EP1165836A2 - Gene, proteine und biallelische markers die mit der schizophrenie assoziert sind - Google Patents

Gene, proteine und biallelische markers die mit der schizophrenie assoziert sind

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Publication number
EP1165836A2
EP1165836A2 EP00914325A EP00914325A EP1165836A2 EP 1165836 A2 EP1165836 A2 EP 1165836A2 EP 00914325 A EP00914325 A EP 00914325A EP 00914325 A EP00914325 A EP 00914325A EP 1165836 A2 EP1165836 A2 EP 1165836A2
Authority
EP
European Patent Office
Prior art keywords
seq
sequence
sbgl
biallelic marker
polynucleotide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00914325A
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English (en)
French (fr)
Inventor
Daniel Cohen
Marta Blumenfeld
Ilya Chumakov
Lydie Bougueleret
Bernard Bihain
Laurent Essioux
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Merck Biodevelopment SAS
Original Assignee
Genset SA
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Filing date
Publication date
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Publication of EP1165836A2 publication Critical patent/EP1165836A2/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • 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 invention concerns the human sbgl, g34665, sbg2, g35017 and g35018 genes, polynucleotides, polypeptides biallelic markers, and human chromosome 13q31-q33 biallelic markers.
  • the invention also concerns the association established between schizophrenia and bipolar disorder and the biallelic markers and the sbgl, g34665, sbg2, g35017 and g35018 genes and nucleotide sequences.
  • the invention provides means to identify compounds useful in the treatment of schizophrenia, bipolar disorder and related diseases, means to determine the predisposition of individuals to said disease as well as means for the disease diagnosis and prognosis.
  • Parkinson's disease Huntington's disease, motor neuron disease, etc.
  • disorders of mood e.g. unipolar depression, bipolar disorder, anxiety, etc.
  • diseases involving the intellect e.g. Alzheimer's disease, Lewy body dementia, schizophrenia, etc.
  • these systems have been implicated in many other disorders, such as coma, head injury, cerebral infarction, epilepsy, alcoholism and the mental retardation states of metabolic origin seen particularly in childhood.
  • Schizophrenia is one of the most severe and debilitating of the major psychiatric diseases. It usually starts in late adolescence or early adult life and often becomes chronic and disabling. Men and women are at equal risk of developing this illness; however, most males become ill between 16 and 25 years old, while females develop symptoms between 25 and 30. People with schizophrenia often experience both "positive" symptoms (e.g., delusions, hallucinations, disorganized thinking, and agitation) and "negative” symptoms (e.g., lack of drive or initiative, social withdrawal, apathy, and emotional unresponsiveness).
  • "positive” symptoms e.g., delusions, hallucinations, disorganized thinking, and agitation
  • negative symptoms e.g., lack of drive or initiative, social withdrawal, apathy, and emotional unresponsiveness.
  • schizophrenia accounts for one fourth of all mental health costs and takes up one in three psychiatric hospital beds. Most schizophrenia patients are never able to work.
  • the cost of schizophrenia to society is enormous. In the United States, for example, the direct cost of treatment of schizophrenia has been estimated to be close to 0.5% of the gross national product.
  • Standardized mortality ratios (SMRs) for schizophrenic patients are estimated to be two to four times higher than the general population, and their life expectancy overall is 20 % shorter than for the general population.
  • SMRs Standardized mortality ratios
  • the most common cause of death among schizophrenic patients is suicide (in 10 % of patients) which represents a 20 times higher risk than for the general population. Deaths from heart disease and from diseases of the respiratory and digestive system are also increased among schizophrenic patients.
  • Bipolar disorders are relatively common disorders with severe and potentially disabling effects. In addition to the severe effects on patients' social development, suicide completion rates among bipolar patients are reported to be about 15%.
  • - bipolar disorder I including patients displaying mania for at least one week
  • - bipolar disorder II including patients displaying hypomania for at least 4 days, characterized by milder symptoms of excitement than mania, who have not previously displayed mania, and have previously suffered from episodes of major depression;
  • bipolar disorder not otherwise specified including patients otherwise displaying features of bipolar disorder II but not meeting the 4 day duration for the excitement phase, or who display hypomania without an episode of major depression; and
  • bipolar disorder As there are currently no cures for bipolar disorder or schizophrenia, the objective of treatment is to reduce the severity of the symptoms, if possible to the point of remission. Due to the similarities in symptoms, schizophrenia and bipolar disorder are often treated with some of the same medicaments. Both diseases are often treated with antipsychotics and neuroleptics. For schizophrenia, for example, antipsychotic medications are the most common and most valuable treatments. There are four main classes of antipsychotic drugs which are commonly prescribed for schizophrenia. The first, neuroleptics, exemplified by chlo ⁇ romazine (Thorazine), has revolutionized the treatment of schizophrenic patients by reducing positive
  • Clozapine has serious limitations. It was originally withdrawn from the market because it can cause agranulocytosis, a potentially lethal inability to produce white blood cells. Agranulocytosis remains a threat that requires careful monitoring and periodic blood tests. Clozapine can also cause seizures and other disturbing side effects (e.g., drowsiness, lowered blood pressure, drooling, bed-wetting, and weight gain). Thus it is usually taken only by patients who do not respond to other drugs.
  • Schizophrenia and bipolar disorder are now considered to be brain diseases, and emphasis is placed on biological determinants in researching the conditions.
  • neuroimaging and neuropathological studies have shown evidence of brain abnormalities in schizophrenic patients. The timing of these pathological changes is unclear but are likely to be a defect in early brain development. Profound changes have also occurred in hypotheses concerning neurotransmitter abnormalities in schizophrenia.
  • the dopamine hypothesis has been extensively revised and is no longer considered as a primary causative model.
  • Oligonucleotide probes or primers hybridizing specifically with a sbgl, g34665, sbg2, g35017 or g35018 genomic or cDNA sequence are also part of the present invention, as well as DNA amplification and detection methods using said primers and probes.
  • the invention also concerns to biallelic markers of the sbgl, g34665, sbg2, g35017 or g35018 gene and the use thereof. Included are probes and primers for use in genotyping biallelic markers of the invention.
  • the invention provides appropriate tools for establishing further genetic associations between alleles of biallelic markers on the 13q31-13q33 locus and a trait.
  • Methods and products are provided for the molecular detection of a genetic susceptibility in humans to schizophrenia and bipolar disorder. They can be used for diagnosis, staging, prognosis and monitoring of this disease, which processes can be further included within treatment approaches.
  • the invention also provides for the efficient design and evaluation of suitable therapeutic solutions including individualized strategies for optimizing drug usage, and screening of potential new medicament candidates.
  • Figure 3 is a table demonstrating the results of a haplotype association analysis between total French Canadian schizophrenia cases and haplotypes which consist of chromosome 13q31 - q33 biallelic markers of the invention.
  • Figure 4 is a table showing the involvement of selected biallelic markers of the invention in statistically significant haplotypes.
  • Figure 5 is a table demonstrating the results of a haplotype association analysis between French Canadian schizophrenia cases and haplotypes which consist of chromosome 13q31 -q33 biallelic markers of the invention.
  • Figure 6 is a table demonstrating the results of a haplotype association analysis between French Canadian schizophrenia cases and haplotypes which consist of chromosome 13q31-q33 biallelic markers of the invention.
  • Figures 7A and 7B show the results of a haplotype association analysis (Omnibus LR test value distribution) between schizophrenia cases and haplotypes comprising Region D biallelic markers of the invention.
  • Figures 8 A and 8B show the results of a haplotype association analysis (HaplotMaxM test value distribution) between schizophrenia cases and haplotypes comprising Region D biallelic markers of the invention.
  • Figures 9A and 9B show the results of a haplotype association analysis (Omnibus LR test value distribution) between bipolar disorder cases and haplotypes comprising Region D biallelic markers of the invention.
  • Figures 10A and 10B show the results of a haplotype association analysis (HaploMaxM test value distribution) between bipolar disorder cases and haplotypes comprising Region D biallelic markers of the invention.
  • Figures 1 IA and 1 IB show the results of a haplotype association analysis (HaploMaxS test value distribution) between bipolar disorder cases and haplotypes comprising Region D biallelic markers of the invention.
  • Figure 12 shows a comparison of the number of significant single and multipoint biallelic marker analyses in subregions DI to D4 of Region D in French Canadian samples.
  • Figure 13 shows a summary of the number of significant single and multipoint biallelic marker analyses across Region D in French Canadian samples.
  • Figure 14 shows a comparison of the number of significant single and multipoint biallelic marker analyses in subregions DI to D4 of Region D in United States schizophrenia samples.
  • Figure 15 shows a summary of the number of significant single and multipoint biallelic marker analyses across Region D in United States schizophrenia samples.
  • Figure 16 shows a comparison of the number of significant single and multipoint biallelic marker analyses in subregions DI to D4 of Region D in Argentinian bipolar disorder samples.
  • Figure 17 shows a summary of the number of significant single and multipoint biallelic marker analyses across Region D in Argentinian bipolar disorder samples.
  • Figure 18 shows the effect of injection of an sbgl peptide on locomotor activity and stereotypy of mice .
  • Figure 19 is a block diagram of an exemplary computer system.
  • Figure 20 is a flow diagram illustrating one embodiment of a process 200 for comparing a new nucleotide or protein sequence with a database of sequences in order to determine the homology levels between the new sequence and the sequences in the database.
  • Figure 21 is a flow diagram illustrating one embodiment of a process 250 in a computer for determining whether two sequences are homologous.
  • Figure 22 is a flow diagram illustrating one embodiment of an identifier process 300 for detecting the presence of a feature in a sequence.
  • SEQ ID No. 1 contains the approximately 319kb of genomic nucleotide sequence comprising sbgl, g34665, sbg2, g35017 and g35018 nucleic acid sequences and the biallelic markers Al to A360 and polymo ⁇ hisms A361 to A489 located on the human chromosome 13q31-q33 locus.
  • SEQ ID Nos. 2 to 26 contain cDNA sequences of the sbgl gene.
  • SEQ ID Nos. 27 to 35 contain amino acid sequences of sbgl polypeptides, encoded by cDNAs of SEQ ID Nos. 2 to 26.
  • SEQ ID No. 36 to 40 contain cDNA sequences of the g35018 gene
  • SEQ ID No. 41 to 43 contain amino acid sequences of an g35018 polypeptides.
  • the following codes have been used in the Sequence Listing to indicate the locations of biallelic markers within the sequences and to identify each of the alleles present at the polymorphic base.
  • the code “r” in the sequences indicates that one allele of the polymo ⁇ hic base is a guanine, while the other allele is an adenine.
  • the code “y” in the sequences indicates that one allele of the polymorphic base is a thymine, while the other allele is a cytosine.
  • the code “m” in the sequences indicates that one allele of the polymo ⁇ hic base is an adenine, while the other allele is an cytosine.
  • the code “k” in the sequences indicates that one allele of the polymo ⁇ hic base is a guanine, while the other allele is a thymine.
  • the code “s” in the sequences indicates that one allele of the polymo ⁇ hic base is a guanine, while the other allele is a cytosine.
  • the code “w” in the sequences indicates that one allele of the polymo ⁇ hic base is an adenine, while the other allele is an thymine.
  • Candidate region on the chromosome 13 (linkage analysis) Genetic link or "linkage” is based on an analysis of which of two neighboring sequences on a chromosome contains the least recombinations by crossing-over during meiosis. To do this, chromosomal markers, like microsatellite markers, have been localized with precision on the genome. Genetic link analysis calculates the probabilities of recombinations on the target gene with the chromosomal markers used, according to the genealogical tree, the transmission of the disease, and the transmission of the markers. Thus, if a particular allele of a given marker is transmitted with the disease more often than chance would have it (recombination level between 0 and 0.5), it is possible to deduce that the target gene in question is found in the neighborhood of the marker.
  • linkage analysis observations have been made, according to which a candidate region for schizophrenia is present on chromosome 13q32 locus (Blouin et al., 1998). Linkage analysis has been successfully applied to map simple genetic traits that show clear Mendelian inheritance patterns and which have a high penetrance, but this method suffers from a variety of drawbacks.
  • linkage analysis is limited by its reliance on the choice of a genetic model suitable for each studied trait.
  • the resolution attainable using linkage analysis is limited, and complementary studies are required to refine the analysis of the typical 20 Mb regions initially identified through this method.
  • linkage analysis have proven difficult when applied to complex genetic traits, such as those due to the combined action of multiple genes and/or environmental factors.
  • biallelic markers located on the human chromosome 13q31-q33 locus associated with schizophrenia are disclosed.
  • the identification of these biallelic markers in association with schizophrenia has allowed for the further definition of the chromosomal region suspected of containing a genetic determinant involved in a predisposition to develop schizophrenia and has resulted in the identification of novel gene sequences disclosed herein which are associated with a predisposition to develop schizophrenia.
  • the present invention thus provides an extensive fine structure map of the 13q31-q33 locus, including novel biallelic markers located on the human 13q31-q33 locus, approximately 319kb of genomic nucleotide sequence of a subregion of the human 13q3 l-q33 locus, and polymo ⁇ hisms including biallelic markers and nucleotide deletions in said 319kb genomic sequence.
  • the biallelic markers of the human chromosome 13q31-q33 locus and the nucleotide sequences, polymo ⁇ hisms and gene sequences located in Region D subregion of the human chromosome 13q3 l-q33 locus are useful as genetic and physical markers for further mapping studies.
  • polymo ⁇ hisms are used in the design of assays for the reliable detection of genetic susceptibility to schizophrenia and bipolar disorder. They can also be used in the design of drug screening protocols to provide an accurate and efficient evaluation of the therapeutic and side-effect potential of new or already existing medicament or treatment regime.
  • nucleotide is also used herein to encompass "modified nucleotides" which comprise at least one modifications (a) an alternative linking group, (b) an analogous form of purine, (c) an analogous form of pyrimidine, or (d) an analogous sugar, for examples of analogous linking groups, purine, pyrimidines, and sugars see for example PCT publication No. WO 95/04064.
  • the polynucleotides of the invention are preferably comprised of greater than 50% conventional deoxyribose nucleotides, and most preferably greater than 90% conventional deoxyribose nucleotides.
  • the polynucleotide sequences of the invention may be prepared by any known method, including synthetic, recombinant, ex vivo generation, or a combination thereof, as well as utilizing any purification methods known in the art.
  • purified is used herein to describe a polynucleotide or polynucleotide vector of the invention which has been separated from other compounds including, but not limited to other nucleic acids, carbohydrates, lipids and proteins (such as the enzymes used in the synthesis of the polynucleotide), or the separation of covalently closed polynucleotides from linear polynucleotides.
  • a polynucleotide is substantially pure when at least about 50 %, preferably 60 to 75% of a sample exhibits a single polynucleotide sequence and conformation (linear versus covalently close).
  • a substantially pure polynucleotide typically comprises about 50 %, preferably 60 to 90% weight/weight of a nucleic acid sample, more usually about 95%, and preferably is over about 99% pure.
  • Polynucleotide purity or homogeneity may be indicated by a number of means well known in the art, such as agarose or polyacrylamide gel electrophoresis of a sample, followed by visualizing a single polynucleotide band upon staining the gel. For certain pu ⁇ oses higher resolution can be provided by using HPLC or other means well known in the art.
  • isolated requires that the material be removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition, and still be isolated in that the vector or composition is not part of its natural environment.
  • probe denotes a defined nucleic acid segment (or nucleotide analog segment, e.g., polynucleotide as defined herein) which can be used to identify a specific polynucleotide sequence present in samples, said nucleic acid segment comprising a nucleotide sequence complementary of the specific polynucleotide sequence to be identified.
  • nucleic acid segment or nucleotide analog segment, e.g., polynucleotide as defined herein
  • trait or “phenotype” are used herein to refer to symptoms of, or susceptibility to schizophrenia or bipolar disorder; or to refer to an individual's response to an agent acting on schizophrenia or bipolar disorder; or to refer to symptoms of, or susceptibility to side effects to an agent acting on schizophrenia or bipolar disorder.
