WO2000058510A2 - Schizophrenia associated genes, proteins and biallelic markers - Google Patents
Schizophrenia associated genes, proteins and biallelic markers Download PDFInfo
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- WO2000058510A2 WO2000058510A2 PCT/IB2000/000435 IB0000435W WO0058510A2 WO 2000058510 A2 WO2000058510 A2 WO 2000058510A2 IB 0000435 W IB0000435 W IB 0000435W WO 0058510 A2 WO0058510 A2 WO 0058510A2
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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
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- 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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; 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
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- 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.
- CNS disorders have complex and poorly understood etiologies, as well as symptoms that are overlapping, poorly characterized, and difficult to measure.
- future treatment regimes and drug development efforts will be required to be more sophisticated and focused on multigenic causes, and will need new assays to segment disease populations, and provide more accurate diagnostic and prognostic information on patients suffering from CNS disorders.
- Neurotransmitters serve as signal transmitters throughout the body. Diseases that affect neurotransmission can therefore have serious consequences. For example, for over 30 years the leading theory to explain the biological basis of many psychiatric disorders such as depression has been the monoamine hypothesis. This theory proposes that depression is partially due to a deficiency in one of the three main biogenic monoamines, namely dopamine, norepinephrine and/or serotonin. In addition to the monoamine hypothesis, numerous arguments tend to show the value in taking into account the overall function of the brain and no longer only considering a single neuronal system. In this context, the value of dual specific actions on the central aminergic systems including second and third messenger systems has now emerged.
- CNS disorders may have an endocrine component; the hypothalamic-pituitary-adrenal (HP A) axis, including the effects of corticotrophin-releasing factor and glucocorticoids, plays an important role in the pathophysiology of CNS disorders.
- HP A hypothalamic-pituitary-adrenal
- the hypothalamus-pituitary-adrenal (HPA) axis the hypothalamus lies at the top of the hierarchy regulating hormone secretion.
- 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.
- 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. Linkage analysis involves the study of families with multiple affected individuals and is useful in the detection of inherited-traits, which are caused by a single gene, or possibly a very small number of genes. But, linkage studies have proven difficult when applied to complex genetic traits. Most traits of medical relevance do not follow simple Mendelian monogenic inheritance. However, complex diseases often aggregate in families, which suggests that there is a genetic component to be found. Such complex traits are often due to the combined action of multiple genes as well as environmental factors. Such complex trait, include susceptibilities to heart disease, hypertension, diabetes, cancer and inflammatory diseases. Drug efficacy, response and tolerance/toxicity can also be considered as multifactoral traits involving a genetic component in the same way as complex diseases.
- the basis for accomplishment of these goals is to use genetic association analysis to detect markers that predict susceptibility for these traits.
- Recently, advances in the fields of genetics and molecular biology have allowed identification of forms, or alleles, of human genes that lead to diseases.
- the development of single gene disorders is determined, or largely influenced, by the alleles of a single gene.
- the alleles that cause these disorders are, in general, highly deleterious (and highly penetrant) to individuals who carry them. Therefore, these alleles and their associated diseases, with some exceptions, tend to be very rare in the human population.
- 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 affects 1% of the world population. There are an estimated 45 million people with schizophrenia in the world, with more than 33 million of them in the developing countries. This disease places a heavy burden on the patient's family and relatives, both in terms of the direct and indirect costs involved and the social stigma associated with the illness, sometimes over generations. Such stigma often leads to isolation and neglect.
- 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 disorders are characterized by phases of excitement and often including depression; the excitement phases, referred to as mania or hypomania, and depression can alternate or occur in various admixtures, and can occur to different degrees of severity and over varying time periods. Because bipolar disorders can exist in different forms and display different symptoms, the classification of bipolar disorder has been the subject of extensive studies resulting in the definition of bipolar disorder subtypes and widening of the overall concept to include patients previously thought to be suffering from different disorders. Bipolar disorders often share certain clinical signs, symptoms, treatments and neurobiological features with psychotic illnesses in general and therefore present a challenge to the psychiatrist to make an accurate diagnosis. Furthermore, because the course of bipolar disorders and various mood and psychotic disorders can differ greatly, it is critical to characterize the illness as early as possible in order to offer means to manage the illness over a long term.
- Bipolar disorders appear in about 1.3% of the population and have been reported to constitute about half of the mood disorders seen in a psychiatric clinic. Bipolar disorders have been found to vary with gender depending of the type of disorder; for example, bipolar disorder I is found equally among men and women, while bipolar disorder II is reportedly more common in women. The age of onset of bipolar disorders is typically in the teenage years and diagnosis is typically made in the patient's early twenties. Bipolar disorders also occur among the elderly, generally as a result of a medical or neurological disorder.
- the DSM-IV classification of bipolar disorder distinguishes among four types of disorders based on the degree and duration of mania or hypomania as well as two types of disorders which are evident typically with medical conditions or their treatments, or to substance abuse. Mania is recognized by elevated, expansive or irritable mood as well as by distractability, impulsive behavior, increased activity, grandiosity, elation, racing thoughts, and pressured speech. Of the four types of bipolar disorder characterized by the particular degree and duration of mania , DSM-IV includes:
- - 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 classified in DSM-VI are disorders evident or caused by various medical disorder and their treatments, and disorders involving or related to substance abuse.
- Medical disorders which can cause bipolar disorders typically include endocrine disorders and cerebrovascular injuries, and medical treatments causing bipolar disorder are known to include glucocorticoids and the abuse of stimulants.
- the disorder associated with the use or abuse of a substance is referred to as "substance induced mood disorder with manic or mixed features".
- Diagnosis of bipolar disorder can be very challenging.
- One particularly troublesome difficulty is that some patients exihibit mixed states, simultaneously manic and dysphoric or depressive, but do not fall into the DSM-IV classification because not all required criteria for mania and major depression are met daily for at least one week.
- Other difficulties include classification of patients in the DSM-IV groups based on duration of phase since patients often cycle between excited and depressive episodes at different rates.
- the use of antidepressants may alter the course of the disease for the worse by causing "rapid- cycling".
- Also making diagnosis more difficult is the fact that bipolar patients, particularly at what is known as Stage III mania, share symptoms of disorganized thinking and behavior with bipolar disorder patients.
- 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
- atypical neuroleptics The well known deficiencies in the standard neuroleptics have stimulated a search for new treatments and have led to a new class of drugs termed atypical neuroleptics.
- Clozapine The first atypical neuroleptic, Clozapine, is effective for about one third of patients who do not respond to standard neuroleptics. It seems to reduce negative as well as positive symptoms, or at least exacerbates negative symptoms less than standard neuroleptics do. Moreover, it has beneficial effects on overall functioning and may reduce the chance of suicide in schizophrenic patients. It does not produce the troubling neurological symptoms of the standard neuroleptics, or raise blood levels of the hormone prolactin, excess of which may cause menstrual irregularities and infertility in women, impotence or breast enlargement in men.
- 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.
- schizophrenia and bipolar disorder are primarily genetic conditions, although environmental risk factors are also involved at some level as necessary, sufficient, or interactive causes.
- schizophrenia occurs in 1% of the general population. But, if there is one grandparent with schizophrenia, the risk of getting the illness increases to about 3%; one parent with Schizophrenia, to about 10%. When both parents have schizophrenia, the risk rises to approximately 40%.
- the present invention stems from the identification of novel polymo ⁇ hisms including biallelic markers located on the human chromosome 13q31-q33 locus, the identification and characterization of novel schizophrenia-related genes located on the human chromosome 13q31- q33 locus, and from the identification of genetic associations between alleles of biallelic markers located on the human chromosome 13q31-q33 locus and disease, as confirmed and characterized in a panel of human subjects.
- the invention furthermore provides a fine structure map of the region which includes the schizophrenia-associated gene sequences.
- the present invention pertains to nucleic acid molecules comprising the genomic sequences of novel human genes encoding sbgl, g34665, sbg2, g35017 and g35018 proteins, proteins encoded thereby, as well as antibodies thereto.
- the sbgl, g34665, sbg2, g35017 and g35018 genomic sequences may also comprise regulatory sequence located upstream (5 '-end) and downstream (3 '-end) of the transcribed portion of said gene, these regulatory sequences being also part of the invention.
- the invention also deals with the cDNA sequence encoding the sbgl and g35018 proteins.
- 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.
- a further object of the invention consists of recombinant vectors comprising any of the nucleic acid sequences described above, and in particular of recombinant vectors comprising a sbgl, g34665, sbg2, g35017 or g35018 regulatory sequence or a sequence encoding a sbgl, g34665, sbg2, g35017 or g35018 protein, as well as of cell hosts and transgenic non human animals comprising said nucleic acid sequences or recombinant vectors.
- 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.
- An embodiment of the invention encompasses any polynucleotide of the invention attached to a solid support polynucleotide may comprise a sequence disclosed in the present specification; optionally, said polynucleotide may comprise, consist of, or consist essentially of any polynucleotide described in the present specification; optionally, said determining may be performed in a hybridization assay, sequencing assay, microsequencing assay, or an enzyme- based mismatch detection assay; optionally, said polynucleotide may be attached to a solid support, array, or addressable array; optionally, said polynucleotide may be labeled.
- the invention is directed to drug screening assays and methods for the screening of substances for the treatment of schizophrenia, bipolar disorder or a related CNS disorder based on the role of sbgl, g34665, sbg2, g35017 and g35018 nucleotides and polynucleotides in disease.
- One object of the invention deals with animal models of schizophrenia, including mouse, primate, non-human primate bipolar disorder or related CNS disorder based on the role of sbgl in disease.
