WO2009101619A2

WO2009101619A2 - Methods for predicting a patient's response to lithium treatment

Info

Publication number: WO2009101619A2
Application number: PCT/IL2009/000162
Authority: WO
Inventors: Ruth Navon; Anat Levit; Gilad Silberberg
Original assignee: Ramot At Tel-Aviv University Ltd.
Priority date: 2008-02-11
Filing date: 2009-02-11
Publication date: 2009-08-20
Also published as: WO2009101619A3

Abstract

The present invention provides use of specific single nucleotide polymorphisms (SNPs) in the calcium channel γ-2 subunit (Cacng2) gene as a tool to predict response to lithium-based therapy in psychiatric conditions, specifically in patients suffering from bipolar disorders. The invention therefore concerns methods for identifying an increased likelihood for positive response to lithium treatment in a subject by determining allelic variants of Cacng2 gene present in a nucleic acid sample obtained from a subject, wherein the presence of certain allelic variants is indicative of an increased likelihood for a positive response of the subject to lithium treatment. The invention further provides oligonucleotides useful in the detection of the SNPs as well as kits for affecting the methods of the invention.

Description

Methods for predicting a patient's response to lithium treatment

FIELD OF THE INVENTION

This invention relates to methods for predicting the response to lithium therapy in patients with mental or psychiatric disorders, by analyzing the presence of specific SNPs in the Cacng2 gene.

BACKGROUND OF THE INVENTION

Bipolar disorder (BPD; also called manic-depressive illness) and schizophrenia are severe, chronic and life threatening illnesses, both characterized by extremely high suicide rates (1). Lithium carbonate (lithium) is considered the treatment of choice for BPD and multiple studies have shown its efficacy in prophylactic treatment of BPD. However, lithium is effective only in 60—80% of BPD patients (2). Long-term lithium treatment has been shown to reduce the risk of suicide and normalize the increased cardiovascular mortality (3). Lithium is known to affect neurotransmitter release, the metabolism of biogenic monoamines and neuronal signal transmission through perturbation of the distribution of sodium, magnesium, and calcium. Lithium can inhibit depolarization-induced and calcium-dependent release of norepinephrine and dopamine and may stimulate the release of serotonin. Direct molecular targets suggested to be inhibited by lithium include inositol monophosphatase phosphomonoesterases, and glycogen synthase kinase-3β (GSK-3β) (4).

The fact that patients with a positive family history of BPD have a good response to lithium prophylaxis (5) suggests that genetic factors play a substantial role in lithium treatment of the disease. Numerous genes have been tested for association with lithium response, but most yielded nonreplicable or negative results. The relatively few genes in which positive association to lithium response was found, include phospholipase C gamma (PLCGl), (6) and tryptophan hydroxylase (TPH) (7).

The calcium channel γ-2 subunit gene (CacngT) located at chromosome 22ql3.1 region was recently reported to be associated with schizophrenia (8). This finding is consistent with several previous studies reporting linkage of the chromosomal region 22ql2-13 to schizophrenia (9). In addition, the 22ql2-13 chromosomal region has been linked with BPD (10). The D22S278 marker, present in that region, revealed convincing evidence of linkage disequilibrium with both BPD (10) and schizophrenia (11).

WO 2007/047634 discloses specific SNPs in the CACNG2 gene associated with response to lithium treatment in bipolar patients.

References

1. Tanney B: Psychiatric diagnoses and suicidal acts. New York, Guilford Press, 2000

2. Manji HK, Bebchuk JM, Moore GJ, Glitz D, Hasanat KA, Chen G: Modulation of CNS signal transduction pathways and gene expression by mood-stabilizing agents: therapeutic implications. J Clin Psychiatry 1999; 60 Suppl 2:27-39; discussion 40-1, 113-6

3. Ahrens B, Muller-Oerlinghausen B, Schou M, Wolf T, Alda M, Grof E, Grof P, Lenz G, Simhandl C, Thau K, et al.: Excess cardiovascular and suicide mortality of affective disorders may be reduced by lithium prophylaxis. J Affect Disord 1995; 33(2):67-75

4. Phiel CJ, Klein PS: Molecular targets of lithium action. Annu Rev Pharmacol Toxicol 2001; 41:789-813

5. Mendlewicz J, Fieve RR, Stallone F: Relationship between the effectiveness of lithium therapy and family history. Am J Psychiatry 1973; 130(9): 1011-3

6. Turecki G, Grof P, Cavazzoni P, Duffy A, Grof E, Ahrens B, Berghofer A, Muller-Oerlinghausen B, Dvorakova M, Libigerova E, Vojtechovsky M, Zvolsky P, Joober R, Nilsson A, Prochazka H, Licht RW, Rasmussen NA, Schou M, Vestergaard P, Holzinger A, Schumann C, Thau K, Rouleau GA, Alda M: Evidence for a role of phospholipase C-gammal in the pathogenesis of bipolar disorder. MoI Psychiatry 1998; 3(6):534-8 7. Serretti A, Lilli R, Lorenzi C, Gasperini M, Smeraldi E: Tryptophan hydroxylase gene and response to lithium prophylaxis in mood disorders. J Psychiatr Res 1999; 33(5):371-7

8. Liu Y, Farm, C.S., Liu, C, Chen, WJ., Wu, J., Hung, S. et al: (2005): SNP Fine Mapping of Chromosome 22ql2 reveals the Novel Vulnerability Gene for Schizophrenia, CACNG2: Association with Impairment of Sustained Attention and Executive Function, in World Congress on Psychiatric Genetics XIII. Boston, US, 2005, p 55

9. Badner JA, Gershon ES: Meta-analysis of whole-genome linkage scans of bipolar disorder and schizophrenia. MoI Psychiatry 2002; 7(4):405-l 1

10. Liang SG, Sadovnick AD, Remick RA, Keck PE, McElroy SL, Kelsoe JR: A linkage disequilibrium study of bipolar disorder and microsatellite markers on 22ql3. Psychiatr Genet 2002; 12(4):231-5

11. Gill M, Vallada H, Collier D, Sham P, Holmans P, Murray R, McGuffin P, Nanko S, Owen M, Antonarakis S, Housman D, Kazazian H, Nestadt G, Pulver AE, Straub RE, MacLean CJ, Walsh D, Kendler KS, DeLisi L, Polymeropoulos M, Coon H, Byerley W, Lofthouse R, Gershon E, Read CM, et al.: A combined analysis of D22S278 marker alleles in affected sib-pairs: support for a susceptibility locus for schizophrenia at chromosome 22ql2. Schizophrenia Collaborative Linkage Group (Chromosome 22). Am J Med Genet 1996; 67(l):40-5

12. Torrey EF, Webster M, Knable M, Johnston N, Yolken RH: The Stanley foundation brain collection and neuropathology consortium. Schizophr Res 2000; 44(2): 151-5 - A -

13. Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real- time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25(4):402-8

14. Myakishev MV, Khripin Y, Hu S, Hamer DH: High-throughput SNP genotyping by allele-specific PCR with universal energy-transfer-labeled primers. Genome Res 2001; l l(l):163-9

15. Rusch TL, Dickinson W., Che J., Fieweger K., Chudyk J., Doktycz M., Yu A., and Weber J.L., : Instrumentation for Continuous Array Genotyping of Short Insert/Deletion Polymorphisms. Proceedings of the SPIE - Microarrays and Combinatorial Technologies for Biomedical Applications: Design, Fabrication, and Analysis, 2003; 4966:138-145

16. Barnett V, Lewis, T.: Outliers in statistical data. Chichester, John Wiley & Sons, 1994

17. Fisher R: Statistical methods for research workers. Edinburgh, Olivier & Boyd, 1932

18. Schaid DJ, Rowland CM, Tines DE, Jacobson RM, Poland GA: Score tests for association between traits and haplotypes when linkage phase is ambiguous. Am J Hum Genet 2002; 70(2):425-34

19. Slatkin M, Excoffier L: Testing for linkage disequilibrium in genotypic data

using the Expectation-Maximization algorithm. Heredity 1996; 76 ( Pt 4):377-83

20. Nuovo GJ et al: In situ amplification using universal energy transfer-labeled primers, J. Histochem. Cytochem. (1999) 47: 273-280. SUMMARY OF THE INVENTION

The present invention provides the use of single nucleotide polymorphisms (SNPs) in the calcium channel γ-2 subunit (Cacng2) gene located on chromosomal region 22ql3.1 as a predictive method of lithium response, wherein the SNP is selected from the group consisting of rs2284017, rs2284018, rs5750285 and any combination thereof.

In accordance with the present invention there is provided a method for identifying an increased likelihood for positive response to lithium treatment in a subject by determining allelic variants of Cacng2 gene present in a nucleic acid sample obtained from a subject, wherein the presence of certain allelic variants is indicative of an increased likelihood for a positive response of the subject to lithium treatment. The allelic variants of Cacng2 gene of the present invention are selected from the group consisting of rs2284017, rs2284018 and rs5750285 and any combination thereof, wherein the C allele is indicative of a positive response to treatment.

