WO2007121319A2

WO2007121319A2 - Compostions and methods for determining and predicting treatment responses for depression and anxiety

Info

Publication number: WO2007121319A2
Application number: PCT/US2007/066562
Authority: WO
Inventors: Ma-Li Wong; Julio Licinio
Original assignee: University Of California
Priority date: 2006-04-14
Filing date: 2007-04-12
Publication date: 2007-10-25
Also published as: WO2007121319A3

Abstract

The invention provides genetic means, including compositions (e.g., kits) and methods, to predict the efficacy of a treatment in depression and anxiety and to predict a patient's response to a particular a specific medication or treatment. The invention provides methods for diagnosing the presence of a psychiatric disorder or determining the outcome of a treatment for a psychiatric disorder. The invention provides methods for methods for diagnosing the presence of a psychiatric disorder in an individual by determining what cyclic nucleotide phosphodiesterase (PDE) protein or transcript isoforms are expressed in an individual.

Description

COMPOSITIONS AND METHODS FOR DETERMINING AND PREDICTING TREATMENT RESPONSES FOR DEPRESSION AND ANXIETY

FEDERAL FUNDING

This invention was produced in part using funds from the Federal government under NIH Grant Nos. NIH grants NIH/NIGMS UOl GM61394; NIH/NIDDK ROl DK063240; NIH/NCRR K24 RR017365; NIH/NHGRI R03 HG02500; NIH/NIGMS R03 HG/CA02500; Error! Unknown document property name.NIH/NCRR M01-RR00865. Accordingly, the Federal government has certain rights in this invention.

TECHNICAL FIELD This invention relates to medicine, psychiatry and molecular cellular. In one aspect, the invention provides genetic means, including compositions and methods, to predict the efficacy of a treatment in depression in a subpopulation of patients and to predict a patient's response to a particular a specific medication or treatment. In one aspect, the invention provides methods for diagnosing the presence of a psychiatric disorder or determining the outcome of a treatment for a psychiatric disorder. In one aspect, the invention provides methods for diagnosing the presence of a psychiatric disorder in an individual by determining what cyclic nucleotide phosphodiesterase (PDE) protein or transcript isoforms are expressed in an individual.

BACKGROUND

Major depression is a common and complex disorder of gene-environment interactions. The specific genetic substrates and precipitating environmental factors have not yet been elucidated. The disorder affects 10% of males and 20% of females and has a point prevalence of 3%. Its cost to the U.S. economy exceeds 100 billion dollars per year. Over twenty drugs are approved by the U.S. Food and Drug Administration for treatment of depression, each one with efficacy of approximately 60%. Various subgroups of patients respond differently to each drug, so that if multiple trials are conducted, eventually 85% of patients will respond. Because there are no clinical or biomarker predictors of treatment response, the assignment of a depressed patient to a drug is based solely on chance or on attempts to minimize side effects that are more likely to occur with a specific medication.

Cyclic nucleotide phosphodiesterases (PDEs) constitute a family of enzymes that degrade cyclic AMP (cAMP) and cyclic guanosine monophosphate (cGMP). Intracellular cyclic nucleotide levels increase in response to extracellular stimulation by hormones, neurotransmitters or growth factors and are downregulated through hydrolysis catalyzed by PDEs, which are therefore plausible therapeutic targets. Cyclic AMP is a second messenger implicated in learning, memory and mood, and cGMP influences in nervous system processes that are controlled by the nitric oxide (N0)/cGMP pathway. Eleven different phosphodiesterase (PDEl-PDEl 1) families have already been identified based on their substrate specificities, kinetic properties, allosteric regulators, inhibitor sensitivities, and amino acid sequences (1-10). Among each family, many genes and splice variants have been identified (2, 11). Each family and members within a family exhibit distinct tissue and cell-expression patterns (1, 3-5, 8, 9, 12). Because the hydrolysis of cyclic monophosphatase (cAMP) and cyclic guanosine monophosphate (cGMP) are controlled by multiple PDEs, they influence numerous pharmacological processes, including mediation of inflammation, ion channel function, muscle contraction, learning, differentiation, apoptosis, lipogenesis, glycogenolysis, and gluconeogenesis (13).

As regulators of the ubiquitous second messengers cAMP and cGMP, PDEs modulate the transduction of various extracellular signals through the activation of cell- surface receptors. Intracellular concentrations of cyclic nucleotides increase and activate their target enzymes, which include protein kinase A (PKA) and protein kinase G (PKG). These protein kinases are responsible for the phosphorylation of a number of substrates, such as ion channels, contractile proteins and transcription factors; thus, PDEs regulate key cellular functions. Therefore, PDEs have fundamental and pharmacological interest, and they have been acknowledged as important drug targets for the treatment of disparate diseases, such as congestive heart disease, depression, asthma, inflammation, and erectile dysfunction (14-17).

The PDE enzymes can be classified by their substrate: PDE4, -7 and -8 selectively hydrolyzes cAMP; PDE5, -6 and -9 are cGMP-specific enzymes, and PDEl, -2, -3, -10 and - 11 hydrolyses both cyclic nucleotides (14, 18, 19). The regulatory N-terminus of these enzymes has considerable variation among PDEs and includes regions that auto-inhibit the catalytic domains and regions that control subcellular localization (20, 21). This region may include a calmodulin binding protein (PDEl), cGMP binding sites (PDE2), phosphorylation sites for several protein kinases (PDE1-5), and a transducin binding domain (PDE6). It has been proposed that impairment of signal transduction that regulates neuroplasticity and cell survival could be an important contributory mechanism to major depressive disorder (MDD) (22). Moreover, cAMP-mediated signaling appears to have a key role in the pathophysiology and pharmacotherapy of depression (23). Several lines of investigation suggest that PDE4, a c AMP- specific enzyme, should be considered to be a prime target for therapeutic intervention in a range of CNS disorders, including depression and impaired cognition (24-26). PDE4 is the predominant mediator of cAMP hydrolysis (27-30), and the selective inhibitor of PDE4 rolipram has been shown to produce antidepressant- like and memory-enhancing effects in animals (23, 24, 26, 31). Behavioral phenotype and pharmacological data of knockout mice support the concept that PDE4D might be an enzyme subtype involved in the mediation of depressive symptoms and antidepressant response (32).

PDE4D knockout mice have an antidepressant-like profile. This strongly suggests that PDE4D-regulated cAMP signaling may play a role in the pathophysiology and pharmacotherapy of depression (25, 26).

Major depressive disorder (MDD) is one of the most common psychiatric disorders with both environmental and genetic factors contributing to its etiology. Heritability of MDD is estimated at 0.36 to 0.7 based on twin studies (33-39). The specific genetic substrates, precipitating environmental factors and predictors of treatment response have not been elucidated yet.

SUMMARY

The invention provides compositions and methods for associating specific cyclic nucleotide phosphodiesterase (PDE) sequence variations, so-called haplotypes, with different antidepressant- mediated responses in a human subpopulation, particularly, a subpopulation having a diagnosis of major depression and having high levels of anxiety. Thus, the compositions and methods of the invention can be used to predict a sub-population of patients (as defined by a haplotypes) response to antidepressant and to aid in monitoring, evaluating and adjusting an on-going regimen of antidepressant drug therapy. In particular, the compositions and methods of the invention can be used to predict and monitor treatment responses in a phenotypic subgroup of depressed patients, e.g., a high-anxiety depressed patient subpopulation, by determining the presence, or absence, of heterozygocity or homozygocity for an identified haplotype of the cyclic nucleotide phosphodiesterase (PDE) gene.

While the invention is not limited to practicing the compositions and methods on any particular individual or subpopulation, because the cyclic nucleotide phosphodiesterase (PDE) haplotypes described herein are relatively frequent in American Latino subpopulations, the compositions and methods of the invention can be particularly useful for predicting antidepressant treatment response in Mexican Americans. Thus, the methods of the invention can be used by a health professional to develop a treatment therapy or regimen, or help evaluate or critique an on-going treatment regimen. In one aspect of the methods, the subject is selected from a subpopulation of patients, e.g., a subpopulation of patients comprising a Mexican-American subpopulation.

In one aspect of the methods, wherein the subject is diagnosed (e.g., as diagnosed according to the Structured Clinical Interview for DSM-IV, as discussed below, or any other accepted diagnostic protocol) as having a psychiatric disorder comprising depression and anxiety. In one aspect, the psychiatric disorder comprises major depression and anxiety disorder. In one aspect, the psychiatric disorder therapy comprises treatment with an antidepressant agent, e.g., tricyclic antidepressants, selective serotonin reuptake inhibitors and/or PDE antagonists or equivalent drugs.

The invention provides methods for determining a subject's responsiveness (or non- responsiveness) to a psychiatric disorder therapy, wherein the subject is diagnosed with depression and/or anxiety, the method comprising the steps of: (a) providing a nucleic acid- comprising sample from the subject; (b) analyzing the sample and detecting whether the subject is homozygous for a cyclic nucleotide phosphodiesterase (PDE) haplotype, wherein the presence of homozygosity for the genotype correlates with responsiveness (or non- responsiveness) to the depression and/or anxiety therapy. The invention provides methods for screening a subject to determine the subject's responsiveness to a psychiatric disorder therapy comprising use of an agent which prevents or treats a psychiatric disorder associated with the cyclic nucleotide phosphodiesterase (PDE) gene, comprising: (a) providing a nucleic acid- comprising sample from the subject; (b) detecting whether the subject is homozygous for a cyclic nucleotide phosphodiesterase (PDE) haplotype, wherein the presence of homozygosity for the cyclic nucleotide phosphodiesterase (PDE) genotype correlates with a level of responsiveness (or non- responsiveness) to the therapy.

In one aspect, the subject is diagnosed as having a psychiatric disorder comprising depression and anxiety, or depression, or anxiety. In one aspect, the psychiatric disorder comprises major depression disorder (MDD), including any psychiatric disorder therapy comprising a treatment (e.g., with a drug) with an antidepressant agent. In one aspect, the antidepressant agent is selected from the group consisting of tricyclic antidepressants, selective serotonin reuptake inhibitors and cyclic nucleotide phosphodiesterase (PDE) antagonists. In one aspect, the cyclic nucleotide phosphodiesterase (PDE) haplotype comprises PDE9A, PDEIlA, PDE2A, PDE5A, PDE6C, PDElOA and/or PDElA, e.g., a PDE9A or PDEIlA, or PDEl IA.

