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WO2003101377A2 - Association des polymorphismes mononucleotidiques dans le locus dgcr2 et les locus voisins avec la schizophrenie - Google Patents

Association des polymorphismes mononucleotidiques dans le locus dgcr2 et les locus voisins avec la schizophrenie Download PDF

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Publication number
WO2003101377A2
WO2003101377A2 PCT/IL2003/000464 IL0300464W WO03101377A2 WO 2003101377 A2 WO2003101377 A2 WO 2003101377A2 IL 0300464 W IL0300464 W IL 0300464W WO 03101377 A2 WO03101377 A2 WO 03101377A2
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snp
kit
dgcr2
seq
allele
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PCT/IL2003/000464
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WO2003101377A3 (fr
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Ariel Darvasi
Naomi Zak
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Idgene Pharmaceuticals Ltd.
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Priority to AU2003231897A priority Critical patent/AU2003231897A1/en
Publication of WO2003101377A2 publication Critical patent/WO2003101377A2/fr
Publication of WO2003101377A3 publication Critical patent/WO2003101377A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to (i) the use of genetic markers, such as single nucleotide polymorphisms (SNPs), in the DCGR2 locus and neighboring loci for the identification of genes related to and for determining a risk of developing schizophrenia, a complex (e.g., multigenic) disease; (ii) methods of identifying novel drugs for the treatment and/or prevention of schizophrenia; (iii) methods of treating and/or preventing schizophrenia; and (iv) pharmacogenomic methods of determining drug responsiveness in mental disorders, schizophrenia in particular.
  • SNPs single nucleotide polymorphisms
  • Common diseases e.g. certain mental disorders, various cancers, heart diseases and diabetes
  • other traits of medical relevance e.g. individual drug responsiveness
  • the genetic component of common diseases is further demonstrated by adoption and twin studies, which, for example, implicate genetic factors as major predisposing factors for most psychiatric diseases [Burmeister, M., (1999). Basic concepts in the study of diseases with complex genetics. Biol. Psychiatry 45: 522-532].
  • these diseases do not follow simple Mendelian inheritance, and their transmission pattern is best explained by models involving several genes, as well as non-genetic (environmental) factors.
  • Schizophrenia (MIM 181500) is a common psychiatric disease that affects about 1% of the world population. It accounts for about 25% of all mental health costs and schizophrenic patients make up 1/3 of all psychiatric hospital beds [Kaplan, H. R. and Sadock, B. J. (1991). Synopsis of psychiatry: behavioral sciences, clinical psychiatry, Library of Congress Cataloging-in Publication Data]. Schizophrenia is used to describe a cluster of symptoms that typically include "positive" symptoms such as delusions, hallucinations and disordered thinking and "negative” symptoms such as social withdrawal, apathy, and emotional unresponsiveness.
  • This psychiatric disease is considered one of the most devastating of mental illnesses; disease onset is early in a patient's life and its symptoms can be destructive to both patients and their immediate environment. Since genetic and molecular factors have not been identified, schizophrenia is diagnosed by phenotypic symptoms only.
  • Schizophrenia like most other psychiatric disorders, is a complex disease that cannot be explained by a single genetic or environmental factor [McGuffin, P., Owen, M. J. and Farmer, A. E. (1995). Genetic basis of schizophrenia. Lancet 346: 678-82]. Although the genes increasing risk for schzophrenia have not been identified, there is strong evidence that schizophrenia has a significant genetic component. While the prevalence of schizophrenia in the general population is around 1%, it increases to 8- 12% in the descendants and siblings of affected individuals, and up to 40% when both parents suffer from schizophrenia [Kaplan, H. R. and Sadock, B. J. (1991).
  • Antipsychotic (neuroleptic) medication is the most important part of the treatment regimen for schizophrenic patients. Treatment with antipsychotics has been demonstrated to reduce rates of relapse and re-hospitalizations in a substantial number of patients over many years. [Moller HJ. (2000) State of the art of drug treatment of schizophrenia and the future position of the novel/atypical antipsychotics. World J Biol Psychiatry 1: 204-14; Bridler R, Umbricht D. (2003) Atypical antipsychotics in the treatment of schizophrenia. Swiss Med Wkly 133: 63-76]. Effective medications for schizophrenia have been available since the 1950s, and have revolutionized the treatment of schizophrenic patients by reducing and preventing recurrence of symptoms [Campbell M.
  • clozapine the first "atypical" drug, was introduced. This drug showed excellent antispychotic efficacy in the absence of EPS.
  • propensity of clozapine to cause agranulocytosis led to its withdrawal in many countries and to restrictions on its use in others [Bridler R, Umbricht D. (2003) Atypical antipsychotics in the treatment of schizophrenia. Swiss Med Wkly 133: 63-76].
  • the high efficacy of clozapine led to the development of clozapine-like compounds without its negative side effects. These are risperidone, olanzapine, quetiapine and amisulpride.
  • Atypical antipsychotics are at least as effective as conventional antipsychotics in the treatment of positive symptoms, and differ in their ability to ameliorate negative symptoms.
  • Most of these new-generation drugs demonstrate a high affinity for serotonin 5HT2 receptors and weak affinity for dopamine D2 receptors, resulting in less EPS than typical neuroleptics.
  • atypical medications cause less tardive dyskinesia than do typical antipsychotics, and most do not cause increased prolactin secretion.
  • other side effects such as weight gain, and abnormal glucose regulation, are associated with many of the newer medications. [Bridler R, Umbricht D. (2003) Atypical antipsychotics in the treatment of schizophrenia. Swiss Med Wkly 133: 63-76].
  • identification of disease associated genes will allow the development of new methods for detecting susceptibility to schizophrenia, as well as for preventing the development of the disease.
  • Early identification of subjects at risk of developing schizophrenia would enable administration of early prophylactic, (pharmacological and environmental) treatment.
  • Knowledge of genetic variations in causative genes will enable the development of pharmacogenomics, in which individualized treatment is genotype-specific.
  • NCFS Velocardiofacial syndrome
  • MLM 192430 Velocardiofacial syndrome
  • the del22qll syndrome is the most common microdeletion syndrome, occurring in 1 out of every 4,000 live births.
  • the symptoms of del22qll are many and diverse and they include cardiovascular, thymic, parathyroid and craniofacial abnormalities.
  • behavioral defects such as deficits of learning and memory, speech and language development, attention-deficit disorder, and psychiatric diseases, especially schizophrenia.
  • Some studies with NCFS patients have found that 20-30% of them developed schizophrenia [Murphy, K. C, et al., (1999).
  • Mouse models provide additional data that supports linkage of the del22qll syndrome critical region with schizophrenia and may further define the chromosomal area involved [Lindsay, E. A. (2001). Chromosomal microdeletions: dissecting del22qll syndrome. Nat Rev Genet 2: 858-68; Paylor, R., et al. (2001). Mice deleted for the DiGeorge/velocardiofacial syndrome region show abnormal sensorimotor gating and learning and memory impairments. Hum Mol Genet 10: 2645-50]. The gene content of the human 22ql l deletion region is highly conserved and syntenous to a region of mouse chromosome 16.
  • a mouse deletion model, Dfl has been generated with a 1 Mb deletion that encompasses about half of the approximately 30 genes which are removed in the majority of del22qll syndrome patients (with the ⁇ 3 Mb deletion).
  • Mice heterozygously deleted for the ⁇ 1 Mb interval have sensorimotor gating, learning and memory impairments.
  • the deficit in senorimotor gating is manifested as a decrease in pre-pulse inhibition of the acoustic startle response. This is of particular significance, since patients with schizophrenia show similar deficits [Paylor, R., et al.. (2001).
  • Mice deleted for the DiGeorge/velocardiofacial syndrome region show abnormal sensorimotor gating and learning and memory impai ⁇ nents.
  • DGCR2 DiGeorge syndrome critical region gene 2
  • This gene encodes a putative adhesion receptor protein which could play a role in neural crest cells migration, a process which has been proposed to be altered in DiGeorge syndrome.
  • the mouse ortholog of DGCR2 (Dgcr2; previously sezl2) is a seizure-related gene, expression of which is transiently down-regulated in the mouse brain by injection of pentylenetetrazol (a seizure-inducer) [Kajiwara K. et al., (1996) Cloning of SEZ-12 encoding seizure-related and membrane-bound adhesion protein. Biochem Biophys Res Commun 222 : 144-8] .
  • Apolipoprotein E in Alzheimer's disease [Hutton M, Perez-Tur J, Hardy J. (1998) Genetics of Alzheimer's disease. Essays Biochem 33:117-131] and human leukocyte antigen (HLA) in type I diabetes [Concannon P. et al., (1998) A second- generation screen of the human genome for susceptibility to insulin-dependent diabetes mellitus. Nat Genet 19:292-6]. h the above cases, despite the polygenic nature of the disease, the detected genes account for a large proportion of the disease risk.
  • Apolipoprotein E Apolipoprotein E
  • HLA human leukocyte antigen
  • association studies may be family-based, such as in the transmission disequililbrium test (TDT), in which trios of affected individual and their two parents are studied [Shman RS, McGinnis RE, and Ewens WJ. (1993) Transmission test for linkage disequilibrium: the insulin gene region and insulin-dependent diabetes mellitus (IDDM). Am J Hum Genet 52: 506-16].
  • TTT transmission disequililbrium test
  • association studies may be population based, as in case-control studies, which compare unrelated, affected individuals and unrelated, unaffected controls.
  • association studies offer greater statistical power than linkage analysis for uncovering such genes [Risch N, Merikangas K. (1996) The future of genetic studies of complex human diseases. Science 273: 1516-7].
  • linkage analysis for uncovering such genes
  • the greater ease of matching cases to unrelated controls without the necessity of enrolling additional family members makes the case-control design simpler and more economically feasable than family-based approaches.
  • population-based association studies have a greater potential than family-based studies for accurate mapping and identification of the actual disease- linked gene confined within a narrow chromosomal region.
  • a method of determining predisposition of a subject to develop schizophrenia comprises determining a presence in a homozygous or heterozygous form, or an absence, of at least one genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, wherein the at least one genotype is associated with schizophrenia, thereby determining the predisposition of the subject to developing schizophrenia.
  • kits for determining a predisposition of a subject to develop schizophrenia comprises a packaging material packaging at least one reagent, the at least one reagent being for determining a presence in a homozygous or heterozygous form, or an absence, of at least one genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, wherein the genotype is associated with schizophrenia.
  • the kit further comprises a notification in or on the packaging material identifying the kit for use in determining a predisposition of developing schizophrenia.
  • the notification also provides for instructions of using the kit in determining a predisposition of developing schizophrenia.
  • a method of assisting in diagnosing schizophrenia in a subject in need thereof comprises determining a presence in a homozygous or heterozygous form, or an absence, of at least one genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, wherein the at least one genotype is associated with schizophrenia, thereby assisting in diagnosing schizophrenia in the subject.
  • kits for assisting in diagnosing schizophrenia in a subject in need thereof comprises a packaging material packaging at least one reagent, the at least one reagent for determining a presence in a homozygous or heterozygous form, or an absence, of a genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, twherein the genotype is associated with schizophrenia.
  • the kit further comprises a notification in or on the packaging material identifying the kit for use in assisting in diagnosing schizophrenia in a subject.
  • the notification also provides for instructions of using the kit in assisting in diagnosing schizophrenia of a subject.
  • a method of determining a predisposition to develop schizophrenia in a male subject comprises determining a presence in a homozygous or heterozygous form, or an absence, of at least one genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, the wherein the at least one genotype is associated with schizophrenia in males in higher association than in females, thereby determining the predisposition of the male subject of developing schizophrenia.