- the invention is also directed to methods for the screening of substances or molecules that inhibit the expression of sbgl, g34665, sbg2, g35017 or g35018, as well as with methods for the screening of substances or molecules that interact with a sbgl, g34665, sbg2, g35017 or g35018 polypeptide, or that modulate the activity of a sbgl, g34665, sbg2, g35017 or g35018 polypeptide.
- certain aspects of the present invention stem from the identification of genetic associations between schizophrenia and bipolar disorder and alleles of biallelic markers located on the human chromosome 13q31-q33 region, and more particularly on a subregion thereof referred to herein as Region D.
- the invention provides appropriate tools for establishing further genetic associations between alleles of biallelic markers on the 13q31- 13q33 locus and either side effects or benefit resulting from the administration of agents acting on schizophrenia or bipolar disorder, or schizophrenia or bipolar disorder symptoms, includng agents like chlorpromazine, clozapine, risperidone, olanzapine, sertindole, quetiapine and ziprasidone.
- 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.
- FIGURES is a diagram showing the exon structure of the sbgl gene.
- Figure 2 is a table demonstrating the statistical significance of allelic frequencies of selected chromosome 13q31-q33 biallelic markers of the invention in sporadic and familial French Canadian schizophrenia cases and controls.
- 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.
- SEQ ID No. 44 to 53 contain primers used to isolate sbgl cDNAs
- SEQ ID No. 54 to 1 1 1 contain genomic nucleotide sequences comprising exons of the sbgl gene from several different primates.
- SEQ ID Nos. 1 12 to 229 respectively contain the nucleotide sequence of the ampl icons which comprise the biallelic markers A243 to A360 located on the human chromosome 13q31- q33 locus.
- SEQ ID No 230 contains a primer containing the additional PU 5' sequence described further in Example 2
- SEQ ID No 231 contains a primer containing the additional RP 5' sequence described further in Example 2.
- 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.
- genomic nucleotide sequence disclosed herein can further serve as a reference in genetic or physical analysis of deletions, substitutions, and insertions in that region. Additionally, the sequence information provides a resource for the further identification of new genes in that region. Additionally, the sequences comprising the schizophrenia-associated genes are useful, for example, for the isolation of other genes in putative gene families, the identification of homologs from other species, treatment of disease and as probes and primers for diagnostic or screening assays as described herein.
- 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.
- oligonucleotides and “polynucleotides” include RNA, DNA, or RNA DNA hybrid sequences of more than one nucleotide in either single chain or duplex form.
- nucleotide as used herein as an adjective to describe molecules comprising RNA, DNA, or RNA/DNA hybrid sequences of any length in single- stranded or duplex form.
- nucleotide is also used herein as a noun to refer to individual nucleotides or varieties of nucleotides, meaning a molecule, or individual unit in a larger nucleic acid molecule, comprising a purine or pyrimidine, a ribose or deoxyribose sugar moiety, and a phosphate group, or phosphodiester linkage in the case of nucleotides within an oligonucleotide or polynucleotide.
- 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.
- primer denotes a specific oligonucleotide sequence which is complementary to a target nucleotide sequence and used to hybridize to the target nucleotide sequence.
- a primer serves as an initiation point for nucleotide polymerization catalyzed by either DNA polymerase, RNA polymerase or reverse transcriptase.
- 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.
- g34665 gene when used herein, encompasses genomic, mRNA and cDNA sequences encoding the g34665 protein, including the untranslated regulatory regions of the genomic DNA.
- sbg2 gene when used herein, encompasses genomic, mRNA and cDNA sequences encoding the sbg2 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.
- the term "13q31-q33-related biallelic marker” relates to a set of biallelic markers residing in the human chromosome 13q31-q33 region.
- the term 13q3 l-q33-related biallelic marker encompasses all of the biallelic markers disclosed in Table 6b and any biallelic markers in linkage disequilibrium therewith ,as well as any biallelic markers disclosed in Table 6c and any biallelic markers in linkage disequilibrium therewith.
- the preferred chromosome 13q31-q33-related biallelic marker alleles of the present invention include each one the alleles described in Tables 6b individually or in groups consisting of all the possible combinations of the alleles listed.
- Region D-related biallelic marker relates to a set of biallelic markers in linkage disequilibrium with the subregion of the chromosome 13q3 l-q33 region referred to herein as Region D.
- Region D-related biallelic marker encompasses the biallelic markers Al to A242, A249 to A251, A257 to A263, A269 to A270, A278, A285 to
- sbgl -related biallelic marker relates to a set of biallelic markers in linkage disequilibrium with the sbgl gene or an sbgl nucleotide sequence.
- the term sbgl -related biallelic marker encompasses the biallelic markers A85 to A219 disclosed in Table 6b and any biallelic markers in linkage disequilibrium therewith.
- 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.
- g35018-related biallelic marker relates to a set of biallelic markers in linkage disequilibrium with the g35018 gene or a g35018 nucleotide sequence.
- the term g35018-related biallelic marker encompasses the biallelic markers Al to A39 disclosed in Table 6b and any biallelic markers in linkage disequilibrium therewith.
- polypeptide refers to a polymer of amino acids without regard to the length of the polymer; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide.
- 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.
- a polypeptide is substantially pure when at least about 50%, preferably 60 to 75% of a sample exhibits a single polypeptide sequence.
- a substantially pure polypeptide typically comprises about 50%, preferably 60 to 90% weight/weight of a protein sample, more usually about 95%, and preferably is over about 99% pure.
- Polypeptide purity or homogeneity is 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 polypeptide band upon staining the gel.
- 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”.
- antibody refers to a polypeptide or group of polypeptides which are comprised of at least one binding domain, where an antibody binding domain is formed from the folding of variable domains of an antibody molecule to form three-dimensional binding spaces with an internal surface shape and charge distribution complementary to the features of an antigenic determinant of an antigen., which allows an immunological reaction with the antigen.
- Antibodies include recombinant proteins comprising the binding domains, as wells as fragments, including Fab, Fab', F(ab)2, and F(ab')2 fragments.
- an "antigenic determinant” is the portion of an antigen molecule, in this case an sbgl polypeptide, that determines the specificity of the antigen-antibody reaction.
- An “epitope” refers to an antigenic determinant of a polypeptide.
- An epitope can comprise as few as 3 amino acids in a spatial conformation which is unique to the epitope. Generally an epitope comprises at least 6 such amino acids, and more usually at least 8-10 such amino acids.
- Methods for determining the amino acids which make up an epitope include x-ray crystallography, 2-dimensional nuclear magnetic resonance, and epitope mapping e.g. the Pepscan method described by Geysen et al. 1984; PCT Publication No. WO 84/03564; and PCT Publication No. WO 84/03506.
- the invention also relates to variants and fragments of the polynucleotides described herein, particularly of a nucleotide sequence of SEQ ID Nos. 1 to 26, 36 to 40 and 54 to 229, and particularly of a nucleotide sequence of SEQ ID Nos. 1 to 26, 36 to 40 and 54 to 229 containing one or more biallelic markers and/or other polymo ⁇ hisms according to the invention.
- 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.
- Variants of polynucleotides according to the invention include, without being limited to, nucleotide sequences which are at least 95%) identical to a polynucleotide selected from the group consisting of the nucleotide sequences SEQ ID Nos. 1 to 26, 36 to 40 and 54 to 229 or to any polynucleotide fragment of at least 8 consecutive nucleotides of a polynucleotide selected from the group consisting of the nucleotide SEQ ID Nos.
- 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.
- Nucleotide changes present in a variant polynucleotide may be silent, which means that they do not alter the amino acids encoded by the polynucleotide. However, nucleotide changes may also result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. The substitutions, deletions or additions may involve one or more nucleotides.
- the variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions.
- 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.
- the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
- Homology is evaluated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85(8):2444-2448; Altschul et al., 1990, J. Mol. Biol. 215(3):403-410; Thompson et al., 1994, Nucleic Acids Res. 22(2):4673-4680; Higgins et al.,
- 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)
- TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.
- 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.
- genomic sequences of sbg2, g35017 and g35018 are indicated by nucleotide position in the 5' to 3' orientation on SEQ ID No 1.
- sbgl and g34665 are transcribed in the opposite direction, ie. from the nucleic acid strand complementary to SEQ ID No 1.
- Genomic sequences of sbgl and g34665 are thus indicated by nucleotide position in the 3' to 5' orientation on SEQ ID No 1.
- 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.
- nucleic acid fragments of any size and sequence may be comprised by the polynucleotides described in this section.
- Another particularly preferred set of 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 such a length is consistent with the lengths of the particular nucleotide position, of SEQ ID No.
- contiguous span comprises at least 1, 2, 3, 5, or 10 nucleotide positions of any one of the following ranges of nucleotide positions, designated posl to pos!66, of SEQ ID No. 1 listed in Table 1 below:
- 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.
- 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 or 200 nucleotides, to the extent that the length of said span is consistent with the nucleotide position range, of nucleotide positions 292653 to 292841, 295555 to 296047 or 295580 to 296047 of SEQ ID No 1.
- 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 or 200 nucleotides, to the extent that the length of said span is consistent with the nucleotide position range, of nucleotide positions 292653 to 292841, 295555 to 296047, or 295580 to 296047 of SEQ ID No
- 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.
- Another object of the invention consists of a purified, isolated, or recombinant nucleic acid that hybridizes with a g34665 nucleotide sequence of any of nucleotide positions 292653 to 296047, 292653 to 292841, 295555 to 296047, 295980 to 296047 and 296048 to 298048 SEQ ID No 1 or a complementary sequence thereto or a variant thereof, under the stringent hybridization conditions as defined above.
- 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
- SEQ ID No 1 a sequence complementary thereto, as well as fragments and variants thereof.
- These polynucleotides may be purified, isolated, or recombinant.