In one aspect, the subject's responsiveness (or non- responsiveness) to a psychiatric disorder therapy comprises remission while on an antidepressant or a psychiatric disorder therapy, and optionally the cyclic nucleotide phosphodiesterase (PDE) haplotype comprises PDElA or PDEl IA.

In one aspect of the methods, the nucleic acid-comprising sample comprises a blood sample, a saliva sample or a cell sample. The cell sample can comprise a cell from any source, e.g., a biopsy, a buccal or a skin scraping or sample.

In one aspect, subject is selected from a subpopulation of patients, e.g., a latino, e.g., a Mexican-American, subpopulation.

In one aspect, the genotype of the subject with respect to the cyclic nucleotide phosphodiesterase (PDE) haplotypes, is determined by amplification genotyping, in situ hybridization techniques, DNA array (biochip) analysis and/or direct DNA sequencing. The amplification genotyping can comprise any amplification genotyping protocol, e.g., a polymerase chain reaction (PCR).

The invention provides kits suitable for determining a subject's responsiveness (or non- responsiveness) to a psychiatric disorder therapy, wherein the subject is diagnosed with depression and/or anxiety, the kit comprising (a) material for determining whether the subject is homozygous for a cyclic nucleotide phosphodiesterase (PDE) haplotype; (b) suitable packaging material; and optionally (c) instructional material for use of said kit. In one aspect, the material in the kit comprises at least one nucleic acid (e.g., an oligonucleotide, such as a labeled probe or a PCR primer) that specifically binds to a cyclic nucleotide phosphodiesterase (PDE) haplotype. In one aspect, the cyclic nucleotide phosphodiesterase (PDE) haplotype comprises PDE9A, PDEIlA, PDE2A, PDE5A, PDE6C, PDElA and/or PDElOA, or the probe or a PCR primer comprises all or part of (a subsequence thereof) of PDE9A, PDEl IA, PDE2A, PDE5A, PDE6C, PDElA and/or PDElOA.

In one aspect, the cyclic nucleotide phosphodiesterase (PDE) haplotype nucleic acid sequence (e.g., probe or a PCR primer) specifically binds to a SNP, and optionally the sequence that specifically binds to SNPs comprises at least one polymerase chain reaction (PCR) primer or a hybridization probe. In one aspect, the kit further comprises material to process a nucleic acid-comprising biological sample. The invention provides kits suitable for determining a subject's responsiveness to a drug therapy for MDD, the kit comprising (a) material for determining whether the subject is homozygous for a PDE haplotype; (b) suitable packaging material; and optionally (c) instructional material for use of said kit. The kit can further comprise material to process a nucleic acid-comprising biological sample.

The invention provides methods for determining a nucleotide polymorphism associated with a specific response (or non-responsive) to a psychiatric disorder therapy in an individual comprising (a) sequencing all or a portion of the nucleotide sequence of chromosome comprising at least one haplotype PDE9A, PDEIlA, PDE2A, PDE5A, PDE6C, PDElA or PDElOA from the individual having psychiatric disorder therapy, comparing the chromosome sequence from the individual to chromosome sequences of other individuals having the same psychiatric disorder and the same psychiatric disorder therapy, and determining the presence of a nucleotide polymorphism in the sequenced haplotype sequences; and, (b) correlating the specific response of the individual to the psychiatric disorder therapy with the presence or absence of the nucleotide polymorphism in the other individuals, wherein optionally the psychiatric disorder therapy is a drug therapy, or depression and anxiety.

The invention provides methods for determining a nucleotide polymorphism associated with the presence (or absence) of a psychiatric disorder in an individual comprising (a) sequencing all or a portion of the nucleotide sequence of chromosome comprising at least one haplotype PDE9A, PDEl IA, PDE2A, PDE5A, PDE6C, PDElA or PDElOA from the individual, comparing the chromosome sequence from the individual to chromosome sequences in individuals having the same psychiatric disorder, and determining the presence or absence of a nucleotide polymorphism in individuals having the same psychiatric disorder; and, (b) correlating the diagnosis of the psychiatric disorder to the presence or absence of the nucleotide polymorphism, wherein optionally the psychiatric disorder therapy is a depression, or depression and anxiety.

The invention provides methods for diagnosing (or the prognosis of) the presence of a psychiatric disorder in an individual by determining a nucleotide polymorphism in an individual comprising (a) sequencing all or a portion of the nucleotide sequence of chromosome 2q31-32 and/or 5ql4-31 from the individual, comparing the 2q31-32 and/or 5ql4-31 sequence from the individual to chromosome 2q31-32 and/or 5ql4-31 sequences in individuals having the same psychiatric disorder, and determining the presence or absence of a nucleotide polymorphism in individuals having the same psychiatric disorder; and, (b) correlating the presence or absence of the nucleotide polymorphism to the presence or absence of (or the prognosis of) a psychiatric disorder, wherein optionally the psychiatric disorder therapy is a depression, or depression and anxiety.

The invention provides methods for determining a cyclic nucleotide phosphodiesterase (PDE) protein or transcript isoform associated with a specific response (or non-response) to a psychiatric disorder therapy in an individual comprising (a) determining the cyclic nucleotide phosphodiesterase (PDE) protein or transcript isoform or isoforms expressed in the individual, comparing the cyclic nucleotide phosphodiesterase (PDE) protein or transcript isoform or isoforms expressed in the individual to cyclic nucleotide phosphodiesterase (PDE) protein or transcript isoform or isoforms expressed in individuals having the same psychiatric disorder therapy; and, (b) correlating the response (or non-response) of the individual to the psychiatric disorder therapy with the presence or absence of the expressed cyclic nucleotide phosphodiesterase (PDE) protein or transcript isoform or isoforms, wherein optionally the psychiatric disorder therapy is a drug therapy. The invention provides methods for determining a cyclic nucleotide phosphodiesterase

(PDE) protein or transcript isoform associated with the presence (or absence) of a psychiatric disorder in an individual comprising (a) determining the cyclic nucleotide phosphodiesterase (PDE) protein or transcript isoform or isoforms expressed the individual, comparing the PDE protein or transcript isoform or isoforms expressed in the individual to PDE protein or transcript isoform or isoforms expressed in individuals having the same psychiatric disorder, and determining the presence or absence of PDE protein or transcript isoform or isoforms in individuals having the same psychiatric disorder; and, (b) correlating the diagnosis (or the prognosis of) of the psychiatric disorder to the presence or absence of the PDE protein or transcript isoform or isoforms expressed in the individuals, wherein optionally the psychiatric disorder therapy is a depression.

The invention provides methods for diagnosing (or the prognosis of) the presence of a psychiatric disorder in an individual by determining what cyclic nucleotide phosphodiesterase (PDE) protein or transcript isoforms are expressed in an individual comprising (a) determining the PDE protein or transcript isoform or isoforms expressed the individual, comparing the PDE protein or transcript isoform or isoforms expressed in the individual to PDE protein or transcript isoform or isoforms expressed in individuals having the same psychiatric disorder, and determining the presence or absence of PDE protein or transcript isoform or isoforms expressed in individuals having the same psychiatric disorder; and, (b) correlating the presence or absence of the PDE protein or transcript isoform or isoforms expressed in the individuals to the presence or absence of (or the prognosis of) a psychiatric disorder, wherein optionally the psychiatric disorder therapy is a depression.

The invention provides an array (biochip) comprising a plurality of nucleic acids each comprising a cyclic nucleotide phosphodiesterase (PDE) haplotype sequence. In one aspect of the array (biochip), the cyclic nucleotide phosphodiesterase (PDE) haplotype sequences comprises PDE9A, PDEIlA, PDE2A, PDE5A, PDE6C, PDElA and/or PDElOA, or, the cyclic nucleotide phosphodiesterase (PDE) haplotype sequences comprises PDE9A, PDEIlA, PDE2A, PDE5A, PDE6C, PDElA and PDElOA. The invention also provides multiplexed systems for determining a subject's responsiveness (or non- responsiveness) to a psychiatric disorder therapy, wherein the subject is diagnosed with depression and/or anxiety, comprising: (a) the array (biochip) of the invention for determining the presence or absence of cyclic nucleotide phosphodiesterase (PDE) haplotype sequences; and (b) a computer system operably linked to the array for analyzing the results of the presence or absence of cyclic nucleotide phosphodiesterase (PDE) haplotype sequences incorporating/ using a method of the invention (e.g., as a computer-implemented method in a computer program product), and outputting that information to a user.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

All publications, patents, patent applications, GenBank sequences and ATCC deposits, cited herein are hereby expressly incorporated by reference for all purposes.

DESCRIPTION OF DRAWINGS

Figures are attached. Figure 1 illustrates data showing the linkage disequilibrium pattern in gene PDEIlA in the "Depressed Group" test group, as discussed in Example 1, below.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

The invention provides compositions and methods for associating cyclic nucleotide phosphodiesterase (PDE) sequence variations, so-called haplotypes, with different antidepressant- mediated responses in a phenotypic subgroup of depressed patients, i.e., a high- anxiety depressed patient subpopulation. In one aspect, the invention provides compositions and methods for identifying individuals with susceptibility to major depressive disorder (MDD) and related mood disorders. In one embodiment, the invention provides compositions and methods for characterizing (categorizing/ prioritizing) treatment responses to antidepressant drugs.

There is no consensus in the field as to the causes of major depressive disorder or related mood conditions. While the invention is not limited by any particular mechanism of action, the invention provides support for the involvement of genes for cyclic nucleotide phosphodiesterase (PDE) in the pathophysiology of major depressive disorder in the response to antidepressant treatment. This invention for the first time establishes and demonstrates a link between susceptibility to major depression (e.g., major depressive disorder (MDD)) and related mood disorders and treatment responses to the PDE enzymes that degrade cyclic guanosine monophosphate (cGMP).

This invention provides compositions, including arrays or probes comprising SNPs or arrays and probes comprising PDE nucleic acid sequences, useful for the identification of individuals having susceptibility to major depressive disorder (MDD) and related mood disorders, and of major depressive disorder in the response to antidepressant treatment. This invention provides compositions and methods to identify individuals with susceptibility to major depressive disorder and related mood disorders. This invention provides compositions and methods to help characterize treatment response to antidepressant drugs.

This invention also provides compositions and methods to help characterize the utility of current drugs that modulate PDEs actions - such as drugs that act by increasing, decreasing, enhancing, or diminishing PDEs actions, directly or indirectly, in the treatment and prevention of major depression and other mood conditions. This invention also provides compositions and methods to aid in the development of new strategies to modulate PDEs actions in the treatment and prevention of major depression and other mood conditions.