  • kits for determining a predisposition to develop schizophrenia in a male subject comprises a packaging material packaging at least one reagent, the at least one reagent for determining a presence in a homozygous or heterozygous form, or an absence, of a genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, twherein the genotype is associated with schizophrenia in males in higher association than in females.
  • the kit further comprises a notification in or on the packaging material, the notification identifying the kit for use in determining a predisposition of a male subject of developing schizophrenia.
  • the notification also provides for instructions of using the kit in determining the predisposition of a male subject of developing schizophrenia.
  • method of assisting in diagnosing schizophrenia in a male subject in need thereof there is provided.
  • the method according to this aspect of the present invention comprises determining a presence in a homozygous or heterozygous form, or an absence, of at least one genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, the wherein the at least one genotype is associated with schizophrenia in males in higher association than in females, thereby assisting in diagnosing schizophrenia in -the male subject.
  • kit for assisting in diagnosing schizophrenia in a male subject in need thereof is provided.
  • the kit comprises a packaging material packaging at least one reagent, the at least one reagent for determining a presence in a homozygous or heterozygous form, or an absence, of a genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, twherein the genotype is associated with schizophrenia in males in higher association than in females.
  • the kit further comprises a notification in or on the packaging material, the notification identifying the kit for use in assisting in diagnosing schizophrenia in a male subject.
  • the notification also provides for instructions of using the kit in assisting in diagnosing schizophrenia of a male subject.
  • a method of predicting drug responsiveness of a subject having schizophrenia to a schizophrenia drug comprises determining a presence in a homozygous or heterozygous form, or an absence, of at least one genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, the wherein the at least one genotype is associated with drug responsiveness to the drug, thereby predicting drug responsiveness of the subject to the drug.
  • a kit for predicting drug responsiveness of a subject having schizophrenia to a schizophrenia drug comprises determining a presence in a homozygous or heterozygous form, or an absence, of at least one genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, the wherein the at least one genotype is associated with drug responsiveness to the drug, thereby predicting drug responsiveness of the subject to the drug.
  • the kit comprises a packaging material packaging at least one reagent, the at least one reagent for determining a presence in a homozygous or heterozygous form, or an absence, of a genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, twherein the genotype is associated with schizophrenia.
  • the kit further comprises a notification in or on the packaging material, the notification identifying the kit for use in predicting drug responsiveness of a subject having schizophrenia to the drug.
  • the notification also provides for instructions of using the kit in assisting in predicting drug responsiveness of a subject having schizophrenia to the drug.
  • a method of predicting drug responsiveness of a subject having a given mental disorder to a mental disorder drug comprises determining a presence in a homozygous or heterozygous form, or an absence, of at least one genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, the wherein the at least one genotype is associated with drug responsiveness to the drug in at least one mental disorder, thereby predicting drug responsiveness of the subject having the given mental disorder to the drug.
  • kits for predicting drug responsiveness of a subject having a given mental disorder to a mental disorder drug comprises a packaging material packaging at least one reagent, the at least one reagent for determining a presence in a homozygous or heterozygous form, or an absence, of a genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, twherein the genotype is associated with drug responsiveness to the drug in at least one mental disorder.
  • the kit further comprises a notification in or on the packaging material, the notification identifying the kit for use in predicting drug responsiveness of a subject having the given mental disorder to the drug.
  • the notification also provides for instructions of using the kit in assisting in predicting drug responsiveness of a subject having the given mental disorder to the drug.
  • a method of identifying a genetic association with, or a genetic cause to, varying drug responsiveness to a schizophrenia drug comprises determining via a population association study an association between a presence in a homozygous or heterozygous form, or an absence, of at least one genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, and responsiveness or non-responsiveness to the schizophrenia drug, thereby identifying a genetic association with, or a genetic cause to, the varying drug responsiveness to the schizophrenia drug.
  • kits for identifying a genetic association with, or a genetic cause to, varying drug responsiveness to a schizophrenia drug comprises a packaging material packaging at least one reagent, the at least one reagent for determining a presence in a homozygous or heterozygous form, or an absence, of a genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, twherein the genotype is associated with schizophrenia.
  • the kit further comprises a notification in or on the packaging material, the notification identifying the kit for use in identifying a genetic association with, or a genetic cause to, varying drug responsiveness to the drug.
  • the notification also provides for instructions of using the kit in identifying a genetic association with, or a genetic cause to, varying drug responsiveness to the drug.
  • a method of identifying a genetic association with, or a genetic cause to, varying drug responsiveness to a mental disorder drug comprises determining via a population association study an association between a presence in a homozygous or heterozygous form, or an absence, of at least one genotype in the DGCR2 locus or in neighboring loci, the neighboring loci in linkage disequilibrium with the DGCR2 locus, and responsiveness or non- responsiveness to the mental disorder drug, thereby identifying a genetic association with, or a genetic cause to, the varying drug responsiveness to the mental disorder drug.
  • kits for identifying a genetic association with, or a genetic cause to, varying drug responsiveness of a subject having a given mental disorder to a mental disorder drug comprises a packaging material packaging at least one reagent, the at least one reagent for determining a presence in a homozygous or heterozygous form, or an absence, of a genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, twherein the genotype is associated with drug responsiveness to the drug in at least one mental disorder.
  • the kit further comprises a notification in or on the packaging material, the notification identifying the kit for use in identifying a genetic association with, or a genetic cause to, varying drug responsiveness of a subject having the given mental disorder to the drug.
  • the notification also provides for instructions of using the kit in identifying a genetic association with, or a genetic cause to, varying drug responsiveness of a subject having the given mental disorder to the drug.
  • the schizophrenia drug is selected from the group consisting of zuclopenthixol and olanzapine.
  • the mental disorder drug is selected from the group consisting thioridazine, clozapine, haloperidol, fluphenazine, cholrpromazine, risperidone, levomepromazine, perhenazine, chlorprothixene, pimozide, sulpiride, olanzapine, zuclopenthixol, amitriptyline, imipramine, clomipramine, desipramine, doxepin, mianserin, maprotiline, phenelzine, fluoxetine, trazodone, fluvoxamine, sertraline, paroxetine, reboxetine, citalopram, nefazodone, venlafoxine, lithium salts, carbamazapine, valproic acid, and clonazepam.
  • the mental disorder is selected from the group consisting of schizophrenia, schizoaffective disorder, bipolar disorder, depression, obsessive compulsive disorder, panic disorder, agoraphobia, specific phobia, social phobia, post-traumatic stress disorder, pain disorder, anxiety, somatization disorder, anorexia nervosa, bulimia, and nervosa.
  • At least one genotype in the DGCR2 locus is a guanine nucleotide — and/or an adenosine nucleotide - containing allele of SNP rs807759 set forth in SEQ ID NO: 13.
  • At least one genotype in the DGCR2 locus is a cytosine nucleotide — and/or a thymine nucleotide - containing allele of SNP rs2072123 set forth in SEQ ID NO:l.
  • at least one genotype in the DGCR2 locus is an adenosine nucleotide — and/or a guanine nucleotide - containing allele of SNP rs2073776 set forth in SEQ ID NO:5.
  • At least one genotype in the DGCR2 locus is an allelic haplotype comprising at least two of: a guanine nucleotide - and/or an adenosine nucleotide - containing allele of SNP rs807759 set forth in SEQ ID NO: 13; a cytosine nucleotide - and/or a thymine nucleotide - containing allele of SNP rs2072123 set forth in SEQ ID NO:l; and an adenosine nucleotide - and/or a guanine nucleotide - containing allele of SNP rs2073776 set forth in SEQ ID NO:5.
  • At least one genotype in the DGCR2 locus is an allelic haplotype including at least one SNP selected from the group consisting of: a guanine nucleotide - and/or an adenosine nucleotide - containing allele of SNP rs807759 set forth in SEQ ID NO: 13; a cytosine nucleotide - and/or a thymine nucleotide - containing allele of SNP rs2072123 set forth in SEQ ID NO:l; an adenosine nucleotide — and/or a guanine nucleotide - containing allele of SNP rs2073776 set forth in SEQ ID NO:5; and/or one of the following SNPs in the COMT locus: a cytosine nucleotide - and/or a thymine nucleotide - containing allele of SNP rs807759 set forth in
  • the at least one oligonucleotide has a sequence selected capable of differentially hybridizing with at least one allele of an SNP selected from the group consisting of rs807759, rs2072123 and rs2073776, wherein the at least one oligonucleotide can differentiate between polymorphs of the SNP via differential hybridization.
  • At least one oligonucleotide has a sequence selected capable of differentially hybridizing with at least one allele of an SNP in linkage disequilibrium with rs807759, rs2072123 or rs2073776, wherein the at least one oligonucleotide can differentiate between polymorphs of the SNP via differential hybridization.
  • At least one oligonucleotide has a sequence selected capable of hybridizing to a sequence region adjacent to an SNP selected from the group consisting of rs807759, rs2072123 and rs2073776, wherein the at least one oligonucleotide can differentiate between polymorphs of the SNP via a differential template dependent primer extension reaction or a differential template dependent ligation reaction.
  • At least one oligonucleotide has a sequence selected capable of hybridizing to a sequence region adjacent to an SNP in linkage disequilibrium with rs807759, rs2072123 or rs2073776, wherein the at least one oligonucleotide can differentiate between polymo hs of the SNP via a differential template dependent primer extension reaction or a differential template dependent ligation reaction.
  • At least one oligonucleotide has a sequence selected capable of hybridizing to a sequence region adjacent to an SNP selected from the group consisting of rs807759, rs2072123 and rs2073776, wherein the the at least one oligonucleotide can be used to amplify polymorphs of the SNP via an amplification reaction.
  • At least one oligonucleotide has a sequence selected capable of hybridizing to a sequence region adjacent to an SNP in linkage disequilibrium with rs807759, rs2072123 or rs2073776, wherein the at least one oligonucleotide can be used to amplify polymorphs of the SNP via an amplification reaction.
  • At least one antibody is differentially interactable with at least one form of a DGCR2 protein encoded by a DGCR2 allele having an SNP rs2072123, wherein the the at least one antibody can differentiate between polymo ⁇ hs of the DGCR2 protein via differential antibody interaction.
  • At least one antibody is differentially interactable with at least one form of a DGCR2 protein encoded by a DGCR2 allele having a non-synonymous SNP in linkage disequilibrium with rs807759, rs2072123 or rs2073776, wherein the the at least one antibody can differentiate between polymo ⁇ hs of the DGCR2 protein via differential antibody interaction.
  • the DNA chip comprises attached thereto at least one oligonucleotide having a sequence selected capable of differentially hybridizing with at least one allele of an SNP selected from the group consisting of rs807759, rs2072123 and rs2073776, wherein the at least one oligonucleotide can differentiate between polymo ⁇ hs of the SNP via differential hybridization.
  • the DNA chip comprises attached thereto at least one oligonucleotide having a sequence selected capable of differentially hybridizing with at least one allele of an SNP in linkage disequilibrium with rs807759, rs2072123 or rs2073776, wherein the at least one oligonucleotide can differentiate between polymo ⁇ hs of the SNP via differential hybridization.
  • At least one reagent is designed for use in a detection method selected from the group consisting of a signal amplification method, a direct detection method and detection of at least one sequence change.
  • the signal amplification method amplifies a molecule selected from the group consisting of a DNA molecule and an RNA molecule.
  • the signal amplification method is selected from the group consisting of PCR, LCR (LAR), Self-Sustained Synthetic Reaction (3SR NASBA) and Q-Beta (Q ⁇ ) Replicase reaction.