  • allele is used herein to refer to variants of a nucleotide sequence.
  • a biallelic polymo ⁇ hism has two forms. Typically the first identified allele is designated as the original allele whereas other alleles are designated as alternative alleles. Diploid organisms may be homozygous or heterozygous for an allelic form.
  • heterozygositv rate is used herein to refer to the incidence of individuals in a population, which are heterozygous at a particular allele. In a biallelic system the heterozygosity rate is on average equal to 2P a (l-Pa) . where P a is the frequency of the least common allele. In order to be useful in genetic studies a genetic marker should have an adequate level of heterozygosity to allow a reasonable probability that a randomly selected person will be heterozygous.
  • genotype refers the identity of the alleles present in an individual or a sample.
  • a genotype preferably refers to the description of the biallelic marker alleles present in an individual or a sample.
  • genotyping a sample or an individual for a biallelic marker involves determining the specific allele or the specific nucleotide(s) carried by an individual at a biallelic marker.
  • haplotype refers to a combination of alleles present in an individual or a sample on a single chromosome. In the context of the present invention a haplotype preferably refers to a combination of biallelic marker alleles found in a given individual and which may be associated with a phenotype.
  • polyvmo ⁇ hism refers to the occurrence of two or more alternative genomic sequences or alleles between or among different genomes or individuals.
  • Polymo ⁇ hic refers to the condition in which two or more variants of a specific genomic sequence can be found in a population.
  • a "polymorphic site” is the locus at which the variation occurs.
  • a polymo ⁇ hism may comprise a substitution, deletion or insertion of one or more nucleotides.
  • a single nucleotide polymo ⁇ hism is a single base pair change. Typically a single nucleotide polymo ⁇ hism is the replacement of one nucleotide by another nucleotide at the polymo ⁇ hic site. Deletion of a single nucleotide or insertion of a single nucleotide, also give rise to single nucleotide polymo ⁇ hisms.
  • single nucleotide polymo ⁇ hism preferably refers to a single nucleotide substitution.
  • the polymo ⁇ hic site may be occupied by two different nucleotides.
  • biaselic polvmo ⁇ hism and “biallelic marker” are used interchangeably herein to refer to a polymo ⁇ hism having two alleles at a fairly high frequency in the population, preferably a single nucleotide polymo ⁇ hism.
  • a "biallelic marker allele” refers to the nucleotide variants present at a biallelic marker site.
  • the frequency of the less common allele of the biallelic markers of the present invention has been validated to be greater than 1%, preferably the frequency is greater than 10%, more preferably the frequency is at least 20% (i.e. heterozygosity rate of at least 0.32), even more preferably the frequency is at least 30% (i.e.
  • a biallelic marker wherein the frequency of the less common allele is 30% or more is termed a "high quality biallelic marker.” All of the genotyping, haplotyping, association, and interaction study methods of the invention may optionally be performed solely with high quality biallelic markers.
  • nucleotides in a polynucleotide with respect to the center of the polynucleotide are described herein in the following manner.
  • the nucleotide at an equal distance from the 3' and 5' ends of the polynucleotide is considered to be "at the center" of the polynucleotide, and any nucleotide immediately adjacent to the nucleotide at the center, or the nucleotide at the center itself is considered to be "within 1 nucleotide of the center.”
  • any of the five nucleotides positions in the middle of the polynucleotide would be considered to be within 2 nucleotides of the center, and so on.
  • the polymorphism, allele or biallelic marker is "at the center" of a polynucleotide if the difference between the distance from the substituted, inserted, or deleted polynucleotides of the polymo ⁇ hism and the 3' end of the polynucleotide, and the distance from the substituted, inserted, or deleted polynucleotides of the polymo ⁇ hism and the 5' end of the polynucleotide is zero or one nucleotide.
  • the polymo ⁇ hism is considered to be "within 1 nucleotide of the center.” If the difference is 0 to 5, the polymorphism is considered to be “within 2 nucleotides of the center.” If the difference is 0 to 7, the polymo ⁇ hism is considered to be "within 3 nucleotides of the center,” and so on.
  • the polymo ⁇ hism, allele or biallelic marker is "at the center" of a polynucleotide if the difference between the distance from the substituted, inserted, or deleted polynucleotides of the polymo ⁇ hism and the 3' end of the polynucleotide, and the distance from the substituted, inserted, or deleted polynucleotides of the polymo ⁇ hism and the 5' end of the polynucleotide is zero or one nucleotide.
  • the polymo ⁇ hism is considered to be "within 1 nucleotide of the center.” If the difference is 0 to 5, the polymo ⁇ hism is considered to be “within 2 nucleotides of the center.” If the difference is 0 to 7, the polymo ⁇ hism is considered to be "within 3 nucleotides of the center,” and so on.
  • upstream is used herein to refer to a location which, is toward the 5' end of the polynucleotide from a specific reference point.
  • base paired and "Watson & Crick base paired” are used interchangeably herein to refer to nucleotides which can be hydrogen bonded to one another be virtue of their sequence identities in a manner like that found in double-helical DNA with thymine or uracil residues linked to adenine residues by two hydrogen bonds and cytosine and guanine residues linked by three hydrogen bonds (See Stryer, L., Biochemistry, 4th edition, 1995).
  • complementary or “complement thereof are used herein to refer to the sequences of polynucleotides which is capable of forming Watson & Crick base pairing with another specified polynucleotide throughout the entirety of the complementary region. This term is applied to pairs of polynucleotides based solely upon their sequences and not any particular set of conditions under which the two polynucleotides would actually bind.
  • sbgl gene when used herein, encompasses genomic, mRNA and cDNA sequences encoding the sbgl protein, including the untranslated regulatory regions of the genomic DNA.
  • g35017 gene when used herein, encompasses genomic, mRNA and cDNA sequences encoding the g35017 protein, including the untranslated regulatory regions of the genomic DNA.
  • g35018_gene when used herein, encompasses genomic, mRNA and cDNA sequences encoding the g35018 protein, including the untranslated regulatory regions of the genomic DNA.
  • g34665-related biallelic marker relates to a set of biallelic markers in linkage disequilibrium with the g34665 gene or an sbgl nucleotide sequence.
  • the term g34665-related biallelic marker encompasses the biallelic markers A230 to A236 disclosed in Table 6b and any biallelic markers in linkage disequilibrium therewith.
  • sbg2-related biallelic marker relates to a set of biallelic markers in linkage disequilibrium with the sbg2 gene or an sbg2 nucleotide sequence.
  • the term sbg2 -related biallelic marker encompasses the biallelic markers A79 to A99 disclosed in Table
  • g35017-related biallelic marker relates to a set of biallelic markers in linkage disequilibrium with the g35017 gene or an g35017 nucleotide sequence.
  • the term g35017-related biallelic marker encompasses biallelic marker A41 disclosed in Table 6b and any biallelic markers in linkage disequilibrium therewith.
  • polypeptides which include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide.
  • polypeptides which contain one or more analogs of an amino acid including, for example, non-naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.
  • polypeptides with substituted linkages as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
  • non-human animal refers to any non-human vertebrate, birds and more usually mammals, preferably primates, farm animals such as swine, goats, sheep, donkeys, and horses, rabbits or rodents, more preferably rats or mice.
  • animal is used to refer to any vertebrate, preferable a mammal. Both the terms “animal” and “mammal” expressly embrace human subjects unless preceded with the term "non-human”.
  • Variants of polynucleotides are polynucleotides that differ from a reference polynucleotide.
  • a variant of a polynucleotide may be a naturally occurring variant such as a naturally occurring allelic variant, or it may be a variant that is not known to occur naturally.
  • Such non-naturally occurring variants of the polynucleotide may be made by mutagenesis techniques, including those applied to polynucleotides, cells or organisms. Generally, differences are limited so that the nucleotide sequences of the reference and the variant are closely similar overall and, in many regions, identical.
  • nucleotide SEQ ID Nos. 1 to 26, 36 to 40 and 54 to 229 and preferably at least 99%) identical, more particularly at least 99.5% identical, and most preferably at least 99.8% identical to a polynucleotide selected from the group consisting of the nucleotide SEQ ID Nos. 1 to 26, 36 to 40 and 54 to 229 or to any polynucleotide fragment of at least 30, 35, 40, 50, 70, 80 ⁇ 100, 250, 500 , 1000 or 2000, to the extent that the length is consistent with the particular sequence ID, consecutive nucleotides of a polynucleotide selected from the group consisting of the nucleotide sequences of SEQ ID Nos. 1 to 26, 36 to 40 and 54 to 229.
  • a polynucleotide fragment is a polynucleotide having a sequence that is entirely the same as part but not all of a given nucleotide sequence, preferably the nucleotide sequence of an sbgl polynucleotide, and variants thereof, or of a polynucleotide of any of SEQ ID Nos 1 to 26, 36 to 40 and 54 to 229, or a polynucleotide comprising one of the biallelic markers Al to A360 or polymo ⁇ hism A361 to A489, or the complements thereof.
  • Such fragments may be "freestanding", i.e.
  • fragments may be present within a single larger polynucleotide.
  • such fragments may comprise, consist of, or consist essentially of a contiguous span of at least 8, 10, 12, 15, 18, 20, 25, 30, 35, 40, 50, 70, 80, 100, 250, 500 , 1000 or 2000 nucleotides in length of any of SEQ ID Nos 1 to 26, 36 to 40 and 54 to 229.
  • percentage of sequence identity and “percentage homology” are used interchangeably herein to refer to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • BLAST Basic Local Alignment Search Tool
  • BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database
  • TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands)
  • the BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as "high-scoring segment pairs," between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database.
  • High-scoring segment pairs are preferably identified (i.e., aligned) by means of a scoring matrix, many of which are known in the art.
  • the scoring matrix used is the BLOSUM62 matrix (Gonnet et al., 1992, Science 256:1443-1445; Henikoff and Henikoff, 1993, Proteins 17:49-61).
  • the PAM or PAM250 matrices may also be used (see, e.g., Schwartz and Dayhoff, eds., 1978, Matrices for Detecting Distance Relationships: Atlas of Protein Sequence and Structure, Washington: National Biomedical Research Foundation).
  • the BLAST programs evaluate the statistical significance of all high- scoring segment pairs identified, and preferably selects those segments which satisfy a user- specified threshold of significance, such as a user-specified percent homology.
  • the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula of Karlin (see, e.g., Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2267-2268).
  • the BLAST programs may be used with the default parameters or with modified parameters provided by the user.
  • procedures using conditions of high stringency are as follows: Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65°C in buffer composed of 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 ⁇ g/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65°C, the preferred hybridization temperature, in prehybridization mixture containing 100 ⁇ g/ml denatured salmon sperm DNA and 5-20 X 10 6 cpm of 32 P-labeled probe.
  • filter washes can be done at 37°C for 1 h in a solution containing 2 x SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA, followed by a wash in 0.1 X SSC at 50°C for 45 min. Following the wash steps, the hybridized probes are detectable by autoradiography.
  • Other conditions of high stringency which may be used are well known in the art and as cited in Sambrook et al., 1989; and Ausubel et al., 1989. These hybridization conditions are suitable for a nucleic acid molecule of about 20 nucleotides in length.
  • hybridization conditions described above are to be adapted according to the length of the desired nucleic acid, following techniques well known to the one skilled in the art.
  • the suitable hybridization conditions may for example be adapted according to the teachings disclosed in the book of Hames and Higgins (1985) or in Sambrook et al.(1989).
  • Genomic Sequences of the polynucleotides of the invention concerns genomic DNA sequences of the sbgl, g34665, sbg2, g35017 and g35018 genes, as well as DNA sequences of the human chromosome 13q31-q33 region, and more particularly, a subregion thereof referred to herein as region D.
  • Preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60,
  • nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, 1000 or 2000 nucleotides, to the extent that the length of said span is consistent with the length of the SEQ ID, of SEQ ID Nos. 112 to 229.
  • said span is at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, 1000 or 2000 nucleotides of SEQ ID Nos. 112 to 1 14, 115 to 117, 119, 121, 125 to 145, 147 to 150, 159 to 170, and 176 to 229.
  • the invention also encompasses a purified, isolated, or recombinant polynucleotide comprising a nucleotide sequence having at least 70, 75, 80, 85, 90, or 95% nucleotide identity with a nucleotide sequence of of nucleotide positions 31 to 292651 and 292844 to 319608 of SEQ ID No. 1, or a complementary sequence thereto or a fragment thereof.
  • Another object of the invention consists of a purified, isolated, or recombinant nucleic acid that hybridizes with a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, 1000 or 2000 nucleotides of SEQ ID No 1 or a complementary sequence thereto or a variant thereof, under the stringent hybridization conditions as defined above.
  • Additional preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100 or 200 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises a biallelic marker.
  • said contiguous span comprises ar biallelic marker selected from the group consisting of Al to A69, A71 to A74, A76 to A94, A96 to A106, A108 to Al 12, Al 14 to A177, A179 to A197, A199 to A222, A224 to A242.
  • allele 2 is present at the biallelic marker.
  • the present invention encompasses the g34665, g34673, g34667, g35017 and g35018 genes and nucleotide sequences.
  • g34665
  • the invention concerns g34665 genomic sequences consisting of, consisting essentially of, or comprising the sequence of nucleotide positions 292653 to 296047 of SEQ ID No 1, a sequence complementary thereto, as well as fragments and variants thereof.
  • polynucleotides may be purified, isolated, or recombinant.
  • said contiguous span comprises a g34665-related biallelic marker.
  • said biallelic marker is selected from the group consisting of A230 to A236. It should be noted that nucleic acid fragments of any size and sequence may also be comprised by the polynucleotides described in this section.
  • the invention also encompasses a purified, isolated, or recombinant polynucleotide comprising a nucleotide sequence having at least 70, 75, 80, 85, 90, 95, 97, 98 or 99% nucleotide identity with a nucleotide sequence of of nucleotide positions 290653 to 292652, 292653 to 296047, 292653 to 292841, 295555 to 296047, 295580 to 296047 and 296048 to 298048 of SEQ ID No 1 or a complementary sequence thereto or a fragment thereof.
  • the nucleotide differences as regards to nucleotide positions 290652 to 292652, 292653 to 296047,
  • 292653 to 292841, 295555 to 296047, 295580 to 296047 and 296048 to 298048 of SEQ ID No 1 may be generally randomly distributed throughout the entire nucleic acid. Nevertheless, preferred nucleic acids are those wherein the nucleotide differences as regards to the nucleotide sequence of SEQ ID No 1 are predominantly located outside the coding sequences contained in the exons. These nucleic acids, as well as their fragments and variants, may be used as oligonucleotide primers or probes in order to detect the presence of a copy of the g34665 gene in a test sample, or alternatively in order to amplify a target nucleotide sequence within the g34665 sequences.
  • the g34665 genomic nucleic acid comprises at least 3 exons.
  • the exon positions in SEQ ID No 1 are detailed below in Table 2.
  • the invention embodies purified, isolated, or recombinant polynucleotides comprising a nucleotide sequence selected from the group consisting of the 3 exons of the g34665 gene, or a sequence complementary thereto.
  • the invention also deals with purified, isolated, or recombinant nucleic acids comprising a combination of two exons of the g34665 gene.
  • Intron B-Ab refers to the nucleotide sequence located between Exon B and Exon Ab, and so on.
  • the position of the introns is detailed in Table 2.
  • the invention embodies purified, isolated, or recombinant polynucleotides comprising a nucleotide sequence selected from the group consisting of the 2 introns of the g34665 gene, or a sequence complementary thereto.
  • nucleic acid fragments of any size and sequence may also be comprised by the polynucleotides described in this section, flanking the genomic sequences of g34665 on either side or between two or more such genomic sequences.
  • a g34665 polynucleotide or gene may further contain regulatory sequences both in the non-coding 5 '-flanking region and in the non-coding 3 '-flanking region that border the region containing said genes or exons.
  • Polynucleotides derived from 5' and 3' regulatory regions are useful in order to detect the presence of at least a copy of a nucleotide sequence comprising a g34665 nucleotide sequence of SEQ ID No. 1 or a fragment thereof in a test sample.