In one aspect, the invention provides specific single nucleotide polymorphisms (SNPs) in PDE genes that have been significantly associated with either major depression and/or antidepressant treatment response. In one aspect, the invention provides the SNPs in PDE genes that are likely to be associated with either major depression and/or antidepressant treatment response. In one aspect, the invention provides the SNPs in PDE genes that have not been associated with either major depression and/or antidepressant treatment response. A detailed description of our phenotypic characterization of major depressive disorder and treatment response, and of our genotyping, and data analyses strategies are provided herein.

To investigate an association between genes encoding cyclic nucleotide phosphodiesterases (PDEs) and susceptibility to Major Depressive Disorder (MDD), we genotyped single-nucleotide polymorphisms (SNPs) in twenty-one genes of this superfamily in 284 depressed Mexican- Americans who participated in a prospective, double-blind, pharmacogenetic study of antidepressant response and 331 sex-, age-, and ethnically-matched controls. We found that polymorphisms in PDE9A and PDEl IA were highly associated with the diagnosis of MDD, and SNPs of the PDE2A, PDE5A, PDE6C, and PDElOA genes were associated with the diagnosis of Major Depressive Disorder (MDD). Remission on antidepressants was significantly associated with polymorphisms in PDElA and PDEIlA. Thus, we found significant associations with both the diagnosis of MDD and remission in response to antidepressants with SNPs in the PDEIlA gene. We conclude that PDEIlA has a role in the pathophysiology of MDD. Our findings demonstrate that cGMP-related PDEs have a role in the biology of depression.

The present invention provides genetic methods and compositions for the diagnosis, prognosis and treatment of psychiatric disorders associated with the PDE gene, including depression and/or anxiety disorders and related pathologies. Anxiety disorders encompassed by this invention include panic disorder, obsessive-compulsive disorder, post-traumatic stress disorder, and phobias, including both specific phobias and social phobias. The methods and compositions of the invention utilize polymorphic variations in the PDE gene to screen patients to assess their responsiveness to particular psychiatric therapies, development of diagnostics and therapies for psychiatric disorders associated with the PDE gene, and development of individualized drug treatments based on an individual's genotypic profile with respect to the PDE gene.

In demonstrating the efficacy of the compositions and methods of the invention, as discussed in Example 1, below, the described data indicate that PDE genes that modulate intracellular levels of cGMP are the predominant class of PDE associated with the diagnosis and treatment outcome of major depression. All but one PDE gene we identified were either cGMP-specific or dual- substrate enzymes. PDE8B, which is cAMP-specific, was associated with treatment response in our fluoxetine treated group, but none of the SNPs we examined in cAMP-specific PDE genes were significantly associated with diagnosis, even though in our study the SNPs density was higher for the class of cAMP-specific genes. We found that polymorphisms in the PDEIlA gene are significantly associated with the diagnosis of MDD and treatment response, which suggests the involvement of this enzyme in the biology of depression. These data therefore demonstrate the involvement of chromosome 2q31-32 in the diagnosis of MDD and in antidepressant response. Generating and Manipulating Nucleic Acids

The invention provides compositions and methods comprising isolating a nucleic acid from a sample from a subject and analyzing genotype, including detecting whether the subject is homozygous for a cyclic nucleotide phosphodiesterase PDE haplotype, e.g., comprising a cyclic nucleotide phosphodiesterase (PDE) haplotype PDE9A, PDEIlA, PDE2A, PDE5A, PDE6C, PDElOA and/or PDElA. The genotype of the subject, e.g., with respect to the PDE haplotype, can be determined by any method or protocol or device known in the art, including amplification genotyping, in situ hybridization techniques, DNA array (biochip) analysis and/or direct DNA sequencing. Haplotypes are groups of two or more SNPs that are functionally and/or spatially linked. The invention can be practiced in conjunction with any method or protocol or device known in the art, which are well described in the scientific and patent literature.

The nucleic acids used to practice this invention, e.g., primers for use in amplification detection, sequencing, Southerns and the like, whether RNA, cDNA, genomic DNA, vectors, viruses or hybrids thereof, may be isolated from a variety of sources, genetically engineered, synthetic, amplified, and/or expressed/ generated recombinantly (recombinant polypeptides can be modified or immobilized to arrays in accordance with the invention). Any recombinant expression system can be used, including bacterial, mammalian, yeast, insect or plant cell expression systems.

Alternatively, these nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Carruthers (1982) Cold Spring Harbor Symp. Quant. Biol. 47:411-418; Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22:1859; U.S. Patent No. 4,458,066. Double stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with a primer sequence. Techniques for the manipulation of nucleic acids, such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), VOIS. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N. Y. (1993). The nucleic acids used to practice this invention, whether RNA, antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybrids thereof, may be isolated from a variety of sources, genetically engineered, amplified, and/or expressed/ generated recombinantly. Any recombinant expression system can be used, including bacterial, mammalian, yeast, insect or plant cell expression systems. Another useful means of obtaining and manipulating nucleic acids used to practice the methods of the invention is to clone from genomic samples, and, if desired, screen and re-clone inserts isolated or amplified from, e.g., genomic clones or cDNA clones. Sources of nucleic acid used in the methods of the invention include genomic or cDNA libraries contained in, e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Patent Nos. 5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld (1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC); bacterial artificial chromosomes (BAC); Pl artificial chromosomes, see, e.g., Woon (1998) Genomics 50:306-316; Pl-derived vectors (PACs), see, e.g., Kern (1997) Biotechniques 23:120-124; cosmids, recombinant viruses, phages or plasmids. In practicing the invention, nucleic acids (e.g., DNA from patient samples) can be detected, sequenced and/or reproduced by amplification. Amplification can also be used to sequence, clone or modify the nucleic acids of the invention. Thus, the invention provides amplification primer sequence pairs (e.g., in kits) for detecting, sequencing or amplifying nucleic acids. One of skill in the art can design amplification primer sequence pairs for any part of or the full length of these sequences.

Amplification reactions can also be used to quantify the amount of nucleic acid in a sample (such as the amount of message in a cell sample), label the nucleic acid (e.g., to apply it to an array or a blot), detect a nucleic acid or sequence (e.g., SNPs), or quantify the amount of a specific nucleic acid in a sample. In one aspect of the invention, message isolated from a cell or a cDNA library are amplified.

The skilled artisan can select and design suitable oligonucleotide amplification primers (e.g., for detecting genotype/ haplotype of nucleic acid in a patient sample). Amplification methods are also well known in the art, and include, e.g., polymerase chain reaction, PCR (see, e.g., PCR Protocols, A Guide to Methods and Applications, ed. Innis, Academic Press, N.Y. (1990) and PCR Strategies (1995), ed. Innis, Academic Press, Inc., N. Y., ligase chain reaction (LCR) (see, e.g., Wu (1989) Genomics 4:560; Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117); transcription amplification (see, e.g., Kwoh (1989) Proc. Natl. Acad. Sci. USA 86:1173); and, self- sustained sequence replication (see, e.g., Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:1874); Q Beta replicase amplification (see, e.g., Smith (1997) /. Clin. Microbiol. 35:1477-1491), automated Q-beta replicase amplification assay (see, e.g., Burg (1996) MoI. Cell. Probes 10:257-271) and other RNA polymerase mediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario); see also Berger (1987) Methods Enzymol. 152:307- 316; Sambrook; Ausubel; U.S. Patent Nos. 4,683,195 and 4,683,202; and Sooknanan (1995) Biotechnology 13:563-564.

Haplotypes (SNPs) can be detected using single-strand conformation polymorphism analysis, which identifies base differences by alteration in electrophoretic migration of single stranded PCR products, as described, e.g., USPN 5,879,884; Orita et al., Proc. Nat. Acad. Sci. USA 86:2766-2770 (1989). Amplified PCR products can be generated as described above, and heated or otherwise denatured, to form single stranded amplification products. Single-stranded nucleic acids may refold or form secondary structures which are partially dependent on the base sequence. Electrophoretic mobility of single- stranded amplification products can detect base- sequence difference between alleles or target sequences. Haplotypes (SNPs) can be detected using allele- specific PCR, which differentiates between alleles differing in the presence or absence of a variation or polymorphism. PCR amplification primers are designed to bind only to certain alleles of a target sequence; see, e.g., Gibbs (1989) Nucleic Acid Res. 17:12427-2448.

Haplotypes (SNPs) can be detected using allele- specific oligonucleotide (ASO) screening methods, e.g., as described by Saiki (1986) Nature 324:163-166. Oligonucleotides with one or more base pair mismatches are designed for any particular allele. ASO screening methods can detect variations between haplotypes. Mismatches between variant haplotypes or PCR amplified DNA can show decreased binding of the oligonucleotide relative to a variant haplotypes (or mutant) oligonucleotide. Oligonucleotide probes can be designed that under low stringency will bind to both polymorphic forms of the allele, but that at higher stringency, will bind detectably more strongly to the allele to which they correspond. Stringency conditions can be devised in which an essentially binary response is obtained, for example, an ASO corresponding to a haplotype will hybridize to that allele, and not to an alternative haplotype allele.

Haplotypes (SNPs) can be detected using ligase-mediated allele detection, e.g., as described in Landegren (1988) Science 241:1077-1080. Ligase may also be used to detect haplotypes SNPs (e.g., mutations) in a ligation amplification reaction, e.g., as described in Wu (1989) Genomics 4:560-569. The ligation amplification reaction (LAR) utilizes amplification of specific DNA sequence using sequential rounds of template dependent ligation, e.g., as in Wu (1990) Proc. Nat. Acad. Sci. USA 88:189-193.

Amplification products generated using the polymerase chain reaction can be analyzed by the use of denaturing gradient gel electrophoresis. Different haplotype alleles can be identified based on the different sequence-dependent melting properties and electrophoretic migration of DNA in solution. DNA molecules melt in segments, termed melting domains, under conditions of increased temperature or denaturation. Each melting domain melts cooperatively at a distinct, base- specific melting temperature (Tm). Melting domains are at least 20 base pairs in length, and may be up to several hundred base pairs in length. Differentiation between haplotypes (SNPs) based on sequence specific melting domain differences can be assessed using polyacrylamide gel electrophoresis.