- 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 94124 to 94964 SEQ ID No 1 or a complementary sequence thereto or a fragment thereof.
- the nucleotide differences as regards to nucleotide positions 94124 to 94964 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.
- 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 g35017 gene in a test sample, or alternatively in order to amplify a target nucleotide sequence within the g35017 sequences.
- Another object of the invention consists of a purified, isolated, or recombinant nucleic acid that hybridizes with a g35017 nucleotide sequence of any of nucleotide positions 94124 to
- 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 or 500 nucleotides of nucleotide position 94124 to 94964 of SEQ ID No 1 or the complements thereof.
- 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 or 500 nucleotides of nucleotide position 94124 to 94964 of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises a g35017 related biallelic marker.
- said biallelic marker is the biallelic marker designated A41 in Table 6b. It should be noted that nucleic acid fragments of any size and sequence may also be comprised by the polynucleotides described in this section.
- nucleic acid fragments of any size and sequence may also be comprised by the polynucleotides described in this section, flanking the genomic sequences of g35017 on either side or between two or more such genomic sequences.
- a g35017 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 g35017 5' and 3' regulatory regions are useful in order to detect the presence of at least a copy of a nucleotide sequence comprising an g35017 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, or a sequence complementary thereto or a biologically active fragment or variant thereof.
- the 5' regulatory region may comprise a nucleotide sequence
- the invention concenrs g35018 genomic sequences consisting of, consisting essentially of, or comprising the sequence of nucleotide positions 1108 to 65853 of SEQ ID No 1 , a sequence complementary thereto, as well as fragments and variants thereof. These polynucleotides may be purified, isolated, or recombinant.
- 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,
- said contiguous span comprises at least 1 , 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 1 : 1 108 to 65853, 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, or the complements thereof.
- 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, or 1000 nucleotides of nucleotide positions 1 108 to 65853, 1108 to 1289, 14877 to 14920, 18778 to 18862, 25593 to 25740, 29388 to 29502, 29967 to 30282, 64666 to 64812 or 65505 to 65853 of SEQ ID No 1, or the complements thereof, wherein said contiguous span comprises a g35018 related biallelic marker.
- said biallelic marker is selected from the group consisting of A 1 to A39.
- 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, or 95% nucleotide identity with a nucleotide sequence of nucleotide positions 31 to 1 107, 1108 to 65853, 1108 to 1289, 14877 to 14920, 18778 to 18862, 25593 to 25740, 29388 to 29502, 29967 to 30282, 64666 to 64812, 65505 to 65853 and 65854 to 67854 of SEQ ID No 1 or a complementary sequence thereto or a fragment thereof.
- nucleotide differences as regards to nucleotide positions 31 to 1107, 1 108 to 65853, 1108 to 1289, 14877 to 14920, 18778 to 18862, 25593 to 25740, 29388 to 29502, 29967 to 30282, 64666 to 64812, 65505 to 65853 and 65854 to 67854 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 nucleotide positions 31 to 1107, 1 108 to 65853, 1108 to 1289, 14877 to 14920,
- 18778 to 18862, 25593 to 25740, 29388 to 29502, 29967 to 30282, 64666 to 64812, 65505 to 65853 and 65854 to 67854 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 g35018 gene in a test sample, or alternatively in order to amplify a target nucleotide sequence within the g35018 sequences.
- Another object of the invention consists of a purified, isolated, or recombinant nucleic acid that hybridizes with a g35018 nucleotide sequence of any of nucleotide positions 31 to 1107, 1108 to 65853, 1108 to 1289, 14877 to 14920, 18778 to 18862, 25593 to 25740, 29388 to 29502, 29967 to 30282, 64666 to 64812, 65505 to 65853 and 65854 to 67854 SEQ ID No 1, or a complementary sequence thereto or a variant thereof, under the stringent hybridization conditions as defined above.
- 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 or 500 nucleotides, to the extent that said span is consistent with the nucleotide position range, of SEQ ID No 1 , wherein said contiguous span comprises at least 1 , 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 1 : 1255 to 1289, 29967 to 301 15, 30225 to 30282, or the complements thereof, as well as polynucleotides having at least 70, 75, 80, 85, 90, or 95% nucleotide identity with said span and polynucleotides capable of hybridizing with said span.
- the g35018 genomic nucleic acid comprises at least 8 exons.
- the exon positions in SEQ ID No 1 are detailed below in Table 3.
- the invention embodies purified, isolated, or recombinant polynucleotides comprising a nucleotide sequence selected from the group consisting of the 8 exons of the g35018 gene, or a sequence complementary thereto.
- the invention also deals with purified, isolated, or recombinant nucleic acids comprising a combination of at least two exons of the 35018 gene, wherein the polynucleotides are arranged within the nucleic acid, from the 5 '-end to the 3 '-end of said nucleic acid, in the same order as in SEQ ID No 1.
- Intron 1 refers to the nucleotide sequence located between Exon 1 and Exon 2, and so on. The position of the introns is detailed in Table 3.
- the invention embodies purified, isolated, or recombinant polynucleotides comprising a nucleotide sequence selected from the group consisting of the 7 introns of the g35018 gene, or a sequence complementary thereto. While this section is entitled “Genomic Sequences of g35018,” it should be noted that nucleic acid fragments of any size and sequence may also be comprised by the polynucleotides described in this section, flanking the genomic sequences of g35018 on either side or between two or more such genomic sequences.
- a g35018 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 g35018 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, or a sequence complementary thereto or a biologically active fragment or variant thereof.
- a 5' regulatory region may comprise an isolated, purified, or recombinant polynucleotide comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35,
- a 3' regulatory region may comprise an isolated, purified, or recombinant polynucleotide comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of nucleotide positions 65854 to 67854 of SEQ ID No 1, or the complements thereof.
- nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising, consisting essentially of, or consisting of a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of nucleotide positions 213818 to 243685 of SEQ ID No 1, or the complements thereof.
- nucleic acids of the invention encompass an sbgl nucleic acid from any source, including primate, non-human primate, mammalian and human sbgl nucleic acids.
- 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, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises an sbgl related biallelic marker.
- said biallelic marker is selected from the group consisting of A85 to A219.
- said biallelic marker is selected from the group consisting of A85 to A94, A96 to A106, A108 to Al 12, Al 14 to A177, A179 to A197 and A199 to A219.
- nucleic acid fragments of any size and sequence may also be comprised by the polynucleotides described in this section.
- the human sbgl gene comprises exons selected from at least 22 different exons or exon forms, referred to herein as exons MSI, Ml , M692, M862, MS2, M1069, M1090, Ml 117, N , N2, Nbis, O, OI , 02, Obis, P, X, Ql , Q, Qbis, Rbis and R Of these, the following exon sets contain sequence overlap and do not occur together in an mRNA: exons Ml, M692, M862, MS2, M1090 M1069 and Ml 117; exons MSI, Ml, M692 and M862; exons N and N2; exons 01 and 02; exons Q and Qbis; exons R and R bis; and exons Q and Ql .
- nucleotide positions of sbgl exons in SEQ ID No. 1 are detailed below in Table 4.
- the exon structure of the sbgl gene is further shown in Figure 1.
- the invention embodies purified, isolated, or recombinant polynucleotides comprising a nucleotide sequence selected from the group consisting of the exons of the sbgl 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 4 selected from the group consisting of the exons MSI, Ml, M692, M862, MS2, M1069, Ml 090, Ml 1 17, N , N2, Nbis, O, OI, 02, Obis, P, X, Ql, Q, Qbis, R and Rbis of the sbgl 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 sbgl gene selected from the group consisting of exons MSI, Ml, M692,
- 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,
- nucleotides of SEQ ID No 1 wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 1 : 213818 to 215818, 215819 to 215941, 215819 to 215975, 216661 to 216952, 216661 to 217061, 217027 to 217061, 229647 to 229742, 230408 to 230721, 231272 to 231412, 231787 to 231880, 231870 to 231879, 234174 to 234321, 237406 to 237428, 239719 to 239807, 239719 to 239853, 240528 to 240569, 240528 to 240596, 240528 to 240617, 240528 to 240644, 240528 to 240824, 240528 to 240994, 240528 to 241685, 240800 to 240993 and 241686 to 2436
- Another object of the invention consists of a purified, isolated, or recombinant nucleic acid that hybridizes with an sbgl nucleotide sequence of nucleotide positions 213818 to 243685,
- the present invention further embodies purified, isolated, or recombinant polynucleotides comprising a nucleotide sequence selected
- the present invention encompasses the sbgl gene as well as sbgl genomic sequences consisting of, consisting essentially of, or comprising the sequence of nucleotide positions 215819 to 241685 of SEQ ID No 1, a sequence complementary thereto, as well as fragments and variants thereof.
- the invention also encompasses a purified, isolated, or recombinant polynucleotide comprising a nucleotide sequence of sbgl having at least 70, 75, 80, 85, 90, or 95%) nucleotide identity with a sequence selected from the group consisting of nucleotide positions 213818 to 215818, 215819 to 215941, 215819 to 215975, 216661 to 216952, 216661 to 217061, 217027 to
- nucleotide differences as regards the nucleotide positions 213818 to 215818, 215819 to 215941, 215819 to 215975, 216661 to 216952, 216661 to 217061, 217027 to 217061, 229647 to 229742, 230408 to 230721 , 231272 to 231412, 231787 to 231880, 231870 to 231879, 234174 to 234321, 237406 to 237428, 239719 to 239807, 239719 to 239853, 240528 to 240569, 240528 to 240596, 240528 to 240617, 240528 to 240644, 240528 to 240824, 240528 to 240994, 240528 to 241685, 240800 to 240993 and
- 241686 to 243685 of SEQ ID No. 1 may generally be distributed throughout the nucleic acid.