  • the direct detection method is selected from the group consisting of a cycling probe reaction (CPR) and a branched DNA analysis.
  • CPR cycling probe reaction
  • detection of at least one sequence change is effected by a method selected from the group consisting of the Allele Refractory Mutation System (ARMS), restriction fragment length polymo ⁇ hism (RFLP analysis), primer extension, mini-sequencing, PyrosequencingTM, Competitive Oligonucleotide Single Base Extension (COSBE), Dynamic Allele Specific Hybridization (DASH), heteroduplex/homoduplex resolution by Denaturing High Performance Liquid Chromatography (DHPLC), heteroduplex/homoduplex resolution by enzymatic cleavage, capillary electrophoresis - Single Strand Conformation Polymo ⁇ hism (CE- SSCP), Conformation Sensitive Gel Electrophoresis (CSGE), Allele specific ligation (OLA), Matrix-Assisted Laser Deso ⁇ tion-Ionization (MALDI) time-of-flight (TOF) Mass Spectrometry (MS), Fluorescence Resonance Energy Transfer (FRET), Fluor
  • the immunological detection method is selected from the group consisting of a radio- immunoassay (RIA), an enzyme linked immunosorbent assay (ELISA), a western blot, an immunohistochemical analysis, and fluorescence activated cell sorting (FACS).
  • RIA radio- immunoassay
  • ELISA enzyme linked immunosorbent assay
  • FACS fluorescence activated cell sorting
  • a method of identifying novel drugs for treatment of schizophrenia comprises incubating an integral membrane protein DGCR2 or any active portion thereof or cells expressing integral membrane protein DGCR2 or any active portion thereof with potential drugs and selecting for at least one drug of the potential drugs that modulates integral membrane protein DGCR2 activity or expression, thereby identifying novel drugs for treatment of schizophrenia.
  • a method of treating schizophrenia comprises administering to a subject in need thereof a therapeutically effective amount of a drug for schizophrenia, the drug for schizophrenia having been identified capable of modulating integral membrane protein DGCR2 activity or expression using the method described hereinabove.
  • the at least one drug of the potential drugs is selected from the group consisting of a peptide, a polynucleotide and a small molecule.
  • the polynucleotide is selected from the group consisting of an antisense oligonucleotide, an siRNA, a ribozymes and a DNAzyme.
  • the peptide is selected from the group consisting of a small peptide, a polypeptide and an antibody.
  • a method of treating and/or preventing schizophrenia comprises administering to a subject in need thereof a therapeutically effective amount of at least one agent capable of modulating DGCR2 protein expression or activity.
  • the at least one agent is selected from the group consisting of an anti-DGCR2 antibody, a polynucleotide encoding an intracellular anti-DGCR2 antibody, an anti-DGCR2 antisense molecule, an anti-DGCR2 siRNA, an anti-DGCR2 ribozyme, an anti-
  • a method of treating and/or preventing schizophrenia comprises administering to a subject in need thereof a therapeutically effective amount of recombinant integral membrane protein DGCR2 or any active portion thereof.
  • the present invention successfully addresses the shortcomings of the presently known configurations by providing methods and kits useful for predisposition, diagnosis and treatment of schizophrenia.
  • all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
  • suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control.
  • the materials, methods, and examples are illustrative only and not intended to be limiting.
  • the following terms and phrases shall have the following meaning:
  • the term "predisposition” refers to a situation of susceptibility to develop a disorder or disease. An individual with a predisposition to a disorder or disease is more likely than an individual without the predisposition to the disorder or disease to develop the disorder or disease.
  • Methods of "determining a predisposition" of an individual to a disorder or disease are used in genetic counseling, especially prior to making a decision to conceive a child. Also, application of methods for detecting genotypes of SNPs in the DCGR2 locus as described herein can be applied to the parents of an individual with schizophrenia.
  • the transmission of the polymo ⁇ hism refers to the distribution of a DCGR2 polymo ⁇ hic allele from a parent to an offspring.
  • the likelihood that the guanine allele will be transmitted is usually the same as the likelihood that the adenosine allele will be transmitted to the offspring.
  • a genetic locus that is linked to another locus shows similar allelic transmission.
  • polymo ⁇ hism refers to the occurrence of two or more genetically determined variant forms (alleles) of a particular nucleic acid, at a frequency where the rarer (or rarest) form could not be maintained by recurrent mutation alone.
  • a single nucleotide polymo ⁇ hism results from a single base difference between related alleles at the same genetic locus.
  • Exemplary nucleotide polymo ⁇ hisms include the G/A polymo ⁇ hism of SNP rs807759 set forth in SEQ ID NO: 13.
  • homozygous refers to an individual having two identical alleles of a certain polymo ⁇ hism.
  • a non-limiting example is an individual having the GG genotype of SNP rs807759. This individual is referred to as a homozygote for this SNP.
  • heterozygous refers to an individual having two different alleles of a certain polymo ⁇ hism.
  • an individual having the A/G genotype of SNP rs807759 is referred as a heterozygote individual for this SNP.
  • absence of genotype is used herein to describe the negative result of a specific genotype determination test.
  • the genotype determination test is suitable for the identification of guanine nucleotide - containing allele of SNP rs807759 set forth in SEQ ID NO: 13, and the individual on which the test is performed is homozygote for the adenosine nucleotide - containing allele of SNP rs807759, then the result of the test will be "absence of genotype".
  • SNPs may be detectable at the protein level if the nucleotide change is in the coding sequence and encodes a different amino acid residue, creating two "polymo ⁇ hs" of the protein.
  • a non-limiting example of an amino acid change is the Val/Ala polymo ⁇ hism at position 473 of the DGCR2 protein set forth in SEQ ID NO:24, which results from the C/T polymo ⁇ hism of SNP rs2072123 set forth in SEQ ID NO:l in the DGCR2 gene.
  • DGCR2 locus refers to the location of a DNA stretch in the human genome corresponding to a gene coding for the DGCR2 protein.
  • neighborhboring loci is used herein to describe DNA sequences (either genes or intergene sequences) that are in close vicinity of the DGCR2 locus and that include other SNPs that are in linkage disequilibrium with SNPs in the DGCR2 locus.
  • linkage disequilibrium (LD) is used to describe the statistical correlation between two neighboring polymo ⁇ hic genotypes.
  • LD refers to the correlation between the alleles of a random gamete at the two loci, assuming Hardy-Weinberg equilibrium (statistical independence) between gametes. LD is quantified with either Lewontin's parameter of association (D') or with Pearson correlation coefficient (r) [Devlin B, Risch N. (1995). A comparison of linkage disequilibrium measures for fine-scale mapping. Genomics. 29: 311-322.]. Two loci with a LD value of 1 are said to be in complete LD. At the other extreme, two loci with a LD value of 0 are termed to be in linkage equilibrium.
  • Linkage disequilibrium is calculated following the application of the expectation maximization algorithm (EM) for the estimation of haplotype frequencies [Slatkin M, Excoffier L. (1996). Testing for linkage disequilibrium in genotypic data using the Expectation- Maximization algorithm. Heredity. 76: 377-83].
  • EM expectation maximization algorithm
  • genetic association refers to a linkage between certain genetic markers such as SNPs, di-nucleotide or tri-nucleotide repeats and the like, with a certain phenotype such as disease, trait, drug responsiveness and the like.
  • genetic cause refers to the genetic basis of a phenomenon such as a disease or disorder or varying responsiveness to a drug.
  • the genetic make-up of an individual determines the individual various traits, including individual's risk of developing a disease and the response exerted in an individual by a specific drug.
  • drug responsiveness is used herein in reference to genotypes in the DGCR2 locus that determine the specific response an individual has to a specific drug.
  • the response to the drug can be high- or low-efficiency, no-effect or adverse- effects.
  • the knowledge of the specific genotype which predicts drug responsiveness can be applied to dosing and/or drug selection, avoiding adverse reactions or therapeutic failure and thus can enhance therapeutic or prophylactic efficiency when treating a subject.
  • mental disorder refers to any disorder affecting an individual behavior, which has a central nervous system etiology.
  • treating refers to inhibiting or arresting the development of a disease, disorder or condition and/or causing the reduction, remission, or regression of a disease, disorder or condition.
  • Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a disease, disorder or condition, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a disease, disorder or condition.
  • the term "preventing” refers to keeping a disease, disorder or condition from occurring in a subject who may be at risk for the disease, but has not yet been diagnosed as having the disease.
  • a "subject in need” refers to an individual who has been diagnosed with or is suspected of having a disease, disorder or condition, or who is predisposed to a disease, disorder or condition.
  • Those of skill in the art will understand that a variety of methods may be used to determine a subject at risk for a disease, and that whether a subject is at risk for a disease, will depend on a variety of factors known to those of skill in the art, including genetic make-up of the subject, age, body weight, sex, diet, general health, occupation, exposure to environmental conditions, marital status, familial history and the like.
  • administering to a subject refers to means for providing a compound that modulates DGCR2 activity and/or expression to a patient, using any suitable route, e.g., oral, sublingual intravenous, subcutaneous, transcutaneous, intramuscular, intracutaneous, intrathecal, epidural, intraoccular, intracranial, inhalation, rectal, vaginal, and the like administration.
  • compositions in the form of creams, lotions, tablets, capsules, pellets, dispersible powders, granules, suppositories, syrups, elixirs, lozenges, injectable solutions, sterile aqueous or non- aqueous solutions, suspensions or emulsions, patches, and the like, is also contemplated.
  • the active ingredients may be compounded with non-toxic, pharmaceutically acceptable carriers including, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, dextrans, and the like.
  • the phrase "therapeutically effective amount”, when used in reference to the invention methods employing compounds that modulate DGCR2 activity and/or expression, refers to a dose of compound sufficient to provide circulating concentrations high enough to impart a beneficial effect on the recipient thereof.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated, the severity of the disorder, the activity of the specific compound used, the route of administration, the rate of clearance of the specific compound, the duration of treatment, the drugs used in combination or coincident with the specific compound, the age, body weight, sex, diet and general health of the patient, and like factors well known in the medical arts and sciences.
  • kits refers to the combination of packaging material, reagents and a notification of use.
  • the kit may be approved by a regulatory agency, such as the FDA, for the use identified in the notification.
  • the notification may also include instructions to use.
  • packaging material refers to paper, plastic, foil, foamed substances and other materials commonly used in the packaging of chemical reagents into kits, in the form of tubes, containers, holders, wrappers, etc.
  • notification refers to the print, in any language, identifying the use of a kit and/or instructions of how to use the kit for that use.
  • active portion refers to any portion of the protein that participates in protein catalytic activity, ligand binding, correct protein folding or protein membrane anchoring.
  • agent capable of modulating DGCR2 protein expression or activity refers to any agent that can modulate transcription of the gene, translation of the transcript and/or post-translational modification of the protein or any agent that is capable of modulating protein catalytic activity, ligand binding to the protein, protein folding or protein membrane anchoring.
  • the present invention is of (i) the use of SNPs in the DGCR2 locus and neighboring loci for the identification of genes related to, and for determining a risk of developing schizophrenia; (ii) methods of identifying novel drugs for the treatment and/or prevention of schizophrenia; (iii) methods of treating and/or preventing schizophrenia; and (iv) pharmacogenomic methods of determining drug responsiveness in mental disorders, schizophrenia in particular.
  • GRR g ⁇ enotype relative risk
  • genotypes of SNPs in the DGCR2 locus are associated with drug responsiveness towards schizophrenia drugs.
  • the present invention discloses methods of predicting, preventing and treating schizophrenia.
  • the present invention is the first to link ' DGCR2 with schizophrenia. It has established a strong and statistically significant . association between genotypes in the DGCR2 locus and increased risk to develop schizophrenia.