Haplotypes (SNPs) can be detected without an amplification step, based on polymorphisms including restriction fragment length polymorphisms in a patient and a family member. Hybridization probes are generally oligonucleotides which bind through complementary base pairing to all or part of a target nucleic acid. Probes can bind target sequences lacking complete complementarity with the probe sequence depending on the stringency of the hybridization conditions. Probes can be labeled directly or indirectly. By assaying for the presence or absence of the probe, the presence or absence of the target sequence can be detected. Direct labeling methods include radioisotope labeling, such as with ³²P or ³⁵S. Indirect labeling methods include fluorescent tags, biotin complexes which may be bound to avidin or streptavidin, or peptide or protein tags. Visual detection methods include photo-luminescence, Texas red, rhodamine and its derivatives, red leuco dye and 3, 3', 5, 5'- tetramethylbenzidine (TMB), fluorescein, and its derivatives, dansyl, umbelliferone and the like or with horse radish peroxidase, alkaline phosphatase and the like.

Haplotypes (SNPs) can be detected using an oligonucleotide ligation assay (OLA), as described, e.g., in U.S. Pat. No. 4,998,617; Landegren (1988) Science 241:1077-1080. The OLA protocol uses two oligonucleotides which are designed to be capable of hybridizing to abutting sequences of a single strand of a target. One of the oligonucleotides is linked to a separation marker, e.g., biotinylated, and the other is detectably labeled. If the precise complementary sequence is found in a target molecule, the oligonucleotides will hybridize such that their termini abut, and create a ligation substrate. Ligation then permits the labeled oligonucleotide to be recovered using avidin, or another biotin ligand. One variant nucleic acid detection assay combines attributes of PCR and OLA, see, e.g., Nickerson (1990) Proc. Natl. Acad. Sci. USA 87:8923-8927. In this method, PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA.

Haplotypes (SNPs) can be detected using a specialized exonuclease-resistant nucleotide, e.g., see USPN 4, 656, 127 '. A primer complementary to the allelic sequence immediately 3' to the polymorphic site is permitted to hybridize to a target molecule obtained from a particular animal or human. If the polymorphic site on the target molecule contains a nucleotide that is complementary to the particular exonuclease-resistant nucleotide derivative present, then that derivative will be incorporated onto the end of the hybridized primer. Such incorporation renders the primer resistant to exonuclease, and thereby permits its detection. Since the identity of the exonuclease-resistant derivative of the sample is known, a finding that the primer has become resistant to exonucleases reveals that the nucleotide present in the polymorphic site of the target molecule was complementary to that of the nucleotide derivative used in the reaction. This method has the advantage that it does not require the determination of large amounts of extraneous sequence data.

Haplotypes (SNPs) can be detected sequence- specific ribozymes, see, e.g., USPN 5,498,531. This method can be used to score SNPs based on the development or loss of a ribozyme cleavage site. Perfectly matched sequences can be distinguished from mismatched sequences by nuclease cleavage digestion assays or by differences in melting temperature. If the SNP affects a restriction enzyme cleavage site, the SNP can be identified by alterations in restriction enzyme digestion patterns, and the corresponding changes in nucleic acid fragment lengths determined by gel electrophoresis. Haplotypes (SNPs) can be detected using Genetic Bit Analysis or GBA™, e.g., see Nikiforov (1994) Nucleic Acids Res. 22(20): 4167-4175. This is a method for typing single nucleotide polymorphisms in DNA. Specific fragments of genomic DNA containing the polymorphic site(s) are first amplified by the polymerase chain reaction (PCR) using one regular and one phosphorothioate-modified primer. The double-stranded PCR product is rendered single-stranded by treatment with the enzyme T7 gene 6 exonuclease, and captured onto individual wells of a 96 well polystyrene plate by hybridization to an immobilized oligonucleotide primer. This primer is designed to hybridize to the single- stranded target DNA immediately adjacent from the polymorphic site of interest. Using the Klenow fragment of E. coli DNA polymerase I or a modified T7 DNA polymerase, the 3' end of the capture oligonucleotide is extended by one base using a mixture of one biotin-labeled, one fluorescein- labeled, and two unlabeled dideoxynucleoside triphosphates. Antibody conjugates of alkaline phosphatase and horseradish peroxidase are then used to determine the nature of the extended base in an ELISA format. In one aspect, haplotypes, or SNPs, are detected using biochips, or arrays. For example, several probes capable of hybridizing specifically to allelic (haplotypes) variants are attached to a solid phase support, e.g., a "chip". Oligonucleotides can be bound to a solid support by a variety of processes, including lithography. For example a chip can hold up to 250,000 oligonucleotides (GeneChip, Affymetrix). Mutation detection analysis using these chips comprising oligonucleotides, also termed "DNA probe arrays" is described e.g., in

Cronin et al. (1996) Human Mutation 7:244 and in Kozal et al. (1996) Nature Medicine 2:753. In one embodiment, a chip comprises all the haplotypes (allelic) variants a gene, e.g., the PDE gene and its sequence variations. The solid phase support can be contacted with a test nucleic acid and hybridization to the specific probes is detected. Thus, the identity of numerous allelic variants of one or more genes can be identified in a simple hybridization experiment.

In practicing the methods of the invention, any known array and/or method of making and using arrays can be incorporated in whole or in part, or variations thereof, as described, for example, in U.S. Patent Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695; 6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174; 5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522; 5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also, e.g., WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also, e.g., Johnston (1998) Curr. Biol. 8:R171-R174; Schummer (1997) Biotechniques 23:1087-1092; Kern (1997) Biotechniques 23:120-124; Solinas-Toldo (1997) Genes, Chromosomes & Cancer 20:399-407; Bowtell (1999) Nature Genetics Supp. 21:25-32. See also published U.S. patent applications Nos. 20010018642; 20010019827; 20010016322; 20010014449; 20010014448; 20010012537; 20010008765.

Haplotypes (SNPs) can be detected using multicomponent integrated systems, such as microfluidic-based systems or "lab on a chip" systems. These systems miniaturize and compartmentalize processes such as PCR and capillary electrophoresis reactions in a single functional device. See, e.g., U.S. Pat. No. 5,589,136, which describes the integration of PCR amplification and capillary electrophoresis in chips.

Haplotypes (SNPs) can be detected using integrated systems, particularly when microfluidic systems are used. These systems can comprise a pattern of microchannels designed onto a glass, silicon, quartz, or plastic wafer included on a microchip. The movements of the samples can be controlled by electric, electroosmotic or hydrostatic forces applied across different areas of the microchip to create functional microscopic valves and pumps with no moving parts. Varying the voltage controls the liquid flow at intersections between the micro-machined channels and changes the liquid flow rate for pumping across different sections of the microchip. In such a system, the containers/compartments of the kit may be embodied as chambers and/or channels of the microfluidic system.

Haplotypes (SNPs) can be detected using mass spectrometry. Mass spectrometry takes advantage of the unique mass of each of the four nucleotides of DNA. SNPs can be unambiguously genotyped by mass spectrometry by measuring the differences in the mass of nucleic acids having alternate SNP alleles. MALDI-TOF (Matrix Assisted Laser Desorption Ionization— Time of Flight) mass spectrometry technology is preferred for extremely precise determinations of molecular mass, such as SNPs. Numerous approaches to SNP analysis have been developed based on mass spectrometry. An exemplary analysis is mini-sequencing primer extension, which can also be utilized in combination with other approaches, such as traditional gel -based formats and microarrays.

If a polymorphic region is located in an exon, either in a coding or non-coding region of the gene, the identity of the allelic variant can be determined by determining the molecular structure of the mRNA, pre-mRNA, or cDNA. The molecular structure can be determined using any of the above described methods for determining the molecular structure of the genomic DNA, e.g., sequencing and single-strand conformational polymorphism (SSCP). Haplotype determination procedures can be performed in situ directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary. Nucleic acid reagents may be used as probes and/or primers for such in situ procedures

Diagnosing and Rating Psychiatric Disorders

The invention provides methods for determining a subject's responsiveness to a psychiatric disorder therapy, wherein the subject is diagnosed with depression and anxiety. The invention also provides methods for screening a subject to determine the subject's responsiveness to a psychiatric disorder (e.g., depression and anxiety) therapy comprising use of an agent which prevents or treats a psychiatric disorder associated with the PDE gene. In practicing the invention any method (e.g., "rating scale") or protocol can be used to diagnose a psychiatric disorder (e.g., depression and anxiety) or assess the progress of treatment for a psychiatric disorder (e.g., depression and anxiety).

The depression diagnosed in practicing the methods and compositions of the invention includes all diseases and conditions which are associated with depression, including those classified in the IDC-10 and Diagnostic and Statistical Manual IV (DSM-IV) rating scales. These diseases or disorders comprise major depression, dysthymic disorder, depressive episodes of bipolar disorders and depressive episodes associated with other mood disorders, including seasonal mood disorders and mood disorders due to a general medical condition and substance induced mood disorder.

For example, in practicing the invention any rating scale can be used to measure the severity of a psychiatric disorder (e.g., depression and anxiety) in a subject. For example, in depression, the most frequently used scales include the Hamilton Depression Rating (HAM-D) Scale, the Beck Depression Inventory (BDI), the Montgomery-Asberg Depression Rating Scale (MADRS), the Geriatric Depression Scale (GDS), and the Zung Self-Rating Depression Scale (ZSRDS). For anxiety, the most frequently used scales include the Hamilton Anxiety Rating (HAM-A) Scale, and the Beck Anxiety Inventory (BAI). These or any art-acceptable means to diagnose and/or access a psychiatric disorder (e.g., depression and anxiety) in a subject can be used.

For example, in the studies described herein, the association of PDE genotypes with the phenotype of antidepressant treatment response was studied in depressed Mexican- Americans who completed a prospective randomized, placebo lead-in, double-blind treatment of fluoxetine or desipramine, with active treatment for eight weeks, where the primary outcome measures of the association analysis were the changes in the Hamilton rating scales for anxiety (HAM-A) and depression (HAM-D).

In alternative embodiments, the term "treatment" refers to partially or completely ameliorating at least one symptom of, partially or completely treating or curing and/or preventing the development of a disease or a condition, for example, depression or anxiety. For example DSM-IV criteria for depression and Clinical Rating Scale for Depression are summarized below:

MAJOR DEPRESSIVE DISORDER: DSM-IV DIAGNOSTIC CRITERIA At least five of the following symptoms are present during the same period. At least (1) depressed mood or (2) loss of interest or pleasure must be present. Symptoms are present most of the day, nearly daily for at least 2 weeks.