In one of its aspects, the present invention is directed to a method for identifying an increased likelihood for positive response to lithium treatment in a subject, the method comprising testing a sample obtained from the subject for the presence of at least one polymorphism of the Cacng2 gene selected from the group consisting of: (i) allele C of the C/T single nucleotide polymorphism (SNP) rs2284017; (ii) allele C of the C/T single nucleotide polymorphism (SNP) rs2284018;

(iii) allele C of the C/G single nucleotide polymorphism (SNP) rs5750285; and

(iv) any combination thereof; wherein presence of at least one of the polymorphisms indicates an increased likelihood for a positive response to lithium treatment in the subject.

In one embodiment, the presence of allele C of the C/T polymorphism rs2284017 and allele C of the C/T polymorphism rs2284018 is indicative of an increased likelihood for a positive response to lithium treatment in the subject. In another embodiment, the presence of allele C of the C/T polymorphism rs2284017 and allele C of the C/T polymorphism rs5750285 is indicative of an increased likelihood for a positive response to lithium treatment in the subject. Additionally, the presence of allele C of the C/T polymorphism rs2284018 and allele C of the C/T polymorphism rs5750285 is indicative of an increased likelihood for a positive response to lithium treatment in the subject. In a specific embodiment, the presence of allele C of the C/T polymorphism rs2284017, allele C of the C/T polymorphism rs2284018, and allele C of the C/T polymorphism rs5750285 is indicative of an increased likelihood for a positive response to lithium treatment in the subject.

In a second aspect, the present invention provides a method for identifying a an increased likelihood for positive response to lithium treatment in a subject, comprising testing a sample obtained from the subject for the presence of at least one genotype of the Cacng2 gene selected from the group consisting of:

(i) C/C genotype of a single nucleotide polymorphism (SNP) rs2284017; (ii) C/C genotype of a single nucleotide polymorphism (SNP) rs2284018; (iii) C/C genotype of a single nucleotide polymorphism (SNP) rs5750285; and

(iv) any combination thereof; wherein the presence of at least one genotype indicates an increased likelihood for a positive response to lithium treatment in the subject.

The methods of the present invention can be performed in combination with testing for the presence of at least one additional marker associated with response to lithium treatment.

The method for identifying an increased likelihood for positive response to lithium treatment can further comprise measuring a clinical symptom of the subject. In one embodiment, the subject of the method is human.

The subject can suffer from a mental disorder, a psychiatric disorder or a psychotic disorder. The mental or psychiatric disorder can be a mood disorder. The mood disorder, for example, is a bipolar disorder.

The methods of the invention for identifying an increased likelihood for positive response to lithium treatment in a subject can be performed prior to treatment of the subject with lithium or during the treatment of the subject with lithium. Optionally, the sample is obtainable from a body fluid. The later can be selected from the group consisting of blood, saliva, cerebrospinal fluid, urine, and sperm.

Testing for the presence of a polymorphism in the present invention can be performed by SNP genotyping, for example, by allele-specifϊc PCR.

In one embodiment, the presence of the polymorphism is determined by contacting nucleic acids obtained from a sample of the subject with a polynucleotide probe which hybridizes with at least one polymorphism of the Cacng2 gene selected from the group consisting of:

(i) allele C of the C/T single nucleotide polymorphism (SNP) rs2284017; (ii) allele C of the C/T single nucleotide polymorphism (SNP) rs2284018;

(iii) allele C of the C/G single nucleotide polymorphism (SNP) rs5750285; and any combination thereof; and determining whether the sample comprises a polynucleotide that hybridizes with the probe, thereby indicating the presence of said polymorphism.

In another aspect, the present invention provides a nucleic acid probe comprising a nucleotide sequence which hybridizes to a polymorphism in the Cacng2 gene, wherein the polymorphism comprises at least one single nucleotide polymorphisms (SNP) selected from the group consisting of:

(i) allele C of the C/T single nucleotide polymorphism (SNP) rs2284017; (ii) allele C of the C/T single nucleotide polymorphism (SNP) rs2284018; (iii) allele C of the C/G single nucleotide polymorphism (SNP) rs5750285; (iv) allele T of the C/T single nucleotide polymorphism (SNP) rs2284017;

(v) allele T of the C/T single nucleotide polymorphism (SNP) rs2284018; (vi) allele G of the C/G single nucleotide polymorphism (SNP) rs5750285; and (vii) any combination thereof.

In one embodiment said nucleic acid probe is used for identifying an increased likelihood for a positive response to lithium treatment in a subject.

The probe of the present invention can further be used in combination with detection of at least one additional marker for response to lithium treatment.

The present invention further provides the use of a nucleotide sequence which hybridizes to a polymorphism in the Cacng2 gene, wherein the polymorphism comprises at least one single nucleotide polymorphisms (SNP) selected from the group consisting of:

(i) allele C of the C/T single nucleotide polymorphism (SNP) rs2284017; (ii) allele C of the C/T single nucleotide polymorphism (SNP) rs2284018; (iii) allele C of the C/G single nucleotide polymorphism (SNP) rs5750285; (iv) allele T of the C/T single nucleotide polymorphism (SNP) rs2284017; (v) allele T of the C/T single nucleotide polymorphism (SNP) rs2284018; (vi) allele G of the C/G single nucleotide polymorphism (SNP) rs5750285; and (vii) any combination thereof. for identifying an increased likelihood for a positive response to lithium treatment in a subject.

The present invention is also directed to an array comprising a substrate having a plurality of segments, wherein at least one of the segments comprises a probe which hybridizes to a polymorphism in the Cacng2 gene, wherein the polymorphism is at least one single nucleotide polymorphisms (SNP) selected from the group consisting of: (i) allele C of the C/T single nucleotide polymorphism (SNP) rs2284017; (ii) allele C of the C/T single nucleotide polymorphism (SNP) rs2284018; (iii) allele C of the C/G single nucleotide polymorphism (SNP) rs5750285; (iv) allele T of the C/T single nucleotide polymorphism (SNP) rs2284017; (v) allele T of the C/T single nucleotide polymorphism (SNP) rs2284018; (vi) allele G of the C/G single nucleotide polymorphism (SNP) rs5750285; and (vii) any combination thereof.

In one embodiment said array is used for identifying an increased likelihood for a positive response to lithium treatment in a subject.

The present invention also provides the use of an array comprising a substrate having a plurality of segments, wherein at least one of the segment comprises a probe which hybridizes to a polymorphism in the Cacng2 gene, in identifying an increased likelihood for a positive response to lithium treatment in a subject, wherein the polymorphism is at least one single nucleotide polymorphisms (SNP) selected from the group consisting of:

The present invention further provides a kit compartmentalized to receive at least one oligonucleotide probe which hybridizes to at least one single nucleotide polymorphisms (SNP) selected from the group consisting of:

(i) allele C of the C/T single nucleotide polymorphism (SNP) rs2284017; (ii) allele C of the C/T single nucleotide polymorphism (SNP) rs2284018; (iii) allele C of the C/G single nucleotide polymorphism (SNP) rs5750285; (iv) allele T of the C/T single nucleotide polymorphism (SNP) rs2284017; (v) allele T of the C/T single nucleotide polymorphism (SNP) rs2284018; (vi) allele G of the C/G single nucleotide polymorphism (SNP) rs5750285; and (vii) any combination thereof and at least one additional reagent.

The kit can further comprise at least one additional oligonucleotide that hybridizes with at least one additional marker for response to lithium treatment. Optionally, the oligonucleotide probe comprises at least 12, 14, 15, or 21 contiguous nucleotides selected from SEQ ID NO: 1-9.

The present invention further comprises a method for obtaining information regarding the increased likelihood for positive response to lithium treatment in a subject, the method comprising testing a sample obtained from the subject for the presence of at least one polymorphism of the Cacng2 gene selected from the group consisting of:

(i) allele C of the C/T single nucleotide polymorphism (SNP) rs2284017; (ii) allele C of the C/T single nucleotide polymorphism (SNP) rs2284018; (iii) allele C of the C/G single nucleotide polymorphism (SNP) rs5750285; and

(iv) any combination thereof; wherein presence of each of the polymorphisms indicates an increased likelihood for a positive response to lithium treatment in the subject. The information can be used to classify a subject in a clinical trial. The information can also be used to classify a population of subjects.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph representing mRNA expression of Cacng2. Normalized mRNA expression levels for Cacng2 were analyzed by real-time PCR for schizophrenia, bipolar and control subjects. The mRNA expression level of every sample (RQ value) was normalized to the average mRNA expression level for the control group (av[RQ]). The y-axis units are arbitrary for relative comparison of individual samples within the three sample groups presented.

Figure 2 is a graph showing Genotypes (A, B) and allele (C, D) counts in lithium response categories in SNPs rs2284017 (A, C) and rs5750285 (B, D) in the Aberdeen population set.