- 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 a gene comprising an sbgl nucleic acid sequence in a test sample, or alternatively in order to amplify a target nucleotide sequence within an sbgl nucleic acid sequence or adjoining region.
- Additional preferred nucleic acids of the invention include isolated, purified, or recombinant sbgl 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 213818 to 215818, 215819 to 215941, 215819 to 215975, 216661 to 216952, 216661 to 217061, 217027 to 217061, 229647 to 229742, 230408 to 230721, 231272 to 231412, 231787 to 231880, 231870 to 231879, 234174 to 234321, 237406 to 237428, 239719 to 239807, 239719 to 239853, 240528 to 240569, 240528 to 240596, 240528 to 240617, 240528 to 240644, 240528 to 240824, 240528 to 240994,
- said contiguous span comprises an sbgl -related biallelic marker.
- 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.
- 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,
- said contiguous span comprises at least 1 , 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 1 : 215820 to 215941, 216661 to 217009, 230409 to 290721, 231272 to 231411, 234202 to 234321, 240528 to 240567, 240528 to 240827 and 240528 to 240996, or the complements thereof, as well as polynucleotides having at least 70, 75, 80, 85, 90, or 95% nucleotide identity with said span, and polynucleotides capable of hybridizing with said span.
- the present invention also comprises a purified or isolated nucleic acid encoding an sbgl protein having the amino acid sequence of any one of SEQ ID Nos 27 to 35 or a peptide fragment or variant thereof.
- 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.
- the invention encompasses a purified, isolated, or recombinant nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID Nos 2 to 26, complementary sequences thereto, splice variants thereof, as well as allelic variants, and fragments thereof.
- preferred polynucleotides of the invention include purified, isolated, or recombinant sbgl cDNAs consisting of, consisting essentially of, or comprising a nucleotide sequence selected from the group consisting of SEQ ID Nos 2 to 26.
- 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.
- the invention also pertains to a purified or isolated nucleic acid comprising a polynucleotide having at least 70, 80, 85, 90 or 95% nucleotide identity with a polynucleotide selected from the group consisting of SEQ ID Nos 2 to 26, 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 SEQ ID Nos 2 to 26, or a sequence complementary thereto or a biologically active fragment thereof.
- Another object of the invention relates to purified, isolated or recombinant nucleic acids comprising a polynucleotide that hybridizes, under the stringent hybridization conditions defined herein, with a polynucleotide selected from the group consisting of SEQ ID Nos 2 to 26, or a sequence complementary thereto or a variant thereof or a biologically active fragment thereof.
- the sbgl cDNA forms of SEQ ID Nos 2 to 26 are further described in Table 5a below. Shown on the Table 5a are the positions of the 5' UTR, the open reading frame (ORF), the 3'
- 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.
- variants may represent rare polymo ⁇ hisms or may be the result of tissue-specific RNA editing.
- some variations may be the result of the presence in the human genome of one or more sbgl -related genes or a small family of sbgl -related genes with strict tissue specificity of expression and small variation in gene structure. The latter hypothesis was tested by applicants for the case where the exon-intron structure of these genes are identical, demonstrating that variations in at least exons M and N are not the result of the presence of related genes.
- the present invention thus further encompasses variant sbgl polynucleotides having at least one nucleotide substitution as described in Table 5d below.
- the nucleotide and amino acid variations as shown in Table 5d are shown in terms of the nucleotide sequence of SEQ ID No. 2, and specify variations as found in exons M862, N, O, Qbis and R.
- 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)
- MS2NOQbisR:296 10 M1069NOQR:75 (10) M1069N2OQlQbisR:75 (10) M1069NOQlQbisR:75 (10) M1069N2OO2QbisR:75 (10) M1069NOO2QbisR:75 (10) M1069N2NbisOO2XQbisR:75 (10) M1069N2OQR:75 (10) M1069N2OQbisR:75 (10) M1069NNbisOQR:75 (10) M1069NNbisOQbisR:75 (10) M1069NOO2XQbisR:75 (10) M1069NOXQR:75 (10) M1069NOQbisRbis:75 (10) M1069NOQbisRbis:75 (10) M1069N2OQbisRbis:75 (10) M1069N2OQbisRbis:75 (10) M1069N2OQbisRbis:75 (10) M1069N2OQbisRbis:75 (10) M1069N2OQ
- the sbgl open reading frame is contained in the corresponding mRNA of a cDNA sequence selected from the group consisting of SEQ ID Nos 2 to 26.
- the effective sbgl coding sequence (CDS) may include several forms as indicated above, in some embodiments encompassing isolated, purified, and recombinant polynucleotides which encode a polypeptide comprising a contiguous span of at least 4 amino acids, preferably 6, more preferably at least 8 or 10 amino acids, yet more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID Nos 27 to 35.
- the effective sbgl coding sequence (CDS) may comprise the region between the first nucleotide of the ATG codon and the end nucleotide of the stop codon of SEQ ID Nos 2 to 26 as indicated in Table 5a above.
- the above disclosed polynucleotide that contains the coding sequence of the sbgl gene may be expressed in a desired host cell or a desired host organism when this polynucleotide is placed under the control of suitable expression signals.
- the expression signals may be either the expression signals contained in the regulatory regions in the sbgl gene of the invention or in contrast the signals may be exogenous regulatory nucleic sequences.
- Such a polynucleotide, when placed under the suitable expression signals may also be inserted in a vector for its expression and/or amplification.
- the genomic sequence of the sbgl gene contains regulatory sequences both in the non-coding 5 '-flanking region and in the non-coding 3 '-flanking region that border the sbgl coding region containing the exons of the gene.
- the 3 '-regulatory sequence of the sbgl gene may comprise the sequence localized between the nucleotide in position 213818 and the nucleotide in position 215818 of the nucleotide sequence of SEQ ID No 1.
- the 5'-regulatory sequence of the sbgl gene may comprise the sequence localized between the 5' end of the particular form of exon M and nucleotide position 243685 of SEQ ID No 1.
- Polynucleotides derived from the 5' and 3' regulatory regions are useful in order to detect the presence of at least a copy of an sbg 1 nuc leotide sequence of SEQ ID No 1 or a fragment thereof in a test sample.
- 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.
- Promoter sequence within the upstream genomic DNA may be further defined by constructing nested 5' and/or 3' deletions in the upstream DNA using conventional techniques such as Exonuclease III or appropriate restriction endonuclease digestion.
- the resulting deletion fragments can be inserted into the promoter reporter vector to determine whether the deletion has reduced or obliterated promoter activity, such as described, for example, by Coles et al.(1998). In this way, the boundaries of the promoters may be defined.
- potential individual regulatory sites within the promoter may be identified using site directed mutagenesis or linker scanning to obliterate potential transcription factor binding sites within the promoter individually or in combination.
- 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.
- Polynucleotides carrying the regulatory elements located at the 5' end and at the 3' end of the sbgl coding region may be advantageously used to control the transcriptional and translational activity of an heterologous polynucleotide of interest.
- the present invention also concerns a purified or isolated nucleic acid comprising a polynucleotide which is selected from the group consisting of the 5' and 3' regulatory regions of sbgl, or a sequence complementary thereto or a biologically active fragment or variant thereof.
- "3' regulatory region” may comprise the nucleotide sequence located between positions 213818 and 215818 of SEQ ID No 1.
- "5 ' regulatory region” may comprise the nucleotide sequence located between the 5' end of a particular variant of exon M and nucleotide position 243685 of SEQ ID No 1.
- the 5' end of particular form of exon M may be selected from the group consisting of nucleotide postions 240569, 241596, 240617, 240644, 240824, 240994, 241685 and 240993 of SEQ ID No 1.
- the 5' regulatory region comprises the nucleotides of nucleotide positions 241686 to 243685 of SEQ ID No l .
- 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.
- Another object of the invention consists of purified, isolated or recombinant nucleic acids comprising a polynucleotide that hybridizes, under the stringent hybridization conditions defined herein, with a polynucleotide selected from the group consisting of the nucleotide sequences of the 5'- and 3' regulatory regions of sbgl, or a sequence complementary thereto or a variant thereof or a biologically active fragment thereof.
- Preferred fragments of the 5' regulatory region have a length of about 1500 or 1000 nucleotides, preferably of about 500 nucleotides, more preferably about 400 nucleotides, even more preferably 300 nucleotides and most preferably about 200 nucleotides.
- Preferred fragments of the 3' regulatory region are at least 50, 100, 150, 200, 300 or
- 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.
- a nucleic acid or polynucleotide is "functional" as a regulatory region for expressing a recombinant polypeptide or a recombinant polynucleotide if said regulatory polynucleotide contains nucleotide sequences which contain transcriptional and translational regulatory information, and such sequences are "operably linked" to nucleotide sequences which encode the desired polypeptide or the desired polynucleotide.
- 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.
- the sbgl regulatory polynucleotides according to the invention may be part of a recombinant expression vector that may be used to express a coding sequence in a desired host cell or host organism.
- the recombinant expression vectors according to the invention are 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 5 '-untranslated region (5'-UTR) of the sbgl cDNA, or a biologically active fragment or variant thereof.
- 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 5' regulatory region, or its biologically active fragments or variants, is operably linked at the 5'-end of the polynucleotide encoding the desired polypeptide or polynucleotide.
- 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 present invention further embodies purified, isolated, or recombinant polynucleotides comprising a nucleotide sequence selected from the group consisting of the introns of the sbg2 gene, or a sequence complementary thereto.
- the position of the introns is detailed in Table 5f.
- Intron S refers to the nucleotide sequence located between Exon S and
- 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.