1. Depressed mood (sometimes irritability in children and adolescents) most of the day, nearly every day

2. Markedly diminished interest or pleasure in almost all activities most of the day, nearly every day (as indicated either by subjective account or observation by others of apathy most of the time) 3. Significant weight loss/gain

4. Insomnia/hypersomnia

5. Psychomotor agitation/retardation

6. Fatigue (loss of energy)

7. Feelings of worthlessness (guilt) 8. Impaired concentration (indecisiveness) 9. Recurrent thoughts of death or suicide

CLINICAL RATING SCALE FOR DEPRESSION

None Score = 0

Meets no DSM criteria for a depressive disorder (Major depression, Dysthymia or

Brief Recurrent Depression)

Has never sought help for depressive symptoms (sx) Has never taken prescription meds for depressive sx

Possible Score = 1

May have some depressive sx

Family members may report depressive sx

Does not meet DSM criteria for a depressive disorder Has never sought help

Has never taken prescription medications for depression

Mild Score = 2

May meet DSM criteria for a depressive disorder May have sought help

Has never taken prescription medications for depression

Depressive symptoms were transient

Moderate Score = 3 Meets DSM criteria

May have sought help

May have taken prescription medications for depression

Depressive episodes were transient but recurrent

Severe Score = 4 Meets DSM criteria

Has sought help

Has usually taken prescription medications for depression

Depressive episodes have been multiple throughout life

If <20 years old, depression started by age 12 Severe and chronic Score = 5

Meets all criteria for "Severe"

Has been diagnosed with depression

Depressive episodes have been chronic throughout life

Kks

The invention provides kits suitable for determining a subject's responsiveness to a psychiatric disorder therapy. For example, kits of the invention can be used to evaluate or determine the optimal treatment, e.g., drug regimen, drug scheduling or treatment protocol, when a subject is diagnosed with depression and anxiety. The kit can comprise material for determining any particular haplotypes, e.g., whether the subject is homozygous for a PDE haplotype, e.g., a cyclic nucleotide phosphodiesterase PZ)E haplotype comprising PDE9A, PDEIlA, PDE2A, PDE5A, PDE6C, PDElOA and/or PDElA, including, for example, PCR or other amplification primers for detecting one, several or all of these particular PDE haplotypes. The kit can comprise suitable packaging material. The kit can comprise instructional material for use of said kit, e.g., instructions on practicing the methods of the invention.

The kit can comprise nucleic acids to determine a particular haplotypes or genotype, as discussed, above. For example, the kit can comprise nucleic acid that specifically binds to htSNPs, e.g., primers such as polymerase chain reaction (PCR) primers or a hybridization probes. The kit also can comprise material or items to retrieve a nucleic acid-comprising sample from a subject, and/or to store or to process the nucleic acid-comprising biological sample. The kits comprise a vial, tube, or any other container which contains one or more oligonucleotides or primers which hybridize to a nucleic acid isolated form a subject, or a nucleic acid derived from a subject, e.g., an amplification product. The kits may also contain components of the amplification system, including PCR reaction materials such as buffers and a thermostable polymerase. A kit of the invention can be used in conjunction with commercially available amplification kits, e.g., from GIBCO BRL (Gaithersburg, Md.) Stratagene (La Jolla, Calif.), Invitrogen (San Diego, Calif.), Schleicher & Schuell (Keene, N.H.), Boehringer Mannheim (Indianapolis, Ind.). A kit of the invention also can comprise positive or negative control reactions or markers, molecular weight size markers for gel electrophoresis, and the like. A kit of the invention also can comprise labeling or instructions indicating the suitability of the kits for diagnosing depression and indicating how the oligonucleotides are to be used for that purpose. Cyclic nucleotide phosphodiesterase (PDE) protein or transcript isoforms The invention provides methods for determining a cyclic nucleotide phosphodiesterase protein or transcript isoform associated with a specific response to a psychiatric disorder therapy (e.g., any PDE-associated psychiatric disorder, such as depression, anxiety, or anxiety and depression) in an individual comprising (a) determining a PDE protein or transcript isoform or isoforms expressed in the individual, comparing the PDE protein or transcript isoform or isoforms expressed in the individual to PDE protein or transcript isoform or isoforms expressed in individuals having the same psychiatric disorder therapy; and, (b) correlating the response of the individual to the psychiatric disorder therapy with the presence or absence of the expressed PDE protein or transcript isoform or isoforms, wherein optionally the psychiatric disorder therapy is a drug therapy, e.g., for depression and/or anxiety. The results of this method can be used by a clinician to determine what therapy or drugs to use, in what combinations and/or in what dosages, or for what patient populations.

The invention provides methods for determining a PDE protein or transcript isoform associated with the presence of a psychiatric disorder ( e.g., any PDE-associated psychiatric disorder, such as depression, anxiety, or anxiety and depression) in an individual comprising (a) determining the PDE protein or transcript isoform or isoforms expressed the individual, comparing the PDE protein or transcript isoform or isoforms expressed in the individual to PDE protein or transcript isoform or isoforms expressed in individuals having the same psychiatric disorder, and determining the presence or absence of PDE protein or transcript isoform or isoforms in individuals having the same psychiatric disorder; and, (b) correlating the diagnosis of the psychiatric disorder to the presence or absence of the PDE protein or transcript isoform or isoforms expressed in the individuals, wherein optionally the psychiatric disorder therapy is a depression and/or anxiety therapy. The invention provides methods for diagnosing the presence of a psychiatric disorder (e.g., e.g., any PDE-associated psychiatric disorder, such as depression, anxiety, or anxiety and depression) in an individual by determining what PDE protein or transcript isoforms are expressed in an individual comprising (a) determining the PDE protein or transcript isoform or isoforms expressed the individual, comparing the PDE protein or transcript isoform or isoforms expressed in the individual to PDE protein or transcript isoform or isoforms expressed in individuals having the same psychiatric disorder, and determining the presence or absence of PDE protein or transcript isoform or isoforms expressed in individuals having the same psychiatric disorder; and, (b) correlating the presence or absence of the PDE protein or transcript isoform or isoforms expressed in the individuals to the presence or absence of a psychiatric disorder, wherein optionally the psychiatric disorder therapy is a depression and/or anxiety therapy. The results of this method can also be used in the prognosis of a psychiatric disorder or predicting the outcome of therapy for a psychiatric disorder, e.g., a therapy for depression and/or anxiety. Determining PDE protein or transcript isoform or isoforms can be accomplished using any method or protocol, e.g., as described herein, all of which are well known in the art, including, e.g., PCR or other amplification protocols, electrophoresis molecular sizing, antibodies specific for particular alternatively spliced protein motifs, and the like. While the invention is not limited by any particular mechanism of action, pharmacological and genetic studies have indicated that cGMP could be the central mediator of the effects nitric oxide/cGMP in several brain regions (63-65). Cyclic GMP has several target proteins, including cGMP-regulated cation channels, and cGMP-dependent protein kinases (PKs). Two cGMP-PKs genes (type 1 and type 2) that have been described in mammals are widely distributed in the brain (64, 66). cGMP has been implicated in neuronal maturation (67- 69), directional guidance of growth cones (70-72) and learning and memory tasks (73-76).

Arrays

The invention provides an array (biochip) comprising a plurality of nucleic acids each comprising a cyclic nucleotide phosphodiesterase (PDE) haplotype sequence. In one aspect of the array (biochip), the cyclic nucleotide phosphodiesterase (PDE) haplotype sequences comprises PDE9A, PDEl IA, PDE2A, PDE5A, PDE6C, PDElA and/or PDElOA

Nucleic acids (e.g., comprising haplotype sequences) used to practice the invention can be immobilized to or applied to an array. One or more, or, all the haplotype sequences can be measured by hybridization of a sample comprising nucleic acids representative of or complementary to a genome or to transcripts of a cell, by hybridization to immobilized nucleic acids on an array, or "biochip." By using an "array" of nucleic acids on a biochip/ microarray/ microchip, some or all of the genome and/or transcripts of a cell can be simultaneously identified and/or quantified. Alternatively, arrays comprising genomic nucleic acid can also be used to determine the genotype and/or haplotype of an individual. Polypeptide arrays" can also be used to simultaneously quantify a plurality of proteins. The present invention can be practiced with any known "array," also referred to as a "microarray" or "nucleic acid array" or "polypeptide array" or "antibody array" or "biochip," or variation thereof. Arrays are generically a plurality of "spots" or "target elements," each target element comprising a defined amount of one or more biological molecules, e.g., oligonucleotides, immobilized onto a defined area of a substrate surface for specific binding to a sample molecule, e.g., mRNA transcripts, a genomic sample or both.

Multiplexed systems The invention also provides multiplexed systems for determining a subject's responsiveness (or non- responsiveness) to a psychiatric disorder therapy, wherein the subject is diagnosed with depression and/or anxiety, comprising the arrays (biochips) of the invention for determining the presence or absence of cyclic nucleotide phosphodiesterase (PDE) haplotype sequences and computer systems operably linked to the array for analyzing the results of the presence or absence of cyclic nucleotide phosphodiesterase (PDE) haplotype sequences incorporating/ using a method of the invention (e.g., as a computer-implemented method in a computer program product), and outputting that information to a user. Computer readable media that can be used to practice the invention include magnetically readable media, optically readable media, electronically readable media and magnetic/optical media. For example, the computer readable media may be a hard disk, a floppy disk, a magnetic tape, CD-ROM, Digital Versatile Disk (DVD), Random Access Memory (RAM), or Read Only Memory (ROM) as well as other types of other media known to those skilled in the art.

Haplotype sequences can be stored as text in a word processing file, such as Microsoft WORD™ or WORDPERFECT™ or as an ASCII file in a variety of database programs familiar to those of skill in the art, such as DB2, SYBASE, or ORACLE. In addition, many computer programs and databases may be used to practice the methods of the invention; e.g., MacPattern™ (EMBL), DiscoveryBase™ (Molecular Applications Group), GeneMine™ (Molecular Applications Group), Look™ (Molecular Applications Group), MacLook™ (Molecular Applications Group), BLAST™ and BLAST2™ (NCBI), BLASTN™ and BLASTX™ (Altschul et al, J. MoI. Biol. 215: 403, 1990), FASTA™ (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85: 2444, 1988); BioByteMasterFile database, the Genbank database, and the Genseqn database.