  • Polynucleotides carrying the regulatory elements located at the 5' end and at the 3' end of the genes comprising the exons of the present invention may be advantageously used to control the transcriptional and translational activity of a heterologous polynucleotide of interest.
  • the present invention also relates to a purified or isolated nucleic acid comprising a polynucleotide which is selected from the group consisting of the 5' and 3' regulatory regions of g34665, or a sequence complementary thereto or a biologically active fragment or variant thereof.
  • the invention concerns g35017 genomic sequences consisting of, consisting essentially of, or comprising the sequence of nucleotide positions 94124 to 94964 of
  • nucleic acid fragments of any size and sequence may also be comprised by the polynucleotides described in this section, flanking the genomic sequences sbgl on either side or between two or more such genomic sequences.
  • nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 8, 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 75, 80, 100, 200 or 500 nucleotides, to the extent that the length of said contiguous span is consistent with the length of the SEQ ID, of a nucleotide sequence selected from the group consisting of SEQ ID Nos 2 to 26, or the complements thereof.
  • nucleic acid fragments of any size and sequence may also be comprised by the polynucleotides described in this section.
  • Primers used to isolate the particular sbgl cDNAs listed above from RNA from various tissues are provided below in Table 5b. Primers designed to hybridize to nucleic acid sequences of exons MSI, M862, Ml 090, Ml 1 17 and MS2, and exons P and R resulted in the cloning of multiple cDNA forms for several sets of primers. The primers used are listed in SEQ ID Nos 44 ot 53. mRNA forms of sbgl were found to differ among tissues; Table 5c lists cDNA forms cloned from various tissues and the relative percentages and numbers of clones found per tissue for each listed sbgl mRNA form.
  • the present inventors have also identified further variations in cDNA sequence as obtained from various tissues and compared with the consensus sbgl genomic nucleotide sequence.
  • the tissues from which cDNA was cloned were obtained from pooled individuals numbering from 1 1 to 60.
  • Table 5d below describes the identities of variants, the nucleotide position of the variation in nucleotide sequence of SEQ ID No 2, and the number of samples having the specified sequence for each respective nucleotide position on the sbgl cDNA sequence of SEQ ID No. 2.
  • amino acid changes in the corresponding sbgl polypeptide sequence (described herein), if any, resulting from the nucleotide sequence variations in the cDNA of SEQ ID No 2.
  • the invention encompasses purified, isolated, or recombinant polynucleotides and polypeptides encoded thereby, wherein the polynucleotides comprise a contiguous span of at least 8, 12, 15, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 100, 150, or 200 nucleotides of SEQ ID No 2 or the complement thereof, and wherein said contiguous span further comprises a nucleotide sequence variation according to
  • the present invention comprises a purified or isolated sbgl cDNA encoding an sbgl protein or a peptide fragment or variant thereof.
  • a purified or isolated nucleic acid encoding an sbgl protein may have the amino acid sequence of any of SEQ ID Nos 27 to 35 or a peptide fragment or variant thereof.
  • Preferred nucleic acids of the invention also include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 8, 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 75, 80, 100, 200 or 500 nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID Nos 2 to 26, or the complements thereof, wherein said span comprises a sbgl-related biallelic marker of the invention.
  • the positions of selected biallelic markers of the invention in sbgl cDNA sequences and polypeptide sequences are listed below in Table 5e.
  • Said contiguous span may comprise a biallelic marker selected from the group of biallelic markers listed in Table 5e; optionally, said biallelic marker is selected from the group consisting of the biallelic markers located in an sbgl cDNA form, as listed in Table 5e; optionally, said biallelic marker is selected from the group consisting of the biallelic markers located in an sbgl coding sequence, as listed in Table 5e.
  • sbgl cDNA Sequences nucleic acid fragments of any size and sequence may also be comprised by the polynucleotides described in this section, flanking the genomic sequences of sbgl on either side or between two or more such genomic sequences.
  • M862 and R testicle 100% M862NOQbisR (188 clones) amygdala 100% M862NOQbisR (42 clones) caudate nucleus 100% M862NOQbisR (39 clones) cerebellum 100% M862NOQbisR (87 clones) hippocampus 100% M862NOQbisR (36 clones) substantia nigra 100% M862NOQbisR (96 clones) thalamus 100% M862NOQbisR (30 clones)
  • M1117N002QbisR (1 clones) amygdala 100% M1117NOQBISR (90 clones) caudate nucleus 100% M1117NOQBISR (94 clones) cerebellum 100% M1117NOQBISR (88 clones) corpus callosum 100% M1117N0QBISR (94 clones) cortex 100% M1117NOQBISR (95 clones) hippocampus 100% M1117N20QR (66 clones) substantia nigra 100% M1117N20QR (90 clones)
  • the promoter activity of the 5' regulatory regions contained in sbgl can be assessed as described below.
  • each of these promoter reporter vectors include multiple cloning sites positioned upstream of a reporter gene encoding a readily assayable protein such as secreted alkaline phosphatase, luciferase, ⁇ galactosidase, or green fluorescent protein.
  • the sequences upstream of the sbgl coding region are inserted into the cloning sites upstream of the reporter gene in both orientations and introduced into an appropriate host cell.
  • the level of reporter protein is assayed and compared to the level obtained from a vector which lacks an insert in the cloning site.
  • the presence of an elevated expression level in the vector containing the insert with respect to the control vector indicates the presence of a promoter in the insert.
  • the upstream sequences can be cloned into vectors which contain an enhancer for increasing transcription levels from weak promoter sequences. A significant level of expression above that observed with the vector lacking an insert indicates that a promoter sequence is present in the inserted upstream sequence.
  • the strength and the specificity of the promoter of the sbgl gene can be assessed through the expression levels of a detectable polynucleotide operably linked to the sbgl promoter in different types of cells and tissues.
  • the detectable polynucleotide may be either a polynucleotide that specifically hybridizes with a predefined oligonucleotide probe, or a polynucleotide encoding a detectable protein, including an sbgl polypeptide or a fragment or a variant thereof. This type of assay is well-known to those skilled in the art and is described in US Patent No. 5,502,176; and US Patent No. 5,266,488. Some of the methods are discussed in more detail below.
  • the invention also pertains to a purified or isolated nucleic acid comprising a polynucleotide having at least 95% nucleotide identity with a polynucleotide selected from the group consisting of the 5' and 3' regulatory regions, advantageously 99 % nucleotide identity, preferably 99.5% nucleotide identity and most preferably 99.8% nucleotide identity with a polynucleotide selected from the group consisting of the 5' and 3' regulatory regions, or a sequence complementary thereto or a variant thereof or a biologically active fragment thereof.
  • Bioly active sbgl polynucleotide derivatives of SEQ ID No 1 are polynucleotides comprising or alternatively consisting in a fragment of said polynucleotide which is functional as a regulatory region for expressing a recombinant polypeptide or a recombinant polynucleotide in a recombinant cell host. It could act either as an enhancer or as a repressor.
  • the regulatory polynucleotides of the invention may be prepared from the nucleotide sequence of SEQ ID No 1 by cleavage using suitable restriction enzymes, as described for example in Sambrook et al.(1989).
  • the regulatory polynucleotides may also be prepared by digestion of SEQ ID No 1 by an exonuclease enzyme, such as Bal31 (Wabiko et al., 1986).
  • exonuclease enzyme such as Bal31 (Wabiko et al., 1986).
  • These regulatory polynucleotides can also be prepared by nucleic acid chemical synthesis, as described elsewhere in the specification.
  • a preferred 5 '-regulatory polynucleotide of the invention includes the 5 '-untranslated region (5'-UTR) of the sbgl cDNA, or a biologically active fragment or variant thereof.
  • a preferred 3 '-regulatory polynucleotide of the invention includes the 3 '-untranslated region (3' -UTR) of the sbgl cDNA, or a biologically active fragment or variant thereof.
  • a further object of the invention consists of a purified or isolated nucleic acid comprising: a) a nucleic acid comprising a regulatory nucleotide sequence selected from the group consisting of: (i) a nucleotide sequence comprising a polynucleotide of the sbgl 5' regulatory region or a complementary sequence thereto;
  • nucleotide sequence comprising a polynucleotide having at least 95% of nucleotide identity with the nucleotide sequence of the sbgl 5' regulatory region or a complementary sequence thereto;
  • nucleotide sequence comprising a polynucleotide that hybridizes under stringent hybridization conditions with the nucleotide sequence of the sbgl 5' regulatory region or a complementary sequence thereto;
  • said nucleic acid includes the 3 '-untranslated region (3'-UTR) of the sbgl cDNA, or a biologically active fragment or variant thereof.
  • the regulatory polynucleotide of the 3' regulatory region, or its biologically active fragments or variants, is advantageously operably linked at the 3 '-end of the polynucleotide encoding the desired polypeptide or polynucleotide.
  • the desired polypeptide encoded by the above-described nucleic acid may be of various nature or origin, encompassing proteins of prokaryotic or eukaryotic origin.
  • the polypeptides expressed under the control of an sbgl regulatory region include bacterial, fungal or viral antigens.
  • eukaryotic proteins such as intracellular proteins, like "house keeping" proteins, membrane-bound proteins, like receptors, and secreted proteins like endogenous mediators such as cytokines.
  • the desired polypeptide may be the sbgl protein, especially the protein of the amino acid sequences of SEQ ID Nos 27 to 35, or a fragment or a variant thereof.
  • the desired nucleic acids encoded by the above-described polynucleotide may be complementary to a desired coding polynucleotide, for example to the sbgl coding sequence, and thus useful as an antisense polynucleotide.
  • Such a polynucleotide may be included in a recombinant expression vector in order to express the desired polypeptide or the desired nucleic acid in host cell or in a host organism.
  • Suitable recombinant vectors that contain a polynucleotide such as described herein are disclosed elsewhere in the specification.
  • Particularly preferred sbg2 nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 nucleotides, to the extent that the length of said span is consistent with said nucleotide position range, of nucleotide positions 201188 to 216915,
  • nucleic acid fragments of any size and sequence may be comprised by the polynucleotides described in this section.
  • the human sbg2 gene comprises exons selected from at least 4 exons, referred to herein as exons S, T, U and V.
  • exons S, T, U and V The nucleotide positions of sbg2 exons in SEQ ID No. 1 are detailed below in Table 5f.
  • the invention embodies purified, isolated, or recombinant polynucleotides comprising a nucleotide sequence selected from the group consisting of the exons of the sbg2 gene, or a sequence complementary thereto.
  • Preferred are purified, isolated, or recombinant polynucleotides comprising at least one exon having the nucleotide position ranges listed in
  • Table 5f selected from the group consisting of the exons S, T, U and N of the sbg2 gene, or a complementary sequence thereto or a fragment or a variant thereof.
  • purified, isolated, or recombinant nucleic acids comprising a combination of at least two exons of the sbg2 gene selected from the group consisting of exons S, T, U and V, wherein the polynucleotides are arranged within the nucleic acid in the same relative order as in
  • the invention embodies purified, isolated, or recombinant polynucleotides comprising a nucleotide sequence selected from the group consisting of the 3 introns of the sbg2 gene, or a sequence complementary thereto.
  • the invention also encompasses a purified, isolated, or recombinant polynucleotide comprising a nucleotide sequence of sbg2 having at least 70, 75, 80, 85, 90, 95, 98 or 99% nucleotide identity with a sequence selected from the group consisting of nucleotide positions 201 188 to 216915, 201188 to 201234, 214676 to 214793, 215702 to 215746 and 216836 to 216915 of SEQ ID No. 1 or a complementary sequence thereto or a fragment thereof.
  • nucleotide differences as regards the nucleotide positions 201188 to 216915, 201 188 to 201234, 214676 to 214793, 215702 to 215746 and 216836 to 216915 of SEQ ID No. 1 may be generally randomly distributed throughout the entire nucleic acid.
  • Additional preferred nucleic acids of the invention include isolated, purified, or recombinant sbg2 polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100 or 200 nucleotides of nucleotide positions 201188 to 216915, 201 188 to 201234, 214676 to 214793, 215702 to 215746 and 216836 to
  • said contiguous span comprises an sbg2-related biallelic marker.
  • said biallelic marker is selected from the group consisting of A79 to A99. It should be noted that nucleic acid fragments of any size and sequence may also be comprised by the polynucleotides described in this section. Either the original or the alternative allele may be present at said biallelic marker.
  • An sbg2 polynucleotide or gene may further contain regulatory sequences both in the non-coding 5 '-flanking region and in the non-coding 3 '-flanking region that border the region containing said genes or exons.
  • Polynucleotides derived from 5' and 3' regulatory regions are useful in order to detect the presence of at least a copy of a nucleotide sequence comprising an sbg2 nucleotide sequence of SEQ ID No. 1 or a fragment thereof in a test sample.
  • Polynucleotides carrying the regulatory elements located at the 5' end and at the 3' end of the genes comprising the exons of the present invention may be advantageously used to control the transcriptional and translational activity of a heterologous polynucleotide of interest.
  • nucleic acid fragments of any size and sequence may also be comprised by the polynucleotides described in this section, flanking the genomic sequences of sbg2 on either side or between two or more such genomic sequences.
  • polynucleotide construct and “recombinant polynucleotide” are used interchangeably herein to refer to linear or circular, purified or isolated polynucleotides that have been artificially designed and which comprise at least two nucleotide sequences that are not found as contiguous nucleotide sequences in their initial natural environment. It should be noted that the present invention embodies recombinant vectors comprising any one of the polynucleotides described in the present invention.
  • the invention also encompasses DNA constructs and recombinant vectors enabling a conditional expression of a specific allele of a nucleotide sequence comprising an sbgl, g34665, sbg2, g35017 or g35018 polynucleotide and also of a copy of a sequence comprising a nucleotide sequence of an sbgl , g34665, sbg2, g35017 or g35018 polynucleotide and also of a copy of a sequence comprising a nucleotide sequence of an sbgl , g
  • nucleotide sequence comprising an sbgl, g34665, sbg2, g35017 or g35018 polynucleotide further comprises a biallelic marker of the present invention.
  • a first preferred DNA construct is based on the tetracycline resistance operon tet from E. coli transposon Tnl 10 for controlling the expression of an sbgl, g34665, sbg2, g35017 or g35018 polynucleotide, such as described by Gossen et al. (1992, 1995) and Furth et al.(1994).
  • Such a DNA construct contains seven tet operator sequences from TnlO (tetop) that are fused to either a minimal promoter or a 5'-regulatory sequence of the sbgl, g34665, sbg2, g35017 or g35018 polynucleotide, said minimal promoter or said sbgl, g34665, sbg2, g35017 or g35018 polynucleotide regulatory sequence being operably linked to a polynucleotide of interest that codes either for a sense or an antisense oligonucleotide or for a polypeptide, including an sbgl, g34665, sbg2, g35017 or g35018 polynucleotide-encoded polypeptide or a peptide fragment thereof.
  • This DNA construct is functional as a conditional expression system for the nucleotide sequence of interest when the same cell also comprises a nucleotide sequence coding for either the wild type (tTA) or the mutant (rTA) repressor fused to the activating domain of viral protein VP16 of herpes simplex virus, placed under the control of a promoter, such as the HCMVIE1 enhancer/promoter or the MMTV-LTR.
  • a preferred DNA construct of the invention comprises both the polynucleotide containing the tet operator sequences and the polynucleotide containing a sequence coding for the tTA or the rTA repressor.
  • conditional expression DNA construct contains the sequence encoding the mutant tetracycline repressor rTA, the expression of the polynucleotide of interest is silent in the absence of tetracycline and induced in its presence.
  • a second preferred DNA construct will comprise, from 5'-end to 3'-end: (a) a first nucleotide sequence comprising an sbgl polynucleotide; (b) a nucleotide sequence comprising a positive selection marker, such as the marker for neomycine resistance (neo); and (c) a second nucleotide sequence comprising a respective sbgl polynucleotide, and is located on the genome downstream of the first sbgl polynucleotide sequence (a). Also encompassed are DNA construct prepared in an analogous manner using g34665, sbg2, g35017 or g35018 nucleotide sequences in place of the sbgl sequences described above.