  • the inventors of the present invention have uncovered that the G allele of SNP rs807759 set forth in SEQ ID NO: 13, the C allele of SNP rs2072123 set forth in SEQ ID NO:l and the A allele of SNP rs2073776 set forth in SEQ ID NO:5 are highly and significantly associated with schizophrenia.
  • a method of determining a predisposition of a subject to develop schizophrenia comprises determining a presence in a homozygous or heterozygous form, or an absence, of at least one genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, the wherein the at least one genotype is associated with schizophrenia, thereby determining the predisposition of the subject of developing schizophrenia.
  • a kit for determining a predisposition of a subject to develop schizophrenia comprises determining a presence in a homozygous or heterozygous form, or an absence, of at least one genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, the wherein the at least one genotype is associated with schizophrenia, thereby determining the predisposition of the subject of developing schizophrenia.
  • the kit according to this aspect of the present invention comprises a packaging material packaging at least one reagent, the at least one reagent is for determining a presence in a homozygous or heterozygous form, or an absence, of at least one genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, twherein the genotype is associated with schizophrenia.
  • the kit further comprises a notification in or on the packaging material identifying the kit for use in determining a predisposition of developing schizophrenia.
  • the notification also provides for instructions of using the kit in determining a predisposition of developing schizophrenia.
  • Genotypes in the DGCR2 locus can also assist in the diagnosis of schizophrenia.
  • a method of assisting in diagnosing schizophrenia in a subject in need thereof comprises determining a presence in a homozygous or heterozygous form, or an absence, of at least one genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, the wherein the at least one genotype is associated with schizophrenia, thereby assisting in diagnosing schizophrenia in the subject.
  • kits for assisting in diagnosing schizophrenia in a subject in need thereof comprises a packaging material packaging at least one reagent, the at least one reagent for determining a presence in a homozygous or heterozygous form, or an absence, of a genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, twherein the genotype is associated with schizophrenia.
  • the kit further comprises a notification in or on the packaging material identifying the kit for use in assisting in diagnosing schizophrenia in a subject.
  • the notification also provides for instructions of using the kit in assisting in diagnosing schizophrenia of a subject.
  • the G allele and the GG genotype of SNP rs807759, the C allele and the CC genotype of SNP rs2072123, and the A allele and AA genotype of SNP rs2073776 are highly and significantly associated with schizophrenia in males.
  • the predisposition probability for schizophrenia is 2.33-fold higher in males having the GG genotype of SNP rs807759 over males having the AA genotype
  • the predisposition probability for schizophrenia is 1.97-fold higher in males having the CC genotype of SNP rs2072123 over males having the TT genotype
  • the predisposition probability for schizophrenia is 2.3-fold higher in males having the AA genotype of SNP rs2073776 over males having the GG genotype.
  • the predisposition probability in males having the GG-CC-AA triple genotype of SNPs rs807759- rs2072123- rs2073776 is 2.49-fold higher than in males having the protective triple genotype.
  • the probability for schizophrenia in males having the GG-CC- AA-CC-GG-GG genotype of SNPs rs807759- rs2072123- rs2073776- rs737865- rs4680- rsl65599 is 7-fold higher than in males having the protective genotype.
  • a method of determining a predisposition to develop schizophrenia in a male subject comprises determining a presence in a homozygous or heterozygous form, or an absence, of at least one genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, the wherein the at least one genotype is associated with schizophrenia in males in higher association than in females, thereby determining the predisposition of the male subject of developing schizophrenia.
  • a kit for determining a predisposition to develop schizophrenia in a male subject comprises determining a presence in a homozygous or heterozygous form, or an absence, of at least one genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, the wherein the at least one genotype is associated with schizophrenia in males in higher association than in females, thereby determining the predisposition of the
  • the kit comprises a packaging material packaging at least one reagent, the at least one reagent for determining a presence in a homozygous or heterozygous form, or an absence, of a genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, twherein the genotype is associated with schizophrenia in males in higher association than in females.
  • the kit further comprises a notification in or on the packaging material, the notification identifying the kit for use in determining a predisposition of a male subject of developing schizophrenia.
  • the notification also provides for instructions of using the kit in determining the predisposition of a male subject of developing schizophrenia.
  • Genotypes in the DGCR2 locus can also assist in the diagnosis of schizophrenia in a male subject.
  • method of assisting in diagnosing schizophrenia in a male subject in need thereof comprises determining a presence in a homozygous or heterozygous form, or an absence, of at least one genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, the wherein the at least one genotype is associated with schizophrenia in males in higher association than in females, thereby assisting in diagnosing schizophrenia in the male subject.
  • kit for assisting in diagnosing schizophrenia in a male subject in need thereof comprises a packaging material packaging at least one reagent, the at least one reagent for determining a presence in a homozygous or heterozygous form, or an absence, of a genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, twherein the genotype is associated with schizophrenia in males in higher association than in females.
  • the kit further comprises a notification in or on the packaging material, the notification identifying the kit for use in assisting in diagnosing schizophrenia in a male subject.
  • the notification also provides for instructions of using the kit in assisting in diagnosing schizophrenia of a male subject.
  • genotypes of SNPs in the DGCR2 locus are highly and significantly associated with drug responsiveness to schizophrenia drugs which are commonly used in treating schizophrenia and other mental disorders. More specifically, as is further shown in Example 2 of the Examples section which follows, the TC genotype of SNP rs2072123 was found to be highly and significantly associated with high efficiency of zuclopenthixol treatment in schizophrenic males. In addition, the AA genotype of SNP rs2073776 was significantly associated with high efficiency of olanzapine in schizophrenic males.
  • a method of predicting drug responsiveness of a subject having schizophrenia to a schizophrenia drug comprises determining a presence in a homozygous or heterozygous form, or an absence, of at least one genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, the wherein the at least one genotype is associated with drug responsiveness to the drug, thereby predicting drug responsiveness of the subject to the drug.
  • a kit for predicting drug responsiveness of a subject having schizophrenia to a schizophrenia drug comprises determining a presence in a homozygous or heterozygous form, or an absence, of at least one genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, the wherein the at least one genotype is associated with drug responsiveness to the drug, thereby predicting drug responsiveness of the subject to the drug.
  • the kit comprises a packaging material packaging at least one reagent, the at least one reagent for determining a presence in a homozygous or heterozygous form, or an absence, of a genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, twherein the genotype is associated with schizophrenia.
  • the kit further comprises a notification in or on the packaging material, the notification identifying the kit for use in predicting drug responsiveness of a subject having schizophrenia to the drug.
  • the notification also provides for instructions of using the kit in assisting in predicting drug responsiveness of a subject having schizophrenia to the drug.
  • a method of predicting drug responsiveness of a subject having a given mental disorder to a mental disorder drug comprises determining a presence in a homozygous or heterozygous form, or an absence, of at least one genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, the wherein the at least one genotype is associated with drug responsiveness to the drug in at least one mental disorder, thereby predicting drug responsiveness of the subject having the given mental disorder to the drug.
  • kits for predicting drug responsiveness of a subject having a given mental disorder to a mental disorder drug comprises a packaging material packaging at least one reagent, the at least one reagent for determining a presence in a homozygous or heterozygous form, or an absence, of a genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, twherein the genotype is associated with drug responsiveness to the drug in at least one mental disorder.
  • the kit further comprises a notification in or on the packaging material, the notification identifying the kit for use in predicting drug responsiveness of a subject having the given mental disorder to the drug.
  • the notification also provides for instructions of using the kit in assisting in predicting drug responsiveness of a subject having the given mental disorder to the drug.
  • a method of identifying a genetic association with, or a genetic cause to, varying drug responsiveness to a schizophrenia drug comprises determining via a population association study an association between a presence in a homozygous or heterozygous form, or an absence, of at least one genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, and responsiveness or non-responsiveness to the schizophrenia drug, thereby identifying a genetic association with, or a genetic cause to, the varying drug responsiveness to the schizophrenia drug.
  • kits for identifying a genetic association with, or a genetic cause to, varying drug responsiveness to a schizophrenia drug comprises a packaging material packaging at least one reagent, the at least one reagent for determining a presence in a homozygous or heterozygous form, or an absence, of a genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus; twherein the genotype is associated with schizophrenia.
  • the kit further comprises a notification in or on the packaging material, the notification identifying the kit for use in identifying a genetic association with, or a genetic cause to, varying drug responsiveness to the drug.
  • the notification also provides for instructions of using the kit in identifying a genetic association with, or a genetic cause to, varying drug responsiveness to the drug.
  • a method of identifying a genetic association with, or a genetic cause to, varying drug responsiveness to a mental disorder drug comprises determining via a population association study an association between a presence in a homozygous or heterozygous form, or an absence, of at least one genotype in the DGCR2 locus or in neighboring loci, the neighboring loci in linkage disequilibrium with the DGCR2 locus, and responsiveness or non- responsiveness to the mental disorder drug, thereby identifying a genetic association with, or a genetic cause to, the varying drug responsiveness to the mental disorder drug.
  • kits for identifying a genetic association with, or a genetic cause to, varying drug responsiveness of a subject having a given mental disorder to a mental disorder drug comprises a packaging material packaging at least one reagent, the at least one reagent for determining a presence in a homozygous or heterozygous form, or an absence, of a genotype in the DGCR2 locus or in neighboring loci, the neighboring loci being in linkage disequilibrium with the DGCR2 locus, twherein the genotype is associated with drug responsiveness to the drug in at least one mental disorder.
  • the kit further comprises a notification in or on the packaging material, the notification identifying the kit for use in identifying a genetic association with, or a genetic cause to, varying drug responsiveness of a subject having the given mental disorder to the drug.
  • the notification also provides for instructions of using the kit in identifying a genetic association with, or a genetic cause to, varying drug responsiveness of a subject having the given mental disorder to the drug.
  • the schizophrenia drug used by the present invention can be zuclopenthixol, and/or olanzapine.
  • Other drugs used in treating a variety of mental disorders, inclusive future drugs, can also be tested pharmacogenomically in accordance with the teachings and the broad spirit of the present invention.
  • the mental disorder drug can be any known antipsychotic and/or neuroleptic drug for the treatment of patients with mental disorder.
  • Examples include, but are not limited to, thioridazine, clozapine, haloperidol, fluphenazine, chohpromazine, risperidone, levomepromazine, perhenazine, chlo ⁇ rothixene, pimozide, sulphide, olanzapine, zuclopenthixol, amitriptyline, imipramine, clomipramine, desipramine, doxepin, mianserin, maprotiline, phenelzine, fluoxetine, trazodone, fluvoxamine, sertraline, paroxetine, reboxetine, citalopram, nefazodone, venlafoxine, lithium salts, carbamazapine, valproic acid, and clonazepam.
  • the mental disorder for which drug responsiveness can be determined using the methods of the present invention include, for example, schizophrenia, schizoaffective disorder, bipolar disorder, depression, obsessive compulsive disorder, panic disorder, agoraphobia, specific phobia, social phobia, post-traumatic stress disorder, pain disorder, anxiety, somatization disorder, anorexia nervosa, bulimia, and/or nervosa.
  • a method of identifying novel drugs for treatment of schizophrenia comprises incubating the integral membrane protein DGCR2, or any active portion thereof, or cells expressing integral membrane protein DGCR2, or any active portion thereof, with potential drugs and selecting for at least one drug of the potential drugs that modulates integral membrane protein DGCR2 activity or expression, thereby identifying novel drugs for treatment of schizophrenia.