The computer/ processor used to practice the methods of the invention can be a conventional general-purpose digital computer, e.g., a personal "workstation" computer, including conventional elements such as microprocessor and data transfer bus. The computer/ processor can further include any form of memory elements, such as dynamic random access memory, flash memory or the like, or mass storage such as magnetic disc optional storage. For example, a conventional personal computer such as those based on an Intel microprocessor and running a Windows operating system can be used. Any hardware or software configuration can be used to practice the methods of the invention. For example, computers based on other well-known microprocessors and running operating system software such as UNIX, Linux, MacOS and others are contemplated. As used herein, the terms "computer," "computer program" and "processor" are used in their broadest general contexts and incorporate all such devices.

The invention will be further described with reference to the following examples; however, it is to be understood that the invention is not limited to such examples. EXAMPLES EXAMPLE 1: Predicting patient responses to antidepressants

The following example describes making and using exemplary compositions of the invention and how to practice the compositions and methods of the invention. The following example also describes studies and data demonstrating that the compositions and methods are effective for predicting the response of a sub-population of patients (e.g., Mexican- Americans) to antidepressants and to aid in monitoring, evaluating and adjusting an on-going regimen of antidepressant drug therapy. In particular, these studies demonstrate that the compositions and methods of the invention can be used to predict and monitor treatment responses in a phenotypic subgroup of depressed patients, a high-anxiety depressed patient subpopulation, by determining the presence, or absence, of homozygosity for various haplotypes of the PDE gene, including a cyclic nucleotide phosphodiesterase (PDE) haplotype PDE9A, PDEIlA, PDE2A, PDE5A, PDE6C, PDElOA and/or PDElA. Methods Study Population. The study population consisted of 284 depressed subjects enrolled in a pharmacogenetic study of antidepressant treatment response to desipramine or fluoxetine. We also studied 331 age- and sex-matched control subjects who were recruited from the same Mexican-American community in Los Angeles, and studied by the same, bilingual, clinical research team at the Center for Pharmacogenomics and Clinical Pharmacology, Jane & Terry Semel Institute for Neuro science and Human Behavior, David Geffen School of Medicine at UCLA (77). Controls were in general good health but were not screened for medical or psychiatric illness. All patients were Mexican-American men and women aged 21-68 years, with a current episode of major depression as diagnosed by DSM-IV (78). In this study, all Mexican-American subjects had at least three grandparents born in Mexico (79). We used diagnostic and ratings instruments that have been fully validated in English and in Spanish, and conducted all assessments in the subjects' primary language.

Inclusion criteria included DSM-IV diagnosis of current, unipolar major depressive episode, with a 21 -Item Hamilton Depression Rating Scale (HAM-D) (80) score of 18 or greater with item number 1 (depressed mood) rated 2 or greater. There was no anxiety threshold for inclusion. Subjects with any primary axis I disorder other than major depressive disorder (e.g., dementia, psychotic illness, bipolar disorder, adjustment disorder), electroconvulsive therapy in the last 6 months or previous lack of response to desipramine or fluoxetine were excluded. As anxiety can be a manifestation of depression, patients who met criteria for depression and also anxiety disorder disorders were not excluded. Exclusion criteria included active medical illnesses that could be etiologically related to the ongoing depressive episode (e.g., untreated hypothyroidism, cardiovascular accident within the past 6 months, uncontrolled hypertension or diabetes), current, active suicidal ideation with a plan and strong intent, pregnancy, lactation, current use of medications with significant central nervous system activity, which interfere with EEG activity (e.g., benzodiazepines) or any other antidepressant treatment within the 2 weeks prior to enrollment, illicit drug use and/or alcohol abuse in the last 3 months or current enrollment in psychotherapy. All patients had an initial comprehensive psychiatric and medical assessment and, if enrolled, had 9 weeks of structured follow-up assessments. The study consists of two phases: a 1-week, single-blind placebo lead-in phase to minimize the impact of placebo responders, followed, if subjects continue to meet the inclusion criteria after phase 1, by random assignment to one of the two treatment groups: fluoxetine 10-40 mg/day or desipramine 50- 200 mg/day, administered in a double-blind manner for 8 weeks, with blind dose escalation based on clinical outcomes. In the depressed group, 230 subjects received treatment in our double-blind clinical trial, of those 122 patients were treated with desipramine (83F, 39M) and 108 were treated with Fluoxetine (7 IF, 37M). 69 patients treated with desipramine (45F, 24M) and 72 treated with fluoxetine (52F, 20M) completed our 8-week treatment with weekly data collection.

Genomic DNA collection. At the initial visit, blood samples were collected into ethylenediaminetetraacetic acid (K₂EDTA) BD VACUTAINER® EDTA tubes (Becton, Dickinson and Co., Franklin Lakes, NJ) and genomic DNA was isolated from those samples using PUREGENE® DNA purification kits (Gentra Systems, Inc., Indianopolis, IN, USA). Antidepressant Treatment Response. Our primary clinical outcome measure within the depressed group receiving antidepressant treatment was the HAM-D. Treatment response was classified into two categories: remission and non-remission status taking in consideration the final (week 8) HAM-D score. Remission was defined as having a final HAM-D score of less than 8. Single-Nucleotide Polymorphism (SNP) Genotyping Methods. Single-nucleotide polymorphisms (SNPs) were selected from 21 of the 25 genes in the PDE family, located across 14 chromosomes. We selected an average of 10 intragenic SNPs per gene from dbSNP (build 121). SNP assays were designed and typed with the GOLDEN GATE™ assay (Illumina, Inc., San Diego, CA) as part of a 1536 multiplex reaction (81). DNAs with poor results (50% GC score below 0.65) were removed as well as loci with a low clustering score (below 0.3). The threshold for retaining individual genotype calls was set to GENECALL™ (Illumina, Inc., San Diego, CA) score of 0.25. Cleaning and Filtering steps:

SNP Quality Control. Our data analysis plan included a series of data cleaning steps followed by a series of filtering steps to identify a list of significantly associated SNPs. Only data generated by SNP assays that were successfully genotyped on at least 80% of samples were included. Data quality was assessed by duplicate DNAs (n=26) across all plates. Genotypes from non-matching duplicates were dropped; they were also dropped if they had one missing data point for either allele.

Hardy-Weinberg Equilibrium (HWE). We used the Hardy- Weinberg equilibrium equation (p²+2pq+q² =1; p is the frequency of the dominant allele and q is the frequency of the recessive allele for a trait controlled by a pair of alleles) to determine the probable genotype frequencies in our study populations. Deviation from Hardy-Weinberg equilibrium was tested separately for control and depressed groups using the ALLELE™ procedure in SAS/GENETICS 9.1.3™ (SAS Institute Inc., Cary, NC) PROC ALLELE™ uses the notation and methods described by Weir (1996) (82). SNPs that were not in Hardy-Weinberg Equilibrium in the control group (p<0.05) and SNPs that were monoallelic in both groups were excluded.

Linkage disequilibrium among SNPs. Pairwise linkage disequilibrium was calculated within each gene for all SNPs that passed the quality control measures using the r² measure. An r cutoff of 80% or higher was used to remove redundant SNPs from the analysis. The Four Gamete rule was used to identify haplotype blocks. This method of haplotype block definition assumes no recombination within a block, but does allow for recombination between blocks (83). Linkage disequilibrium measures were assessed using version HAPLOVIEW 3.2™ (84)

Statistical Analyses

SNP Analyses. Allele, genotype and allelic trend association tests were performed using PROC CASE CONTROL™ in SAS/GENETICS 9.1.3™. PROC CASE CONTROL™ is designed to test for differences in frequency of marker data when random samples are available from populations who are affected and unaffected by disease and is based on case- control tests for biallelic markers described by P. D. Sasieni (85). The following criteria were used to identify a list of SNPs statistically associated with a diagnoses of depression: 1) SNPs were in HWE equilibrium in the control group; 2) The minor allele frequency in the control group was > 5%; 3). Multiple testing was corrected using Bonferroni correction, which set the significance level atp value <0.0006 for tests between control and depressed groups. We tested our secondary hypothesis using similar criteria in order to identify a list of SNPs associated with treatment response: 1) SNPs were in HWE equilibrium in the control group; 2) The minor allele frequency in the control group was > 5%; 3) Thep value < 0.05 for allele test between remitter and non-remitter groups was used, as due to small sample size this part of the analyses are preliminary. Odds ratios. We compared the odds of having depression given the homozygous major, homozygous minor, or heterozygous genotype for SNPs associated with diagnoses of depression. Similarly, odds of attaining remission given the homozygous major, homozygous minor, or heterozygous genotype for SNPs associated with treatment response. Odds ratios were calculated using PROC FREQ™ in SAS/GENETICS 9.1.3™.

Results

Cleaning and Filtering steps

Quality control. The distribution of SNPs and genes across the chromosome after quality control can be seen in Table 1. In summary, 159 SNPs (80%) passed 7/7 plates; 15 (7%) passed 6/7 plates; 2 (1%) passed 5/7 plates, and 24 (12%) passed less than 5 of 7 plates. 92.6% of the quality control duplicates matched; 7.4% did not match. We eliminated 2.3 % of observations because they had missing data on either allele. Most (87.5%) of our original dataset remained after this step of cleaning.

Hardy- Weinberg Equilibrium (HWE). We examined HWE in control and depressed groups separately. We excluded 18 SNPs that were not in Hardy- Weinberg Equilibrium in the control group; we also excluded 5 SNPs that were monoallelic in at least one of the groups. After quality control and HWE steps, 153 SNPs remained for linkage disequilibrium analyses.

Linkage disequilibrium. We assessed linkage disequilibrium (LD) in each gene for control and depressed groups separately. We removed 75 of the 153 SNPs from further analysis because they were in LD with an r² of 80% or greater with other SNPs within a specific gene. Figure 1 illustrates the typical LD and haplotype block structure that were obtained. Data on 78 SNPs (out of the initial 200) were used for further data analyses after our quality control steps. The density of SNPs per PDE family and class were as follows: 10.7 SNPs per family of c AMP- specific PDEs, 6.6 SNPs per family of dual substrate (cAMP and cGMP) PDEs, and 4.3 SNPs per family of cGMP- specific PDE. SNP Association with MDD. Two SNPs (rs729861 in PDE9A and rs3770018 in

PDEIlA) were significant at the Bonferroni corrected significance level of < 0.0006 for the test between control and depressed groups (Table 2).