DETAILED DESCRIPTION OF EMBODIMENTS

As used herein, the singular forms "a," "and," and "the" include plural referents unless the context clearly indicates to the contrary. For example, "a polynucleotide" includes a plurality of polynucleotides and "the SNP" includes reference to one or more SNPs.

Lithium is a known medication in the psychiatric setting for use in treating bipolar disorders. In particular, it is used as a mood-altering drug. Lithium is typically suitable for both mania and depression. In some circumstances, Lithium is used to augment other psychiatric drugs.

Some individuals respond positively to lithium treatment. However, others do not enjoy the beneficial effects of this treatment. Additionally, it is not uncommon for physicians in the psychiatric arena to try plurality of medications until they select the most suitable treatment for their particular patients. This trial and error approach suffers from several disadvantages. Prolonged periods of treatment sometimes elapse before the physician reaches the conclusion that Lithium, for example, is not suitable for a particular individual. In addition, negative side effects can appear after many months of the treatment. Therefore, the need arises to provide methods for determining a priori the putative outcome of lithium treatment. Indeed, the present invention provides a method for prediction of response to lithium treatment.

Definitions:

An "allele" is a particular form of a genetic locus, distinguished from other forms by its particular nucleotide sequence, or one of the alternative polymorphisms found at a polymorphic site.

"Bipolar disorder" is a mood disorder characterized by alternating periods of extreme moods. Bipolar disorders shall encompass those characteristics and symptoms provided, for example, in DSM IV (American Psychiatric Association, 1994) criteria.

A "genotype" shall have its ordinary meaning in the art and encompass one or more nucleotide pair(s) found at a set of one or more polymorphic sites in a locus on a pair of homologous chromosomes in an individual. "Genotyping" shall encompass a process for determining a genotype.

A "haplotype" shall have its ordinary meaning in the art and shall encompass one or more nucleotides found at a set of one or more polymorphic sites in a locus on a single chromosome in an individual. "Haplotyping" shall mean a process for determining one or more haplotypes. The terms "mental illness or disorder" or "psychiatric disease or illness or disorder" can be used herein interchangeably and refer to mood disorders, bipolar disorder, euphoric mania, Bipolar I, Rapid Cycling, dysphoric mania, PTSD, Panic Attacks or Panic Disorder, alcohol or substance dependence, History of Suicide Attempt, and any combination thereof.

"A psychotic disorder" refers to a condition that affects the mind, resulting in at least some loss of contact with reality. Symptoms of a psychotic disorder include are provided for example in DSM IV (American Psychiatric Association, 1994) criteria. Schizophrenia, schizophreniform disorder, delusional disorder, and brief psychotic disorder are examples of psychotic disorders.

A "mood disorder" refers to disruption of feeling tone or emotional state experienced by a subject for an extensive period of time. Mood disorders include major depression disorder (i.e., unipolar disorder), mania, dysphoria, bipolar disorder, dysthymia, and cyclothymia.

" A polymorphism" shall encompass a sequence variation observed in a subject at a polymorphic site. "Polymorphic site" or "PS" is a position on a chromosome or DNA molecule at which at least two alternative sequences are found in a populaattiion.

Both "probe" or "an oligonucleotide probe" or "a primer" refers herein to a nucleic acid molecule or a sequence complementary therewith, when used to detect the presence of complementary sequences in a sample. The detection is carried out by identification of hybridization complexes between the probe and the assayed sequence. The probe, in some embodiments, may be attached to a solid support or to a detectable label. The probe will generally be single stranded. The probe(s) or primer(s) typically comprise 10 to 50 nucleotides. By way of non-limiting example, a probe or primer typically comprise 15 to 30 nucleotides. The particular properties of a probe will depend upon the particular use and are within the competence of one of ordinary skill in the art to determine.

A "subject" encompasses mammalian subject or human. As used herein a "sample" refers to any biological sample obtained from a subject which is suitable for isolation of nucleic acids. Such biological sample may be obtained from e.g. blood, saliva, cerebrospinal fluid, urine, feces or sperm. Such sample comprises nucleic acid(s).

SNPs are single nucleotide variations at a polymorphic site that occur in a population. Information concerning SNPs is obtainable from various repositories such as Ensembl, a joint project between EMBL - European Bioinformatics Institute (EBI) and the Wellcome Trust Sanger Institute (WTSI), or the dbSNP, the SNP repository maintained by NCBI, The Human Genetic Bi-Allelic Sequences Database (HGVBase) and The SNP Consortium Ltd. (TSC).

International collaborations have envisaged classifying SNPs for genomes of numerous species including Homo sapiens, Mus musculus, and plant genomes. As a mere example, HapMap project seek to identify the genetic patterns of human DNA sequence variation. Information such as SNP genotypes, recombination rates may be downloaded from the HapMap website (www.hapmap.org).

SNPs are conventionally identified by their relative position within a nucleotide sequence. Typically, following identification of a SNP a database reference is provided, "rs" number/SNP ID number. Consequently, sequence and other information related with a given "rs" number/SNP ID number may be obtained by browsing, for example, the dbSNP of the Entrez SNP which is provided by the NCBI, at www.ncbi.nlm.nih.gov.

The present invention discloses genetic polymorphisms which are associated with response to lithium treatment. Specifically, the invention provides alleles, haplotypes and genotypes of the Cacng2 gene that are associated with positive response to lithium treatment of a subject.

As described herein, the polymorphisms of the present invention can be used in combination with additional known polymorphisms and optionally other known clinical tests. The polymorphisms which are associated with dichotomized response to lithium treatment disclosed hereinbelow comprise at least one of the following single nucleotides polymorphisms (SNPs): (a) rs2284017; (b) rs2284018; and (c) rs5750285.

SEQ ID NO:1 is a portion of the Cacng2 nucleotide sequence that comprises rs2284017. The C/T single nucleotide polymorphism is denoted as (Y) in the sequence below. Position: at chromosome 22 at pos 35426873, band: 22ql2.3. The sequence includes chromosome 22: positions 35426473-35427273 (Strand: +).

CTAGTCTCTT GGCTACCTAT CTCCCTCCAT CCATCTAACT AAAATCCAAG GAAAGCAAAT TGCCACTATG ATCATTTCCC TAAAGCGGTC CTCATTCACT TTGCTTTTCT TCTCTTTCGG AATAAAAGTC TGTCATAAAT GTTTAATTAA ATGGAAGTCT TTCCACTGTT AATGACTAGC TGTATGAAAG GCTTTTCATT TTTTTAAGGA GGGGGCGTGT GTGTGCATGT GTTTAAAACA GTCCCTATTA ATTTCTTGAT ATTGATTTTC TGGCTGGGGA AGAGCATATA GTATTCAGCA TAGAGCTCCT CTGCAGAATA TGTTAAAGAC AGCGGTTTGG ATTGAGAAAG GGCACCAAAT GCAGTATGCG TCAAATAGAC CATCGCTCAA TGAAGTCACT Y

AGCTATTCAA AACATGTTCA GCCATCACTC AACCGTTTGC ACCAAGAATG TTGGGAGGGG GGCATGGGAT TTGTGCTCTT TCCCAGATGT AATCATTTCC GAAAGTGTGG CTTTTGCTCA GTTTGGGTTA GACCCTGGGG CCACATCAGT TTCTCTCTCA CCCATCCATT TCCCTCTCTT CTCCCCCATC TCCTAGTCTC AACTCCCTGC ATCACCATCC CATGTCGAGC TTAACAAACA ATCCTGCAGT CCTTGCTTCC CGACGCTGCC TCAAACGGCC CCCAGTATTG GTTCATAACA GAAGCAAAAC ACATAAGCCT CTTTTTTTCC CCCACATGGA AGCATATGTA TCTCTCTGTG CACGTCTATC TATGGGTGGC TGTAGCTTGC ACACACAGAC

SEQ ID NO:2 is a portion of the Cacng2 nucleotide sequence that comprises rs2284018. The C/T single nucleotide polymorphism is denoted as (Y) in the sequence below. Position: at chromosome 22 at pos 35427510, band: 22ql2.3. The sequence includes chromosome 22: positions 35427210-35427810 (Strand: +).