  • a method of treating schizophrenia comprises administering to a subject in need thereof a therapeutically effective amount of a drug for schizophrenia, the drug for schizophrenia having been identified capable of modulating integral membrane protein DGCR2 activity or expression.
  • the potential drug can be a peptide, a polynucleotide and/or a small molecule.
  • the polynucleotide used by the present invention can be an antisense oligonucleotide, an siRNA, a ribozymes and/or a DNAzyme.
  • the peptide used by the present invention can be a small peptide, a polypeptide and/or an antibody.
  • Assays of integral membrane protein DGCR2 expression may include monitoring integral membrane protein DGCR2 levels using an anti- integral membrane protein DGCR2 antibody, in assays such as, for example, in situ immunochemistry staining, Western blot, ELISA, fluorescence activated cell sorting, RIA, etc., and/or monitoring integral membrane protein DGCR2 mRNA levels using any one of a plurality of molecular methods based on hybridization of nucleic acids, such as, for example, in situ hybridization, Northern blot, dot blot, RNA protection assays, etc.
  • determining the various alleles of SNPs in the DGCR2 locus is effected by any one of a variety of methods including, but not limited to, a signal amplification method, a direct detection method and detection of at least one sequence change. These methods can be employed to determine the genotype of the DGCR2 locus in a subject. As will be explained hereinbelow, determination of the DGCR2 genotype may also be accomplished directly by analysis of the DGCR2 gene products, and more particularly, analysis of the Val/Ala polymo ⁇ hism at position 473 of the DGCR2 protein set forth in SEQ ID NO:24
  • amplification of, for example, a DNA molecule or an RNA molecule is used.
  • Signal amplification methods which might be used as part of the methods and kits of the present invention include, but are not limited to PCR, LCR (LAR), Self-Sustained Synthetic Reaction (3SR/NASBA) or a Q-Beta (Q ⁇ ) Replicase reaction.
  • PCR Polymerase Chain Reaction
  • PCR The polymerase chain reaction (PCR), as described in U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis and Mullis et al, is a method of increasing the concentration of a segment of target sequence in a mixture of genomic DNA without cloning or purification.
  • This process for amplifying the target sequence involves the introduction of a molar excess of two oligonucleotide primers, which are complementary to their respective strands of the double-stranded target sequence to the DNA mixture containing the desired target sequence.
  • the mixture is denatured and then allowed to hybridize.
  • the primers are extended with polymerase so as to form complementary strands.
  • the steps of denaturation, hybridization (annealing), and polymerase extension (elongation) can be repeated as often as needed, in order to obtain relatively high concentrations of a segment of the desired target sequence. Because the desired segments of the target sequence become the dominant sequences (in terms of concentration) in the mixture, they are said to be "PCR-amplified”.
  • Ligase Chain Reaction (LCR or LAR): In the ligase chain reaction [LCR; sometimes referred to as “Ligase Amplification Reaction” (LAR)] described by Barany [Proc. Natl. Acad. Sci., 88:189 (1991); Barany, PCR Methods and Applic, 1:5 (1991)] and Wu and Wallace [Genomics 4:560 (1989)] four oligonucleotides, two adjacent oligonucleotides which uniquely hybridize to one strand of target DNA, and a complementary set of adjacent oligonucleotides, which hybridize to the opposite strand, are mixed and DNA ligase is added to the mixture.
  • LCR ligase chain reaction
  • LAR Ligase Amplification Reaction
  • ligase will covalently link each set of hybridized molecules.
  • two probes are ligated together only when they base-pair with sequences in the target sample, without gaps or mismatches. Repeated cycles of denaturation, and ligation amplify a short segment of DNA. LCR has also been used in combination with PCR to achieve enhanced detection of single- base changes [Segev, PCT Publication No. W09001069 Al (1990)].
  • the self-sustained sequence replication reaction (3SR) (Guatelli et al, Proc. Natl. Acad. Sci., 87:1874- 1878, 1990), with an erratum at Proc. Natl. Acad. Sci., 87:7797, (1990) is a transcription-based in vitro amplification system (Kwok et al., Proc. Natl. Acad. Sci., 86:1173-1177, 1989) that can exponentially amplify RNA sequences at a uniform temperature. The amplified RNA can then be utilized for mutation detection (Fahy et al, PCR Meth.
  • an oligonucleotide primer is used to add a phage RNA polymerase promoter to the 5' end of the sequence of interest.
  • a cocktail of enzymes and substrates that includes a second primer, reverse transcriptase, RNase H, RNA polymerase and ribo-and deoxyribonucleoside triphosphates, the target sequence undergoes repeated rounds of transcription, cDNA synthesis and second-strand synthesis to amplify the area of interest.
  • Q-Beta (Q ⁇ ) Replicase In this method, a probe that recognizes the sequence of interest is attached to the replicatable RNA template for Q ⁇ replicase.
  • a previously identified major problem with false positives resulting from the replication of unhybridized probes has been addressed through use of a sequence-specific ligation step.
  • available thermostable DNA ligases are not effective on this RNA substrate, so the ligation must be performed by T4 DNA ligase at low temperatures (37 degrees C). This prevents the use of high temperature as a means of achieving specificity as in the LCR, the ligation event can be used to detect a mutation at the junction site, but not elsewhere.
  • Direct detection methods are possible to detect a mutation at the junction site, but not elsewhere.
  • the direct detection method may be, for example, a cycling probe reaction (CPR) or a branched DNA analysis.
  • CPR cycling probe reaction
  • a branched DNA analysis e.g., a method that does not amplify the signal exponentially is more amenable to quantitative analysis. Even if the signal is enhanced by attaching multiple dyes to a single oligonucleotide, the co ⁇ elation between the final signal intensity and amount of target is direct.
  • Such a system has an additional advantage that the products of the reaction will not themselves promote further reaction, so contamination of lab surfaces by the products is not as much of a concern.
  • Traditional methods of direct detection including Northern and Southern band RNase protection assays usually require the use of radioactivity and are not amenable to automation.
  • Recently devised techniques have sought to eliminate the use of radioactivity and/or improve the sensitivity in automateable formats. Two examples are the "Cycling Probe Reaction” (CPR), and "Branched DNA” (bDNA).
  • Cycling probe reaction The cycling probe reaction (CPR) (Duck et al., BioTech., 9:142, 1990), uses a long chimeric oligonucleotide in which a central portion is made of RNA while the two termini are made of DNA. Hybridization of the probe to a target DNA and exposure to a thermostable RNase H causes the RNA portion to be digested. This destabilizes the remaining DNA portions of the duplex, releasing the remainder of the probe from the target DNA and allowing another probe molecule to repeat the process. The signal, in the form of cleaved probe molecules, accumulates at a linear rate.
  • Branched DNA Branched DNA (bDNA), described by Urdea et al., Gene 61:253-264 (1987), involves oligonucleotides with branched structures that allow each individual oligonucleotide to carry 35 to 40 labels (e.g., alkaline phosphatase enzymes). While this enhances the signal from a hybridization event, signal from nonspecific binding is similarly increased.
  • labels e.g., alkaline phosphatase enzymes
  • Allele-specific amplification For differential amplification of SNP alleles, a PCR primer is designed, where the 3' base co ⁇ esponds to the polymo ⁇ hic site. Consequently, only one of the two alleles will allow perfect matching and be amplified. In this case, SNP detection is concomitant to the amplification step.
  • This strategy has been termed ARMS or Allele Refractory Mutation System [Newton CR, et al., (1989). Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Research 17, 2503-16, 1989].
  • the allele specific primers are designed to differ significantly in their melting temperature (Tm), by addition of a GC-tail to one of the primers. Allele detection can be achieved by Tm shift measurement [Germer S, et al. (1999). Single-tube genotyping without oligonucleotide probes. Genome Research 9, 72-8, 1999].
  • Restriction Fragment Langth Polymorphism When a nucleotide substitution creates or eliminates a restriction site, the alleles can be identified by appropriate restriction, followed by electrophoresis for resolution of the digestion products [Middle F, et al. (2000). Bipolar disorder and variation at a common polymo ⁇ hism (A1832G) within exon 8 of the Wolfram gene. American Journal of Medical Genetics 96, 154-7, 2000].
  • Primer-extension and minisequencing After amplification of a fragment of the SNP-containing DNA, an oligonucleotide primer is annealed immediately upstream to the polymo ⁇ hism. In the presence of the appropriate nucleotides, the primer is extended by one or more bases.
  • the alleles can then be distinguished on the basis of size or mass of the extended product either by mass spectrometry or DHPLC (Denaturing High Performance Liquid Cliromatography), according to specific fluorescence of the inco ⁇ orated nucleotide (see TDI below), by regular electrophoresis-based sequencing techniques, or through hybridization on a high density oligonucleotide a ⁇ ay [Yershov G, et al. (1996). DNA analysis and diagnostics on oligonucleotide microchips. Proc. Natl. Acad. Sci. USA 93, 4913-8, 1996].
  • the PyrosequencingTM strategy [Ahmadian et al. (2000).
  • Allele-specific hybridization The amplified fragments are hybridized over an array of sequence-specific oligonucleotides, using high-stringency hybridization conditions to allow single-base mismatch detection [Saiki RK, et al. (1989). Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes. Proc. Nat. Acad. Sci. USA 86, 6230-4, 1989]; [Sapolsky RJ, Hsie L, Berno A, Ghandour G, Mittmann M, Fan JB (1999). High-throughput polymo ⁇ hism screening and genotyping with high-density oligonucleotide a ⁇ ays. Genetic Analysis 14, 187-92, 1999].
  • DASH Dynamic Allele Specific Hybridization
  • Heteroduplex/homoduplex resolution If the two allelic forms are present during PCR amplification, heteroduplexes will be formed as a result of annealing between the alternative alleles. Homoduplexes can be distinguished from heteroduplexes, either through DHPLC [Giordano M, et al. (1999). Identification by denaturing high-performance liquid cliromatography of numerous polymo ⁇ hisms in a candidate region for multiple sclerosis susceptibility. Genomics 56, 247-53, 1999], or by enzymatic cleavage of the mismatched molecule [Youil R, et al. (1995). Screening for mutations by enzyme mismatch cleavage with T4 endonuclease VII.
  • reaction products are analyzed by ELISA [Tobe VO, et al. (1996). Single-well genotyping of diallelic sequence variations by a two-color ELISA- based oligonucleotide ligation assay. Nucleic Acids Research 24, 3728-32, 1996] or through FRET detection. Allele detection
  • the amplified product is usually necessary, and can be achieved either homogeneously, or in an allele-specific way: Fluorimetric, colorimetric detection, and phosphorimaging are based on homogeneous internal labeling (fluorescent or radioactive, respectively) of the amplified product, or inco ⁇ oration of a fluorescent dye (e.g. Ethidium Bromide, or SYBR green) to the final product.
  • a fluorescent dye e.g. Ethidium Bromide, or SYBR green
  • Electrophoresis has for long been a choice separation technique for DNA molecular studies. Agarose, or acrylamide gel electrophoresis are used for low- throughput analysis of restriction digestion products, or sequencing reactions, respectively. These basic methods have been adapted to higher throughput demands, by means of MADGE (Microtiter Array Diagonal Electrophoresis), [Bolla MK, et al. (1995). High-throughput method for determination of apolipoprotein E genotypes with use of restriction digestion analysis by microplate a ⁇ ay diagonal gel electrophoresis. Clinical Chemistry Al, 1599-604, 1995], or microchip electrophoresis [Schmalzing D, et al. (2000). Microchip electrophoresis: a method for high-speed SNP detection. Nucleic Acids Research 28, E43, 2000]. Acrylamide gels tend to be replaced by capillary electrophoresis.