  • this DNA construct also comprises a negative selection marker located upstream the nucleotide sequence (a) or downstream the nucleotide sequence
  • the negative selection marker comprises the thymidine kinase (tk) gene (Thomas et al., 1986), the hygromycine beta gene (Te Riele et al., 1990), the hprt gene ( Van der Lugt et al., 1991; Reid et al., 1990) or the Diphteria toxin A fragment (Dt-A) gene (Nada et al., 1993; Yagi et al.1990).
  • tk thymidine kinase
  • Dt-A Diphteria toxin A fragment
  • the positive selection marker is located within and exon of an sbgl, g34665, sbg2, g35017 or g35018 polynucleotide so as to interrupt the sequence encoding the sbgl, g34665, sbg2, g35017 or g35018 protein.
  • sbgl, g34665, sbg2, g35017 or g35018 polynucleotide so as to interrupt the sequence encoding the sbgl, g34665, sbg2, g35017 or g35018 protein.
  • the first and second nucleotide sequences (a) and (c) may be indifferently located within an sbgl, g34665, sbg2, g35017 or g35018 polynucleotide regulatory sequence, an intronic sequence, an exon sequence or a sequence containing both regulatory and/or intronic and/or exon sequences.
  • the size of the nucleotide sequence of (a) and (c) ranges from 1 to 50 kb, preferably from 1 to 10 kb, more preferably from 2 to 6 kb and most preferably from 2 to 4 kb. DNA Constructs Allowing Homologous Recombination: Cre-LoxP System.
  • the PI phage possesses a recombinase called Cre which interacts specifically with a 34 base pairs lox? site.
  • the loxP site is composed of two palindromic sequences of 13 bp separated by a 8 bp conserved sequence (Hoess et al., 1986).
  • the recombination by the Cre enzyme between two loxP sites having an identical orientation leads to the deletion of the DNA fragment.
  • the Cre-ZojcP system used in combination with a homologous recombination technique has been first described by Gu et al.(1993, 1994). Briefly, a nucleotide sequence of interest to be inserted in a targeted location of the genome harbors at least two loxP sites in the same orientation and located at the respective ends of a nucleotide sequence to be excised from the recombinant genome. The excision event requires the presence of the recombinase (Cre) enzyme within the nucleus of the recombinant cell host.
  • Re recombinase
  • the recombinase enzyme may be brought at the desired time either by (a) incubating the recombinant cell hosts in a culture medium containing this enzyme, by injecting the Cre enzyme directly into the desired cell, such as described by Araki et al.(1995), or by lipofection of the enzyme into the cells, such as described by Baubonis et al.(1993); (b) transfecting the cell host with a vector comprising the Cre coding sequence operably linked to a promoter functional in the recombinant cell host, which promoter being optionally inducible, said vector being introduced in the recombinant cell host, such as described by Gu et al.(1993) and Sauer et al.(1988); (c) introducing in the genome of the cell host a polynucleotide comprising the Cre coding sequence operably linked to a promoter functional in the recombinant cell host, which promoter is optionally inducible, and said polynucleotide being inserted
  • the vector containing the sequence to be inserted in an sbgl, g34665, sbg2, g35017 or g35018 gene sequence by homologous recombination is constructed in such a way that selectable markers are flanked by lox? sites of the same orientation, it is possible, by treatment by the Cre enzyme, to eliminate the selectable markers while leaving the sbgl, g34665, sbg2, g35017 or g35018 polynucleotide sequences of interest that have been inserted by an homologous recombination event.
  • a further preferred DNA construct of the invention comprises, from 5'-end to 3'-end: (a) a first nucleotide sequence that is comprised by an sbgl polynucleotide; (b) a nucleotide sequence comprising a polynucleotide encoding a positive selection marker, said nucleotide sequence comprising additionally two sequences defining a site recognized by a recombinase, such as a loxP site, the two sites being placed in the same orientation; and (c) a second nucleotide sequence comprising an sbgl polynucleotide, and is located on the genome downstream of the first sbgl polynucleotide sequence (a). Also encompassed are DNA construct prepared in an analogous manner using g34665, sbg2, g35017 or g35018 nucleotide sequences in place of the sbgl sequences described above.
  • sequences defining a site recognized by a recombinase are preferably located within the nucleotide sequence (b) at suitable locations bordering the nucleotide sequence for which the conditional excision is sought.
  • two loxP sites are located at each side of the positive selection marker sequence, in order to allow its excision at a desired time after the occurrence of the homologous recombination event.
  • the excision of the polynucleotide fragment bordered by the two sites recognized by a recombinase, preferably two loxP sites is performed at a desired time, due to the presence within the genome of the recombinant host cell of a sequence encoding the Cre enzyme operably linked to a promoter sequence, preferably an inducible promoter, more preferably a tissue-specific promoter sequence and most preferably a promoter sequence which is both inducible and tissue-specific, such as described by Gu et al.(1994).
  • a promoter sequence preferably an inducible promoter, more preferably a tissue-specific promoter sequence and most preferably a promoter sequence which is both inducible and tissue-specific, such as described by Gu et al.(1994).
  • the presence of the Cre enzyme within the genome of the recombinant cell host may result from the breeding of two transgenic animals, the first transgenic animal bearing the sbgl, g34665, sbg2, g35017 or g35018 polynucleotide -derived sequence of interest containing the loxP sites as described above and the second transgenic animal bearing the Cre coding sequence operably linked to a suitable promoter sequence, such as described by Gu et al.(1994).
  • Spatio-temporal control of the Cre enzyme expression may also be achieved with an adenovirus based vector that contains the Cre gene thus allowing infection of cells, or in vivo infection of organs, for delivery of the Cre enzyme, such as described by Anton and Graham (1995) and Kanegae et al.(1995).
  • the DNA constructs described above may be used to introduce a desired nucleotide sequence of the invention, preferably an sbgl, g34665, sbg2, g35017 or g35018 polynucleotide, and most preferably an altered copy an sbgl , g34665, sbg2, g35017 or g35018 polynucleotide sequence, within a predetermined location of the targeted genome, leading either to the generation of an altered copy of a targeted gene (knock-out homologous recombination) or to the replacement of a copy of the targeted gene by another copy sufficiently homologous to allow an homologous recombination event to occur (knock-in homologous recombination).
  • the DNA constructs described above may be used to introduce an sbgl, g34665, sbg2, g35017 or g35018 polynucleotide.
  • the antisense tools are chosen among the polynucleotides (15-200 bp long) that are complementary to the 5'end of an sbgl, g34665, sbg2, g35017 or g35018 polynucleotide mRNA.
  • a combination of different antisense polynucleotides complementary to different parts of the desired targeted gene are used.
  • the antisense polynucleotides of the invention have a 3' polyadenylation signal that has been replaced with a self-cleaving ribozyme sequence, such that RNA polymerase II transcripts are produced without poly(A) at their 3' ends, these antisense polynucleotides being incapable of export from the nucleus, such as described by Liu et al.(1994).
  • Probes and primers of the invention also include isolated, purified, or recombinant polynucleotides having at least 70, 75, 80, 85, 90, or 95% nucleotide identity with a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, 1000 or 2000 nucleotides of nucleotide positions 31 to 292651 and 292844 to 319608 of SEQ ID No. 1.
  • Preferred probes and primers of the invention also include isolated, purified, or recombinant polynucleotides comprising an sbgl, g34665, sbg2, g35017 or g35018 nucleotide sequence having at least 70, 75, 80, 85, 90, or 95% nucleotide identity with at least one sequence selected from the group consisting of the following nucleotide positions of SEQ ID No. 1 : (a) 290653 to 292652, 292653 to 296047, 292653 to 292841, 295555 to 296047,
  • probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35,
  • the invention also relates to nucleic acid probes characterized in that they hybridize specifically, under the stringent hybridization conditions defined above, with a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, 1000 or 2000 nucleotides of nucleotide positions 31 to 292651 and 292844 to 319608 of SEQ ID No. 1 , or a variant thereof or a sequence complementary thereto.
  • nucleic acid probes characterized in that they hybridize specifically, under the stringent hybridization conditions defined above, with a nucleic acid selected from the group consisting of nucleotide positions:
  • Tm melting temperature
  • the GC content in the probes of the invention usually ranges between 10 and 75 %, preferably between 35 and 60 %, and more preferably between 40 and 55 %.
  • a probe or a primer according to the invention may be between 8 and 2000 nucleotides in length, or is specified to be at least 12, 15, 18, 20, 25, 35, 40, 50, 60, 70, 80, 100, 250, 500 , 1000 nucleotides in length. More particularly, the length of these probes can range from 8, 10,
  • nucleotides preferably from 10 to 50, more preferably from 15 to 30 nucleotides.
  • Shorter probes tend to lack specificity for a target nucleic acid sequence and generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. Longer probes are expensive to produce and can sometimes self-hybridize to form hairpin structures.
  • the appropriate length for primers and probes under a particular set of assay conditions may be empirically determined by one of skill in the art.
  • the primers and probes can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences and direct chemical synthesis by a method such as the phosphodiester method of Narang et al.(1979), the phosphodiester method of Brown et al .( 1979), the diethylphosphoramidite method of Beaucage et al .( 1981 ) and the solid support method described in EP 0 707 592.
  • Detection probes are generally nucleic acid sequences or uncharged nucleic acid analogs such as, for example peptide nucleic acids which are disclosed in International Patent Application WO 92/20702, mo ⁇ holino analogs which are described in U.S. Patents Numbered 5,185,444; 5,034,506 and 5,142,047.
  • the probe may have to be rendered "non-extendable" in that additional dNTPs cannot be added to the probe.
  • analogs usually are non-extendable and nucleic acid probes can be rendered non-extendable by modifying the 3' end of the probe such that the hydroxyl group is no longer capable of participating in elongation.
  • any of the polynucleotides of the present invention can be labeled, if desired, by inco ⁇ orating a label detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • useful labels include radioactive substances ( 32 P, 35 S, 3 H, 125 I), fluorescent dyes (5-bromodesoxyuridin, fluorescein, acetylaminofluorene, digoxigenin) or biotin.
  • polynucleotides are labeled at their 3' and 5' ends. Examples of non-radioactive labeling of nucleic acid fragments are described in the French patent No. FR-7810975 or by Urdea et al (1988) or Sanchez-Pescador et al (1988).
  • the probes according to the present invention may have structural characteristics such that they allow the signal amplification, such structural characteristics being, for example, branched DNA probes as those described by Urdea et al. in 1991 or in the European patent No. EP 0 225 807 (Chiron).
  • a label can also be used to capture the primer, so as to facilitate the immobilization of either the primer or a primer extension product, such as amplified DNA, on a solid support.
  • a capture label is attached to the primers or probes and can be a specific binding member which forms a binding pair with the solid's phase reagent's specific binding member (e.g. biotin and streptavidin).
  • a polynucleotide or a probe may be employed to capture or to detect the target DNA.
  • the polynucleotides, primers or probes provided herein may, themselves, serve as the capture label.
  • a solid phase reagent's binding member is a nucleic acid sequence
  • it may be selected such that it binds a complementary portion of a primer or probe to thereby immobilize the primer or probe to the solid phase.
  • a polynucleotide probe itself serves as the binding member those skilled in the art will recognize that the probe will contain a sequence or "tail" that is not complementary to the target.
  • a polynucleotide primer itself serves as the capture label
  • at least a portion of the primer will be free to hybridize with a nucleic acid on a solid phase.
  • DNA Labeling techniques are well known to the skilled technician.
  • the probes of the present invention are useful for a number of pu ⁇ oses. They can be notably used in Southern hybridization to genomic DNA. The probes can also be used to detect PCR amplification products.
  • any of the polynucleotides, primers and probes of the present invention can be conveniently immobilized on a solid support.
  • Solid supports are known to those skilled in the art and include the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, sheep (or other animal) red blood cells, duracytes and others.
  • the solid support is not critical and can be selected by one skilled in the art. Thus, latex particles, microparticles, magnetic or nonmagnetic beads, membranes, plastic tubes, walls of microtiter wells, glass or silicon chips, sheep (or other suitable animal's) red blood cells and duracytes are all suitable examples. Suitable methods for immobilizing nucleic acids on solid phases include ionic, hydrophobic, covalent interactions and the like.
  • a solid support refers to any material which is insoluble, or can be made insoluble by a subsequent reaction. The solid support can be chosen for its intrinsic ability to attract and immobilize the capture reagent. Alternatively, the solid phase can retain an additional receptor which has the ability to attract and immobilize the capture reagent.
  • the additional receptor can include a charged substance that is oppositely charged with respect to the capture reagent itself or to a charged substance conjugated to the capture reagent.
  • the receptor molecule can be any specific binding member which is immobilized upon (attached to) the solid support and which has the ability to immobilize the capture reagent through a specific binding reaction. The receptor molecule enables the indirect binding of the capture reagent to a solid support material before the performance of the assay or during the performance of the assay.
  • the solid phase thus can be a plastic, derivatized plastic, magnetic or non-magnetic metal, glass or silicon surface of a test tube, microtiter well, sheet, bead, microparticle, chip, sheep (or other suitable animal's) red blood cells, duracytes and other configurations known to those of ordinary skill in the art.
  • the polynucleotides of the invention can be attached to or immobilized on a solid support individually or in groups of at least 2, 5, 8, 10, 12, 15, 20, or 25 distinct polynucleotides of the invention to a single solid support.
  • polynucleotides other than those of the invention may be attached to the same solid support as one or more polynucleotides of the invention.
  • the invention also comprises a method for detecting the presence of a nucleic acid comprising a nucleotide sequence selected from a group consisting of SEQ ID Nos. 1 to 26, 36 to 40 and 54 to 229, a fragment or a variant thereof or a complementary sequence thereto in a sample, said method comprising the following steps of: a) bringing into contact a nucleic acid probe or a plurality of nucleic acid probes which can hybridize with a nucleotide sequence included in a nucleic acid selected form the group consisting of the nucleotide sequences of SEQ ID Nos. 1 to 26, 36 to 40 and 54 to 229, a fragment or a variant thereof or a complementary sequence thereto and the sample to be assayed; and b) detecting the hybrid complex formed between the probe and a nucleic acid in the sample.
  • the invention further concerns a kit for detecting the presence of a nucleic acid comprising a nucleotide sequence selected from a group consisting of SEQ ID Nos. 1 to 26, 36 to 40 and 54 to 229, a fragment or a variant thereof or a complementary sequence thereto in a sample, said kit comprising: a) a nucleic acid probe or a plurality of nucleic acid probes which can hybridize with a nucleotide sequence included in a nucleic acid selected form the group consisting of the nucleotide sequences of SEQ ID Nos. 1 to 26, 36 to 40 and 54 to 229, a fragment or a variant thereof or a complementary sequence thereto; and b) optionally, the reagents necessary for performing the hybridization reaction.
  • a nucleic acid probe or a plurality of nucleic acid probes which can hybridize with a nucleotide sequence included in a nucleic acid selected form the group consisting of the nucleotide sequences of SEQ ID
  • said nucleic acid probe or the plurality of nucleic acid probes are labeled with a detectable molecule.
  • said nucleic acid probe or the plurality of nucleic acid probes has been immobilized on a substrate.
  • a substrate comprising a plurality of oligonucleotide primers or probes of the invention may be used either for detecting or amplifying targeted sequences in a nucleotide sequence of SEQ ID No. 1, more particularly in an sbgl, g34665, sbg2, g35017 or g35018 polynucleotide, or in genes comprising an sbgl, g34665, sbg2, g35017 or g35018 polynucleotide and may also be used for detecting mutations in the coding or in the non-coding sequences of an sbgl, g34665, sbg2, g35017 or g35018 nucleic acid sequence, or genes comprising an sbgl, g34665, sbg2, g35017 or g35018 nucleic acid sequence.
  • probes are specifically designed to have a nucleotide sequence allowing their hybridization to the genes that carry known mutations (either by deletion, insertion or substitution of one or several nucleotides).