TGGAAGCATA TGTATCTCTC TGTGCACGTC TATCTATGGG TGGCTGTAGC TTGCACACAC AGACATATAT AGCCATTCAA ATGCAGTCCC TCATACTAAA GTCTGTTGCC AAATGCAGGT TGGATGACCC ACAGGAGTTT TTTTTTCTGT GCTTATTAAT GTGTCCCAGA AGTACATGCC TTCATCCATA GCGTGATCCC CCAGCCTTTA CACCGTGGCG GCTCCTGTGC CCACAGCACA GACACACATG CCTCAACACT CCGCAAGATC AAGGCAAAGA GCTGACACCC CCACTCCCCC Y

TCAACCTCCC CATGCCCTCC CCTCCCTCCC TCCCTGCCTG CCTGCACAAT GTCTGATTCT CAAGCCGGCT GCAATGCTGA CTCTACTAGC TTCTGGCTGA CAGACATGTC ACTCTGTGGA AGCCTAACCC GCAAGTGAAT TGCTGTTTAT TAGAACTTAG GCTTGTTTCC TGTGAGAGAC GTATGTATTT TTAGACTTTG TAAACGTTAA AATACACAAA TACACACACG CAGCCAGCCA CCTCGAGTGA TATGTGTGGC TCATAGACAT TACGAAATGC TCTTGAACTC AGCTTGAGAT

SEQ ID NO:3 is a portion of the Cacng2 nucleotide sequence that comprises rs5750285. The A/G single nucleotide polymorphism is denoted as (S) in the sequence below. Position: at chromosome 22 at pos 35434194, band: 22ql2.3. The sequence includes chromosome 22: positions 35433794-35434594 (Strand: +).

GACATCTTCC CTGAGGACAG GATATTTAAA CTGACATGTA GGAATGAGAA AGAGGTACCT AGGCCAAGCT GGGGGTGGTT AGAGGGTGCA AGGGAAGCTG AGAAGAGGAA CATTTCAGGC TGGGAGAGTA GTTTGTGCAA AGGTTCAGAA GTGTGAGTAA GATGATTGGC CAGTTTTGGG GGAAAAATAC TAAGCATGGC CGATCCTAAG ACAATGATAG GAAATGTGTT AGGCCAAGCA CCTGGAGACC ATGGAGGGGA GGTTGGAGGA TGAGCAACAA CCTGGAAGAA GCTGGTGGGA TATGGAAGGA GAGAACCAAC CCTggaggca ggaagacact gggagacagg aaggcaattg ctgctgagct ctaagcaaga agtgatggtg gcttggacca S cacaatggca atgggatggg gaaaagtgAA GGTAATCGGG GTATTTGGGA AGAAATTGAT GACTACTGGT GTCCATTACT AAGATGGGCA ACTGTGAGGC AGGAGCAGGT TGGGGTGTGT GTGTGGGGCA GGGGATGATG CAGGAAGGTG GGGAAAGCCA AGAATTCTGC TTTGGCCGTG TATCTAGTTT TGCTACGTTC TATTACCTCT TCACTCCCTG CTCCCAGCCT ATATCTTCTT GGCAAGTGAA TTAATTAATT AGCAATGTAA AGAACTCATG AAAGAAGAGA TTTGGGCTGT TCACATTGTA TTTCCAACAC TTGGAACACA GCCTAGCACA CAGTAGGTGT TCAATCAAAA TGAATATTTT TCTTGCTATA AAATTTCAAA TGCATGTCTT

It should be noted that rs2284017, rs2284018, and rs5750285 are single nucleotide polymorphisms located at an intronic non-coding region.The testing for the presence of one or more polymorphisms of these SNPs in a sample obtained from a subject indicates an increased likelihood for a positive response to lithium treatment in the subject.

The term "an increased likelihood for a positive response to lithium treatment", as used herein, refers to a statistically significant increase in the probability of manifesting a positive response to lithium treatment in a subject having a polymorphism of the present invention selected from: allele C of the C/T single nucleotide polymorphism (SNP) rs2284017; allele C of the C/T single nucleotide polymorphism (SNP) rs2284018; allele C of the C/G single nucleotide polymorphism (SNP) rs5750285, compared with the probability in an individual lacking the polymorphisms.

The term "lithium treatment", as used herein, refers to administration of a lithium based therapeutic substance intended to ameliorate symptoms associated with a psychiatric disease or illness or disorder or a psychotic disorder, to lessen the severity or, or to prevent at least partially the symptoms of the disease, illness or disorder.

The term "positive response" with respect to lithium treatment refers to at least partial good response. Partial good response is identified by at least a marked reduction in frequency of episodes or admissions or significant morbidity, and is well within the understanding of a person skilled in the art (for example, see Mendelewicz J et al., 1973).

Preparation of Probes and Arrays

SNP detection in the context of the present invention can be accomplished in a variety of different ways. In some embodiments, the detection of the presence or absence of the at least one polymorphism involves contacting a polymorphic site of polymorphisms associated with lithium response with a probe. Typically, the probe is an oligonucleotide probe, where the probe selectively hybridizes with the polymorphic site. Selective hybridization may be typically provided by way of achieving conditions of either low stringency, medium stringency, or high stringency. Low stringency can be provided by use of 0.03M sodium chloride and 0.03M sodium citrate at approximately 4O⁰C. Medium stringency can be achieved with same at approximately 50⁰C, while high stringency can be at approximately 60⁰C. Normally, high stringency conditions are used.

In the present context, a nucleotide sequence which is capable of selective hybridizing will generally have at least 60%, 70%, 80%, 90%, 95% or 99% sequence identity with any of the nucleotide sequences of the present invention, SEQ ID NOS: 1- 9, spanning over a region of at least 8, at least 15, at least 20, at least 30, at least 50, or at least 100 contiguous nucleotides, or their entire length. Oligonucleotide primers and probes of the present invention can be prepared by various methods known in the art. Cloning and restriction of appropriate sequences and direct chemical synthesis can be utilized for that purpose. The probes and primers can comprise nucleic acid analogs and the like, such as, for example, locked nucleic acid analogs and morpholino analogs. The 3' end of a probe can be functionalized with either a capture or otherwise detectable label.

The probes and primers of the present invention can be labeled. This is performed by incorporating a label measurable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. By way of non-limiting example, such labels can comprise radioactive substances (³²P, ³⁵S, ³H, ¹²⁵I), fluorescent dyes (digoxigenin, fluorescein, 5-bromodesoxyuridin, acetylaminofluorene), biotin, nanoparticles, and the like. Such oligonucleotides are typically labeled at their 3' and 5' ends.

An oligonucleotide probe may span two or more polymorphic sites. Unless otherwise specified, an oligonucleotide probe can include one or more nucleic acid analogs, labels or other substituents or moieties as long as the base-pairing function is retained. Accordingly, the present invention provides a nucleic acid probe comprising a nucleotide sequence which hybridizes to a polymorphism in the Cacng2 gene, wherein the polymorphism comprises at least one single nucleotide polymorphisms (SNP) selected from the group consisting of:

(i) allele C of the C/T single nucleotide polymorphism (SNP) rs2284017; (ii) allele C of the C/T single nucleotide polymorphism (SNP) rs2284018; (iii) allele C of the C/G single nucleotide polymorphism (SNP) rs5750285; (iv) allele T of the C/T single nucleotide polymorphism (SNP) rs2284017; (v) allele T of the C/T single nucleotide polymorphism (SNP) rs2284018;

(vi) allele G of the C/G single nucleotide polymorphism (SNP) rs5750285; and (vii) any combination thereof.

The present invention discloses the use of the probe in identifying an increased likelihood for positive response to lithium treatment in a subject.

In particular, the present invention implicates those specific allelic variants which are indicative of positive response to lithium treatment. The specific alleles indicating positive response to lithium treatment can be selected from the group consisting of:

(i) allele C of the C/T single nucleotide polymorphism (SNP) rs2284017; (ii) allele C of the C/T single nucleotide polymorphism (SNP) rs2284018; (iii) allele C of the C/G single nucleotide polymorphism (SNP) rs5750285;

Accordingly, in one aspect, the present invention provides a nucleic acid probe or primer comprising a nucleotide sequence which hybridizes to a polymorphism in the Cacng2 gene, wherein the polymorphism comprises at least one single nucleotide polymorphisms (SNP) selected from the group consisting of:

(vi) allele G of the C/G single nucleotide polymorphism (SNP) rs5750285; and

(vii) any combination thereof. for use in identifying an increased likelihood for positive response to lithium treatment in a subject.

In another embodiment, one or more additional marker(s) for response to lithium treatment can be analyzed. For example, in addition, detection of polymorphisms in beta-adrenergic receptor kinase (ADRBK2), neurotrophic tyrosine kinase receptor type 2 (NTRK2), brain derived neurotrophic factor (BDNF), synthase kinase 3 beta (GSK3B), G protein receptor kinase 3 (GRK3), Inositol phosphatases (INPPl, IMPAl, IMPA2), gene(s) may be additionally performed.

In one embodiment, an oligonucleotides probe or primer comprises a sequence selected from the following group:

ATAGACCATCGCTCAATGAAGTCACTfC/TlAGCTATTCAAAACATGTTCAGCCAT (per rs2284017, denoted as SEQ ID NO: 4 and 5, respectively)

CAAAGAGCTGACACCCCCACTCCCCCfC/TITCAACCTCCCCATGCCCTCCCCTCC (per rs2284018, denoted as SEQ ID NO: 6 and 7, respectively)

GCAAGAAGTGATGGTGGCTTGGACCAfC/GICACAATGGCAATGGGATGGGGAAAA (per rs5750285, denoted as SEQ ID NO: 8 and 9, respectively) The bold/underlined nucleotides identify the polymorphic sites or allelic variants of each of the respective single nucleotide polymorphisms (SNPs) of the present invention. In one specific example, the oligonucleotide probes comprise at least 8 contiguous nucleotides sequence of any of SEQ ID NO: 1-9, wherein the nucleotides comprise the respective underlined polymorphic site.