  • a streamlined mutation detection system multicolor post-PCR fluorescence labeling and single-strand conformational polymo ⁇ hism analysis by capillary electrophoresis.
  • Heteroduplexes are identified according to the molecular conformation by CSGE (Conformation Sensitive Gel Electrophoresis), recently adapted to higher throughput by capillary CSGE [Rozycka M, et al. (2000).
  • CSGE Conformation Sensitive Gel Electrophoresis
  • DPLC Denaturing High Performance Liquid Cliromatography
  • Heterozygotes give two peaks and homozygotes give one peak when tested alone.
  • Homozygotes give two peaks after addition of the reference allele only if they possess the alternative allele.
  • DHPLC is also efficient for minisequencing resolution [Hoogendoom B, et al. (1999). Genotyping single nucleotide polymo ⁇ hisms by primer extension and high performance liquid cliromatography. Human Genetics 104,
  • MALDI-TOF Mass Spectrometry Mass spectrometry is used to measure the mass-to-charge ratio of ions - from which their molecular weight is infe ⁇ ed. A few MS techniques are applicable to DNA analysis.
  • MALDI Microx-Assisted Laser
  • MALDI -TOF time-of-flight
  • TOF time-of-flight
  • SNPs are accurately analyzed by MALDI -TOF, in various allele discrimination formats [Griffin TJ, Smith LM (2000).
  • FRET or Fluorescence Resonance Energy Transfer occurs between a fluorescence donor (D) and a fluorescence acceptor (A) in close proximity, if the emission spectrum of the donor overlaps with the excitation spectrum of the acceptor.
  • acceptor If the acceptor is non-fluorescent, it acts as a quencher, resulting in a net decrease in photon emission from the donor. If A is also fluorescent, the decrease in emission from
  • D is concomitant with an increase in fluorescence at the emission wavelength of A.
  • the efficiency of energy transfer decreases very rapidly with increasing distance between the donor and acceptor, allowing its use for allele detection in a variety of applications [De Angelis DA (1999). Why FRET over genomics? [In Process Citation].
  • Fluorescence polarization takes advantage of the mobility of molecules in solution: fluorescent free nucleotides are more mobile and will therefore show no polarization, while nucleotides that have been inco ⁇ orated, in a primer extension reaction, to a larger oligonucleotide primer, will be detected as polarized molecules, resulting in an amplification of the fluorescence signal.
  • TaqMan (5' exonuclease assay): [Livak KJ, et al. (1995). Towards fully automated genome-wide polymo ⁇ hism screening [letter]. Nature Genetics 9, 341-2, 1995].
  • the probes are short oligonucleotides probes that undergo FRET in their intact state because they are labeled at each end with a fluorescence donor and acceptor pair.
  • the probe is designed to hybridize to a sequence being amplified in a PCR reaction. If there is a perfect match between the probe and the target, the 5 '-exonuclease activity of Taq polymerase will digest the hybridized TaqMan probe during the elongation cycle, separating D from A and resulting in a decrease in FRET.
  • Molecular beacons [Tyagi S, et al. (1998). Multicolor molecular beacons for allele discrimination. Nature Biotechnology 16, 49-53, 1998] These oligonucleotide probes are chemically modified with a fluorescence donor D at their 5' end and a non- fluorescent quenching acceptor Q at their 3' end. In the absence of a perfectly matched target, they assume a stem-and-loop structure in solution. This hai ⁇ in conformation positions D and Q in extremely close proximity, much closer than a pair of fluorophores at opposite ends of a randomly coiled oligonucleotide, resulting in an effective fluorescence quenching.
  • the hai ⁇ in loop hybridizes to the target, thus preventing the folding of the probe and separating D from Q and resulting in a fluorescence increase.
  • the ability of molecular beacons to form hai ⁇ in structures significantly enhances their specificity compared with standard oligonucleotide probes of the same size.
  • Sco ⁇ ion primers [Thelwell N, et al. (2000). Mode of action and application of sco ⁇ ion primers to mutation detection Nucleic Acids Research 28, 3752-61, 2000] combine the properties of a PCR primer and of a FRET probe similar to the molecular beacons.
  • Sunrise probes [Nazarenko IA, et al. (1997).
  • DOL Dye-labeled Oligonucleotide Ligation
  • TDI Template-directed Dye Inco ⁇ oration - [Chen X, (1997). Fluorescence energy transfer detection as a homogeneous DNA diagnostic method. Proc. Natl. Acad. Sci. USA 94, 10756-61, 1997] is a primer-extension strategy, based on the FRET detection assay.
  • the primer to be extended is labeled with a fluorescent donor. Inco ⁇ oration of an acceptor-labelled nucleotide, by the primer extension reaction, brings the fmorophores in close proximity, thus resulting in FRET.
  • PNA hybridization probes Peptide Nucleic Acid (PNA - [Corey DR (1997). Peptide nucleic acids: expanding the scope of nucleic acid recognition. Trends in Biotechnology 15, 224-9, 1997] is a DNA analog containing the four normal nucleotides of DNA attached to a neutrally charged amide backbone. The neutral backbone confers unique characteristics on the hybridization of PNA with DNA, resulting in improved hybridization specificity for complementary DNA sequences.
  • Two similar SNP genotyping approaches using PNA have been independently developed recently [Griffin TJ, et al. (1997). Genetic analysis by peptide nucleic acid affinity MALDI-TOF mass spectrometry [see comments].
  • InvaderTM assay involves the sequence-specific hybridization of two oligonucleotides to form a one-base overlapping structure at the polymo ⁇ hic position. This is followed by enzymatic cleavage of an allele specific downstream probe, using a "flap" endonuclease. A large excess of the overlapping probe in the reaction results in an amplification of the cleaved product, relative to the original DNA sample. The cleaved product serves as an invader upstream oligonucleotide in a secondary reaction, in which cleavage of the probe results in FRET.
  • Padlock probes [Nilsson M, et al. (1994). Padlock probes: circularizing oligonucleotides for localized DNA detection. Science 265, 2085-8, 1994]: The 5' and 3' end regions of these linear oligonucleotides are designed to base-pair next to each other on a target strand. If properly hybridized, then the ends can be joined by enzymatic ligation, converting the probes to circularly closed molecules that are catenated to the target sequences. Because the reaction products remain localized at their target, these probes are suitable for simultaneous analysis of many genes.
  • Light-up probes [Isacsson J, et al. (2000). Rapid and specific detection of PCR products using light-up probes [In Process Citation].
  • Molecular and Cellular Probes 14, 321-8, 2000] are PNA probes labeled with a fluorescent dye which has low fluorescence in its unbound state, but generates a readily detectable signal when hybridized to its target sequence. This eliminates the need to separate unbound probes from the hybridization complex prior to detection.
  • Real Time PCR This approach allows simultaneous allele discrimination and detection [Higuchi R, et al. (1993). Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology (N Y) 11, 1026-30, 1993]; [Nitsche A, et al. (1999). Different real-time PCR formats compared for the quantitative detection of human cytomegalovirus DNA. Clinical Chemisti ⁇ 45, 1932-7, 1999].
  • a few • techniques can be applied, and include: Taqman, Molecular beacons, Allele Specific Hybridization, and Allele Specific Amplification. Detection is achieved by FRET, or by fluorescence detection of SYBR-green (a fluorescent dye that binds to double- stranded DNA).
  • Kinetic PCR is based on the differential amplification of the alternative alleles, due to mismatch pairing of an allele-specific primer.
  • the mismatched product will be amplified at a lower rate, and thus its real-time detection will occur at a later cycle, than the full-match allele.
  • RealTime PCR is conveniently performed on the LightCycler (Roche Molecular Biochemicals) or the ABI PRISM 7700 (Applied Biosystems), which combine a thermocycler with a fluorimeter.
  • the Val/Ala polymo ⁇ hism of the DGCR2 enzyme encoded by SNP rs2072123 may affect the activity of the DGCR2 protein.
  • the present invention discloses a strong association of one polymo ⁇ h of the DGCR2 protein exhibiting the Ala amino acid at position 473 of SEQ ID NO:24 to schizophrenia. Therefore, it is possible that effective amounts of DGCR2 modulatory agents that affect DGCR2 expression or activity can be used to treat and/or prevent schizophrenia.
  • a method of treating and/or preventing schizophrenia comprises administering to a subject in need thereof a therapeutically effective amount of at least one agent capable of modulating
  • the agent used in context of the present invention to inhibit DGCR2 activity or expression can be an anti-DGCR2 antibody, a polynucleotide encoding an intracellular anti-DGCR2 antibody, an anti-DGCR2 antisense molecule, an anti-DGCR2 siRNA, an anti-DGCR2 ribozyme, an anti-DGCR2 DNAzyme, and/or a DGCR2 inhibitor.
  • the agent used in context of the present invention to increase DGCR2 activity or expression can be recombinant DGCR2 protein or any active portion thereof.
  • Anti-DGCR2 antibody and/or recombinant protein An anti-DGCR2 antibody can be prepared by conventional antibody preparation methods.
  • Antibodies of the present invention can be used for recognition of the various antibodies
  • DGCR2 polymo ⁇ hs as well as for the inhibition of the DGCR2 activity and/or expression.
  • Methods of making these antibodies are known in the art. See for example,
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Methods for humanizing non-human antibodies are well known in the art.
  • humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the co ⁇ esponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al, J. Mol. Biol., 222:581 (1991)].
  • the techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. hnmunol., 147(l):86-95 (1991)].
  • human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos.
  • Intracellular anti-DGCR2 antibody or recombinant protein The polynucleotide encoding an anti-DGCR2 antibody or recombinant protein, obtained as described above, is inserted into a suitable mammalian expression vector dedicated for intracellular expression of a protein encoded thereby. To generate such an expression vector, a polynucleotide segment encoding a anti-DGCR2 antibody or recombinant protein, devoid of extracellular secretion signal peptide sequence, is ligated into, for example, a commercially available expression vector system suitable for transforming mammalian cells and for directing the expression of the anti-DGCR2 antibody within the transformed cells.
  • Suitable mammalian expression vectors for use with the present invention include, but are not limited to, pcDNA3, pcDNA3.1 (+/-), pZeoSV2(+/-), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, which are available from Invitrogen, pCI which is available from Promega, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.
  • Viral expression vectors can be particularly useful for introducing an anti- DGCR2 antibody polynucleotide or recombinant protein into a cell.
  • the host cell can be a cell in a subject, a cell in vivo, or a cell ex vivo (see, for example U.S. Pat. No. 5,399, 346).
  • the expression vector can be delivered into cells using a variety of delivery approaches, including, but not limited to, microinjection, electroporation, liposomes, epidermal patches, iontophoresis or receptor-mediated endocytosis. The selection of a particular method will depend, for example, on the cell into which the polynucleotide is to be introduced, as well as whether the cell is isolated in culture, or is in a tissue or organ in culture or in situ.
  • Anti-DGCR2 antisense oligonucleotides/vectors In human gene therapy, antisense nucleic acid technology has been one of the major tools of choice to inactivate genes where expression causes disease and is thus undesirable [Walton et al., 1999, Biotechnol Bioeng 65(1): 1-9; Matveeva et al., 1998, Nature Biotechnology 16, 1374 - 1375].
  • the antisense oligonucleotides used by the present invention can be expressed from a nucleic acid construct administered into the tissue, in which case constitutive or preferably inducible promoters are preferly used such that antisense expression can be switched on and off, or alternatively such oligonucleotides can be chemically synthesized and administered directly into the tissue, as part of, for example, a pharmaceutical composition.
  • antisense oligonucleotides can be effective in vivo.