  • known mutations in an sbgl, g34665, sbg2, g35017 or g35018 polynucleotide it is meant, mutations in an sbgl, g34665, sbg2, g35017 or g35018 polynucleotide that have been identified according; the technique used by Huang et al.(1996) or Samson et al.(1996), for example, may be used to identify such mutations.
  • nucleic acid target of length L is scanned for mutations with a tiled array containing 4L probes, the whole probe set containing all the possible mutations in the known wild reference sequence.
  • the hybridization signals of the 15- mer probe set tiled array are perturbed by a single base change in the target sequence.
  • the invention concerns an array of nucleic acid molecules comprising at least one polynucleotide described above as probes and primers.
  • the invention concerns an array of nucleic acid comprising at least two polynucleotides described above as probes and primers.
  • polypeptides are used herein to embrace all of the proteins and polypeptides encoded by the respective sbgl, g34665, sbg2, g35017 and g35018 polypeptides of the present invention.
  • Forming part of the invention are polypeptides encoded by the polynucleotides of the invention, as well as fusion polypeptides comprising such polypeptides.
  • the invention embodies proteins from humans, mammals, primates, non-human primates, and includes isolated or purified sbgl proteins consisting, consisting essentially, or comprising the sequence of SEQ ID Nos 27 to 35, isolated or purified g34665, g35017 and sbg2 proteins encoded by the g34665, g35017 and sbg2 polynucleotide sequence of SEQ ID No 1, and isolated or purified g35018 proteins consisting, consisting essentially, or comprising the sequence of SEQ ID Nos 41 to 43.
  • sbgl, g34665, sbg2, g35017 and g35018 proteins of the invention also comprise naturally-occurring variants of the amino acid sequence of the respective human sbgl, g34665, sbg2, g35017 and g35018 proteins.
  • the present invention embodies isolated, purified, and recombinant polypeptides comprising a contiguous span of at least 4 amino acids, preferably at least 6, more preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids, to the extent that said span is consistent with the length of a particular SEQ ID, of SEQ ID Nos 27 to 35 and 41 to 43.
  • the contiguous stretch of amino acids comprises the site of a mutation or functional mutation, including a deletion, addition, swap or truncation of the amino acids in an sbgl, g34665, sbg2, g35017 and g35018 protein sequence.
  • the invention also embodies isolated, purified, and recombinant sbgl polypeptides comprising a contiguous span of at least 4 amino acids, preferably at least 6 or at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID Nos 27 to 35, wherein said contiguous span comprises an amino acid variation according to Table 5e.
  • the polypeptide of SEQ ID No 32 contains a protease cleavage site at amino acid positions 63 to 64; the polypeptide of SEQ ID No 33 contains a protease cleavage site at amino acid positions 54 to 55 and 57 to 58; the polypeptide of SEQ ID No 34 contains a protease cleavage site at amino acid positions 63 to 64 and 122 to 123; and the polypeptide of SEQ ID No 35 contains a protease cleavage site at amino acid positions 62 to 63 and 63 to 64.
  • sbgl polypeptides of SEQ ID Nos 30, 32 and 34 contain cysteine residues predicted to be capable of forming a disulfide bridge at amino acid positions 82 and 104 of SEQ ID No 30, amino acid positions 82 and 106 and SEQ ID No 32, and amino acid positions 132 and 142 of SEQ ID No 34.
  • the invention comprises isolated, purified, and recombinant sbgl peptides comprising a contiguous span of at least 4 amino acids, preferably at least 6 or at least 8 to 10 amino acids, more preferably at least 12 or 15 amino acids of an amino acid position range selected from the group consisting of amino acid positions: 1 to 63 and 64 to 102 of SEQ ID No 29; 1 to 64, 65 to 111 and 112 to 1 19 of SEQ ID No 30; 1 to 64 and 65 to 126 of SEQ ID No 32; 1 to 64, 65 to 123 and 124 to 153 of SEQ ID No 34; and 1 to 61 and 65 to 106 of SEQ ID No 35.
  • the invention further embodies sbgl, g34665, sbg2, g35017 and g35018 polypeptides, including isolated and recombinant polypeptides, encoded respectively by sbgl, g34665, sbg2, g35017 and g35018 polynucleotides consisting, consisting essentially, or comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or 500 nucleotides, to the extent that the length of said span is consistent with the nucleotide position range, of SEQ ID No 1, wherein said contiguous span comprises at least 1, 2, 3, 4, 5, 7 or 10 of the following nucleotide positions of SEQ ID No 1 :
  • the present invention further embodies isolated, purified, and recombinant polypeptides encoded by an sbgl polynucleotide or gene comprising at least one sbgl nucleotide sequence selected from the group consisting of the following sbgl exons: MSI, Ml, M692, M862, MS2, Ml 069, Ml 090, Ml 117, N , N2, Nbis, O, OI, 02, Obis, P, X, Ql, Q, Qbis, R and Rbis.
  • proteins of the invention is extracted from cells or tissues of humans or non- human animals.
  • Methods for purifying proteins include the use of detergents or chaotropic agents to disrupt particles followed by differential extraction and separation of the polypeptides by ion exchange chromatography, affinity chromatography, sedimentation according to density, and gel electrophoresis.
  • any sbgl, g34665, sbg2, g35017 or g35018 cDNA or fragment thereof, including the respective cDNA sequences of SEQ ID Nos 2 to 26 and 36 to 40 is used to express sbgl, g34665, sbg2, g35017 or g35018 proteins and polypeptides.
  • the nucleic acid encoding the sbgl, g34665, sbg2, g35017 or g35018 protein or polypeptide to be expressed is operably linked to a promoter in an expression vector using conventional cloning technology.
  • the purified fragment obtained from the resulting PCR reaction is digested with Pstl, blunt ended with an exonuclease, digested with Bgl II, purified and ligated to pXTl, now containing a poly A signal and digested with Bgl ⁇ .
  • the ligated product is transfected into mouse NIH 3T3 cells using Lipofectin (Life Technologies).
  • nucleic acids encoding the sbgl, g34665, sbg2, g35017 or g35018 protein or a portion thereof is cloned into pED6dpc2 (Genetics Institute, Cambridge, MA).
  • the resulting pED6dpc2 constructs is transfected into a suitable host cell, such as COS 1 cells. Methotrexate resistant cells are selected and expanded.
  • the above procedures may also be used to express a mutant sbgl, g34665, sbg2, g35017 or g35018 protein responsible for a detectable phenotype or a portion thereof.
  • the expressed proteins are purified using conventional purification techniques such as ammonium sulfate precipitation or chromatographic separation based on size or charge.
  • the protein encoded by the nucleic acid insert may also be purified using standard immunochromatography techniques.
  • a solution containing the expressed sbgl, g34665, sbg2, g35017 or g35018 protein or portion thereof, such as a cell extract, is applied to a column having antibodies against the sbgl, g34665, sbg2, g35017 or g35018 protein or portion thereof is attached to the chromatography matrix.
  • the expressed protein is allowed to bind the immunochromatography column. Thereafter, the column is washed to remove non-specifically bound proteins. The specifically bound expressed protein is then released from the column and recovered using standard techniques.
  • the band will have the mobility expected for the sbgl, g34665, sbg2, g35017 or g35018 protein or portion thereof.
  • the band may have a mobility different than that expected as a result of modifications such as glycosylation, ubiquitination, or enzymatic cleavage.
  • Antibodies capable of specifically recognizing the expressed sbgl, g34665, sbg2, g35017 or g35018 protein or a portion thereof are described below.
  • the nucleic acids encoding the sbgl, g34665, sbg2, g35017 or g35018 protein or a portion thereof is inco ⁇ orated into expression vectors designed for use in purification schemes employing chimeric polypeptides.
  • the nucleic acid encoding the sbgl, g34665, sbg2, g35017 or g35018 protein or a portion thereof is inserted in frame with the gene encoding the other half of the chimera.
  • the other half of the chimera is ⁇ - globin or a nickel binding polypeptide encoding sequence.
  • a chromatography matrix having antibody to ⁇ -globin or nickel attached thereto is then used to purify the chimeric protein.
  • Protease cleavage sites is engineered between the ⁇ -globin gene or the nickel binding polypeptide and the sbgl, g34665, sbg2, g35017 or g35018 protein or portion thereof.
  • the two polypeptides of the chimera is separated from one another by protease digestion.
  • pSG5 which encodes rabbit ⁇ -globin.
  • Intron II of the rabbit ⁇ -globin gene facilitates splicing of the expressed transcript, and the polyadenylation signal inco ⁇ orated into the construct increases the level of expression.
  • Standard methods are published in methods texts such as Davis et al., (1986) and many of the methods are available from Stratagene, Life Technologies, Inc., or Promega.
  • Polypeptide may additionally be produced from the construct using in vitro translation systems such as the In vitro ExpressTM Translation Kit (Stratagene).
  • an antibody composition to specifically bind to a variant sbgl, g34665, sbg2, g35017 or g35018 protein it must demonstrate at least a 5%, 10%, 15%, 20%, 25%, 50%, or 100% greater binding affinity for the respective full length variant sbgl, g34665, sbg2, g35017 or g35018 protein than for the respective reference sbgl, g34665, sbg2, g35017 or g35018 full length protein in an ELISA, RIA, or other antibody-based binding assay.
  • One antibody composition of the invention is capable of specifically binding or specifically binds to the respective sbgl org35018 proteins of SEQ ID Nos 27 to 35 and 41 to 43.
  • the invention also concerns a purified or isolated antibody capable of specifically binding to a mutated sbgl, g34665, sbg2, g35017 or g35018 protein or to a fragment or variant thereof comprising an epitope of the mutated sbgl, g34665, sbg2, g35017 or g35018 protein.
  • the present invention concerns an antibody capable of binding to a polypeptide comprising at least 10 consecutive amino acids of an sbgl, g34665, sbg2, g35017 or g35018 protein and including at least one of the amino acids which can be encoded by the trait causing mutations.
  • Non-human animals and more particularly non-human mammals and non-human primates, whether wild-type or transgenic, which express a different species of sbgl , g34665, sbg2, g35017 or g35018 than the one to which antibody binding is desired, and animals which do not express sbgl, g34665, sbg2, g35017 or g35018 (i.e. an sbgl, g34665, sbg2, g35017 or g35018 knock out animal as described in herein) are particularly useful for preparing antibodies.
  • the humoral immune system of animals which produce a species of sbgl, g34665, sbg2, g35017 or g35018 that resembles the antigenic sequence will preferentially recognize the differences between the animal's native sbgl, g34665, sbg2, g35017 or g35018 species and the antigen sequence, and produce antibodies to these unique sites in the antigen sequence.
  • Such a technique will be particularly useful in obtaining antibodies that specifically bind to any one of the sbgl, g34665, sbg2, g35017 or g35018 proteins.
  • Antibody preparations prepared according to either protocol are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample.
  • the invention is also directed to a method for detecting specifically the presence of an sbgl, g34665, sbg2, g35017 or g35018 polypeptide according to the invention in a biological sample, said method comprising the following steps: a) bringing into contact the biological sample with a polyclonal or monoclonal antibody that specifically binds an sbgl, g34665, sbg2, g35017 or g35018 polypeptide, or to a peptide fragment or variant thereof; and b) detecting the antigen-antibody complex formed.
  • the invention also concerns a diagnostic kit for detecting in vitro the presence of an sbgl, g34665, sbg2, g35017 or g35018 polypeptide according to the present invention in a biological sample, wherein said kit comprises: a) a polyclonal or monoclonal antibody that specifically binds an sbgl, g34665, sbg2, g35017 or g35018 polypeptide, or to a peptide fragment or variant thereof, optionally labeled; b) a reagent allowing the detection of the antigen-antibody complexes formed, said reagent carrying optionally a label, or being able to be recognized itself by a labeled reagent, more particularly in the case when the above-mentioned monoclonal or polyclonal antibody is not labeled by itself.
  • single nucleotide polymo ⁇ hism occur at a greater frequency and with greater uniformity than RFLP or VNTR markers which means that there is a greater probability that such a marker will be found in close proximity to a genetic locus of interest.
  • Single nucleotide polymo ⁇ hisms are less variable than VNTR markers but are mutationally more stable.
  • the biallelic markers of the present invention offer the possibility of rapid, high-throughput genotyping of a large number of individuals.
  • Biallelic markers are densely spaced in the genome, sufficiently informative and can be assayed in large numbers. The combined effects of these advantages make biallelic markers extremely valuable in genetic studies.
  • Biallelic markers can be used in linkage studies in families, in allele sharing methods, in linkage disequilibrium studies in populations, in association studies of case-control populations.
  • An important aspect of the present invention is that biallelic markers allow association studies to be performed to identify genes involved in complex traits. Association studies examine the frequency of marker alleles in unrelated case- and control-populations and are generally employed in the detection of polygenic or sporadic traits. Association studies may be conducted within the general population and are not limited to studies performed on related individuals in affected families (linkage studies).
  • the invention concerns biallelic markers associated with schizophrenia.
  • the invention comprises chromosome 13q31-q33-related biallelic markers, region D-related biallelic markers, sbgl-related biallelic markers, g34665-related biallelic markers, sbg2-related biallelic markers, g35017-related biallelic markers and g35018-related biallelic markers.
  • the markers and polymo ⁇ hisms are generally referred to herein as Al, A2, A3 and so on.
  • the polymo ⁇ hisms and biallelic markers of the invention comprise the biallelic markers designated Al to A360 in Table 6b.
  • the polymo ⁇ hisms of the invention also comprise the polymo ⁇ hisms designated A361 to A489 in Table 6c. Also included are biallelic markers in linkage disequilibrium with the biallelic markers of the invention.
  • Region D Details of chromosome 13q31-q33-related biallelic markers on the subregions designated Region D including subregions thereof designated Regions DI, D2 ,D3 and D4, and adjacent regions referred to as Region E and Region G are shown below and in Tables 6B and 6c. Regions D, G and E of the chromosome 13q31-q33 locus are also shown in Figure 2. References to the corresponding SEQ ID number, to alternative marker designations, and positions of the sequence features within the SEQ ID are given in Tables 6b and 6c for biallelic markers Al to A242 and 361 to 489 located in Region D3 and D4.
  • biallelic markers from the group designated A243 to A360 in Tables 6b and 6c are located in Regions DI, D2, G and E.
  • the relative positions of biallelic markers on Region G and E are further detailed below in Table 5g; the relative positions of biallelic markers on Region DI and D2 are further detailed below in Table 5h.
  • polynucleotides of the present invention are not limited to having the exact flanking sequences surrounding the polymo ⁇ hic bases which, are enumerated in the Sequence Listing. Rather, it will be appreciated that the flanking sequences surrounding the biallelic markers and other polymo ⁇ hisms of the invention, or any of the primers of probes of the invention which, are more distant from the markers, may be lengthened or shortened to any extent compatible with their intended use and the present invention specifically contemplates such sequences. It will be appreciated that the polynucleotides of SEQ ID Nos. 1 to 26, 36 to 40 and 54 to 229 may be of any length compatible with their intended use.
  • flanking regions outside of the contiguous span need not be homologous to native flanking sequences which actually occur in human subjects.
  • the addition of any nucleotide sequence, which is compatible with the nucleotides intended use is specifically contemplated.
  • the contiguous span may optionally include the biallelic markers of the invention in said sequence.
  • Biallelic markers generally comprise a polymo ⁇ hism at one single base position. Each biallelic marker therefore corresponds to two forms of a polynucleotide sequence which, when compared with one another, present a nucleotide modification at one position.
  • the nucleotide modification involves the substitution of one nucleotide for another.
  • allele 1 or allele 2 of the biallelic markers disclosed in Table 6b may be specified as being present at the biallelic marker of the invention.
  • the contiguous span may optionally include a nucleotide at a polymo ⁇ hism position described in Table 6c, including single nucleotide substitutions, deletions as well as multiple nucleotide deletions.
  • the polymo ⁇ hisms of Table 6c have not been validated as biallelic markers, but are expected to be mostly biallelic and may also be referred to as biallelic markers herein.