The oligonucleotide probes can further comprise any of the foregoing sequences where a T is substituted for a U; or a complement sequence of any of the foregoing sequences.

In an embodiment of the present invention, the probe or primer used for the detection of any of the SNP of the present invention is chosen to complement the contiguous nucleotide sequences upstream and downstream from any of the polymorphic sites disclosed herein. By way of non-limiting example, about 10 nucleotides upstream and about 10 nucleotides downstream of the polymorphic site are utilized.

It should be understood that the present invention further contemplates that the presence of a specific haplotype or genotype may be determined using a nucleotide sequence comprising 12, 17, 24, 28, 40 or more nucleotides upstream, or indeed any number within these ranges, and 12, 17, 24, 28, 40 or more nucleotides downstream or any number within these ranges, with respect to a polymorphic site disclosed herein.

The probes and primers can be immobilized on a solid support. Solid supports are known to those skilled in the art. By way of non-limiting example, solid support includes magnetic beads, the walls of wells in a reaction tray, nitrocellulose strips, membranes, glass and the like. The probes of the present invention can also be immobilized on a substrate, e.g. a chip.

Therefore, the present invention also contemplates an array comprising a substrate having a plurality of segments, wherein at least one of said segments comprises a probe which hybridizes to a polymorphism in the Cacng2 gene, wherein said polymorphism is at least one single nucleotide polymorphisms (SNP) from the group consisting of:

(i) allele C of the C/T single nucleotide polymorphism (SNP) rs2284017;

(ii) allele C of the C/T single nucleotide polymorphism (SNP) rs2284018; (iii) allele C of the C/G single nucleotide polymorphism (SNP) rs5750285; (iv) allele T of the C/T single nucleotide polymorphism (SNP) rs2284017; (v) allele T of the C/T single nucleotide polymorphism (SNP) rs2284018; (vi) allele G of the C/G single nucleotide polymorphism (SNP) rs5750285; and

Suitable methods for immobilizing oligonucleotides on a solid phase include covalent bonding and the like which are known in the art. The oligonucleotide probes or primers of the invention can be immobilized on a solid support either individually or in groups of distinct oligonucleotides on a single solid support.

The oligonucleotide probes or primers of the invention may be linked in an array wherein each oligonucleotide is attached to a distinct segment of the solid support. Such oligonucleotide arrays typically enable access to the distinct segments in the array and the recordings of hybridization assay.

According to a particular example, the oligonucleotide probes can be used in an oligonucleotide chip as disclosed in US 5,143,854 or WO 92/10092.

The synthesis of materials such as oligonucleotides of the present invention on the surface of a substrate may be carried out using light-directed methods as described in., e.g. US 5,143,854 and WO 92/10092, or mechanical synthesis methods as described in 5,384,261.

In particular, these light-directed or photolithographic synthesis methods involve a photolysis step and a chemistry step. Briefly, the substrate surface comprises functional groups on its surface. These functional groups are photo-protected by photo labile protecting groups such that in a photolysis step, exposure to light or other activators, activates the functional groups to remove photoprotecting groups. Chemical monomers thereafter bind to the activated portion of the substrate through an unprotected functional group.

In another embodiment, an array of oligonucleotides complementary to a polymorphism in the Cacng2 gene comprising any of: allele C of the C/T single nucleotide polymorphism (SNP) rs2284017; allele C of the C/T single nucleotide polymorphism (SNP) rs2284018; and allele C of the C/G single nucleotide polymorphism (SNP) rs5750285; is used to determine the particular allelic variance in a sample obtained from an subject.

Optionally, a 4L tiled array is employed. In general, the tiled array contains 4L probes (one for each of 4 bases, L denoted the number of sets of the 4 probes). Accordingly, a perfect complement obtained for the subject will hybridize more strongly in comparison to a mismatched pairing. The hybridization is indicative of the particular allelic variance of the subject tested.

The appropriate conditions upon which hybridization assays should be performed are known by the person skilled in the art.

PCR can also be used for the detection of allelic variants of the present application. To that end, primers include those oligonucleotides comprising sequences that flank the underlined sequence i.e. the polymorphic site above. Optionally, the primers can include a sequence that contains the underlined nucleotide.

In some specific embodiments, testing for the presence of a polymorphism of the present invention is performed by allele-specific PCR.

Therefore, in one embodiment, a polymorphism of the present invention can be tested by employing primers as follows: two tailed allele-specific primers, a reverse primer, and two universal energy-transfer (ET) labeled primers of which can be labeled with a green dye (fluorescein) and the other a red dye (sulforhodamine), as disclosed for example in Myakishev MV et al (14).

ET-labeled primers are known in the art and are commercially available, for example, Amplifluor® (by CHEMICON International, Inc. Temecula, CA) see also Nuovo GJ et al (20).

Each of the allele-specific primers comprises a single allele-specific nucleotide, i.e. the above underlined polymorphic site, at the 3' terminus. The later can be preceded by 16 to 21 bases complementary to the Cacng2 gene which immediately precede the polymorphic site, i.e. the tail of the allele-specific primer. The reverse primers for such reaction can be chosen to complement the Cacng2 gene at a suitable distance so as to avoid overlaps with the allele-specific and the ET-labeled primers (which could result with poor performance). The distance between the allele-specific primer(s) and the respective reverse primer(s) can vary from 7 to 160 bp.

The structure of the ET-labeled primer involves a 3' primer sequence and a 5' hairpin region that is labeled with a unique energy transfer pair. The 3' primer sequence comprises the tail of the corresponding allele-specific primer.

Following amplification, the products are analyzed for the fluorescence in a plate reader. Analysis and detailed discussion of Allele-specific PCR assays can be found at Myakishev MV et al (14).

By way of non-limiting example, detection of a polymorphism can further be performed by enzymatic mutation detection, hybridization assay involving allele- specific probes, primer extension assays, a nucleotide amplification assay , genotyping using mass spectrometry, sequencing, and enzymatic cleavage such as cleavage of single base mismatches and alike. These methods are known to the person skilled in the art.

Testing for the presence of the polymorphisms as described herein facilitates the prediction of the response of a subject to lithium treatment, and therefore can be used by a physician to determine whether a lithium based medication is suitable for a subject.

Accordingly, the present invention provides a method for identifying an increased likelihood for positive response to lithium treatment in a subject, the method comprising testing a sample obtained from the subject for the presence of at least one polymorphism of the Cacng2 gene selected from the group consisting of: (i) allele C of the C/T single nucleotide polymorphism (SNP) rs2284017; (ii) allele C of the C/T single nucleotide polymorphism (SNP) rs2284018;

(iii) allele C of the C/G single nucleotide polymorphism (SNP) rs5750285; and (iv) any combination thereof; wherein presence of at least one of said polymorphisms indicates an increased likelihood for a positive response to lithium treatment in the subject. In particular, the presence of allele C of the C/T polymorphism rs2284017, and allele C of the C/T polymorphism rs2284018 is indicative of an increased likelihood for a positive response to lithium treatment in the subject. The presence of allele C of the C/T polymorphism rs2284017, and allele C of the C/T polymorphism rs5750285 is indicative of an increased likelihood for a positive response to lithium treatment in the subject. The presence of allele C of the C/T polymorphism rs2284018, and allele C of the C/T polymorphism rs5750285 is indicative of an increased likelihood for a positive response to lithium treatment in the subject.

In addition, the presence of allele C of the C/T polymorphism rs2284017, allele C of the C/T polymorphism rs2284018, and allele C of the C/T polymorphism rs5750285 is indicative of an increased likelihood for a positive response to lithium treatment in the subject.

In one embodiment of the invention, a polynucleotide sample (such as DNA or RNA) is obtained from a subject's body fluid (e.g. blood or saliva). The person skilled in the art would recognize that there are other manners by which the nucleotide sample can be obtained. The subject's sample can then used to test for the presence of a polymorphism in a Cacng2 gene the polymorphism is selected from the allelic variants of polymorphisms rs2284017, rs2284018 and rs5750285 discloses herein.

In one embodiment, the subject is either diagnosed as or susceptible to a psychiatric disease, illness or disorder, or a psychotic disorder selected from the group consisting of mood disorder, bipolar disorder, euphoric mania, dysphoric mania, Bipolar I, Rapid Cycling, History of Suicide Attempt, PTSD, Panic Attacks/Panic Disorder, Alcohol or Substance Dependence, and any combination thereof.

Kits

The invention also contemplates kits useful for testing for the presence of a polymorphism associated with response to lithium treatment in subjects. The kit can include instructions for use and reagents such as, but not limited to, labels, agents for attaching a label to the probes disclosed herein and more.