  • antisense molecules have been used to a ⁇ est hematopoietic cell proliferation (Szczylik et al., Science 253: 562 1991), growth (Calabretta et al. Proc. Natl. Acad. Sci. USA 88:2351, 1991), or entry into the S phase of the cell cycle (Heikhila et al., Nature, 328:445, 1987) and to prevent receptor mediated responses (Burch and Mahan, J. Clin. Invest. 88:1190, 1991).
  • Antisense therapeutics has the potential to treat many life-threatening diseases with a number of advantages over traditional drugs.
  • Traditional drugs intervene after a disease-causing protein is formed.
  • Antisense therapeutics block mRNA transcription/translation and intervene before a protein is formed, and since antisense molecules target only one specific mRNA, such therapeutic agents should be more effective with fewer side effects than cu ⁇ ent protein-inhibiting therapy.
  • a second option for disrupting gene expression at the level of transcription uses synthetic oligonucleotides capable of hybridizing with double stranded DNA. A triple helix is formed. Such oligonucleotides may prevent binding of transcription factors to the gene's promoter and therefore inhibit transcription. Alternatively, they may prevent duplex unwinding and, therefore, transcription of genes within the triple helical structure.
  • RNA interference an approach which utilizes small interfering dsRNA (siRNA) molecules that are homologous to the target mRNA and lead to its degradation [Carthew, 2001, Cu ⁇ Opin Cell Biol 13(2):244-8].
  • RNAi is an evolutionarily conserved surveillance mechanism that responds to double-stranded RNA by sequence-specific silencing of homologous genes (Fire et al., 1998, Nature 391, 806-811; Zamore et al., 2000, Cell 101, 25-33).
  • RNAi is initiated by the dsRNA-specific endonuclease dicer, which promotes cleavage of long dsRNA into double- stranded fragments between 21 and 25 nucleotides long, termed small interfering RNA (siRNAs) (Zamore et al., 2000, Cell 101, 25-33; Elbashir et al., 2001, Genes Dev. 15, 188-200; Hammond et al., 2000, Nature 404, 293-296; Bernstein et al., 2001, Nature 409, 363-366). siRNA are inco ⁇ orated into a protein complex that recognizes and cleaves target mRNAs (Nykanen et al., 2001, Cell 107, 309-321).
  • RNAi has been increasingly used for the sequence-specific inhibition of gene expression. The possibility of interfering with any specific target RNA has rendered RNAi a valuable tool in both basic research and therapeutic applications. RNAi was first used for gene silencing in nematodes (Fire et al., 1998, Nature 391, 806-811). Recent scientific publications have validated the efficacy of such short double stranded RNA molecules in inhibiting target mRNA expression and thus have clearly demonstrated the therapeutic potential of such molecules.
  • RNAi has been utilized to inhibit expression of hepatitis C (McCaffrey et al., 2002, Nature 418, 38-39), fflV-1 (Jacque et al., 2002, Nature 418, 435-438), HPV in cervical cancer cells (Jiang and Milner 2002, Oncogene 21, 6041-8) and BCR/ABL fusion gene in leukemic cells (Wilda et al., 2002, Oncogene 21, 5716-24).
  • siRNA used by the present invention can be transcribed in vitro from plasmids and administered into the tissue.
  • Transcripts that include two self- complementary siRNAs annealed to form a loop region can be further processed by single-stranded ribonucleases and/or other proteins into a functional duplex siRNA molecule (Leirdal and Sioud, 2002, Biochem Biophys Res Commun 295, 744-8).
  • siRNA can also be prepared from dsRNA by Escherichia coli RNase III cleavage into endoribonuclease-prepared siRNA (esiRNA).
  • siRNA molecules utilized by the present invention are preferably delivered into cell using retro viruses. Delivery of siRNA using retro viruses provides several advantages over methods, such as lipofection, since retroviral delivery is more efficient, uniform and immediately selects for stable "knock-down" cells.
  • siRNA molecules of the present invention are preferably transcribed from expression vectors which can facilitate stable expression of the siRNA transcripts once introduced into a host cell.
  • These vectors are engineered to express small hai ⁇ in RNAs (shRNAs), which are processed in vivo into siRNA molecules capable of ca ⁇ ying out gene-specific silencing [Brurnmelkamp, T.R., et al., (2002) Science 296: 550-53; Paddison, P.J., et al., (2002) Genes Dev. 16:948-58; Paul et al. (2002) Nature Biotech. 20: 505-08, Yu, J.Y et al, (2002) Proc. Natl. Acad. Sci. USA 99: 6047-52].
  • shRNAs small hai ⁇ in RNAs
  • pSUPERTM which includes the polymerase-i ⁇ HI -RNA gene promoter with a well defined start of transcription and a termination signal consisting of five thymidines in a row (T5) (Brummelkamp, 2002, Science 296: 550-53).
  • siRNA expression vector encodes the sense and antisense siRNA under the regulation of separate polIII promoters (Miyagishi and Taira (2002) Nature Biotech. 20:497-500).
  • the siRNA, generated by this vector also includes a 5 thymidine termination signal.
  • catalytic nucleic acid molecules have shown promise as therapeutic agents for suppressing gene expression, and are widely discussed in the literature (Haseloff, J. & Gerlach, W.A. Nature 1988;334: 585; Breaker, R.R. and Joyce, G. Chemistry and Biology 1994; 1:223; Koizumi, M., et al. Nucleic Acids Research 1989;17:7059; Otsuka, E.
  • a catalytic nucleic acid molecule functions by binding to and actually cleaving its target mRNA. Cleavage of the target sequence depends on complementation of the target with the hybridizing regions of the catalytic nucleic acid, and the presence of a specific cleavage sequence.
  • enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, minizyme, leadzyme, oligozyme or DNA enzyme, as used in the art. All of these terminologies describe nucleic acid molecules with enzymatic activity.
  • Anti-DGCR2 ribozymes are being increasingly used for the sequence-specific inhibition of gene expression by the cleavage of mRNAs encoding proteins of interest. The possibility of designing ribozymes to cleave any specific target RNA has rendered them valuable tools in both basic research and therapeutic applications. In the therapeutics area, ribozymes have been exploited to target viral RNAs in infectious diseases, dominant oncogenes in cancers and specific somatic mutations in genetic disorders. Most notably, several ribozyme gene therapy protocols for HIV patients are already in Phase 1 trials (Welch et al., 1998, Curr. Opin. Biotechnol., 9:486-496).
  • ribozymes have been used for transgenic animal research, gene target validation and pathway elucidation.
  • Several ribozymes are in various stages of clinical trials.
  • ANGIOZYME was the first chemically synthesized ribozyme to be studied in human clinical trials.
  • ANGIOZYME specifically inhibits formation of the VEGF-r (Vascular Endothelial Growth Factor receptor), a key component in the angiogenesis pathway.
  • Ribozyme Pharmaceuticals, Inc. as well as other firms have demonstrated the importance of anti-angiogenesis therapeutics in animal models.
  • HEPTAZYME a ribozyme designed to selectively destroy Hepatitis C Virus (HCV) RNA, was found effective in decreasing Hepatitis C viral RNA in cell culture assays (Ribozyme Pharmaceuticals, Inco ⁇ orated).
  • HCV Hepatitis C Virus
  • novel ribozymes can be designed to cleave known substrate, using either random variants of a known ribozyme or random-sequence RNA- as a starting point (Pan, T. and Uhlenbeck, O.C. Biochemistry 1996;31:3887; Tsang, J. and Joyce, G.F. Biochemistry 1994;33:5966; Breaker, R.R. and Joyce, G. Chemistry and Biology 1994; 1:223).
  • DNAzymes are single-stranded, and cleave RNA.
  • a general model (the "10-23" model) for the DNAzyme has been proposed.
  • “10-23” DNAzymes have a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate-recognition domains of seven to nine deoxyribonucleotides each.
  • This type of DNAzyme can effectively cleave its substrate RNA at purine:pyrimidine junctions (Santoro, S.W. & Joyce, G.F. Proc. Natl, Acad. Sci. USA 199; for rev of DNAzymes see Khachigian, LM Cu ⁇ Opin Mol Ther 2002;4:119-21).
  • DNAzymes recognizing single and double-stranded target cleavage sites have been disclosed in U.S. Pat. No. 6,326,174 to Joyce et al. DNAzymes of similar design directed against the human Urokinase receptor were recently observed to inhibit Urokinase receptor expression, and successfully inhibit colon cancer cell metastasis in vivo (Itoh et al., 20002, Abstract 409, Ann Meeting Am Soc Gen Ther www.asgt.org.). In another application, DNAzymes complementary to bcr-abl oncogenes were successful in inhibiting the oncogenes expression in leukemia cells, and lessening relapse rates in autologous bone ma ⁇ ow transplant in cases of CML and ALL.
  • Study subjects - Study subjects were Ashkenazi Jewish with known ethnic origin of at least two generations. Subjects were recruited to study upon signing an informed consent form with approved Institutional Review Board (IRB) protocols.
  • IRS Institutional Review Board
  • Schizophrenic cases - Diagnosis included a direct interview using structured clinical interview for personality disorder (SCID), a questionnaire with inclusion and exclusion criteria and cross-references to medical records.
  • the inclusion criteria specified diagnosis according to the Diagnostic and Statistical Manual of Mental Disorders (DSM-TV).
  • Questionnaires also included information regarding drugs taken and their effects on the patient. Control individuals - Healthy Jewish Ashkenazi individuals.
  • Sample preparation - Genomic DNA was prepared from peripheral blood samples using the NucleonTM kit (Pharmacia, North Peapack, New Jersey, USA) according to manufacturer's instructions. DNA was diluted to 100 ng/ ⁇ l and equal aliquots of DNA samples were pooled together according to disease classification or were used for individual genotyping. Prior to genotyping reactions, pool and individual DNA samples were diluted to 10 and 2 ng/ ⁇ l, respectively.
  • SNP selection - SNPs were selected from the National Center for Biotechnology Information (NCBI) SNP data base
  • SNPs were potentially functional (e.g., non-synonymous, SNPs in regulatory regions) and had records of being validated on genomic DNA samples. SNPs were relatively equally spaced to reach maximal coverage of the gene. SNPs were analyzed by the Pyrosequencing software (PyrosequencingTM, Uppsala, Sweden) for primer design.
  • F forward primer
  • R reverse primer
  • P pyro primer
  • rs2072123 SEQ 1; non synonymous
  • rs2073776 SEQ 5
  • rsl0160 SEQ 9
  • rs807759 SEQ 13
  • rsl557845 SEQ 17
  • PCR primers flanking the SNP were selected using the Primer3 software (http://www.genome.wi.mit.edu) to yield an average PCR product of 150 base pairs (bp).
  • the flanking primers was 5 '-conjugated to a biotin molecule.
  • PCR reactions included 45 cycles of denaturation (20 seconds at 94 °C), annealing (30 seconds at 55 °C) and elongation (30 seconds at 72 °C) using the HotStar Taq Polymerase kit (Qiagen, Valencia, CA, USA) according to manufacturer's instructions. Pool DNA PCR products were subsequently subjected to Pyrosequencing analysis using the Pyrosequencing specific internal primers on the PSQTM 96 System (PyrosequencingTM, Uppsala, Sweden) according to manufacturer's protocol.
  • Restriction Fragment Length Polymorphism (RFLP) analysis - PCR products of SNPs rs2072123 and rs807759 were digested using restriction enzymes Acil and Nlalll (both from NEBiolabs, MA, USA) respectively, and were further analyzed on 3 % agarose (BMA, ME, USA) gels.