- Another object of the invention relates to purified, isolated or recombinant nucleic acids comprising a polynucleotide that hybridizes, under the stringent hybridization conditions defined herein, with a polynucleotide selected from the group consisting of nucleotide positions 201 188 to 216915, 201188 to 201234, 214676 to 214793, 215702 to 215746 and 216836 to
- 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.
- compositions containing a vector of the invention comprise an oligonucleotide fragment of the sbgl, g34665, sbg2, g35017 or g35018 polynucleotide sequences of SEQ ID No.l respectively, as an antisense tool that inhibits the expression of the corresponding gene.
- Preferred methods using antisense polynucleotide according to the present invention are the procedures described by Sczakiel et al.(1995) or those described in PCT Application No WO 95/24223.
- 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).
- these sbgl, g34665, sbg2, g35017 or g35018 antisense polynucleotides also comprise, within the ribozyme cassette, a histone stem-loop structure to stabilize cleaved transcripts against 3'-5' exonucleolytic degradation, such as the structure described by Eckner et al .( 1991 ).
- the polynucleotides of the invention are useful in order to detect the presence of at least a copy of a nucleotide sequence of SEQ ID No. 1 or of the respective sbgl, g34665, sbg2, g35017 and g35018 polynucleotide or gene, or a fragment, complement, or variant thereof in a test sample.
- 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, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, 1000 or 2000 nucleotides, to the extent that said span is consistent with the length of 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
- 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.
- the 3' end of the probe can be functional ized with the capture or detection label to thereby consume or otherwise block the hydroxyl group.
- the 3' hydroxyl group simply can be cleaved, replaced or modified;
- 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.
- the nucleic acid probe or the plurality of nucleic acid probes comprise either a sequence which is selected from the group consisting 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, or a nucleotide sequence comprising a biallelic marker selected from the group consisting of Al to A360 or a polymo ⁇ hism selected from the group consisting of A361 to A489, or the complements thereto.
- 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.
- any polynucleotide provided herein may be attached in overlapping areas or at random locations on the solid support.
- the polynucleotides of the invention may be attached in an ordered array wherein each polynucleotide is attached to a distinct region of the solid support which does not overlap with the attachment site of any other polynucleotide.
- such an ordered array of polynucleotides is designed to be "addressable" where the distinct locations are recorded and can be accessed as part of an assay procedure.
- Addressable polynucleotide arrays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations.
- VLSIPSTM Very Large Scale Immobilized Polymer Synthesis
- an oligonucleotide probe matrix may advantageously be used to detect mutations occurring in an sbgl, g34665, sbg2, g35017 or g35018 polynucleotide, including in genes comprising an sbgl, g34665, sbg2, g35017 or g35018 polynucleotide and preferably in an sbgl, g34665, sbg2, g35017 or g35018 polynucleotide regulatory region.
- 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.
- Another technique that is used to detect mutations in an sbgl , g34665, sbg2, g35017 or g35018 polynucleotide is the use of a high-density DNA array.
- Each oligonucleotide probe constituting a unit element of the high density DNA array is designed to match a specific subsequence of an sbgl, g34665, sbg2, g35017 or g35018 polynucleotide.
- an array consisting of oligonucleotides complementary to subsequences of the target gene sequence is used to determine the identity of the target sequence with the wild-type gene sequence, measure its amount, and detect differences between the target sequence and the reference wild-type nucleic acid sequence of an sbgl, g34665, sbg2, g35017 or g35018 polynucleotide.
- 4L tiled array is implemented a set of four probes (A, C, G, T), preferably 15- nucleotide oligomers. In each set of four probes, the perfect complement will hybridize more strongly than mismatched probes.
- 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 present inventors have further identified potential cleavage sites in the sbgl polypeptides, and several specific sbgl peptides.
- An sbgl peptide has further been tested in behavioral studies by injection in mice, as further detailed in Example 7.
- the polypeptide of SEQ ID No 29 contains a protease cleavage site at amino acid positions 62 to 63;
- the polypeptide of SEQ ID No 30 contains a protease cleavage site at amino acid positions 63 to
- 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.
- the invention also encompasses a purified, isolated, or recombinant polypeptides comprising an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 98 or 99% amino acid identity with the amino acid sequence of SEQ ID Nos 27 to 35 and 41 to 43 or a fragment thereof.
- Sbgl, g34665, sbg2, g35017 and g35018 proteins are preferably isolated from human or mammalian tissue samples or expressed from human or mammalian genes.
- the sbgl, g34665, sbg2, g35017 and g35018 polypeptides of the invention can be made using routine expression methods known in the art.
- the polynucleotide encoding the desired polypeptide is ligated into an expression vector suitable for any convenient host. Both eukaryotic and prokaryotic host systems is used in forming recombinant polypeptides, and a summary of some of the more common systems.
- polypeptide is then isolated from lysed cells or from the culture medium and purified to the extent needed for its intended use. Purification is by any technique known in the art, for example, differential extraction, salt fractionation, chromatography, centrifugation, and the like. See, for example, Methods in Enzymology for a variety of methods for purifying proteins. In addition, shorter protein fragments can be produced by chemical synthesis.
- 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 sbgl , g34665, sbg2, g35017 or g35018 insert in the expression vector may comprise the full coding sequence for the respective sbgl, g34665, sbg2, g35017 or g35018 protein or a portion thereof.
- the sbgl or g35018 derived insert may encode a polypeptide comprising at least 10 consecutive amino acids of the respective sbgl or g35018 protein of SEQ ID Nos 27 to 35 and 41 to 43.
- the expression vector is any of the mammalian, yeast, insect or bacterial expression systems known in the art.
- the entire coding sequence of the sbgl, g34665, sbg2, g35017 or g35018 cDNA through the poly A signal of the cDNA are operably linked to a promoter in the expression vector.
- the nucleic acid encoding a portion of the sbgl, g34665, sbg2, g35017 or g35018 protein lacks a methionine to serve as the initiation site, an initiating methionine can be introduced next to the first codon of the nucleic acid using conventional techniques.
- this sequence can be added to the construct by, for example, splicing out the Poly A signal from pSG5 (Stratagene) using Bgll and Sail restriction endonuclease enzymes and inco ⁇ orating it into the mammalian expression vector pXTl (Stratagene).
- pXTl contains the LTRs and a portion of the gag gene from Moloney Murine Leukemia Virus. The position of the LTRs in the construct allow efficient stable transfection.
- the vector includes the He ⁇ es Simplex Thymidine Kinase promoter and the selectable neomycin gene.
- the nucleic acid encoding the sbgl, g34665, sbg2, g35017 or g35018 protein or a portion thereof is obtained by PCR from a bacterial vector containing the a nucleotide sequence of an exon of an sbgl, g34665, sbg2, g35017 or g35018 gene as described herein and in SEQ ID No 1, or from an sbgl or g35018 cDNA comprising a nucleic acid of SEQ ID No 2 to 26 and 36 to 40 using oligonucleotide primers complementary to the sbgl, g34665, sbg2, g35017 or g35018 nucleic acid or portion thereof and containing restriction endonuclease sequences for Pst I inco ⁇ orated into the 5' primer and BgHI at the
- 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 proteins expressed from host cells containing an expression vector containing an insert encoding the sbgl, g34665, sbg2, g35017 or g35018 protein or a portion thereof can be compared to the proteins expressed in host cells containing the expression vector without an insert.
- the presence of a band in samples from cells containing the expression vector with an insert which is absent in samples from cells containing the expression vector without an insert indicates that the sbgl, g34665, sbg2, g35017 or g35018 protein or a portion thereof is being expressed.
- 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).
- any sbgl, g34665, sbg2, g35017 or g35018 polypeptide or whole protein may be used to generate antibodies capable of specifically binding to an expressed sbgl, g34665, sbg2, g35017 and g35018 protein or fragments thereof.
- an antibody composition to specifically bind to an 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 full length sbgl, g34665, sbg2, g35017 or g35018 protein than for any full length protein in an ELISA, RIA, or other antibody-based binding assay.
- 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.
- antibody compositions of the invention are capable of specifically binding or specifically bind to an sbgl, sbg2 or g35018 protein variant.
- said sbgl protein variant may be a natural variant provided in Tables 5d or 5e.
- the invention concerns antibody compositions, either polyclonal or monoclonal, capable of selectively binding, or selectively bind to an epitope-containing a polypeptide comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of an sbgl, g34665, sbg2, g35017 or g35018 polypeptide.
- 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.
- the invention concerns the use in the manufacture of antibodies of a polypeptide comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of any of SEQ ID Nos 27 to 35 and 41 to 43.
- 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.
- sbgl, g34665, sbg2, g35017 or g35018 knock out animals will recognize all or most of the exposed regions of an sbgl, g34665, sbg2, g35017 or g35018 protein as foreign antigens, and therefore produce antibodies with a wider array of sbgl, g34665, sbg2, g35017 or g35018 epitopes.
- smaller polypeptides with only 10 to 30 amino acids may be useful in obtaining specific binding to any one of the sbgl , g34665, sbg2, g35017 or g35018 proteins.
- 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 antibodies may also be used in therapeutic compositions for killing cells expressing the protein or reducing the levels of the protein in the body.
- the invention comprises the use of an antibody capable of specifically recognizing sbgl , g34665, sbg2, g35017 or g35018 for the treatment of schizophrenia or bipolar disorder.
- the antibodies of the invention may be labeled by any one of the radioactive, fluorescent or enzymatic labels known in the art.
- 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.
- the biallelic marker of the inventions of the present invention offer a number of important advantages over other genetic markers such as RFLP (Restriction fragment length polymo ⁇ hism) and VNTR (Variable Number of Tandem Repeats) markers.
- the first generation of markers were RFLPs, which are variations that modify the length of a restriction fragment. But methods used to identify and to type RFLPs are relatively wasteful of materials, effort, and time.