  • allele 1 or allele 2 of the polymo ⁇ hisms of Table 6c may be specified as being present at the polymo ⁇ hism of the invention.
  • Preferred polynucleotides may consist of, consist essentially of, or comprise a contiguous span of nucleotides of a sequence from SEQ ID Nos. 1 to 26, 36 to 40 and 54 to 229 as well as sequences which are complementary thereto.
  • the "contiguous span” may be at least 8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 70, 80, 100, 250, 500, 1000 or 2000 nucleotides in length, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID.
  • a preferred probe or primer comprises a nucleic acid comprising a polynucleotide selected from the group of the nucleotide sequences of PI to P360 and the complementary sequence thereto, Bl to B229, CI to C229, DI to D360, El to E360.
  • the invention encompasses polynucleotides for use in hybridization assays, sequencing assays, and enzyme-based mismatch detection assays for determining the identity of the nucleotide at a chromosome 13q31 -q33-related biallelic marker, region D-related biallelic marker, or sbgl-, g34665-, sbg2-, g35017- or g35018 -related biallelic marker in any of SEQ ID Nos.
  • WO 95/11995 which describe methods for forming oligonucleotide arrays through techniques such as light-directed synthesis technique.
  • further presentation strategies were developed to order and display the oligonucleotide arrays on the chips in an attempt to maximize hybridization patterns and sequence information. Examples of such presentation strategies are disclosed in PCT Publications WO 94/12305, WO 94/11530, WO 97/29212 and WO 97/31256.
  • the oligonucleotide array may comprise at least one of the sequences selecting from the group consisting of SEQ ID Nos.
  • a further object of the invention relates to an array of nucleic acid sequences comprising either at least one of the sequences selected from the group consisting of PI to P360, Bl to B229, CI to C229, DI to D360 El to E360 or the sequences complementary thereto or a fragment thereof of at least 8, 10, 12, 15, 18, 20, 25, 30, or 40 consecutive nucleotides thereof, or at least one sequence comprising at least 1, 2, 3, 4, 5, 10, 20 biallelic markers selected from the group consisting of Al to A489 or the complements thereof.
  • the invention also pertains to an array of nucleic acid sequences comprising either at least 1, 2, 3, 4, 5, 10, 20 of the sequences selected from the group consisting of PI to P360, Bl to B229, CI to C229, DI to D360, El to E360 or the sequences complementary thereto or a fragment thereof of at least 8 consecutive nucleotides thereof, or at least two sequences comprising a biallelic marker selected from the group consisting of Al to A360 or the complements thereto.
  • a biallelic marker may optionally comprise:
  • a biallelic marker selected from the group consisting of sbgl -related markers A85 to A219, or more preferably a biallelic marker selected from the group consisting of sbgl -related markers A85 to A94, A96 to A106, A108 to Al 12, Al 14 to A177, A179 to A197 and A199 to A219;
  • a biallelic marker selected from the group consisting of g34665-related markers A230 to A236;
  • a biallelic marker selected from the group consisting of sbg2-related markers A79 to A99; (d) the g35017-related marker A41;
  • a biallelic marker selected from the group consisting of g35018-related markers Al to A39;
  • a biallelic marker selected from the group consisting of A239, A227, A198, A228, A223, A107, A218, A270, A75, A62, A65 and A70;
  • a biallelic marker selected from the group consisting of A48, A60, A61 , A62, A65,
  • a biallelic marker selected from the group consisting of A304, A307, A305, A298, A292, A293, A291, A287, A286, A288, A289, A290, 99- A295 A299.
  • A241, A239, A228, A227, A223, A221, A218, A198, A178, 99-24649/186 A108, A107, A80, A75, A70, A65, and
  • a biallelic marker selected from the group consisting of A304, A307, A305, A298, A292, A293, A291, A287, A286, A288, A289, A290, A295 A299, A241, A239, A228, A227, A223, A221, A218, A198, A178, A108, A107, A80, A76, A75, A70, A65, A62, A61, A60 A48.
  • a Region D- or chromosome 13q31-q33-related biallelic marker may be selected from the group consisting of Al to A69, A71 to A74, A76 to A94, A96 to A106, A108 to Al 12, Al 14 to A177, A179 to A197, A199 to A222, A224 to A242, A250 to A251, A259, A269 to A270, A278, A285 to A299, A303 to A307, A330, A334 to A335, A346 to 357 and 361 to 489.
  • a chromosome 13q31-q33-related biallelic marker may be selected from the group consisting of A 1 to A69, A71 to A74, A76 to A94, A96 to Al 06, A 108 to Al 12, Al 14 to A177, A179 to A197, A199 to A222, A224 to A246, A250, A251, A253, A255, A259, A266, A268 to A232 and A328 to A489.
  • a set of said Region D-related biallelic markers or chromosome 13q31-q33-related biallelic markers may comprise at least 1, 2, 3, 4, 5, 10, 20, 40, 50, 100 or 200 of said biallelic markers, respectively.
  • any of the compositions of methods described herein may specifically exclude at least 1, 2, 3, 4, 5, 10, 20 biallelic markers, or all of the biallelic markers selected from the group consisting of: A70, A75, A95, A107, Al 13, A178, A198, A223, A247 to A249, A252, A254, A256 to A258, A260 to A265, A267, A324 to A328.
  • a set of chromosome 13q31-q33-related biallelic markers, Region D-related biallelic markers, or sbgl-, g34665-, sbg2-, g35017- or g35018 -related biallelic markers may comprise at least 1, 2, 3, 4, 5, 10, 20, 40, 50, 100 or 200 of said biallelic markers.
  • Any of a variety of methods can be used to screen a genomic fragment for single nucleotide polymorphisms such as differential hybridization with oligonucleotide probes, detection of changes in the mobility measured by gel electrophoresis or direct sequencing of the amplified nucleic acid.
  • a preferred method for identifying biallelic markers involves comparative sequencing of genomic DNA fragments from an appropriate number of unrelated individuals.
  • DNA samples from unrelated individuals are pooled together, following which the genomic DNA of interest is amplified and sequenced.
  • the nucleotide sequences thus obtained are then analyzed to identify significant polymo ⁇ hisms.
  • One of the major advantages of this method resides in the fact that the pooling of the DNA samples substantially reduces the number of DNA amplification reactions and sequencing reactions, which must be carried out.
  • this method is sufficiently sensitive so that a biallelic marker obtained thereby usually demonstrates a sufficient frequency of its less common allele to be useful in conducting association studies.
  • the frequency of the least common allele of a biallelic marker identified by this method is at least 10%.
  • the genomic DNA samples from which the biallelic markers of the present invention are generated are preferably obtained from unrelated individuals corresponding to a heterogeneous population of known ethnic background.
  • the number of individuals from whom DNA samples are obtained can vary substantially, preferably from about 10 to about 1000, more preferably from about 50 to about 200 individuals.
  • DNA samples are collected from at least about 100 individuals in order to have sufficient polymo ⁇ hic diversity in a given population to identify as many markers as possible and to generate statistically significant results.
  • test samples include biological samples, which can be tested by the methods of the present invention described herein, and include human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk, white blood cells, myelomas and the like; biological fluids such as cell culture supematants; fixed tissue specimens including tumor and non-tumor tissue and lymph node tissues; bone marrow aspirates and fixed cell specimens.
  • the preferred source of genomic DNA used in the present invention is from peripheral venous blood of each donor. Techniques to prepare genomic DNA from biological samples are well known to the skilled technician. Details of a preferred embodiment are provided in Example 1. The person skilled in the art can choose to amplify pooled or unpooled DNA samples.
  • biallelic markers are identified using genomic sequence information generated by the inventors. Genomic DNA fragments, such as the inserts of the BAC clones described above, are sequenced and used to design primers for the amplification of 500 bp fragments. These 500 bp fragments are amplified from genomic DNA and are scanned for biallelic markers. Primers may be designed using the OSP software (Hillier L. and Green P., 1991). All primers may contain, upstream of the specific target bases, a common oligonucleotide tail that serves as a sequencing primer. Those skilled in the art are familiar with primer extensions, which can be used for these pu ⁇ oses.
  • the amplification products generated as described above, are then sequenced using any method known and available to the skilled technician.
  • Methods for sequencing DNA using either the dideoxy-mediated method (Sanger method) or the Maxam-Gilbert method are widely known to those of ordinary skill in the art. Such methods are for example disclosed in Maniatis et al. (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Second Edition, 1989). Alternative approaches include hybridization to high-density DNA probe arrays as described in Chee et al. (Science 274, 610, 1996).
  • the amplified DNA is subjected to automated dideoxy terminator sequencing reactions using a dye-primer cycle sequencing protocol. The products of the sequencing reactions are run on sequencing gels and the sequences are determined using gel image analysis.
  • the polymorphism search is based on the presence of superimposed peaks in the electrophoresis pattern resulting from different bases occurring at the same position. Because each dideoxy terminator is labeled with a different fluorescent molecule, the two peaks corresponding to a biallelic site present distinct colors corresponding to two different nucleotides at the same position on the sequence. However, the presence of two peaks can be an artifact due to background noise. To exclude such an artifact, the two DNA strands are sequenced and a comparison between the peaks is carried out. In order to be registered as a polymorphic sequence, the polymo ⁇ hism has to be detected on both strands.
  • the above procedure permits those amplification products, which contain biallelic markers to be identified.
  • the detection limit for the frequency of biallelic polymo ⁇ hisms detected by sequencing pools of 100 individuals is approximately 0.1 for the minor allele, as verified by sequencing pools of known allelic frequencies.
  • more than 90% of the biallelic polymo ⁇ hisms detected by the pooling method have a frequency for the minor allele higher than 0.25. Therefore, the biallelic markers selected by this method have a frequency of at least 0.1 for the minor allele and less than 0.9 for the major allele.
  • At least 0.2 for the minor allele and less than 0.8 for the major allele Preferably at least 0.2 for the minor allele and less than 0.8 for the major allele, more preferably at least 0.3 for the minor allele and less than 0.7 for the major allele, thus a heterozygosity rate higher than 0.18, preferably higher than 0.32, more preferably higher than 0.42.
  • biallelic markers are detected by sequencing individual DNA samples, the frequency of the minor allele of such a biallelic marker may be less than 0.1.
  • the group contains at least three individuals, more preferably the group contains five or six individuals, so that a single validation test will be more likely to result in the validation of more of the biallelic markers that are being tested. It should be noted, however, that when the validation test is performed on a small group it may result in a false negative result if as a result of sampling error none of the individuals tested carries one of the two alleles. Thus, the validation process is less useful in demonstrating that a particular initial result is an artifact, than it is at demonstrating that there is a bonafide biallelic marker at a particular position in a sequence. All of the genotyping, haplotyping, association, and interaction study methods of the invention may optionally be performed solely with validated biallelic markers.
  • the validated biallelic markers are further evaluated for their usefulness as genetic markers by determining the frequency of the least common allele at the biallelic marker site.
  • the determination of the least common allele is accomplished by genotyping a group of individuals by a method of the invention and demonstrating that both alleles are present.
  • This determination of frequency by genotyping step may be performed on individual samples derived from each individual in the group or by genotyping a pooled sample derived from more than one individual.
  • the group must be large enough to be representative of the population as a whole.
  • the group contains at least 20 individuals, more preferably the group contains at least 50 individuals, most preferably the group contains at least 100 individuals. Of course the larger the group the greater the accuracy of the frequency determination because of reduced sampling error.
  • a biallelic marker wherein the frequency of the less common allele is 30% or more is termed a "high quality biallelic marker.” All of the genotyping, haplotyping, association, and interaction study methods of the invention may optionally be performed solely with high quality biallelic markers.
  • Methods of Genotyping An Individual For Biallelic Markers are provided to genotype a biological sample for one or more biallelic markers of the present invention, all of which may be performed in vitro.
  • Such methods of genotyping comprise determining the identity of a nucleotide at an biallelic marker of the invention by any method known in the art. These methods find use in genotyping case-control populations in association studies as well as individuals in the context of detection of alleles of biallelic markers which, are known to be associated with a given trait, in which case both copies of the biallelic marker present in individual's genome are determined so that an individual may be classified as homozygous or heterozygous for a particular allele.
  • These genotyping methods can be performed nucleic acid samples derived from a single individual or pooled DNA samples.
  • amplification of DNA fragments comprising biallelic markers may be used in various methods and for various pu ⁇ oses and is not restricted to genotyping. Nevertheless, many genotyping methods, although not all, require the previous amplification of the DNA region carrying the biallelic marker of interest. Such methods specifically increase the concentration or total number of sequences that span the biallelic marker or include that site and sequences located either distal or proximal to it. Diagnostic assays may also rely on amplification of DNA segments carrying a biallelic marker of the present invention. Amplification of DNA may be achieved by any method known in the art. The established PCR (polymerase chain reaction) method or by developments thereof or alternatives.
  • Methods well-known to those skilled in the art that can be used to detect biallelic polymo ⁇ hisms include methods such as, conventional dot blot analyzes, single strand conformational polymo ⁇ hism analysis (SSCP) described by Orita et al. (1989), denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis, mismatch cleavage detection, and other conventional techniques as described in Sheffield, V.C. et al. (1991), White et al. (1992), Grompe, M. et al. (1989) and Grompe, M. (1993).
  • SSCP single strand conformational polymo ⁇ hism analysis
  • DGGE denaturing gradient gel electrophoresis
  • heteroduplex analysis mismatch cleavage detection
  • mismatch cleavage detection and other conventional techniques as described in Sheffield, V.C. et al. (1991), White et al. (1992), Grompe, M. et al. (1989) and Grompe
  • the detection of radiolabeled ddNTPs can be achieved through scintillation-based techniques.
  • the detection of fluorescein-linked ddNTPs can be based on the binding of antifluorescein antibody conjugated with alkaline phosphatase, followed by incubation with a chromogenic substrate (such as 7-nitrophenyl phosphate).
  • reporter-detection pairs include: ddNTP linked to dinitrophenyl (DNP) and anti-DNP alkaline phosphatase conjugate (Harju et al., 1993) or biotinylated ddNTP and horseradish peroxidase-conjugated streptavidin with o- phenylenediamine as a substrate (WO 92/15712).
  • DNP dinitrophenyl
  • biotinylated ddNTP and horseradish peroxidase-conjugated streptavidin with o- phenylenediamine as a substrate WO 92/15712
  • Nyren et al. (1993) described a method relying on the detection of DNA polymerase activity by an enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA).
  • ELIDA enzymatic luminometric inorganic pyrophosphate detection assay
  • the present invention provides polynucleotides and methods to genotype one or more biallelic markers of the present invention by performing a microsequencing assay.
  • microsequencing primers include those being featured Table 6d. It will be appreciated that the microsequencing primers listed in Table 6d are merely exemplary and that, any primer having a 3' end immediately adjacent to a polymo ⁇ hic nucleotide may be used. Similarly, it will be appreciated that microsequencing analysis may be performed for any biallelic marker or any combination of biallelic markers of the present invention.
  • One aspect of the present invention is a solid support which includes one or more microsequencing primers listed in Table 6d, or fragments comprising at least 8, at least 12, at least 15, or at least 20 consecutive nucleotides thereof and having a 3' terminus immediately upstream of the corresponding biallelic marker, for determining the identity of a nucleotide at biallelic marker site.
  • the present invention provides polynucleotides and methods to determine the allele of one or more biallelic markers of the present invention in a biological sample, by mismatch detection assays based on polymerases and/or ligases. These assays are based on the specificity of polymerases and ligases. Polymerization reactions places particularly stringent requirements on correct base pairing of the 3' end of the amplification primer and the joining of two oligonucleotides hybridized to a target DNA sequence is quite sensitive to mismatches close to the ligation site, especially at the 3' end.
  • the terms "enzyme based mismatch detection assay” are used herein to refer to any method of determining the allele of a biallelic marker based on the specificity of ligases and polymerases. Preferred methods are described below.
  • OLA Oligonucleotide Ligation Assay
  • OLA uses two oligonucleotides which are designed to be capable of hybridizing to abutting sequences of a single strand of a target molecules.