  • RFLP Restriction Fragment Length Polymorphism
  • Linkage disequilibrium estimates To measure the linkage disequilibrium (LD) between SNPs, haplotype frequencies were estimated using the expectation maximization (EM) [Slatkin M, Excoffier L. (1996). Testing for linkage disequilibrium in genotypic data using the Expectation-Maximization algorithm. Heredity. 76: 377-83] algorithm on 24 individual genotypes and calculating the Lewontin's (D') and Pearson co ⁇ elation (r).
  • EM expectation maximization
  • Test for association - Association of a specific SNP to a disease was evaluated in a case-control study by comparing the frequency of a specific allele and/or genotype in the case population (i.e., patients) with those of the control population, using standard ⁇ and proportion tests under normal approximation.
  • Odds ratios - is an estimate of the relative risk, i.e., the increased probability of disease in populations exposed to the risk allele. OR and approximate confidence intervals, were computed in a standard way [Alan Agresti (1990). Categorical data analysis. New York: Wiley, pp. 54-55] in order to examine the structure and strength of association between genotype and disease.
  • Genotype relative risk is the increased chance of an individual with a particular genotype to develop the disease.
  • the GRR of the risk genotype G with respect to the protective genotype Go, is the ratio between the risk of an individual carrying genotype G to develop the disease, and the risk of an individual ca ⁇ ying genotype Go to develop the disease.
  • the GRR used herein is represented in terms of an appropriate odds ratio (OR) of G versus Go in cases and controls. Computation of the GRR of the haplotypes was based on a multiplicative model in which the GRR of an homozygote individual was the square of the GRR of an heterozygote individual. For further details see Risch and Merikangas, 1996 [The future of genetic studies of complex human diseases. Science 273: 1516-1517].
  • PAR Population attributable risk
  • Haplotype-Trait Association- was analyzed by a haplotype-specific chi-square test for association.
  • the p-value computation is based on an asymptotic approximation (ChiSq).
  • P-value computation is based on a simulation applying 10,000 iterations.
  • GRR Genetic Relative Risk
  • the multiplicative model was assumed. This leads to the conclusion the GRR of a heterozygote for the risk haplotype is equal to the odds ratio associated with the haplotype.
  • the GRR of a homozygote for the risk haplotype is the square of the GRR for an heterozygote. More generally, the GRR of a heterozygote with neither of the protective haplotypes is the product of the associated heterozygote with protective GRR's. Only the GRR for homozygotes with risk hapltypes were computed. Note: in DGCR2 the associated haplotype A-T-G is actually a protective haplotype. For the computation, risk haplotypes are considered all haplotypes other than that haplotype.
  • the protective haplotype is A-T-G. All other haplotypes were collapsed into a single class and computed the GRR of that class. This GRR co ⁇ esponds to the risk associated with a heterozygote which has a single protective haplotype. Based on this GRR, and based on the multiplicative assumption, the PAR was computed using the following formula:
  • PCR products were prepared from pooled DNA samples of 660 schizophrenia cases (SZP) and 2771 control individuals (CTL), and allele frequency was determined using the PyrosequencerTM. The calculated difference in allele frequency between cases and controls was tested for significance using a standard ⁇ 2 analysis.
  • SNPs in the DGCR2 locus are associated with increased risk for schizophrenia and suggest their further analysis by large-scale individual genotyping.
  • SNP genotyping was performed on DNA samples from 24 control individuals.
  • SNPs rs807759, rs2072123, rs2073776 were subjected to individual genotyping to validate the association of the DGCR2 gene locus with schizophrenia.
  • the G allele and the GG genotype of SNP rs807759 are risk factors for schizophrenia
  • SNP rs807759 Odds ratios, Genotype Related Risk (GRR), and Population
  • the C-allele and the CC genotype of SNP rs2072123 are associated with schizophrenia
  • the risk of schizophrenia is 66% higlier in CC homozygotes and 1.14- fold higher in heterozygotes than in individuals with the protective genotype (TT), (GRR, Table 7). This tendency is more prominent in affected males: there is a 1.97-fold and 1.21-fold higher probability for homozygous and heterozygous genotypes respectively. Furthermore, 12% of all schizophrenia patients and 17% of male patients would not have been affected if the population were monomo ⁇ hic for the protective allele and genotype (PAR, Table 7). The allele and genotype Odds Ratios are 1.26 (95% CI: 1.08-1.48) and 1.54 (95% CI: 1.12-2.11) in patients vs.
  • SNP rs2072123 Odds ratios, Genotype Related Risk (GRR), and Population
  • the A-allele and AA homozygote of the SNP rs2073776 are associated with schizophrenia
  • SNP rs2073776 Odds ratios, Genotype Related Risk (GRR), and Population
  • G-C-A homozygotes are 2.12-fold more likely, and heterozygotes (G-C-A/A-T-G) are 1.46-fold more likely to be affected with schizophrenia, than homozygotes for the protective haplotype (A-T-G).
  • A-T-G homozygotes for the protective haplotype
  • Cathecol-O-Methyl Transferase has previously been shown to be associated with schizophrenia [Shifrnan et al., (2002). A highly significant association between a COMT haplotype and Schizophrenia. Am. J. Hum. Genet. 71:1296-1302, 2002]; [Darvasi, Zak, PCT Patent Application No. PCT/IL03/00140, 2003].
  • This "risk” haplotype consists of two “risk” haplotypes: G-C-A in DGCR2, see above; and C-G-G in COMT [Shifrnan et al., (2002). A highly significant association between a COMT haplotype and Schizophrenia. Am. J. Hum. Genet. 71:1296-1302, 2002; Darvasi, Zak, PCT Patent Application No. PCT/TL03/00140, 2003].
  • the Odds Ratio value for the A-T-G- T-A-A haplotype was found to be 0.33 (95% CI: 0.2-0.54), meaning that this haplotype is a protective haplotype.
  • Genotype subclasses in the DGCR2 locus display different responsiveness towards schizophrenia treatment -

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  • General Health & Medical Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Cette invention se rapporte à des procédés et à des kits utilisés pour déterminer la prédisposition et/ou le diagnostic de la schizophrénie, au moyen de génotypes dans le locus DGCR2. Cette invention concerne également des procédés et des médicaments servant à traiter la schizophrénie, ainsi que des procédés et des kits servant à prédire la réaction du sujet à des médicaments contre les troubles mentaux et, plus spécifiquement, la réaction du sujet à des médicaments contre la schizophrénie.
PCT/IL2003/000464 2002-06-03 2003-06-03 Association des polymorphismes mononucleotidiques dans le locus dgcr2 et les locus voisins avec la schizophrenie WO2003101377A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003231897A AU2003231897A1 (en) 2002-06-03 2003-06-03 Association of snps in the dgcr2 locus and neighboring loci with schizophrenia

Applications Claiming Priority (2)

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US38441302P 2002-06-03 2002-06-03
US60/384,413 2002-06-03

Publications (2)

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WO2003101377A2 true WO2003101377A2 (fr) 2003-12-11
WO2003101377A3 WO2003101377A3 (fr) 2005-01-27

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AU (1) AU2003231897A1 (fr)
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005118848A1 (fr) * 2004-06-04 2005-12-15 Novartis Ag Biomarqueurs de prediction de la reactivite au traitement a la clozapine
WO2009089120A2 (fr) * 2008-01-02 2009-07-16 Suregene Llc Marqueurs génétiques de maladie mentale
WO2009092032A1 (fr) * 2008-01-17 2009-07-23 Suregene Llc Marqueurs génétiques de maladie mentale
US7932042B1 (en) 2010-10-13 2011-04-26 Suregene, Llc Methods and compositions for the treatment of psychotic disorders through the identification of the olanzapine poor response predictor genetic signature

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE SNP [Online] 09 June 2004 Retrieved from NCBI Database accession no. rs807759 *

Cited By (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005118848A1 (fr) * 2004-06-04 2005-12-15 Novartis Ag Biomarqueurs de prediction de la reactivite au traitement a la clozapine
JP2008505060A (ja) * 2004-06-04 2008-02-21 ノバルティス アクチエンゲゼルシャフト クロザピン処置に対する応答を予測するためのバイオマーカー
EP2570497A3 (fr) * 2008-01-02 2013-03-27 Suregene Llc Marqueurs génétiques de maladie mentale
EP2570499A3 (fr) * 2008-01-02 2013-04-03 Suregene Llc Marqueurs génétiques de maladie mentale
WO2009089120A3 (fr) * 2008-01-02 2009-09-24 Suregene Llc Marqueurs génétiques de maladie mentale
EP2570501A3 (fr) * 2008-01-02 2013-04-10 Suregene Llc Marqueurs génétiques de maladie mentale
EP2514840A1 (fr) * 2008-01-02 2012-10-24 Suregene Llc Système de génération d'énergie éolienne et procédé d'installation supplémentaire de générateur d'énergie éolienne dans celui-ci
EP2570493A1 (fr) * 2008-01-02 2013-03-20 Suregene LLC Marqueurs génétiques de maladie mentale
EP2570494A1 (fr) * 2008-01-02 2013-03-20 Suregene Llc Marqueurs génétiques de maladie mentale
EP2570495A1 (fr) * 2008-01-02 2013-03-20 Suregene Llc Marqueurs génétiques de maladie mentale
EP2570496A3 (fr) * 2008-01-02 2013-03-27 Suregene Llc Marqueurs génétiques de maladie mentale
EP2570498A3 (fr) * 2008-01-02 2013-03-27 Suregene Llc Marqueurs génétiques de maladie mentale
WO2009089120A2 (fr) * 2008-01-02 2009-07-16 Suregene Llc Marqueurs génétiques de maladie mentale
EP2570500A3 (fr) * 2008-01-02 2013-04-03 Suregene Llc Marqueurs génétiques de maladie mentale
EP2581456A3 (fr) * 2008-01-17 2013-07-17 Suregene Llc Marqueurs génétiques de maladie mentale
EP2581454A3 (fr) * 2008-01-17 2013-07-10 Suregene LLC Marqueurs génétiques de maladie mentale
WO2009092032A1 (fr) * 2008-01-17 2009-07-23 Suregene Llc Marqueurs génétiques de maladie mentale
EP2581455A3 (fr) * 2008-01-17 2013-07-10 Suregene LLC Marqueurs génétiques de maladie mentale
EP2581453A3 (fr) * 2008-01-17 2013-07-10 Suregene Llc Marqueurs génétiques de maladie mentale
EP2581459A3 (fr) * 2008-01-17 2013-07-17 Suregene LLC Marqueurs génétiques de maladie mentale
EP2581457A3 (fr) * 2008-01-17 2013-07-17 Suregene LLC Marqueurs génétiques de maladie mentale
EP2581460A3 (fr) * 2008-01-17 2013-07-17 Suregene LLC Marqueurs génétiques de maladie mentale
EP2581458A3 (fr) * 2008-01-17 2013-07-17 Suregene LLC Marqueurs génétiques de maladie mentale
US8586308B2 (en) 2008-01-17 2013-11-19 Suregene, Llc Genetic markers of mental illness
US9040241B2 (en) 2008-01-17 2015-05-26 Suregene Llc Genetic markers of mental illness
US9738934B2 (en) 2008-01-17 2017-08-22 Clinical Reference Laboratory, Inc. Genetic markers of mental illness
US7932042B1 (en) 2010-10-13 2011-04-26 Suregene, Llc Methods and compositions for the treatment of psychotic disorders through the identification of the olanzapine poor response predictor genetic signature

Also Published As

Publication number Publication date
WO2003101377A3 (fr) 2005-01-27
AU2003231897A8 (en) 2003-12-19
AU2003231897A1 (en) 2003-12-19

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