The instructions can provide particulars of how to perform the test for predicting a subject's response to lithium treatment, as described herein. The instructions can further refer to the manner of attaching a label to the probe, and to the manner by which to obtain a sample for the analysis from a subject. In some embodiments, the kit comprises a labeled oligonucleotide that hybridizes to a portion of Cacng2 gene. Therefore, the present invention provides a kit compartmentalized to receive a reagent for measuring a single nucleotide polymorphism (SNP) of the Cacng2 gene in a nucleic acid sample obtained from a subject, said reagent comprising at least one oligonucleotide which hybridizes to at least one single nucleotide polymorphisms (SNP) from the group consisting of:

(i) allele C of the C/T single nucleotide polymorphism (SNP) rs2284017; (ii) allele C of the C/T single nucleotide polymorphism (SNP) rs2284018; (iii) allele C of the C/G single nucleotide polymorphism (SNP) rs5750285; (iv) allele T of the C/T single nucleotide polymorphism (SNP) rs2284017; (v) allele T of the C/T single nucleotide polymorphism (SNP) rs2284018; (vi) allele G of the C/G single nucleotide polymorphism (SNP) rs5750285; and (vii) any combination thereof.

In some embodiments, the kit can comprise additional oligonucleotide(s) that hybridizes with at least one additional marker for response to lithium treatment. In that respect, the present invention facilitates combined use of known markers associated with response to lithium treatment and at least one single nucleotide polymorphisms (SNP) from the group consisting of:

(iv) allele T of the C/T single nucleotide polymorphism (SNP) rs2284017; (v) allele T of the C/T single nucleotide polymorphism (SNP) rs2284018; (vi) allele G of the C/G single nucleotide polymorphism (SNP) rs5750285;

The reagent of the kit can comprise at least one oligonucleotide selected from SEQ ID NO: 1-9 or any of the aforementioned oligonucleotides probes. The kit can also include additional probe(s) that hybridize to other oligonucleotides.

The oligonucleotides in the kit of the invention can also be immobilized on or directly synthesized on a solid surface such as a microchip, bead, or glass slide (for methods of immobilizing oligonucleotides or synthesizing on a solid surface see, e.g., WO 98/20020 and WO 98/20019).

When the oligonucleotides of the invention are used as allele-specific probes the kits of the invention may also contain other components such as hybridization buffer. The kit optionally further contains dideoxynucleotide triphosphates (ddNTPs) when the alleles at the polymorphic sites are detected by primer extension. Therefore, in one embodiment, the kit contains primer-extension oligonucleotides. The kit may also contain a polymerase and a reaction buffer optimized for primer-extension mediated by the polymerase. In some embodiments, the kits optionally comprise detection reagents, e.g. biotin- or fluorescent-tagged oligonucleotides, ddNTPs, an enzyme-labeled antibody, and substrates capable of generating a detectable signal when acted on by the enzyme.

It is contemplated that the kit of the present invention can, in some embodiments, be used for self-testing. Therefore, such test kits can include devices and instructions that enable a subject to obtain the sample. For example, buccal cells or blood may thus be obtained without the assistance of a health care provider.

The kit can include container(s) for the sample, or the sample can be in a standard blood collection vial.

In another aspect, the methods of the present invention can be utilized during clinical trials. By way of a non-limiting example, the present invention allows monitoring of the correlation between the polymorphisms disclosed herein and a potentially dichotomized response to lithium based medication(s) or combination treatments comprising lithium. Accordingly, the response can be monitored before, and at various points during the treatment of a subject with the medication.

To that end, the present invention further encompasses a method for obtaining information regarding an increased likelihood for positive response to lithium treatment in a subject, said method comprising testing a sample obtained from the subject for the presence of at least one polymorphism of the Cacng2 gene selected from the group consisting of:

(iii) allele C of the C/G single nucleotide polymorphism (SNP) rs5750285; and (iv) any combination thereof; wherein presence of each of said polymorphisms indicates an increased likelihood for a positive response to lithium treatment in the subject, thereby obtaining information regarding a prediction of response to lithium treatment in a subject.

This information can thus be used to at least minimize or avoid therapeutic failure of lithium treatment. A physician or clinician can apply the generated information in a clinical trial. The information can therefore classify the subject in a clinical trial. It may also classify a population of subjects in a similar manner.

The clinical trial may be designed to test the efficacy of a lithium-based medication or the efficacy of a pharmaceutical composition comprising lithium. The clinical trial can be designed to test the efficacy of a pharmaceutical composition which does not comprise lithium in combination with a pharmaceutical composition which comprises lithium. Efficacy in the present context encompasses the result of the treatment, discontinuance rates of the treatment, and dose response curves.

Thus the generated information can be used in correlating a polymorphism selected from rs2284017, rs2284018, and rs5750285 with an outcome of a lithium treatment.

Examples Materials and Methods

Cacng2 mRNA Quantification

Brain samples - Total RNA prepared from frozen human Post-mortem dorsolateral prefrontal cortex (DLPFC) brain samples was obtained from the Stanley Foundation Brain Collection and Neuropathology Consortium, Bethesda, Maryland (to R.N). All samples were from Brodmann's area 46. The collection is well matched for age, pH and gender (12). Schizophrenic and bi-polar patients were diagnosed according to DSM-IIIR (American Psychiatric Association, 1987) or DSM-IV (American Psychiatric Association, 1994) criteria.

RNA and reverse transcription - A total of 105 RNA samples (35 schizophrenia patients, 35 bipolar patients and 35 matched control subjects) were used in this study. The samples were reverse transcribed using 1 μg of DNase-treated RNA, 0.2 μl random hexamer primers (0.5 μg/μl; Promega, Madison, WI), 10 units of Ribonuclease Inhibitor (Takara, Shiga, Japan) and 9 units of avian myeloblastosis virus (AMV) reverse transcriptase (Promega). mRNA quantification - Cacng2 mRNA quantification was performed by realtime PCR (RT-PCR) on the ABI Prism^® 7000 Sequence Detection System (Applied Biosystems, Foster City, CA) using the SYBR Green I assay, with β-2 microglobulin as an internal control.

Primer pairs for Cacng2 and β-2 microglobulin were designed such that at least one of the primers was located on an exon-exon junction of the cDNA sequence. The cDNA sequences were derived from the UCSC Genome Browser Database (http://genome.ucsc.edu), and the primers were designed according to Applied Biosystems recommendations using the Primer Express 2.0 software and synthesized by Integrated DNA Technologies (Coralville, Iowa). Primer^*pairs for^?(?acng2 were:|@)^g

^•(F)

IDi NOTl-WMNHB ;©AT^ΑτMAΑAfe^^AgA^^!7sMlβθ:13). Reactions were carried out in a solution containing 350 nM of primers, 10 μl of SYBR Green I PCR Master Mix (Applied Biosystems) and 5 μl cDNA template in a final volume of 20 μl. Each sample was analyzed in duplicate. The PCR cycling conditions were: 2 min at 50°C, 10 min at 95°C followed by 45 cycles of 15 s at 95°C and 1 min at 60°C. To make sure that the amplification efficiencies were close to 100% and comparable between assays, standard curves were constructed for the Cacng2 and β-2 microglobulin assays, prior to analysis of samples.

Cacng2 mRNA quantity was expressed as the amount of target normalized to an endogenous reference and relative to a calibrator according to the formula 2^"MCt (13), where Ct is the threshold cycle, ΔCt = Ct (target) - Ct (β-2 microglobulin), and ΔΔCt = ΔCt (sample) - ΔCt (calibrator sample). The calibrator sample used was a calculated value of the mean of all control samples values.

DNA samples. DNA was extracted from 10 ml whole blood samples using Nucleon II kits (Scotlab, UK). The samples were obtained from two population sets: the Aberdeen set which included 213 BPD patients and healthy controls (n=197), and the Cagliari set which included 194 BPD patients scored for lithium response. Subjects in the Aberdeen set were recruited through psychiatric hospitals in Scotland, met DSM-IV criteria for bipolar I disorder, and had been receiving lithium therapy for at least 3 years. All subjects were Caucasian with at least three of their four grandparents born in Scotland. Consensus diagnosis was determined by trained psychologists and psychiatrists based on case-note review and clinical interview using semistructured diagnostic questionnaires. The Operational Criteria Checklist for Psychotic Illness program was used to define diagnoses. Patients with schizoaffective disorder were excluded. Final decisions on any diagnostic disagreements (for reasons of missing data or discrepancies between DSM-IV and International Classification of Diseases-10 diagnoses [N=31]) were made by one of the inventors (D.St.C.) and corroborated by another psychiatrist. Lithium response was classified into 5 categories: 0- very bad (complete lack of response), 1- bad, 2- medium, 3- good and 4- very good (no further episodes). The classification was done by extensive case note review.