- the second generation of genetic markers were VNTRs, which can be categorized as either minisatellites or microsatellites. Minisatellites are tandemly repeated DNA sequences present in units of 5-50 repeats which are distributed along regions of the human chromosomes ranging from 0.1 to 20 kilobases in length. Since they present many possible alleles, their informative content is very high. Minisatellites are scored by performing
- RFLPs and VNTRs but offer several advantages.
- Single nucleotide polymorphisms are densely spaced in the human genome and represent the most frequent type of variation.
- An estimated number of more than 10 7 sites are scattered along the 3x109 base pairs of the human genome.
- 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.
- biallelic markers of the present invention are often easier to distinguish and can therefore be typed easily on a routine basis.
- Biallelic markers have single nucleotide based alleles and they have only two common alleles, which allows highly parallel detection and automated scoring.
- 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).
- Biallelic markers in different genes can be screened in parallel for direct association with disease or response to a treatment.
- This multiple gene approach is a powerful tool for a variety of human genetic studies as it provides the necessary statistical power to examine the synergistic effect of multiple genetic factors on a particular phenotype, drug response, sporadic trait, or disease state with a complex genetic etiology.
- 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.
- the polynucleotide of the invention may consist of, consist essentially of, or comprise a contiguous span of nucleotides of a sequence from any of SEQ ID Nos. 1 to 26, 36 to 40 and 54 to 229 as well as sequences which are complementary thereto ("complements thereof).
- 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.
- the present invention encompasses polynucleotides for use as primers and probes in the methods of the invention.
- These polynucleotides may consist of, consist essentially of, or comprise a contiguous span of nucleotides of a sequence from any of SEQ ID Nos. 1 to 26, 36 to 40 and 54 to 229 as well as sequences which are complementary thereto ("complements thereof).
- 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.
- 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 also relates to polynucleotides that hybridize, under conditions of high or intermediate stringency, to a polynucleotide of any of SEQ ID Nos. 1 to 26, 36 to 40 and 54 to 229 as well as sequences, which are complementary thereto.
- polynucleotides are at least 20, 25, 35, 40, 50, 70, 80, 100, 250, 500 , 1000 or 2000 nucleotides in length, to the extent that a polynucleotide of these lengths is consistent with the lengths of the particular
- Preferred polynucleotides comprise a polymorphism of the invention.
- either allele 1 or allele 2 of the polymo ⁇ hism disclosed in Table 6c may be specified as being present at the polymo ⁇ hism of the invention.
- Particularly preferred polynucleotides comprise a biallelic marker of the invention.
- either 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 primers of the present invention may be designed from the disclosed sequences for any method known in the art.
- a preferred set of primers is fashioned such that the 3' end of the contiguous span of identity with the sequences of any of SEQ ID Nos. 1 to 26, 36 to 40 and 54 to 229 is present at the 3' end of the primer.
- Such a configuration allows the 3' end of the primer to hybridize to a selected nucleic acid sequence and dramatically increases the efficiency of the primer for amplification or sequencing reactions.
- the contiguous span is found in one of the sequences described in Table 6a.
- Allele specific primers may be designed such that a biallelic marker or other polymo ⁇ hism of the invention is at the 3' end of the contiguous span and the contiguous span is present at the 3' end of the primer. Such allele specific primers tend to selectively prime an amplification or sequencing reaction so long as they are used with a nucleic acid sample that contains one of the two alleles present at said marker.
- the 3' end of primer of the invention may be located within or at least 2, 4, 6, 8, 10, 12, 15, 18, 20, 25, 50, 100, 250, 500, or 1000 nucleotides upstream of a biallelic marker of the invention in said sequence or at any other location which is appropriate for their intended use in sequencing, amplification or the location of novel sequences or markers.
- Primers with their 3' ends located 1 nucleotide upstream of an biallelic marker of the invention have a special utility as microsequencing assays. Preferred microsequencing primers are described in Table 6d.
- the probes of the present invention may be designed from the disclosed sequences for any method known in the art, particularly methods which allow for testing if a particular sequence or marker disclosed herein is present.
- a preferred set of probes may be designed for use in the hybridization assays of the invention in any manner known in the art such that they selectively bind to one allele of a biallelic marker or other polymo ⁇ hism, but not the other under any particular set of assay conditions.
- Preferred hybridization probes may consists of, consist essentially of, or comprise a contiguous span which ranges in length from 8, 10, 12, 15,
- either of allele 1 or 2 disclosed in Table 6b or 6c may be specified as being present at the biallelic marker site.
- said biallelic marker may be within 6, 5, 4, 3, 2, or 1 nucleotides of the center of the hybridization probe or at the center of said probe.
- the invention encompasses isolated, purified, and recombinant polynucleotides comprising, consisting of, or consisting essentially of a contiguous span of 8 to 50 nucleotides of any one of SEQ ID Nos 1 to 26, 36 to 40 and 54 to 229 and the complement thereof, wherein said span includes a polymo ⁇ hism of the invention, a chromosome 13q31- q33-related biallelic marker, region D-related biallelic marker, or sbgl-, g34665-, sbg2-, g35017- or g35018 -related biallelic marker in said sequence; optionally, wherein said polymo ⁇ hism, chromosome 13q31-q33-related biallelic marker, region D-related biallelic marker, or sbgl-, g34665-, sbg2-, g35017- or g35018 -related biallelic marker selected from the group consisting of A 1 to A
- said probes comprise, consists of, or consists essentially of a sequence selected from the following sequences: PI to P360 and the complementary sequences thereto.
- the invention encompasses isolated, purified and recombinant polynucleotides comprising, consisting of, or consisting essentially of a contiguous span of 8 to 50 nucleotides of any one of SEQ ID Nos 1 to 26, 36 to 40 and 54 to 229, or the complement thereof, wherein the 3' end of said contiguous span is located at the 3' end of said polynucleotide, and wherein the 3' end of said polynucleotide is located within 20 nucleotides upstream of a polymo ⁇ hism of the invention, chromosome 13q31-q33-related biallelic marker, region D-related biallelic marker, or sbgl-, g34665-, sbg2-, g35017- or g35018 -related biallelic marker in said sequence; optionally, wherein said chromosome 13q31-q33-related biallelic marker, region D-related biallelic marker, or sbgl-
- chromosome 13q3 l-q33-related biallelic marker, region D-related biallelic marker, or sbgl-, g34665-, sbg2-, g35017- or g35018 -related biallelic marker is selected from the group consisting of A 1 to A69, A71 to A74, A76 to A94, A96 to A 106, A 108 to A 112, A 114 to A 177,
- chromosome 13q31-q33-related biallelic marker, region D-related biallelic marker, or sbgl-, g34665-, sbg2-, g35017- or g35018 -related biallelic marker is selected from the group consisting of optionally, wherein said chromosome 13q31-q33-related biallelic marker, region D-related biallelic marker, or sbgl-, g34665-, sbg2-, g35017- or g35018 -related biallelic marker is 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, A27
- the invention encompasses isolated, purified, or recombinant polynucleotides comprising, consisting of, or consisting essentially of a sequence selected from the following sequences: Bl to B229 and CI to C229.
- 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.
- 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 1 to 26, 36 to 40 and 54 to 229 or the complement thereof; optionally, wherein said chromosome 13q31-q33-related biallelic marker, region D-related biallelic marker, or sbgl-, g34665-, sbg2-, g35017- or g35018 -related biallelic marker is selected from the group consisting of Al to A489, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, where
- arrays may generally be produced using mechanical synthesis methods or light directed synthesis methods, which incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis (Fodor et al., Science, 251 :767-777, 1991).
- the immobilization of arrays of oligonucleotides on solid supports has been rendered possible by the development of a technology generally identified as "Very Large Scale Immobilized Polymer Synthesis” (VLSIPSTM) in which, typically, probes are immobilized in a high density array on a solid surface of a chip. Examples of VLSIPSTM technologies are provided in US Patents 5,143,854 and 5,412,087 and in PCT Publications WO 90/15070, WO 92/10092 and
- 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.
- Oligonucleotide arrays may comprise at least one of the sequences selected from the group consisting of SEQ ID Nos. 1 to 26, 36 to 40 and 54 to 229; and the sequences complementary thereto or a fragment thereof of at least 8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 70, 80, 100, 250, 500 , 1000 or 2000 consecutive nucleotides, to the extent that fragments of these lengths is consistent with the lengths of the particular Sequence ID, for determining whether a sample contains one or more alleles of the biallelic markers of the present invention. Oligonucleotide arrays may also comprise at least one of the sequences selected from the group consisting of SEQ ID Nos. 1 to 26, 36 to 40 and 54 to 229; and the sequences complementary thereto or a fragment thereof of at least 8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 70, 80, 100, 250,
- arrays may also comprise at least one of the sequences selected from the group consisting of SEQ ID Nos.
- 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.
- the present invention also encompasses diagnostic kits comprising one or more polynucleotides of the invention, optionally with a portion or all of the necessary reagents and instructions for genotyping a test subject by determining the identity of a nucleotide at an biallelic marker of the invention.
- the polynucleotides of a kit may optionally be attached to a solid support, or be part of an array or addressable array of polynucleotides.
- the kit may provide for the determination of the identity of the nucleotide at a marker position by any method known in the art including, but not limited to, a sequencing assay method, a microsequencing assay method, a hybridization assay method, or enzyme-based mismatch detection assay.
- 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 DNA samples are not pooled and are therefore amplified and sequenced individually.
- This method is usually preferred when biallelic markers need to be identified in order to perform association studies within candidate genes.
- highly relevant gene regions such as promoter regions or exon regions may be screened for biallelic markers.