  • One of the oligonucleotides is biotinylated, and the other is detectably labeled. If the precise complementary sequence is found in a target molecule, the oligonucleotides will hybridize such that their termini abut, and create a ligation substrate that can be captured and detected.
  • OLA is capable of detecting biallelic markers and may be advantageously combined with PCR as described by Nickerson D.A. et al. (1990). In this method, PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA.
  • LCR ligase chain reaction
  • GLCR Gap LCR
  • LCR uses two pairs of probes to exponentially amplify a specific target. The sequences of each pair of oligonucleotides, is selected to permit the pair to hybridize to abutting sequences of the same strand of the target. Such hybridization forms a substrate for a template- dependant ligase.
  • LCR can be performed with oligonucleotides having the proximal and distal sequences of the same strand of a biallelic marker site.
  • Ligase/Polymerase-mediated Genetic Bit Analysis is another method for determining the identify of a nucleotide at a preselected site in a nucleic acid molecule (WO 95/21271).
  • This method involves the inco ⁇ oration of a nucleoside triphosphate that is complementary to the nucleotide present at the preselected site onto the terminus of a primer molecule, and their subsequent ligation to a second oligonucleotide.
  • the reaction is monitored by detecting a specific label attached to the reaction's solid phase or by detection in solution.
  • Hybridization assay methods A preferred method of determining the identity of the nucleotide present at a biallelic marker site involves nucleic acid hybridization.
  • hybridization probes which can be conveniently used in such reactions, preferably include the probes defined herein.
  • Any hybridization assay may be used including Southern hybridization, Northern hybridization, dot blot hybridization and solid-phase hybridization (see Sambrook et al., Molecular Cloning - A Laboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y., 1989).
  • the hybridized probes are detectable by autoradiography.
  • procedures using conditions of intermediate stringency are as follows: Filters containing DNA are prehybridized, and then hybridized at a temperature of 60°C in the presence of a 5 x SSC buffer and labeled probe.
  • wash steps may be employed to wash away excess target DNA or probe.
  • Standard heterogeneous assay formats are suitable for detecting the hybrids using the labels present on the primers and probes.
  • Two recently developed assays allow hybridization-based allele discrimination with no need for separations or washes (see Landegren U. et al.,1998).
  • the TaqMan assay takes advantage of the 5' nuclease activity of Taq DNA polymerase to digest a DNA probe annealed specifically to the accumulating amplification product.
  • TaqMan probes are labeled with a donor-acceptor dye pair that interacts via fluorescence energy transfer.
  • Chips of various formats for use in detecting biallelic polymo ⁇ hisms can be produced on a customized basis by Affymetrix (GeneChipTM), Hyseq (HyChip and HyGnostics), and Protogene Laboratories. In general, these methods employ arrays of oligonucleotide probes that are complementary to target nucleic acid sequence segments from an individual which, target sequences include a polymo ⁇ hic marker.
  • the array is tiled to include a number of detection blocks, each detection block being specific for a specific biallelic marker or a set of biallelic markers.
  • a detection block may be tiled to include a number of probes, which span the sequence segment that includes a specific polymorphism. To ensure probes that are complementary to each allele, the probes are synthesized in pairs differing at the biallelic marker. In addition to the probes differing at the polymo ⁇ hic base, monosubstituted probes are also generally tiled within the detection block.
  • the chips may comprise an array of nucleic acid sequences of fragments of about 15 nucleotides in length.
  • the chip may comprise an array including at least one of the sequences selected from the group consisting of SEQ ID
  • Another technique which may be used to analyze polymo ⁇ hisms, includes multicomponent integrated systems, which miniaturize and compartmentalize processes such as PCR and capillary electrophoresis reactions in a single functional device.
  • multicomponent integrated systems which miniaturize and compartmentalize processes such as PCR and capillary electrophoresis reactions in a single functional device.
  • An example of such technique is disclosed in US patent 5,589,136, which describes the integration of PCR amplification and capillary electrophoresis in chips.
  • microfluidic systems can be envisaged mainly when microfluidic systems are used. These systems comprise a pattern of microchannels designed onto a glass, silicon, quartz, or plastic wafer included on a microchip. The movements of the samples are controlled by electric, electroosmotic or hydrostatic forces applied across different areas of the microchip.
  • the microfluidic system may integrate nucleic acid amplification, microsequencing, capillary electrophoresis and a detection method such as laser-induced fluorescence detection.
  • the search for disease-susceptibility genes is conducted using two main methods: the linkage approach in which evidence is sought for cosegregation between a locus and a putative trait locus using family studies, and the association approach in which evidence is sought for a statistically significant association between an allele and a trait or a trait causing allele (Khoury J. et al, 1993).
  • the biallelic markers of the present invention find use in any method known in the art to demonstrate a statistically significant correlation between a genotype and a phenotype.
  • the biallelic markers may be used in parametric and non-parametric linkage analysis methods.
  • the biallelic markers of the present invention are used to identify genes associated with detectable traits using association studies, an approach which does not require the use of affected families and which permits the identification of genes associated with complex and sporadic traits.
  • the genetic analysis using the biallelic markers of the present invention may be conducted on any scale.
  • the whole set of biallelic markers of the present invention or any subset of biallelic markers of the present invention may be used.
  • a subset of biallelic markers corresponding to one or several candidate genes of the present invention may be used.
  • a subset of biallelic markers of the present invention localised on a specific chromosome segment may be used.
  • any set of genetic markers including a biallelic marker of the present invention may be used.
  • the biallelic markers of the present invention may be included in any complete or partial genetic map of the human genome.
  • Linkage analysis is based upon establishing a correlation between the transmission of genetic markers and that of a specific trait throughout generations within a family.
  • the aim of linkage analysis is to detect marker loci that show cosegregation with a trait of interest in pedigrees.
  • non-parametric methods for linkage analysis are that they do not require specification of the mode of inheritance for the disease, they tend to be more useful for the analysis of complex traits.
  • non-parametric methods one tries to prove that the inheritance pattern of a chromosomal region is not consistent with random Mendelian segregation by showing that affected relatives inherit identical copies of the region more often than expected by chance. Affected relatives should show excess "allele sharing" even in the presence of incomplete penetrance and polygenic inheritance.
  • the degree of agreement at a marker locus in two individuals can be measured either by the number of alleles identical by state (IBS) or by the number of alleles identical by descent (IBD).
  • the biallelic markers of the present invention may be used in both parametric and non- parametric linkage analysis. Preferably biallelic markers may be used in non-parametric methods which allow the mapping of genes involved in complex traits.
  • the biallelic markers of the present invention may be used in both IBD- and IBS- methods to map genes affecting a complex trait. In such studies, taking advantage of the high density of biallelic markers, several adjacent biallelic marker loci may be pooled to achieve the efficiency attained by multi-allelic markers (Zhao et al., 1998).
  • the present invention comprises methods for identifying one or several genes among a set of candidate genes that are associated with a detectable trait using the biallelic markers of the present invention.
  • the present invention comprises methods to detect an association between a biallelic marker allele or a biallelic marker haplotype and a trait.
  • the invention comprises methods to identify a trait causing allele in linkage disequilibrium with any biallelic marker allele of the present invention.
  • the biallelic markers of the present invention may further be inco ⁇ orated in any map of a specific candidate region of the genome (a specific chromosome or a specific chromosomal segment for example).
  • association studies may be conducted within the general population and are not limited to studies performed on related individuals in affected families. Association studies are extremely valuable as they permit the analysis of sporadic or multifactor traits.
  • association studies represent a powerful method for fine-scale mapping enabling much finer mapping of trait causing alleles than linkage studies. Studies based on pedigrees often only narrow the location of the trait causing allele. Association studies using the biallelic markers of the present invention can therefore be used to refine the location of a trait causing allele in a candidate region identified by Linkage Analysis methods.
  • Biallelic markers of the present invention can be used to identify the involved gene; such uses are specifically contemplated in the present invention and claims. 1) Determining the frequency of a biallelic marker allele or of a biallelic marker haplotype in a population
  • Another embodiment of the present invention encompasses methods of estimating the frequency of a haplotype for a set of biallelic markers in a population, comprising the steps of: a) genotyping each individual in said population for at least one 13q3 l-q33-related biallelic marker, b) genotyping each individual in said population for a second biallelic marker by determining the identity of the nucleotides at said second biallelic marker for both copies of said second biallelic marker present in the genome; and c) applying a haplotype determination method to the identities of the nucleotides determined in steps a) and b) to obtain an estimate of said frequency.
  • haplotypes Determining the frequency of a haplotype in a population
  • the gametic phase of haplotypes is unknown when diploid individuals are heterozygous at more than one locus. Using genealogical information in families gametic phase can sometimes be inferred (Perlin et al., 1994). When no genealogical information is available different strategies may be used. One possibility is that the multiple-site heterozygous diploids can be eliminated from the analysis, keeping only the homozygotes and the single-site heterozygote individuals, but this approach might lead to a possible bias in the sample composition and the underestimation of low-frequency haplotypes.
  • the pattern or curve of disequilibrium between disease and marker loci is expected to exhibit a maximum that occurs at the disease locus. Consequently, the amount of linkage disequilibrium between a disease allele and closely linked genetic markers may yield valuable information regarding the location of the disease gene.
  • fine-scale mapping of a disease locus it is useful to have some knowledge of the patterns of linkage disequilibrium that exist between markers in the studied region. As mentioned above the mapping resolution achieved through the analysis of linkage disequilibrium is much higher than that of linkage studies.
  • the high density of biallelic markers combined with linkage disequilibrium analysis provides powerful tools for fine-scale mapping. Different methods to calculate linkage disequilibrium are described below under the heading "Statistical Methods".
  • typical examples of inclusion criteria include affection by schizophrenia.
  • Association analysis The general strategy to perform association studies using biallelic markers derived from a region carrying a candidate gene is to scan two groups of individuals (case-control populations) in order to measure and statistically compare the allele frequencies of the biallelic markers of the present invention in both groups. If a statistically significant association with a trait is identified for at least one or more of the analysed biallelic markers, one can assume that: either the associated allele is directly responsible for causing the trait (the associated allele is the trait causing allele), or more likely the associated allele is in linkage disequilibrium with the trait causing allele.
  • the specific characteristics of the associated allele with respect to the gene function usually gives further insight into the relationship between the associated allele and the trait (causal or in linkage disequilibrium). If the evidence indicates that the associated allele within the gene is most probably not the trait causing allele but is in linkage disequilibrium with the real trait causing allele, then the trait causing allele can be found by sequencing the vicinity of the associated marker.
  • Another embodiment of the present invention encompasses methods of detecting an association between a haplotype and a phenotype, comprising the steps of: a) estimating the frequency of at least one haplotype in a trait positive population according to a method of estimating the frequency of a haplotype of the invention; b) estimating the frequency of said haplotype in a control population according to the method of estimating the frequency of a haplotype of the invention; and c) determining whether a statistically significant association exists between said haplotype and said phenotype.
  • a haplotype frequency analysis the frequency of the possible haplotypes based on various combinations of the identified biallelic markers of the invention is determined.
  • the haplotype frequency is then compared for distinct populations of trait positive and control individuals.
  • the number of trait positive individuals, which should be, subjected to this analysis to obtain statistically significant results usually ranges between 30 and 300, with a preferred number of individuals ranging between 50 and 150. The same considerations apply to the number of unaffected individuals (or random control) used in the study.
  • the results of this first analysis provide haplotype frequencies in case-control populations, for each evaluated haplotype frequency a p- value and an odd ratio are calculated. If a statistically significant association is found the relative risk for an individual carrying the given haplotype of being affected with the trait under study can be approximated.
  • Linkage disequilibrium between any pair of biallelic markers comprising at least one of the biallelic markers of the present invention (M Thread M j ) having alleles (a,/b,) at marker M, and alleles (afo_) at marker M, can be calculated for every allele combination (asourceda, , a supplementb J; b caviaa j and b consultationb,), according to the Piazza formula :
  • Linkage disequilibrium (LD) between pairs of biallelic markers (M Thread M.) can also be calculated for every allele combination (a ⁇ aj, ai,bj _ b charginga j and b beneficiaryb j ), according to the maximum-likelihood estimate (MLE) for delta (the composite genotypic disequilibrium coefficient), as described by Weir (Weir B.S., 1996).
  • pr(a) is the probability of allele a. and (a) is the probability of allele ⁇ , and where pr(haplotype (arada a)) is estimated as in Equation 3 above.
  • Methods for determining the statistical significance of a correlation between a phenotype and a genotype may be determined by any statistical test known in the art and with any accepted threshold of statistical significance being required. The application of particular methods and thresholds of significance are well with in the skill of the ordinary practitioner of the art.
  • Testing for association is performed by determining the frequency of a biallelic marker allele in case and control populations and comparing these frequencies with a statistical test to determine if their is a statistically significant difference in frequency which would indicate a correlation between the trait and the biallelic marker allele under study.
  • a haplotype analysis is performed by estimating the frequencies of all possible haplotypes for a given set of biallelic markers in case and control populations, and comparing these frequencies with a statistical test to determine if their is a statistically significant correlation between the haplotype and the phenotype (trait) under study.
  • Any statistical tool useful to test for a statistically significant association between a genotype and a phenotype may be used.
  • the statistical test employed is a chi-square test with one degree of freedom. A P-value is calculated (the P-value is the probability that a statistic as large or larger than the observed one would occur by chance).
  • genotyping data from case-control individuals are pooled and randomised with respect to the trait phenotype.
  • Each individual genotyping data is randomly allocated to two groups, which contain the same number of individuals as the case-control populations used to compile the data obtained in the first stage.
  • a second stage haplotype analysis is preferably run on these artificial groups, preferably for the markers included in the haplotype of the first stage analysis showing the highest relative risk coefficient. This experiment is reiterated preferably at least between 100 and 10000 times. The repeated iterations allow the determination of the percentage of obtained haplotypes with a significant p-value level.
  • the association between a risk factor in genetic epidemiology the risk factor is the presence or the absence of a certain allele or haplotype at marker loci) and a disease is measured by the odds ratio (OR) and by the relative risk (RR). If P(R ) is the probability of developing the disease for individuals with R and P(R " ) is the probability for individuals without the risk factor, then the relative risk is simply the ratio of the two probabilities, that is:
  • F + is the frequency of the exposure to the risk factor in cases and F " is the frequency of the exposure to the risk factor in controls.
  • F + and F " are calculated using the allelic or haplotype frequencies of the study and further depend on the underlying genetic model (dominant, recessive, additive).
  • AR Attributable risk
  • AR P ⁇ (RR-1) / (P E (RR-1)+1) AR is the risk attributable to a biallelic marker allele or a biallelic marker haplotype.
  • P E is the frequency of exposure to an allele or a haplotype within the population at large; and RR is the relative risk which, is approximated with the odds ratio when the trait under study has a relatively low incidence in the general population.
  • AR is the risk attributable to a biallelic marker allele or a biallelic marker haplotype.
  • PE is the frequency of exposure to an allele or a haplotype within the population at large; and RR is the relative risk which, is approximated with the odds ratio when the trait under study has a relatively low incidence in the general population.
  • Identification of additional markers in linkage disequilibrium with a given marker involves: (a) amplifying a genomic fragment comprising a first biallelic marker from a plurality of individuals; (b) identifying of second biallelic markers in the genomic region harboring said first biallelic marker; (c) conducting a linkage disequilibrium analysis between said first biallelic marker and second biallelic markers; and (d) selecting said second biallelic markers as being in linkage disequilibrium with said first marker. Subcombinations comprising steps (b) and (c) are also contemplated.
  • the associated candidate gene sequence can be scanned for mutations by comparing the sequences of a selected number of trait positive and trait negative individuals.
  • functional regions such as exons and splice sites, promoters and other regulatory regions of the gene are scanned for mutations.
  • trait positive individuals carry the haplotype shown to be associated with the trait and trait negative individuals do not carry the haplotype or allele associated with the trait.
  • the mutation detection procedure is essentially similar to that used for biallelic site identification.