Seven other SNPs had ap value < 0.05. Those SNPs were located in 4 genes PDE2A (rs376724), PDE5A (rs3775845), PDE6C (rs650058, rs701865), and PDElOA (rs220818, rs676389 and rs717602). The presence of multiple independent signals in PDE6C and PDElOA further strengthens the likelihood of an association with MDD. Table 2 shows genotype frequencies for significant SNPs in the depressed and control groups, and the odds ratio of depression given a certain genotype. An odds ratio of 1.4 indicates that a person with the minor allele is 40% times more likely to be in the depressed group than not. Likewise, an odds ratio of 0.5 indicates that a person is half as likely to be depressed than not. Thus, odds ratio for being depressed was 2.1 (95% CI 1.3 to 3.3) for individuals who are homozygous (AA) for the major allele for rs3770018 in the PDEIlA gene and 0.6 (95% CI 0.4 to 0.8) for individuals who are homozygous (TT) for the major allele for rs729861 in the gene PDE9A gene. SNP Association with Antidepressant Response. Two SNPs in the PDE family had a p value < 0.05 when tested for association with attaining remitters and non-remitter status within the depressed group (table 3). They were located in the PDElA (rsl549870) and PDEIlA (rsl880916) genes. Interestingly, the PDEIlA gene was associated both with drug response and depression, but different SNPs were associated with diagnosis and drug response (see above). Odds ratio for attaining remitter status was 4.6 (95% CI 1.6 to 13.6) for individuals who are homozygous (G/G) for the major allele for rsl880916 in the PDElA gene and 3.2 (95% CI 1.2722 to 8.0092) for individual who are heterozygous (A/G) for rsl880916 in the PDEIlA gene.

Although each group was small, we also analyzed antidepressant response by drug and found that different SNPs and genes were associated with attaining remitter status in fluoxetine and desipramine treatment groups.

Fluoxetine Treatment. Five SNPs located in four genes were associated with remission during fluoxetine treatment (Table 3). SNPs in PDElA (rsl549870), PDE6A (rs2544934), PDE8B (rs884162) and PDEIlA (rsl880916 and rs3770018) had a difference in allele frequency with a p value < 0.05 for remitters and non-remitters within the subjects treated with fluoxetine. Both SNPs associated with remission within the depression group were also associated within the fluoxetine treated subjects. The odds ratio for remission in the fluoxetine treatment for rsl549870 was 8.8 (1.7118 to 45.2382) for major genotype, for rsl880916 was 5.12 (95% CI 1.0602 to 24.738) for heterozygous genotype, and for rs2544934 was 4.4 (95% CI 1.1608 to 17.0161) for heterozygous genotype. These confidence intervals are wide and these results await confirmation in larger samples.

Desipramine Treatment. Two SNPs were associated with remission during desipramine treatment (Table 3). These SNPs (rs30585, rs992185) were located in the PDElC gene. Odds ratio for remission with desipramine treatment for rs30585 was 5.16 (95%CI 1.0258 to 26.0228) for the minor genotype, and for rs992185 was 4.6 (95% CI 1.66 to 12.7) for the heterozygous genotype.

Discussion We found that SNPs in PDE genes are associated with MDD and antidepressant treatment response. PDEs constitute a complex family of enzymes that are essential regulators of intracellular cyclic nucleotide signaling which have a central role in the signal transduction process of neurons. Through a series of rigorous process of data cleaning, filtering steps, and analyses, we have identified 2 SNPs (in PDE9A and -HA genes) that were associated with diagnosis of MDD and 2 other SNPs (in PDElA and -1 IA genes) were associated with treatment response. Most all of the PDEs that we identified as relevant for disease or drug response catalyze cGMP; only one gene (PDE8A) identified in our study is a cAMP-specific PDE gene.

Association with MDD. Two SNPs in the PDE gene family have significantly different allele frequencies between control and depressed groups. Those SNPs were located in PDE9A and -HA genes. PDE9A belongs to the class of cGMP- specific enzymes and PDEIlA catalyzes both cAMP and cGMP. Our data also indicate that 2 other members of the cGMP- specific enzymes (PDE5A and -6C) and 2 other members of dual substrate (cAMP and cGMP) class of PDEs are (PDE2A and -10A) (14, 18, 19) are also likely to be associated to MDD. Interestingly, 5 out of 6 of these PDEs (PDE2A, -5 A, -6C, -1OA, and -1 IA) are classified as GAF (cGMP binding and stimulated phosphodiesterase, Anabaena adenylate cyclases, and Escherichia coli FhIA) -PDEs (40). High amino acid sequence similarity (42-51%) is found in the catalytic region of GAF-PDEs and catalytic domain phylogenic tree analysis of human PDEs demonstrates evolutionary relatedness among GAF-PDE family and suggests that these genes have a common ancestor gene. Furthermore, analyses of genomic structure reveal that PDE5A, PDE6C and PDEIlA are quite similar, which suggest that these genes have a close evolutionary relatedness and a common ancestral gene (41).

Association with Drug Response. Two SNPs (rs30585 in PDElA and rs992185 in PDEIlA) have significantly different allele frequencies between remitters and non-remitters within the depressed group. PDElA and -HA hydrolyze cAMP and cGMP (14, 18, 19); PDEl is calcium/calmodulin dependent. These two SNPs also have significantly different allele frequencies between remitters and non-remitters within the fluoxetine treated group, but not within the desipramine group (Table 5). Different SNPs and genes were significantly associated with remitters and non-remitters in fluoxetine and desipramine treated patients. Three additional SNPs (rs2544934 in PDE6A, rs884162 in PDE8B, and Rs3770018 in PDEIlA) were also significantly associated with drug response in the fluoxetine group. Genes associated with response to fluoxetine are located in two chromosomal regions: 2q31-32 and 5ql4-31. Two SNPs (rs30585 and rs992185) in the PDElC gene were associated with treatment response in the desipramine group.

CNS Localization of Significant Genes. Cyclic GMP hydrolyzing PDE genes we identified as significantly associated with MDD or antidepressant response; namely, PDEl, -9 and -11 are all expressed in the brain but in different locations (42-44). PDE8B, the only cAMP-specific gene that had significant associations to fluoxetine treatment, is also present in the brain (45).

PDElA is highly present in the brain and it is tightly associated to calmodulin and will be therefore permanently activated (46). PDElC mRNA is mainly expressed in the brain and in the heart (47) and it is the major type expressed in the mouse cerebellar granular cells (48). Interestingly, PDElC down-regulates glucose-induced insulin secretion (49).

PDE8 is insensitive to rolipram, milrinone, and IBMX. There are at least 4 isoforms of these gene; PDE8B1 is expressed abundantly in the thyroid (5) and PDE8B3 is the most abundant variant in the brain (45). PDE9 is IBMX-insensitive; it is expressed in the brain (3, 6). The pattern of PDE9A expression in the brain closely resemble that of soluble guanaylyl cyclase, suggesting a functional association in the regulation of cGMP levels that may play an important role in behavioral state regulation and learning (43). It is widely distributed in the rodent brain and it is present in Purkinje cells (50). PDE9 are expressed in the brain during development and predominantly in neuronal cells bodies (43, 51). PDE9 mRNA has the widest distribution in the CNS and could maintain low basal cellular cGMP levels.

PDEI l is present in the pituitary and low protein levels were detected in neurons (52) and brain (53).

The PDEs that we have identified as genes associated with MDD are also expressed in the CNS. PDE2 has high expression levels were found in brain cortex (54, 55), habenula, olfactory cortices, hippocampus, and basal ganglia (56). PDE5A is expressed in the adult and fetal brain (57). PDE2 and -5 are expressed in the brain during development and predominantly in neuronal cells bodies (43, 51). PDE6 is transducin-activated; it is expressed in retina (1) and in the pineal gland (58) and it is a key component of the visual transduction cascade (59). PDElO transcripts are particularly abundant in the brain tissue (putamen and caudate nucleus) (57, 60). PDElOA is specifically expressed in the striatum (1, 8, 41). The PDElO family was recently shown to be associated with the progressive neurodegenerative form of Huntington's disease (HD) (61, 62).

Figure 1 illustrates linkage disequilibrium pattern in gene PDEI lA in Depressed Group. Standard color scheme white: D' <1 and LOD <2, Blue: D' =1 and LOD <2, shades of pink/red: D'<1 and LOD >2, Bright Red: D' =1 and LOD >2. D prime values of 1.0 are never shown (the box is empty). At the top of the figure the PDEIlA gene structure is illustrated schematically by a thick horizontal line. Short vertical lines indicate genotyped SNPs. Long vertical lines indicate exons (20 total).

Table 1. Distribution of genotyped SNPs by PDE families

Family Genes Substrate SNPs

PDEl IA, IB, 1C cAMP/cGMP 11

PDE2 2A cAMP/cGMP 5

PDE3 3A cAMP/cGMP 2

PDE4 4A, 4B, 4C, 4D cAMP 21

PDE5 5A cGMP 1

PDE6 6A, 6C, 6D, cGMP 8

6G

PDE7 7A, 7B cAMP 6

PDE8 8A, 8B cAMP 5

PDE9 9A cGMP 4

PDElO 1OA cAMP/cGMP 10

PDEI l HA cAMP/cGMP 5

Total 21 78 Table 2. Odds Ratio and allele frequency for SNPs significantly (*) associated and likely [(-), p <0.05] to be associated with depression (MDD) when compared to control (CT)

Gene SNP P value PDE class Allele Minor allele frequency

MDD CT

PDEIlA rs3770018(*) 0.0005 cAMP/cGMP A^C 0.058 0.11

PDE9A rs729861(*) 0.0006 cAMP/cGMP T^C 0.39 0.29

PDE5A rs3775845(~) 0.007 cGMP A^G 0.33 0.26

PDElOA rs717602(~) 0.009 cAMP/cGMP A^G 0.46 0.38

PDE2A rs370013(~) 0.01 cGMP A^G 0.50 0.43

PDE6C rs650058(~) 0.01 cGMP C^T 0.41 0.48

PDElOA rs220818(~) 0.01 cAMP/cGMP T^C 0.29 0.23

PDElOA rs676389(~) 0.03 cGMP T^C 0.24 0.23

PDE6C rs701865(~) 0.03 cAMP/cGMP T^A 0.46 0.40

Table 3. Allele frequency table between Remitter (R) and Non-Remitter (NR) Groups for SNPs significantly associated to drug response atp<0.05

Overall Gene SNP P value Allele Minor allele frequency

R (n=82) NR (n=61)

PDElA rs 1549870 0.005 G^A 0.03 0.12

PDEIlA rsl880916 0.04 G^A 0.16 0.074

Fluoxetine Gene SNP P value Allele Minor allele frequency

R (n=46) NR (n=28)

PDElA rs 1549870 0.007 G^A 0.022 0.14

PDE8B rs884162 0.02 C^T 0.09 0.0

PDE6A rs2544934 0.03 A^T 0.17 0.054

PDEIlA rsl880916 0.03 G^A 0.16 0.036

PDEIlA rs3770018 0.04 A^T 0.076 0.0

Desipramine Gene SNP P value Allele Minor allele frequency

R (n=36) NR (n=33)

PDElC rs992185 0.006 A^C 0.47 0.24

PDElC rs30585 0.02 T^G 0.47 0.26

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Claims

WHAT IS CLAIMED IS:

1. A method for determining a subject's responsiveness (or non- responsiveness) to a psychiatric disorder therapy, wherein the subject is diagnosed with depression and/or anxiety, the method comprising the steps of: (a) providing a nucleic acid-comprising sample from the subject;

(b) analyzing the sample and detecting whether the subject is homozygous for a cyclic nucleotide phosphodiesterase (PDE) haplotype, wherein the presence of homozygosity for the genotype correlates with responsiveness (or non- responsiveness) to the depression and/or anxiety therapy.