- a biallelic marker obtained using this method may show a lower degree of informativeness for conducting association studies, e.g. if the frequency of its less frequent allele may be less than about 10%.
- Such a biallelic marker will however be sufficiently informative to conduct association studies and it will further be appreciated that including less informative biallelic markers in the genetic analysis studies of the present invention, may allow in some cases the direct identification of causal mutations, which may, depending on their penetrance, be rare mutations.
- 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.
- DNA samples can be pooled or unpooled for the amplification step.
- DNA amplification techniques are well known to those skilled in the art.
- Various methods to amplify DNA fragments carrying biallelic markers are further described hereinafter herein.
- the PCR technology is the preferred amplification technique used to identify new biallelic markers.
- 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.
- genomic sequences of candidate genes are available in public databases allowing direct screening for biallelic markers.
- Preferred primers, useful for the amplification of genomic sequences encoding the candidate genes focus on promoters, exons and splice sites of the genes.
- a biallelic marker present in these functional regions of the gene have a higher probability to be a causal mutation.
- 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.
- Validation of the biallelic markers of the present invention The polymo ⁇ hisms are evaluated for their usefulness as genetic markers by validating that both alleles are present in a population. Validation of the biallelic markers is accomplished by genotyping a group of individuals by a method of the invention and demonstrating that both alleles are present. Microsequencing is a preferred method of genotyping alleles. The validation 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 can be as small as one individual if that individual is heterozygous for the allele in question.
- 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.
- Another embodiment of the invention comprises methods of estimating the frequency of an allele in a population comprising genotyping individuals from said population for a 13q31-q33-related biallelic marker and determining the proportional representation of said biallelic marker in said population.
- the methods of estimating the frequency of an allele in a population encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination:
- said 13q31- q33-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ Nos 1 to 26, 36 to 40 and 54 to 229; and the complements thereof;
- said 13q3 l-q33-related biallelic marker may be selected from the biallelic markers described in Table 6b or 6c;
- determining the frequency of a biallelic marker allele in a population may be accomplished by determining the identity of the nucleotides for both copies of said biallelic marker present in the genome of each individual in said population and calculating the proportional representation of
- 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.
- Genotyping can be performed using similar methods as those described above for the identification of the biallelic markers, or using other genotyping methods such as those further described below.
- the comparison of sequences of amplified genomic fragments from different individuals is used to identify new biallelic markers whereas microsequencing is used for genotyping known biallelic markers in diagnostic and association study applications.
- Another embodiment of the invention encompasses methods of genotyping a biological sample comprising determining the identity of a nucleotide at a 13q31-q33-related biallelic marker.
- the genotyping methods of the invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination:
- said 13q31-q33-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID Nos 1 to 26, 36 to 40 and 54 to 229, and the complements thereof;
- said 13q3 l-q33-related biallelic marker may be selected individually or in any combination from the biallelic markers described in Table 6b and 6c;
- said method further comprises determining the identity of a second nucleotide at said biallelic marker, wherein said first nucleotide and second nucleotide are not base paired (by Watson & Crick base pairing) to one another; optionally, said biological sample is derived from a single individual or subject;
- Source of DNA for genotyping Any source of nucleic acids, in purified or non-purified form, can be utilized as the starting nucleic acid, provided it contains or is suspected of containing the specific nucleic acid sequence desired.
- DNA or RNA may be extracted from cells, tissues, body fluids and the like as described herein. While nucleic acids for use in the genotyping methods of the invention can be derived from any mammalian source, the test subjects and individuals from which nucleic acid samples are taken are generally understood to be human.
- Amplification Of DNA Fragments Comprising Biallelic Markers Methods and polynucleotides are provided to amplify a segment of nucleotides comprising one or more biallelic marker of the present invention.
- 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.
- Amplification methods which can be utilized herein include but are not limited to Ligase Chain Reaction (LCR) as described in EP A 320 308 and EP A 439 182, Gap LCR (Wolcott, M.J.), the so-called “NASBA” or “3SR” technique described in Guatelli J.C. et al. (1990) and in Compton J. (1991), Q-beta amplification as described in EP A 4544 610, strand displacement amplification as described in Walker et al. (1996) and EP A 684 315 and, target mediated amplification as described in PCT Publication WO 9322461.
- LCR Ligase Chain Reaction
- NASBA so-called "NASBA” or “3SR” technique described in Guatelli J.C. et al. (1990) and in Compton J. (1991)
- Q-beta amplification as described in EP A 4544 610
- strand displacement amplification as described in Walker et al. (1996) and EP
- LCR and Gap LCR are exponential amplification techniques, both depend on DNA ligase to join adjacent primers annealed to a DNA molecule.
- probe pairs are used which include two primary (first and second) and two secondary (third and fourth) probes, all of which are employed in molar excess to target.
- the first probe hybridizes to a first segment of the target strand and the second probe hybridizes to a second segment of the target strand, the first and second segments being contiguous so that the primary probes abut one another in 5' phosphate-3 'hydroxyl relationship, and so that a ligase can covalently fuse or ligate the two probes into a fused product.
- a third (secondary) probe can hybridize to a portion of the first probe and a fourth (secondary) probe can hybridize to a portion of the second probe in a similar abutting fashion.
- the secondary probes also will hybridize to the target complement in the first instance.
- the third and fourth probes which can be ligated to form a complementary, secondary ligated product. It is important to realize that the ligated products are functionally equivalent to either the target or its complement. By repeated cycles of hybridization and ligation, amplification of the target sequence is achieved.
- Gap LCR is a version of LCR where the probes are not adjacent but are separated by 2 to 3 bases.
- RT-PCR polymerase chain reaction
- AGLCR is a modification of GLCR that allows the amplification of RNA.
- Some of these amplification methods are particularly suited for the detection of single nucleotide polymo ⁇ hisms and allow the simultaneous amplification of a target sequence and the identification of the polymo ⁇ hic nucleotide as it is further described herein.
- PCR technology is the preferred amplification technique used in the present invention.
- a variety of PCR techniques are familiar to those skilled in the art. For a review of
- PCR primers on either side of the nucleic acid sequences to be amplified are added to a suitably prepared nucleic acid sample along with dNTPs and a thermostable polymerase such as Taq polymerase, Pfu polymerase, or Vent polymerase.
- a thermostable polymerase such as Taq polymerase, Pfu polymerase, or Vent polymerase.
- the nucleic acid in the sample is denatured and the PCR primers are specifically hybridized to complementary nucleic acid sequences in the sample.
- the hybridized primers are extended. Thereafter, another cycle of denaturation, hybridization, and extension is initiated. The cycles are repeated multiple times to produce an amplified fragment containing the nucleic acid sequence between the primer sites.
- PCR has further been described in several patents including US Patents 4,683,195, 4,683,202 and 4,965,188.
- Primers can be prepared by any suitable method. As for example, direct chemical synthesis by a method such as the phosphodiester method of Narang S.A. et al. (1979), the phosphodiester method of Brown E.L. et al. (1979), the diethylphosphoramidite method of Beaucage et al. (1981) and the solid support method described in EP 0 707 592.
- the present invention provides primers for amplifying a DNA fragment containing one or more biallelic markers of the present invention. It will be appreciated that the primers listed are merely exemplary and that any other set of primers which produce amplification products containing one or more biallelic markers of the present invention.
- amplified segments carrying biallelic markers can range in size from at least about 25 bp to 35 kbp. Amplification fragments from 25-3000 bp are typical, fragments from 50-1000 bp are preferred and fragments from 100-600 bp are highly preferred. It will be appreciated that amplification primers for the biallelic markers may be any sequence which allow the specific amplification of any DNA fragment carrying the markers. Amplification primers may be labeled or immobilized on a solid support as described in the section titled "Oligonucleotide Probes and Primers".
- Genotyping DNA samples for Biallelic Markers Any method known in the art can be used to identify the nucleotide present at a biallelic marker site. Since the biallelic marker allele to be detected has been identified and specified in the present invention, detection will prove simple for one of ordinary skill in the art by employing any of a number of techniques. Many genotyping methods require the previous amplification of the DNA region carrying the biallelic marker of interest. While the amplification of target or signal is often preferred at present, ultrasensitive detection methods which do not require amplification are also encompassed by the present genotyping methods.
- 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
- Another method for determining the identity of the nucleotide present at a particular polymo ⁇ hic site employs a specialized exonuclease-resistant nucleotide derivative as described in US patent 4,656,127.
- Preferred methods involve directly determining the identity of the nucleotide present at a biallelic marker site by sequencing assay, enzyme-based mismatch detection assay, or hybridization assay. The following is a description of some preferred methods.
- a highly preferred method is the microsequencing technique.
- the term "sequencing assay” is used herein to refer to polymerase extension of duplex primer/template complexes and includes both traditional sequencing and microsequencing.
- the nucleotide present at a polymo ⁇ hic site can be determined by sequencing methods.
- DNA samples are subjected to PCR amplification before sequencing as described above.
- DNA sequencing methods are described in herein.
- the amplified DNA is subjected to automated dideoxy terminator sequencing reactions using a dye-primer cycle sequencing protocol. Sequence analysis allows the identification of the base present at the biallelic marker site.
- a nucleotide at the polymo ⁇ hic site that is unique to one of the alleles in a target DNA is detected by a single nucleotide primer extension reaction.
- This method involves appropriate microsequencing primers which, hybridize just upstream of a polymo ⁇ hic base of interest in the target nucleic acid.
- a polymerase is used to specifically extend the 3' end of the primer with one single ddNTP (chain terminator) complementary to the selected nucleotide at the polymo ⁇ hic site.
- the identity of the inco ⁇ orated nucleotide is determined in any suitable way.