  • Candidate polymorphisms and mutations of the sbgl nucleic acid sequences suspected of being involved in a predisposition to schizophrenia can be confirmed by screening a larger population of affected and unaffected individuals using any of the genotyping procedures described herein. Preferably the microsequencing technique is used. Such polymo ⁇ hisms are considered as candidate "trait-causing" mutations when they exhibit a statistically significant correlation with the detectable phenotype.
  • the nucleic acid sample is contacted with one or more allele specific oligonucleotide probes which, specifically hybridize to one or more human chromosome 13q31-q33 region, Region D, sbgl, g34665, sbg2, g35017 or g35018-related alleles associated with a detectable phenotype.
  • the probes used in the hybridization assay may include the probes listed in Table 6c.
  • the nucleic acid sample is contacted with a second oligonucleotide capable of producing an amplification product when used with the allele specific oligonucleotide in an amplification reaction.
  • the presence of an amplification product in the amplification reaction indicates that the individual possesses one or more human chromosome 13q31-q33 region, Region D, sbgl, g34665, sbg2, g35017 or g35018-related alleles associated with a detectable phenotype.
  • Diagnostics which analyze and predict response to a drug or side effects to a drug, may be used to determine whether an individual should be treated with a particular drug. For example, if the diagnostic indicates a likelihood that an individual will respond positively to treatment with a particular drug, the drug may be administered to the individual. Conversely, if the diagnostic indicates that an individual is likely to respond negatively to treatment with a particular drug, an alternative course of treatment may be prescribed. A negative response may be defined as either the absence of an efficacious response or the presence of toxic side effects.
  • Clinical drug trials represent another application for the markers of the present invention. One or more markers indicative of response to an agent acting against schizophrenia or to side effects to an agent acting against schizophrenia may be identified using the methods described above.
  • the invention thus comprises use of a compound capable of increasing or decreasing sbgl expression or sbgl protein activity in the preparation or manufacture of a medicament.
  • said compound is used for the treatment of a psychiatric disease, preferably for the treatment of schizophrenia or bipolar disorder.
  • said compound acts directly by binding to sbgl or an sbgl receptor.
  • the invention further encompasses said methods for the prevention, treatment, and diagnosis of disease using any of the g34665, sbg2, g35017 or g35018 nucleic acids of proteins of the invention in analogous methods.
  • the biallelic markers and other polymo ⁇ hisms of the present invention can also be used to develop diagnostics tests capable of identifying individuals who express a detectable trait as the result of a specific genotype or individuals whose genotype places them at risk of developing a detectable trait at a subsequent time.
  • the trait analyzed using the present diagnostics may be used to diagnose any detectable trait, including predisposition to schizophrenia or bipolar disorder, age of onset of detectable symptoms, a beneficial response to or side effects related to treatment against schizophrenia or bipolar disorder. Such a diagnosis can be useful in the monitoring, prognosis and/or prophylactic or curative therapy for schizophrenia or bipolar disorder.
  • Diagnostics which analyze and predict response to a drug or side effects to a drug, may be used to determine whether an individual should be treated with a particular drug. For example, if the diagnostic indicates a likelihood that an individual will respond positively to treatment with a particular drug, the drug may be administered to the individual. Conversely, if the diagnostic indicates that an individual is likely to respond negatively to treatment with a particular drug, an alternative course of treatment may be prescribed. A negative response may be defined as either the absence of an efficacious response or the presence of toxic side effects.
  • Clinical drug trials represent another application for the markers of the present invention.
  • One or more markers indicative of response to an agent acting against schizophrenia or to side effects to an agent acting against schizophrenia may be identified using the methods described above. Thereafter, potential participants in clinical trials of such an agent may be screened to identify those individuals most likely to respond favorably to the drug and exclude those likely to experience side effects. In that way, the effectiveness of drug treatment may be measured in individuals who respond positively to the drug, without lowering the measurement as a result of the inclusion of individuals who are unlikely to respond positively in the study and without risking undesirable safety problems.
  • a second preferred embodiment of the recombinant vectors according to the invention comprises expression vectors comprising either a regulatory polynucleotide or a coding nucleic acid of the invention, or both.
  • expression vectors are employed to express an sbgl, g34665, sbg2, g35017 or g35018 polypeptide which can be then purified and, for example be used in ligand screening assays or as an immunogen in order to raise specific antibodies directed against an sbgl , g34665, sbg2, g35017 or g35018 protein.
  • the expression vectors are used for constructing transgenic animals and also for gene therapy.
  • Recombinant vectors comprising a nucleic acid containing a human chromosome 13q31-33-related biallelic marker, preferably a Region D-related biallelic marker or more preferably an sbgl-, g34665-, sbg2-, g35017- or g35018-related biallelic marker is also part of the invention.
  • said biallelic marker is selected from the group consisting of Al to A489, and the complements thereof.
  • recombinant expression vectors will include origins of replication, selectable markers permitting transformation of the host cell, and a promoter derived from a highly expressed gene to direct transcription of a downstream structural sequence.
  • the heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably a leader sequence capable of directing secretion of the translated protein into the periplasmic space or the extracellular medium.
  • preferred vectors will comprise an origin of replication in the desired host, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5'-flanking non- transcribed sequences.
  • DNA sequences derived from the SV40 viral genome for example SV40 origin, early promoter, enhancer, splice and polyadenylation sites may be used to provide the required non-transcribed genetic elements.
  • This genetic material may be introduced in vitro in a cell that has been previously extracted from the organism, the modified cell being subsequently reintroduced in the said organism, directly in vivo into the appropriate tissue.
  • the suitable promoter regions used in the expression vectors according to the present invention are chosen taking into account the cell host in which the heterologous gene has to be expressed.
  • the particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of directing the expression of the nucleic acid in the targeted cell.
  • a human cell it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell, such as, for example, a human or a viral promoter.
  • Promoter regions can be selected from any desired gene using, for example, CAT (chloramphenicol transferase) vectors and more preferably pKK232-8 and pCM7 vectors.
  • CAT chloramphenicol transferase
  • Preferred bacterial promoters are the Lad, LacZ, the T3 or T7 bacteriophage RNA polymerase promoters, the gpt, lambda PR, PL and t ⁇ promoters (EP 0036776), the polyhedrin promoter, or the plO protein promoter from baculovirus (Kit Novagen) (Smith et al., 1983; O'Reilly et al., 1992), the lambda PR promoter or also the trc promoter.
  • polyadenylation signal to effect proper polyadenylation of the gene transcript.
  • the nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed such as human growth hormone and SV40 polyadenylation signals.
  • a terminator is also contemplated as an element of the expression cassette. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
  • g35017 genomic DNA or cDNAs comprised in the nucleic acids of any of nucleotide positions 94124 to 94964 of SEQ ID No. 1, and the complements thereof;
  • g35018 genomic DNA or cDNAs comprised in the nucleic acids of any of nucleotide positions 1108 to 1289, 14877 to 14920, 18778 to 18862, 25593 to 25740, 29388 to 29502, 29967 to 30282, 64666 to 64812, and 65505 to 65853 of SEQ ID No. 1, and the complements thereof.
  • useful expression vectors for bacterial use can comprise a selectable marker and a bacterial origin of replication derived from commercially available plasmids comprising genetic elements of pBR322 (ATCC 37017).
  • Such commercial vectors include, for example, pKK223-3 (Pharmacia, Uppsala, Sweden), and GEM1 (Promega Biotec, Madison, WI, USA).
  • PI bacteriophage vectors such as pi 58 or pl58/neo8 are notably described by Sternberg ( 1992, 1994).
  • Recombinant PI clones comprising sbgl polynucleotide sequences may be designed for inserting large polynucleotides of more than 40 kb (Linton et al., 1993).
  • McCormick et al.(1994) Briefly, E. coli (preferably strain NS3529) harboring the PI plasmid are grown overnight in a suitable broth medium containing 25 ⁇ g/ml of kanamycin. The PI DNA is prepared from the E.
  • PI DNA is purified from the bacterial lysate on two Qiagen-tip 500 columns, using the washing and elution buffers contained in the kit. A phenol/chloroform extraction is then performed before precipitating the DNA with 10% ethanol. After solubilizing the DNA in TE (10 mM Tris-HCl, pH 7.4, 1 mM EDTA), the concentration of the DNA is assessed by spectrophotometry.
  • Ultrafree-MC Filter Unit (Millipore, Bedford, MA, USA - 30,000 molecular weight limit) and then dialyzed against microinjection buffer (10 mM Tris-HCl, pH 7.4; 250 ⁇ M EDTA) containing 100 mM NaCl, 30 ⁇ M spermine, 70 ⁇ M spermidine on a microdyalisis membrane (type VS, 0.025 ⁇ M from Millipore).
  • microinjection buffer 10 mM Tris-HCl, pH 7.4; 250 ⁇ M EDTA
  • microinjection buffer 10 mM Tris-HCl, pH 7.4; 250 ⁇ M EDTA
  • microinjection buffer 10 mM Tris-HCl, pH 7.4; 250 ⁇ M EDTA
  • a microdyalisis membrane type VS, 0.025 ⁇ M from Millipore
  • the vector is derived from an adenovirus.
  • adenovirus vectors according to the invention are those described by Feldman and Steg (1996) or Ohno et al.(1994).
  • Another preferred recombinant adenovirus according to this specific embodiment of the present invention is the human adenovirus type 2 or 5 (Ad 2 or Ad 5) or an adenovirus of animal origin (French patent application N° FR-93.05954).
  • retroviruses for the preparation or construction of retroviral in vitro or in vitro gene delivery vehicles of the present invention include retroviruses selected from the group consisting of Mink-Cell Focus Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis virus and Rous Sarcoma virus.
  • retroviruses selected from the group consisting of Mink-Cell Focus Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis virus and Rous Sarcoma virus.
  • Particularly preferred Murine Leukemia selected from the group consisting of Mink-Cell Focus Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis virus and Rous Sarcoma virus.
  • Murine Leukemia selected from the group consisting
  • Viruses include the 4070A and the 1504A viruses, Abelson (ATCC No VR-999), Friend (ATCC No VR-245), Gross (ATCC No VR-590), Rauscher (ATCC No VR-998) and Moloney Murine Leukemia Virus (ATCC No VR-190; PCT Application No WO 94/24298).
  • Particularly preferred Rous Sarcoma Viruses include Bryan high titer (ATCC Nos VR-334, VR-657, VR- 726, VR-659 and VR-728).
  • BAC bacterial artificial chromosome
  • a preferred BAC vector comprises pBeloBACl 1 vector that has been described by Kim et al.(1996).
  • BAC libraries are prepared with this vector using size-selected genomic DNA that has been partially digested using enzymes that permit ligation into either the Bam HI or Hindlll sites in the vector. Flanking these cloning sites are T7 and SP6 RNA polymerase transcription initiation sites that can be used to generate end probes by either RNA transcription or PCR methods.
  • BAC DNA is purified from the host cell as a supercoiled circle. Converting these circular molecules into a linear form precedes both size determination and introduction of the BACs into recipient cells.
  • the cloning site is flanked by two Not I sites, permitting cloned segments to be excised from the vector by Not I digestion.
  • the DNA insert contained in the pBeloBACl 1 vector may be linearized by treatment of the BAC vector with the commercially available enzyme lambda terminase that leads to the cleavage at the unique cosN site, but this cleavage method results in a full length BAC clone containing both the insert DNA and the BAC sequences. 5. Delivery Of The Recombinant Vectors
  • these constructs must be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cell lines, or in vivo or ex vivo, as in the treatment of certain diseases states.
  • One mechanism is viral infection where the expression construct is encapsulated in an infectious viral particle.
  • non-viral methods for the transfer of polynucleotides into cultured mammalian cells include, without being limited to, calcium phosphate precipitation (Graham et al., 1973; Chen et al., 1987), DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984), direct microinjection (Harland et al., 1985), DNA-loaded liposomes (Nicolau et al., 1982; Fraley et al., 1979), and receptor-mediate transfection (Wu and Wu, 1987; 1988). Some of these techniques may be successfully adapted for in vivo or ex vivo use.
  • the expression polynucleotide may be stably integrated into the genome of the recipient cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non specific location (gene augmentation).
  • the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle.
  • One specific embodiment for a method for delivering a protein or peptide to the interior of a cell of a vertebrate in vivo comprises the step of introducing a preparation comprising a physiologically acceptable carrier and a naked polynucleotide operatively coding for the polypeptide of interest into the interstitial space of a tissue comprising the cell, whereby the naked polynucleotide is taken up into the interior of the cell and has a physiological effect.
  • This is particularly applicable for transfer in vitro but it may be applied to in vivo as well.
  • compositions for use in vitro and in vivo comprising a "naked" polynucleotide are described in PCT application N° WO 90/11092 (Nical Inc.) and also in PCT application No.
  • the transfer of a naked polynucleotide of the invention, including a polynucleotide construct of the invention, into cells may be proceeded with a particle bombardment (biolistic), said particles being DNA-coated microprojectiles accelerated to a high velocity allowing them to pierce cell membranes and enter cells without killing them, such as described by Klein et al.(1987).
  • a particle bombardment biolistic
  • said particles being DNA-coated microprojectiles accelerated to a high velocity allowing them to pierce cell membranes and enter cells without killing them, such as described by Klein et al.(1987).
  • the polynucleotide of the invention may be entrapped in a liposome (Ghosh and Bacchawat, 1991 ; Wong et al., 1980; Nicolau et al., 1987).
  • the invention provides a composition for the in vivo production of the sbgl, g34665, sbg2, g35017 and g35018 protein or polypeptide described herein. It comprises a naked polynucleotide operatively coding for this polypeptide, in solution in a physiologically acceptable carrier, and suitable for introduction into a tissue to cause cells of the tissue to express the said protein or polypeptide.

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EP00914325A 1999-03-30 2000-03-30 Gene, proteine und biallelische markers die mit der schizophrenie assoziert sind Withdrawn EP1165836A2 (de)

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US14392899P 1999-07-14 1999-07-14
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US14645299P 1999-07-29 1999-07-29
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US146452P 1999-07-29
US16228899P 1999-10-28 1999-10-28
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UA79927C2 (en) * 2000-12-05 2007-08-10 Serono Genetics Inst Sa Polynucleotide, coding a polypeptide of potential-depending portal ionic human channel (canion), polypeptyde, antibody, method for identification of candidate modulator of canion-polypeptyde, method for treatment of bipolar disorder or schizophrenia and use of an antibody for production of drugs for treatment of schizophrenia or bipolar disorder
KR20030066813A (ko) * 2001-01-16 2003-08-09 쟝세 Ｄ-아미노산 산화효소와 ｄ-아스파르테이트 산화효소길항제를 이용한 ｃｎｓ 질환의 치료
WO2002086147A2 (en) * 2001-04-24 2002-10-31 Pharmacia & Upjohn Company Single nucleotide polymorphisms diagnostic for schizophrenia
JP2003038198A (ja) * 2001-07-27 2003-02-12 Univ Niigata 精神分裂病により発現量が変化する遺伝子を規定する核酸を解析する方法
IL162481A0 (en) * 2001-12-12 2005-11-20 Genset Sa Method and use of determining genotype
EP1495041A4 (de) * 2002-02-20 2006-02-01 Sirna Therapeutics Inc DURCH RNA-INTERFERENZ VERMITTELTE INHIBIERUNG DER GENEXPRESSION VON G72 UND D-AMINOSÄUREOXIDASE (DAAO) UNTER VERWENDUNG KURZER INTERFERIERENDER NUKLEINSÄURE (siNA)
EP1492885A2 (de) * 2002-04-05 2005-01-05 University Court of the University of Edinburgh Gene, die mit schizophrenie assoziiert sind
US7410759B2 (en) * 2002-11-01 2008-08-12 The Board Of Trustees Of The Leland Stanford Junior University Compositions and methods for diagnosing and treating mood disorders
US8415298B2 (en) 2004-06-21 2013-04-09 The Board Of Trustees Of The Leland Stanford Junior University Of Stanford Administration of FGF2 for treamtent of anxiety

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