used. Typically, the salt concentration is no more than IM, and the temperature is at least 25°C. Suitable conditions are 5XSSPE (750 mM NaCI, 5OmM NaPhosphate, 5mM EDTA pH 7.4) and a temperature of 25-30°C.

In a further aspect, there is provided a host cell comprising an isolated nucleic acid molecule according to invention. The nucleic acid molecule may be provided in a vector. The host cell may comprise an expression vector, or naked DNA encoding the nucleic acid molecules of the invention. A wide variety of suitable host cells are available, both eukaryotic and prokaryotic. Examples include bacteria such as E. coli, yeast, filamentous fungi, insect cells, mammalian cells, preferably immortalised, such as mouse, CHO,

HeLa, myeloma, neuronal cell lines or Jurkat cell lines, human and monkey cell lines and derivatives thereof.

The host cells are preferably capable of expression of the nucleic acid sequence to produce a gene product (i. e. RNA or protein). Such host cells are useful in drug screening systems to identify agents for use in diagnosis or treatment of subjects having, or being susceptible to diseases as defined above.

The method by which said nucleic acid molecules are introduced into a host cell will usually depend upon the nature of both the vector/DNA and the target cell, and will

include those known to a person skilled in the art. Suitable known methods include but are not limited to fusion, conjugation, liposomes, immunoliposomes, lipofectin, transfection, transduction, eletroporation or injection, as described in Sambrook et al.

In a yet further aspect of the invention there is provided a kit for diagnosis of disease or predisposition to disease, comprising a means for determining the presence or absence of

a variant form of one or more of the gene sequences shown in Figures 1 to 11 wherein the presence of a variant is indicative of disease or susceptibility to disease. The variant may be a polymorphism, preferably a risk allele of a polymorphism, of Tables 1 to 12. Alternatively the variant may be AGC repeat instability.

In particular, the kit comprises means for determining the presence or absence of a risk allele of a SNP as represented in Tables 1 to 12.

Preferably the kit will comprise the components necessary to determine the presence or absence of a risk allele, in accordance with the methods of the invention. Such components include PCR primers and/or probes, for example those described above, PCR enzymes, restriction enzymes, and DNA or RNA purification means. Preferably, the kit will contain at least one pair of primers, or probes, preferably as described above in accordance with the tenth aspect of the invention. The primers are preferably allele specific primers. Other components include labelling means, buffers for the reactions. In addition, a control nucleic acid sample may be included, which comprises a wild type or variant nucleic acid sequence as defined above, or a PCR product of the same. The kit will usually also comprise instructions for carrying out the diagnostic method, and a key detailing the correlation between the results and the likelihood of disease. The kit may also comprise an agent for the prevention or treatment of disease.

In a further aspect of the invention, there is provided a method of identifying a compound for treatment of disease, comprising (a) administration of a compound to a host cell comprising an isolated nucleic acid molecule comprising a variant form of one or more of

the sequences shown in Figures 1 to 11, for example at a position which corresponds to an rs mraker or a chromosomal position shown in Tables 1 to 11 ; and (b) determining whether the agent modulates effects of the variant. In this aspect, a nucleic acid molecule of the invention, and/or a cell line according to an aforementioned aspect, may be used to screen for agents which are capable of modulating the effect of a variant, for example an SNP. Potential agents are those which react differently with a risk allele and non-risk allele.

The invention further provides a method of treating or preventing disease in a subject comprising

(i) determining the presence or absence of a variant form of one or more of the nucleic acid sequences shown in Figures 1 to 11 in an isolated sample from the subject; and

(ii) if the variant is present, administering treatment in order to prevent, delay or reduce the disease.

The invention provides for agents to be administered either as DNA or RNA and thus as a form of gene therapy.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features and embodiments described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

The invention will now be described with reference to the following Tables and Figures, and examples which are included for the purposes of illustration only and are not to be construed as being limiting on the invention.

Tables 1 to 11 and 12 to 24 detail polymorphisms identified within the genes encoding DRIP 1-11 (Figures 1 to 11 respectively) and the respective probable chromosomal positions of the polymorphisms. With reference to Tables 1 to 11 and 12 to 24, polymorphisms include deletion/insertion polymorphisms (DIPs) and single nucleotide polymorphism (SNPs). Those polymorphism shown between "start" and "end" correspond to polymorphisms found within the gene. Those polymorphisms shown outside the gene correspond to polymorphisms found within the regions flanking the gene, typically about 2kb. "rs" corresponds to "reference SNP";

TABLES 1 and 14 show polymorphisms identified in the gene encoding DRIPl, C14ORF28, and their position in chromosome 14q21.1;

TABLES 2 and 15 show polymorphisms identified in the gene encoding DRIP2, DOCKlO, and their position in chromosome 2q36.3;