2. A method for screening a subject to determine the subject's responsiveness to a psychiatric disorder therapy comprising use of an agent which prevents or treats a psychiatric disorder associated with the cyclic nucleotide phosphodiesterase (PDE) gene, comprising:

(a) providing a nucleic acid-comprising sample from the subject;

(b) detecting whether the subject is homozygous for a cyclic nucleotide phosphodiesterase (PDE) haplotype, wherein the presence of homozygosity for the cyclic nucleotide phosphodiesterase (PDE) genotype correlates with a level of responsiveness (or non- responsiveness) to the therapy.

3. The method of claim 2, wherein the subject is diagnosed as having a psychiatric disorder comprising depression and anxiety, or depression, or anxiety.

4. The method of claim 1 or claim 2, wherein the psychiatric disorder comprises major depression disorder (MDD).

5. The method of claim 1 or claim 2, wherein the psychiatric disorder therapy comprises treatment with an antidepressant agent.

6. The method of claim 5, wherein the antidepressant agent is selected from the group consisting of tricyclic antidepressants, selective serotonin reuptake inhibitors and cyclic nucleotide phosphodiesterase (PDE) antagonists.

7. The method of claim 1 or claim 2, wherein the cyclic nucleotide phosphodiesterase (PDE) haplotype comprises PDE9A, PDEIlA, PDE2A, PDE5A, PDE6C, PDElOA and/or PDElA.

8. The method of claim 7, wherein the cyclic nucleotide phosphodiesterase (PDE) haplotype comprises PDE9A or PDEl IA.

9. The method of claim 8, wherein the cyclic nucleotide phosphodiesterase (PDE) haplotype comprises PDEIlA.

10. The method of claim 1 or claim 2, wherein the subject's responsiveness (or non- responsiveness) to a psychiatric disorder therapy comprises remission while on an antidepressant or a psychiatric disorder therapy.

11. The method of claim 10, wherein the cyclic nucleotide phosphodiesterase (PDE) haplotype comprises PDElA or PDEIlA.

12. The method of claim 1, wherein the nucleic acid-comprising sample comprises a blood sample, a saliva sample or a cell sample.

13. The method of claim 12, wherein the cell sample comprises a cell from a biopsy, a buccal or a skin scraping or sample.

14. The method of claim 1 or claim 2, wherein the subject is selected from a subpopulation of patients.

15. The method of claim 14, wherein the subpopulation of patients comprises a Mexican-American subpopulation.

16. The method of claim 1 or claim 2, wherein the genotype of the subject with respect to the cyclic nucleotide phosphodiesterase (PDE) haplotypes, is determined by amplification genotyping, in situ hybridization techniques, DNA array (biochip) analysis and/or direct DNA sequencing.

17. The method of claim 16, wherein the amplification genotyping comprises polymerase chain reaction (PCR).

18. A kit suitable for determining a subject's responsiveness (or non- responsiveness) to a psychiatric disorder therapy, wherein the subject is diagnosed with depression and/or anxiety, said kit comprising (a) material for determining whether the subject is homozygous for a cyclic nucleotide phosphodiesterase (PDE) haplotype; (b) suitable packaging material; and optionally (c) instructional material for use of said kit.

19. The kit of claim 18, wherein the material comprises at least one nucleic acid that specifically binds to a cyclic nucleotide phosphodiesterase (PDE) haplotype.

20. The kit of claim 18, wherein the cyclic nucleotide phosphodiesterase (PDE) haplotype comprises PDE9A, PDEIlA, PDE2A, PDE5A, PDE6C, PDElA and/or PDElOA.

21. The kit of claim 18, wherein the nucleic acid that specifically binds to the htSNPs comprises at least one polymerase chain reaction (PCR) primer or a hybridization probe.

22. The kit of claim 18, further comprises material to process a nucleic acid- comprising biological sample.

23. A method for determining a nucleotide polymorphism associated with a specific response (or non-responsive) to a psychiatric disorder therapy in an individual comprising

(a) sequencing all or a portion of the nucleotide sequence of chromosome comprising at least one haplotype PDE9A, PDEl IA, PDE2A, PDE5A, PDE6C, PDElA or PDElOA from the individual having psychiatric disorder therapy, comparing the chromosome sequence from the individual to chromosome sequences of other individuals having the same psychiatric disorder and the same psychiatric disorder therapy, and determining the presence of a nucleotide polymorphism in the sequenced haplotype sequences; and, (b) correlating the specific response of the individual to the psychiatric disorder therapy with the presence or absence of the nucleotide polymorphism in the other individuals, wherein optionally the psychiatric disorder therapy is a drug therapy, or depression and anxiety.

24. A method for determining a nucleotide polymorphism associated with the presence (or absence) of a psychiatric disorder in an individual comprising

(a) sequencing all or a portion of the nucleotide sequence of chromosome comprising at least one haplotype PDE9A, PDEIlA, PDE2A, PDE5A, PDE6C, PDElA or PDElOA from the individual, comparing the chromosome sequence from the individual to chromosome sequences in individuals having the same psychiatric disorder, and determining the presence or absence of a nucleotide polymorphism in individuals having the same psychiatric disorder; and,

(b) correlating the diagnosis of the psychiatric disorder to the presence or absence of the nucleotide polymorphism, wherein optionally the psychiatric disorder therapy is a depression, or depression and anxiety.

25. A method for diagnosing the presence of a psychiatric disorder in an individual by determining a nucleotide polymorphism in an individual comprising

(a) sequencing all or a portion of the nucleotide sequence of chromosome 2q31-32 and/or 5ql4-31 from the individual, comparing the 2q31-32 and/or 5ql4-31 sequence from the individual to chromosome 2q31-32 and/or 5ql4-31 sequences in individuals having the same psychiatric disorder, and determining the presence or absence of a nucleotide polymorphism in individuals having the same psychiatric disorder; and,

(b) correlating the presence or absence of the nucleotide polymorphism to the presence or absence of a psychiatric disorder, wherein optionally the psychiatric disorder therapy is a depression, or depression and anxiety.

26. A method for determining a cyclic nucleotide phosphodiesterase (PDE) protein or transcript isoform associated with a specific response (or non-response) to a psychiatric disorder therapy in an individual comprising (a) determining the cyclic nucleotide phosphodiesterase (PDE) protein or transcript isoform or isoforms expressed in the individual, comparing the cyclic nucleotide phosphodiesterase (PDE) protein or transcript isoform or isoforms expressed in the individual to cyclic nucleotide phosphodiesterase (PDE) protein or transcript isoform or isoforms expressed in individuals having the same psychiatric disorder therapy; and, (b) correlating the response (or non-response) of the individual to the psychiatric disorder therapy with the presence or absence of the expressed cyclic nucleotide phosphodiesterase (PDE) protein or transcript isoform or isoforms, wherein optionally the psychiatric disorder therapy is a drug therapy.

27. A method for determining a cyclic nucleotide phosphodiesterase (PDE) protein or transcript isoform associated with the presence (or absence) of a psychiatric disorder in an individual comprising

(a) determining the cyclic nucleotide phosphodiesterase (PDE) protein or transcript isoform or isoforms expressed the individual, comparing the PDE protein or transcript isoform or isoforms expressed in the individual to PDE protein or transcript isoform or isoforms expressed in individuals having the same psychiatric disorder, and determining the presence or absence of PDE protein or transcript isoform or isoforms in individuals having the same psychiatric disorder; and,

(b) correlating the diagnosis of the psychiatric disorder to the presence or absence of the PDE protein or transcript isoform or isoforms expressed in the individuals, wherein optionally the psychiatric disorder therapy is a depression.

28. A method for diagnosing the presence of a psychiatric disorder in an individual by determining what cyclic nucleotide phosphodiesterase (PDE) protein or transcript isoforms are expressed in an individual comprising

(a) determining the PDE protein or transcript isoform or isoforms expressed the individual, comparing the PDE protein or transcript isoform or isoforms expressed in the individual to PDE protein or transcript isoform or isoforms expressed in individuals having the same psychiatric disorder, and determining the presence or absence of PDE protein or transcript isoform or isoforms expressed in individuals having the same psychiatric disorder; and,

(b) correlating the presence or absence of the PDE protein or transcript isoform or isoforms expressed in the individuals to the presence or absence of a psychiatric disorder, wherein optionally the psychiatric disorder therapy is a depression.

29. An array (biochip) comprising a plurality of nucleic acids each comprising a cyclic nucleotide phosphodiesterase (PDE) haplotype sequence.

30. The array (biochip) claim 29, wherein the cyclic nucleotide phosphodiesterase (PDE) haplotype sequences comprises PDE9A, PDEIlA, PDE2A, PDE5A, PDE6C, PDElA and/or PDElOA.

31. The array (biochip) claim 30, wherein the cyclic nucleotide phosphodiesterase (PDE) haplotype sequences comprises PDE9A, PDEl IA, PDE2A, PDE5A, PDE6C, PDElA and PDElOA.

32. A multiplexed system for determining a subject's responsiveness (or non- responsiveness) to a psychiatric disorder therapy, wherein the subject is diagnosed with depression and/or anxiety, comprising: (a) the array of any of claims 29 to 31, for determining the presence or absence of cyclic nucleotide phosphodiesterase (PDE) haplotype sequences; and

(b) a computer system operably linked to the array for analyzing the results of the presence or absence of cyclic nucleotide phosphodiesterase (PDE) haplotype sequences as set forth in claim 1, and outputting that information to a user.