The Cagliari sample included patients treated at the Lucio Bini Center for mood disorders, affiliated with the University of Cagliari, in Sardinia. Response to lithium was evaluated using prospective data in adult, DSM-IV bipolar I, or II disorder patients providing written, informed consent. Clinical assessments were made by research psychiatrists, during follow-up visits at intervals of 2-3 months, following semi- structured examination protocols and life-charts. Illness-onset and course prior to the study was defined as the time of first psychiatric intervention or by consensus from subjects and family members. Treatment consisted of uninterrupted, closely monitored maintenance therapy, using lithium as a primary option. Serum lithium was assayed quarterly. Treatment response measures were based on percent-time-ill during maintenance treatment as the primary outcome and were divided into 4 categories: 1 - no response (no improvement or worsening of illness), 2 - was poor response (minor or modest improvement in frequency of episodes or admissions; significant morbidity), 3 - partial good response (marked improvement but not episode-free), and 4 - good response (complete remission).

The classification was done by prospective classification in the Cagliari set. Informed consent was obtained from each participant recruited for the study. Ethical approval for the study was granted by the appropriate local ethical committees and by the IOP/SLAM ethical committee. Power analysis was performed using the genetic power calculator (http://statgen.iop.kcl.ac.uk/gpc/). The Aberdeen set had >90% power to detect a heterozygote odds ratio of 2 associated with BPD at the 0.05 level for a risk allele frequency of 0.2.

SNPs selection. A total of 12 SNPs in the Cacng2 gene were selected for genotyping. SNPs were selected according to the linkage disequilibrium (LD) map of the CEU (Caucasian) population in the HapMap Database (http://www.hapmap.org/index.html.en) only from LD blocks adjacent to exons. From each such block, tagging SNPs (the minimal group of SNPs required to define all haplotypes) were selected for genotyping. Thus, the chosen 12 SNPs defined 2 LD blocks.

Genotyping. Allele-specific PCR assays were designed as previously described (14) and genotyping was performed under contract by Prevention Genetics (Marshfield, USA). These assays are based on competitive allele-specific PCR that allows the simultaneous amplification and detection of DNA within a closed reaction vessel. The homogeneous assay utilizes two different fluorescently labeled universal primers; two unlabeled and tailed allele-specific primers, and a common reverse primer in a single well reaction. Submicroliter PCR reactions were carried out with ArrayTape instrumentation and allele calls were generated based on the clustering of fluorescent signals (15), [http://www.global-array.com]. Statistical analyses. Gene expression distribution normality was evaluated using the Shapiro-Wilk test. The Student's t-test was used to compare Cacng2 expression level between patients and controls using the SPSS 10.0 program (SPSS, Chicago, IL).

Outliers were detected by the median of the absolute deviation about the median (MAD) method (http://www.cee.vt.edu/ewr/environmental/teach/smprimer/outlier/ outlier.html and (16)). Correlations between genotypes and lithium response were estimated by Kendall's tau-b or by Fisher's exact χ² test for the dichotomized data (with 10⁶ permutations). Combination (meta-analysis) of P-values from tests in different population sets was performed using Fisher's method (17) for 2x3 tables, and the Mantel-Haenzel method was used to combine P-values of 2x2 tables and their odds ratios with the Epilnfo Statcalc program verion 6 (CDC, USA/WHO, Switzerland). The expectation-maximization (EM) algorithm (18) was used to estimate haplotype frequencies with the R environment for statistical computing (http ://www.r-pro j ect. or gΛ (with 10⁵ permutations). Pairwise LD between SNP markers was measured by Lewontin's coefficient (D') and Pearson's correlation coefficient (r) (19).

Results

Expression analysis. RT-PCR analysis showed that the Cacng2 gene is expressed in the dorsolateral prefrontal cortex (DLPFC) of both patient groups and controls. The mean relative expression levels (±standard deviation) in BPD, SCZ (schizophrenia), and control groups were: 4.486±1.873 (N=31), 2.593±1.393 (N=33), 2.775±1.438 (N=34), respectively. Real-time expression analysis revealed that Cacng2 is 1.61 -fold overexpressed in BPD patients compared to control subjects (P = 0.00013). No significant difference was observed between schizophrenic patients and control subjects (figure 1). Namely, Cacng2 mRNA was found to be over-expressed in brains of BPD patients indicating that this gene is involved with the pathophysiology of BPD.

Correlations with clinical data. Interestingly, a strong significant negative correlation was found between Cacng2 expression and antipsychotic drug (represented as fluphenazine equivalents in mg) administration in both patient groups (bipolar and schizophrenic: Pearson's r= -0.432, P= 0.00045, n=62; BPD only: r= -0.325, P=0.08, n= 30; schizophrenic: r= -0.332, P=0.063, n= 32). In addition, alcohol and drug use among patients and controls were found to positively correlate with Cacng2 expression (Kendall's tau b=0.166, P= 0.028, n=96; Kendall's tau b=0.205, P=0.0086, n=95, respectively).

Association with BPD. All SNPs were in Hardy-Weinberg equilibrium. No association was found between the 12 genotyped SNPs and BPD in the Aberdeen set. Haplotype analysis of a subset of 3 SNPs analyzed herein for Lithium response (proximal to the 3' terminus) found no association with the disease either.

Association with lithium response. Genotypes of two SNPs from the same LD block, rs2284017 and rs5750285 were found to be associated with lithium response levels in the Aberdeen set when the 0 level Lithium response was excluded (Kendall's tau-b P= 0.022, 0.023 respectively). Alleles of the same SNPs were also associated with lithium response both when the 0 level was included (χ² P= 0.040, 0.033) and excluded (χ² P= 0.022, 0.023) (Fig. 2). We further tested these 2 SNPs along with rs2284018, which is located between them in the same LD block, for dichotomized lithium response, i.e. responders (index >2) vs. non-responders (index 0 to 2). That revealed significant associations in the Aberdeen set between SNPs rs2284017 and rs5750285 and lithium response, while in the Cagliari sample similar trends were observed in genotype distribution of these SNPs, and rs2284018, showed significant association with dichotomized lithium response. Most notably, all 3 SNPs were in association with dichotomized lithium response in a combined analysis of both sample sets of bipolar patients: (1) rs2284017 association to lithium response included a combined set of 339 bipolar patients, 188 of the Aberdeen group and the rest from the Cagliari group; (2) rs2284018 association to lithium response included a combined set of 358 bipolar patients, 197 of the Aberdeen group and the rest from the Cagliari group; (3) rs5750285 association to lithium response included a combined set of 329 bipolar patients, 195 of the Aberdeen group and the rest from the Cagliari group. In all 3 SNPs the C allele is "pro-responsive" (Table Ia, b). Table Ia: Genotypic and allelic distribution of rs2284017, rs2284018 and rs5750285, associations and odds ratios in the dichotomized lithium response groups: good vs. poor or no response (indices 3, 4 versus 0, 1, 2 respectively).

Table Ib: Genotypic and allelic distribution of rs2284017 and rs5750285, associations and odds ratios in the dichotomized lithium response groups in the Aberdeen set when O-responders were excluded (good response vs. poor response).

AH haplotypes constructed from the 3 SNPs, as well as all combinations of 2- SNP haplotypes, showed a significant association with dichotomized Lithium response (Table 2).

Although the rs2284018 SNP was significantly associated with dichotomized lithium response only in the Cagliari sample, while the other 2 distal SNPs in the same LD block (rs2284017 and rs5750285) were much more strongly associated in the Aberdeen sample, the trend of the "response-protective" and "response-inhibitive" alleles in all 3 SNPs was maintained in both samples. This consistency was reflected by the positive results obtained for the combined sample analysis. Haplotype analysis has generally yielded stronger associations than those of the single markers, but did not improve the odds ratios except for the Cagliari group, in which the markers seem to be more weakly associated with the disorder than in the Aberdeen group. In order to account for possible population differences, haplotype analysis of the combined set was performed covarying for population membership, which still yielded positive results. Response to lithium treatment was much better in the Cagliari sample than in the Aberdeen sample (good-response rates: 71.76%, 18.59%, respectively; P< 10^"24). This difference may be explained largely by the fact that different criteria for response were used in the different sample sets, since no consensus on defining response has emerged to date. However, the results show that the same haplotypes show significant (or in some cases a trend to) association with lithium response in both groups.

Table 2: Haplotype analysis of dichotomized Lithium response

Frequency OR Frequency OR Frequency OR

+ P + - + +

Ηaplotype - + P +

Aberdeen Cagliari " "Both Sets

1 - 2 - 3: Global 0.089 Global Global 0.011

.103

T- T- G .174 .256 0.175 .63 0.0133

CΛ .166 .274 .032 .90 .167 .259 .74

C DO C- C - C .688 .513 0.0096 .09 0.0046 CΛ .615 .536 .150 .38 .633 .517 .61

Iz2ι Global 0.022 Global Global 0.011

.104 m C - C .24 0.003 .