TABLEs 3 and 16 show polymorphisms identified in the gene encoding DRIP3, YRDC, and their position in chromosome Ip34.3;

TABLES 4 and 17 show polymorphisms identified in the gene encoding DRIP4, PDCD6IP, and their position in chromosome 3p22.3;

TABLES 5 and 18 show polymorphisms identified in the gene encoding DRJP5, TERF2IP, and their position in chromosome 16q22.3;

TABLES 6 and 19 show polymorphisms identified in the gene encoding DRIP6, LM02, and their position in chromosome 1 IpI 3;

TABLES 7 and 20 show polymorphisms identified in the gene encoding DRIP7, GNB2L1, and their position in chromosome 5q35.3;

TABLES 8 and 21 show polymorphisms identified in the gene encoding DRIP8, MLLT3, and their position in chromosome 9p22; TABLES 9 and 22 show polymorphisms identified in the gene encoding DRIP9, PIM2, and their position in chromosome xpl 1.23;

TABLES 10 and 23 show polymorphisms identified in the gene encoding DRIPlO, P4HA1, and their position in chromosome 10q21.3-q23.1; TABLES 11 and 24 show polymorphisms identified in the gene encoding DRIPl 1, KIFlA, and their position in chromosome 2q37.3;

TABLE 12 shows a list of SNPs identified and their associated DRIP gene .

TABLE 13 shows the relationship between DRIPS, the genes encoding them and the chromosomal location of the genes;

FIGURES 1 to 11 show (a) the accession numbers of the genomic DNA sequences encoding the dopamine receptor interacting proteins (DRIPs) 1 to 11 respectively, and (b) the full length cDNA sequences of the dopamine receptor interacting proteins (DRIPs) 1 to 11 respectively.

EXAMPLES

GENOTYPING STUDY MATERIALS AND METHODS

A case-control study was undertaken. High throughput genotyping was performed as described in previously published research (Kutyavin et al., Nucleic Acids Res. 2006;34(19):el28. Epub 2006 Sep 29) based on an endonuclease IV genotyping system. PCR reactions were set up using high throughput liquid handling robots in conjunction with the ABI 7900HT sequence detection system.

SNP

Single nucleotide polymorphisms (SNPs) were selected from a range of sources, including HapMap, dbSNP, PubMed and Ensembl. Additional SNPs were identified through direct DNA sequencing of DRIPS from various samples. Tagging SNPs (Tables 1 to 12 and 14 to 24) were identified using "tagger", "SNP Browser" and other publically available software as well as in silico tools and methods we developed. Patterns of linkage disequilibrium (LD) and genomic organisation across genes corresponding to each of the eleven DPJPs were analysed in detail in all populations, including Caucasians. 100 SNPs were tested for genotyping. nsSNPs (coding) and allele frequencies were examined in detail. Tagging SNPs were selected based on minor allele frequencies, LD measures (pairwise r ² and D values), distance between SNPs, comparison of genomic sequences from different species and other parameters. One SNP (rs3209771) was not polymorphic and another (rsl 1032438) failed the assay.

SAMPLES DNA was derived from buccal swabs or blood. DNA was analysed from a sample consisting of approximately 500 cases and 500 controls of Danish origin. The samples were carefully selected to ensure they were genetically and clinically homogeneous. The patients, recruited from hospitals in Denmark, were chronic schizophrenics who had been suffering from schizophrenia for at least 15 years. AU the patients were matched to at least two controls from Danish blood donors according to gender and age.

RESULTS

809 out of 811 DNA samples worked. Overall, 99.2% of genotyping was successful yielding 74,547 genotypes. A number of checks were performed to assess the quality of the data. To check for differences between plates, chi-squared tests of allele distributions and two-marker allele differences (for markers within 50 kb) were performed to compare patterns of linkage disequilibrium across plates. Chi-squared tests of Hardy- Weinberg equilibrium were also carried out, using a cutoff of p< Ie- 10.

Data were analysed using a 1 -sided Fisher's Exact Test to establish p-values as this provides a better estimate of the p-value than a standard chi-square test when the number of cases is less than 1000. A number of markers appeared to show evidence for positive association. The p-values and odds ratios were at least as significant as those published

elsewhere. 14 markers showed strong statistical significance (p<0.05, df=l). These markers are clustered into three genes (DRIP-2, DRIP-6, DRIP-11) providing further evidence that the results are not random [Table 12]. A further 12 markers representing DRP-I, DRIP-3, DRIP-5, DRff-7 and DRIP-8 showed less significant evidence for association (p<0.1) mostly attributable to low allele frequencies. Genotyping further samples and analysis of additional SNPs listed herein (or their SNP equivalents) should enhance their statistical significance.