.709 .521 0.0079 .620 .536 .152 .41 .642 .524 .63 I

CO 1

X T- C 0.038 .50 0.266 UJ m .101 .219 .059 .050 .669 .19 .187 .213 .18 C^ m T- T 0.227 .51 0.019

.189 .26 .166 .274 .032 .90 .171 .262 .73 c Global 0.059 Global Global 0.0036 i- .055 m 1

C - C 0.00065

.726 .564 0.0149

.05 .702 .56 .016 .85 .708 .563 .89

T - G 0.186

.175 .256 0.0202

.62 .168 .267 .05 .80 .169 .266 ,79

1 - 3: Global 0.0644 Global Global 0.012

.124 r

T - G 0.0012

.257 .428 0.01

.16 .294 .424 .04 .78 .285 .426 .87

(

C - C 0.0104 0.0056

.687 .512 .09 .618 .539 .166 .38 .635 .518 .62 aSNPs representation: 1 - rs2284017; 2 - rs2284018 ; 3 - rs5750285. +: responders. -: non-respoaders. ⁶TlIe combined set analysis was performed with a population covariant; OR, Odds Ratios; P: 2-sided P value. Significant p-values are in bold.

Claims

CLAIMS:

1. A method for identifying an increased likelihood for positive response to lithium treatment in a subject, said method comprising testing a sample obtained from the subject for the presence of at least one polymorphism of the Cacng2 gene selected from the group consisting of:

(iii) allele C of the C/G single nucleotide polymorphism (SNP) rs5750285; and (iv) any combination thereof; wherein presence of at least one of said polymorphisms indicates an increased likelihood for a positive response to lithium treatment in the subject.

2. A method according to claim 1, wherein the presence of allele C of the C/T polymorphism rs2284017, and allele C of the C/T polymorphism rs2284018 is indicative of an increased likelihood for a positive response to lithium treatment in the subject.

3. A method according to claim 1, wherein the presence of allele C of the C/T polymorphism rs2284017, and allele C of the C/T polymorphism rs5750285 is indicative of an increased likelihood for a positive response to lithium treatment in the subject.

4. A method according to claim 1 , wherein the presence of allele C of the C/T polymorphism rs2284018, and allele C of the C/T polymorphism rs5750285 is indicative of an increased likelihood for a positive response to lithium treatment in the subject.

5. A method according to claim 1, wherein the presence of allele C of the C/T polymorphism rs2284017, allele C of the C/T polymorphism rs2284018, and allele C of the C/T polymorphism rs5750285 is indicative of a an increased likelihood for a positive response to lithium treatment in the subject.

6. A method according to claim 1 , further comprising testing for the presence of at least one additional marker for response to lithium treatment.

7. A method for identifying an increased likelihood for positive response to lithium treatment in a subject, comprising testing a sample obtained from the subject for the presence of at least one genotype of the Cacng2 gene selected from the group consisting of:

(i) C/C genotype of a single nucleotide polymorphism (SNP) rs2284017; (ii) C/C genotype of a single nucleotide polymorphism (SNP) rs2284018;

(iii) C/C genotype of a single nucleotide polymorphism (SNP) rs5750285; and (iv) any combination thereof; wherein the presence of at least one genotype indicates an increased likelihood for a positive response to lithium treatment in the subject.

8. The method of claim 7, further comprising testing for the presence of at least one additional marker for response to lithium treatment.

9. A method according to claims 1-8, wherein said subject is human.

10. A method according to claims 1-9, wherein said method is performed prior to treatment of the subject with lithium or during the treatment of the subject with lithium.

11. A method according to claims 1-10, wherein said sample is a body fluid.

12. A method according to claim 11 , wherein said body fluid is selected from the group consisting of blood, saliva, cerebrospinal fluid, urine, and sperm.

13. A method according to claims 1-12, wherein said testing for polymorphism is performed by SNP genotyping.

14. A method according to claims 1-13, wherein the presence of the polymorphism is determined by contacting nucleic acids obtained from a sample of the subject with a polynucleotide probe which hybridizes with at least one polymorphism of the Cacng2 gene selected from the group consisting of:

(iv) allele C of the C/T single nucleotide polymorphism (SNP) rs2284017; (v) allele C of the C/T single nucleotide polymorphism (SNP) rs2284018;

(vi) allele C of the C/G single nucleotide polymorphism (SNP) rs5750285; and any combination thereof; and determining whether the sample comprises a polynucleotide that hybridizes with the probe, thereby indicating the presence of said polymorphism.

15. A method according to claim 13, wherein said SNP genotyping is performed by a method selected from the group consisting of (a) a primer extension assay; (b) PCR assay; (c) an allele-specific PCR assay; (d) a nucleic acid amplification assay; (e) a hybridization assay; (f) a mismatch-detection assay, (g) an enzymatic nucleic acid cleavage assay, and (h) a sequencing assay.

16. A method according to claims 1-15, further comprising measuring a clinical symptom of the subject.

17. The method of claims 1-16, wherein the subject is having a mental disorder, a psychiatric disorder, or a psychotic disorder.

18. The method of claim 17, wherein said psychiatric disorder is a mood disorder.

19. The method of claim 18, wherein said mood disorder is a bipolar disorder.

20. A nucleic acid probe comprising a nucleotide sequence which hybridizes to a polymorphism in the Cacng2 gene, wherein said polymorphism comprises at least one single nucleotide polymorphisms (SNP) selected from the group consisting of:

(i) allele C of the C/T single nucleotide polymorphism (SNP) rs2284017; (ii) allele C of the C/T single nucleotide polymorphism (SNP) rs2284018; (iii) allele C of the C/G single nucleotide polymorphism (SNP) rs5750285; (iv) allele T of the C/T single nucleotide polymorphism (SNP) rs2284017; (v) allele T of the C/T single nucleotide polymorphism (SNP) rs2284018; (vi) allele G of the C/G single nucleotide polymorphism (SNP) rs5750285; and

(vii) any combination thereof. for use in identifying a an increased likelihood for a positive response to lithium treatment in a subject.

21. A nucleic acid probe of claim 20 for use in combination with at least one additional marker for response to lithium treatment.

22. Use of a nucleotide sequence which hybridizes to a polymorphism in the Cacng2 gene, wherein said polymorphism comprises at least one single nucleotide polymorphisms (SNP) selected from the group consisting of:

23. An array comprising a substrate having a plurality of segments, wherein at least one of said segments comprises a probe which hybridizes to a polymorphism in the Cacng2 gene, wherein said polymorphism is at least one single nucleotide polymorphisms (SNP) from the group consisting of:

(i) allele C of the C/T single nucleotide polymorphism (SNP) rs2284017; (ii) allele C of the C/T single nucleotide polymorphism (SNP) rs2284018; (iii) allele C of the C/G single nucleotide polymorphism (SNP) rs5750285; (iv) allele T of the C/T single nucleotide polymorphism (SNP) rs2284017; (v) allele T of the C/T single nucleotide polymorphism (SNP) rs2284018; (vi) allele G of the C/G single nucleotide polymorphism (SNP) rs5750285; and (vii) any combination thereof. for use in identifying an increased likelihood for a positive response to lithium treatment in a subject.

24. Use of an array comprising a substrate having a plurality of segments, wherein at least one of said segment comprises a probe which hybridizes to a polymorphism in the Cacng2 gene in identifying an increased likelihood for a positive response to lithium treatment in a subject, wherein said polymorphism is at least one single nucleotide polymorphisms (SNP) from the group consisting of:

25. A kit for use in identifying an increased likelihood for a positive response to lithium treatment in a subject compartmentalized to receive a reagent for measuring single nucleotide polymorphism (SNP) of the Cacng2 gene in a nucleic acid sample obtained from a subject, said reagent comprising at least one oligonucleotide which hybridizes to at least one single nucleotide polymorphisms (SNP) from the group consisting of:

26. The kit of claim 25, wherein said kit comprises at least one additional oligonucleotide that hybridizes with at least one additional marker for response to lithium treatment.

27. The kit of claim 25, wherein said reagent comprising at least 12, 14, 15, or 21 contiguous nucleotides selected from SEQ ID NO: 1-9.

28. A method for obtaining information regarding the increased likelihood for positive response to lithium treatment in a subject, said method comprising testing a sample obtained from the subject for the presence of at least one polymorphism of the Cacng2 gene selected from the group consisting of:

(i) allele C of the C/T single nucleotide polymorphism (SNP) rs2284017; (ii) allele C of the C/T single nucleotide polymorphism (SNP) rs2284018; (iii) allele C of the C /G single nucleotide polymorphism (SNP) rs5750285; and (iv) any combination thereof; wherein presence of each of said polymorphisms indicates an increased likelihood for a positive response to lithium treatment in the subject, thereby obtaining information regarding the increased likelihood for positive response to lithium treatment in the subject.

29. The method of claim 28, wherein said information classifies a subject in a clinical trial.

30. The method of claim 29, wherein said information classifies a population of subjects.

31. The method of claim 28, wherein said information correlates a polymorphism with an outcome of a lithium treatment.