- microsequencing reactions are carried out using fluorescent ddNTPs and the extended microsequencing primers are analyzed by electrophoresis on ABI 377 sequencing machines to determine the identity of the inco ⁇ orated nucleotide as described in EP 412 883.
- capillary electrophoresis can be used in order to process a higher number of assays simultaneously.
- An example of a typical microsequencing procedure that can be used in the context of the present invention is provided in example 4.
- a homogeneous phase detection method based on fluorescence resonance energy transfer has been described by Chen and Kwok (1997) and Chen et al. (1997).
- amplified genomic DNA fragments containing polymorphic sites are incubated with a 5'- fluorescein-labeled primer in the presence of allelic dye-labeled dideoxyribonucleoside triphosphates and a modified Taq polymerase.
- the dye-labeled primer is extended one base by the dye-terminator specific for the allele present on the template.
- the fluorescence intensities of the two dyes in the reaction mixture are analyzed directly without separation or purification.
- the extended primer may be analyzed by MALDI-TOF Mass Spectrometry.
- the base at the polymo ⁇ hic site is identified by the mass added onto the microsequencing primer (see Haff L.A. and Smirnov I.P., 1997).
- Microsequencing may be achieved by the established microsequencing method or by developments or derivatives thereof.
- Alternative methods include several solid-phase microsequencing techniques.
- the basic microsequencing protocol is the same as described previously, except that the method is conducted as a heterogenous phase assay, in which the primer or the target molecule is immobilized or captured onto a solid support.
- oligonucleotides are attached to solid supports or are modified in such ways that permit affinity separation as well as polymerase extension.
- the 5' ends and internal nucleotides of synthetic oligonucleotides can be modified in a number of different ways to permit different affinity separation approaches, e.g., biotinylation. If a single affinity group is used on the oligonucleotides, the oligonucleotides can be separated from the inco ⁇ orated terminator regent. This eliminates the need of physical or size separation. More than one oligonucleotide can be separated from the terminator reagent and analyzed simultaneously if more than one affinity group is used.
- the affinity group need not be on the priming oligonucleotide but could alternatively be present on the template.
- immobilization can be carried out via an interaction between biotinylated DNA and streptavidin-coated microtitration wells or avidin-coated polystyrene particles.
- oligonucleotides or templates may be attached to a solid support in a high-density format.
- inco ⁇ orated ddNTPs can be radiolabeled (Syvanen, 1994) or linked to fluorescein (Livak and Hainer, 1994).
- 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.
- Discrimination between the two alleles of a biallelic marker can also be achieved by allele specific amplification, a selective strategy, whereby one of the alleles is amplified without amplification of the other allele. This is accomplished by placing a polymo ⁇ hic base at the 3' end of one of the amplification primers. Because the extension forms from the 3'end of the primer, a mismatch at or near this position has an inhibitory effect on amplification. Therefore, under appropriate amplification conditions, these primers only direct amplification on their complementary allele. Designing the appropriate allele-specific primer and the corresponding assay conditions are well with the ordinary skill in the art. Ligation/amplification based methods
- 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.
- either oligonucleotide will be designed to include the biallelic marker site.
- the reaction conditions are selected such that the oligonucleotides can be ligated together only if the target molecule either contains or lacks the specific nucleotide(s) that is complementary to the biallelic marker on the oligonucleotide.
- the oligonucleotides will not include the biallelic marker, such that when they hybridize to the target molecule, a "gap" is created as described in WO 90/01069. his gap is then "filled" with complementary dNTPs (as mediated by DNA polymerase), or by an additional pair of oligonucleotides.
- each single strand has a complement capable of serving as a target during the next cycle and exponential allele-specific amplification of the desired sequence is obtained.
- 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).
- Hybridization refers to the formation of a duplex structure by two single stranded nucleic acids due to complementary base pairing. Hybridization can occur between exactly complementary nucleic acid strands or between nucleic acid strands that contain minor regions of mismatch. Specific probes can be designed that hybridize to one form of a biallelic marker and not to the other and therefore are able to discriminate between different allelic forms.
- Allele-specific probes are often used in pairs, one member of a pair showing perfect match to a target sequence containing the original allele and the other showing a perfect match to the target sequence containing the alternative allele.
- Hybridization conditions should be sufficiently stringent that there is a significant difference in hybridization intensify between alleles, and preferably an essentially binary response, whereby a probe hybridizes to only one of the alleles.
- Stringent, sequence specific hybridization conditions, under which a probe will hybridize only to the exactly complementary target sequence are well known in the art (Sambrook et al., Molecular Cloning - A Laboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y., 1989). Stringent conditions are sequence dependent and will be different in different circumstances.
- stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
- Tm thermal melting point
- 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
- the hybridization step can be performed at 65°C in the presence of SSC buffer, 1 x SSC corresponding to 0.15M NaCl and 0.05 M Na citrate. Subsequently, filter washes can be done at 37°C for 1 h in a solution containing 2X SSC, 0.01% PVP, 0.01% Ficoll, and 0.01%
- 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.
- hybridizations can be performed in solution, it is preferred to employ a solid-phase hybridization assay.
- the target DNA comprising a biallelic marker of the present invention may be amplified prior to the hybridization reaction.
- the presence of a specific allele in the sample is determined by detecting the presence or the absence of stable hybrid duplexes formed between the probe and the target DNA.
- the detection of hybrid duplexes can be carried out by a number of methods.
- Various detection assay formats are well known which utilize detectable labels bound to either the target or the probe to enable detection of the hybrid duplexes.
- hybridization duplexes are separated from unhybridized nucleic acids and the labels bound to the duplexes are then detected.
- 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.
- Hybridization assays based on oligonucleotide arrays rely on the differences in hybridization stability of short oligonucleotides to perfectly matched and mismatched target sequence variants. Efficient access to polymorphism information is obtained through a basic structure comprising high-density arrays of oligonucleotide probes attached to a solid support (the chip) at selected positions. Each DNA chip can contain thousands to millions of individual synthetic DNA probes arranged in a grid-like pattern and miniaturized to the size of a dime. The chip technology has already been applied with success in numerous cases. For example, the screening of mutations has been undertaken in the BRCA1 gene, in S.
- 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.
- EP785280 describes a tiling strategy for the detection of single nucleotide polymorphisms.
- arrays may generally be "tiled” for a large number of specific polymo ⁇ hisms.
- tilting is generally meant the synthesis of a defined set of oligonucleotide probes which is made up of a sequence complementary to the target sequence of interest, as well as preselected variations of that sequence, e.g., substitution of one or more given positions with one or more members of the basis set of monomers, i.e. nucleotides. Tiling strategies are further described in PCT application No. WO 95/11995.
- arrays are tiled for a number of specific, identified biallelic marker sequences.
- 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.
- These monosubstituted probes have bases at and up to a certain number of bases in either direction from the polymo ⁇ hism, substituted with the remaining nucleotides (selected from A, T, G, C and U).
- the probes in a tiled detection block will include substitutions of the sequence positions up to and including those that are 5 bases away from the biallelic marker.
- the monosubstituted probes provide internal controls for the tiled array, to distinguish actual hybridization from artefactual cross-hybridization. Upon completion of hybridization with the target sequence and washing of the array, the array is scanned to determine the position on the array to which the target sequence hybridizes.
- hybridization data from the scanned array is then analyzed to identify which allele or alleles of the biallelic marker are present in the sample.
- Hybridization and scanning may be carried out as described in PCT application No. WO 92/10092 and WO 95/11995 and US patent No. 5,424,186.
- 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
- the chip may comprise an array of at least 2, 3, 4, 5, 6, 7, 8 or more of these polynucleotides of the invention.
- 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.
- loci When data are available from successive generations there is the opportunity to study the degree of linkage between pairs of loci.
- Estimates of the recombination fraction enable loci to be ordered and placed onto a genetic map. With loci that are genetic markers, a genetic map can be established, and then the strength of linkage between markers and traits can be calculated and used to indicate the relative positions of markers and genes affecting those traits (Weir, B.S., 1996).
- the classical method for linkage analysis is the logarithm of odds (lod) score method (see Morton N.E., 1955; Ott J, 1991). Calculation of lod scores requires specification of the mode of inheritance for the disease (parametric method). Generally, the length of the candidate region identified using linkage analysis is between 2 and 20Mb.
- 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.
- biallelic markers of the present invention may be inco ⁇ orated in any map of genetic markers of the human genome in order to perform genome-wide association studies. Methods to generate a high-density map of biallelic markers has been described in US Provisional Patent application serial number 60/082,614.
- 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.
- the methods of estimating the frequency of a haplotype of the invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: optionally said haplotype determination method is selected from the group consisting of asymmetric PCR amplification, double PCR amplification of specific alleles, the Clark method, or an expectation maximization algorithm; optionally, said second biallelic marker is a 13q31-q33-related biallelic marker in a sequence selected from the group consisting of SEQ ID Nos 1 to 26, 36 to 40 and 54 to 229, and the complements thereof; optionally, said 13q31-q33-related biallelic marker may be selected individually or in any combination from the biallelic markers described in Tables 6b and 6c; optionally, the identity of the nucleotides at the biallelic markers in everyone of the sequences of SEQ ID Nos 1 to 26, 36 to 40 and 54 to 229 is determined in steps a) and b). Association studies explore the relationships among frequencies for sets of alleles between loci
- Allelic frequencies of the biallelic markers in a population can be determined using one of the methods described above under the heading "Methods for genotyping an individual for biallelic markers", or any genotyping procedure suitable for this intended pu ⁇ ose.
- Genotyping pooled samples or individual samples can determine the frequency of a biallelic marker allele in a population.
- One way to reduce the number of genotypings required is to use pooled samples.
- a major obstacle in using pooled samples is in terms of accuracy and reproducibility for determining accurate DNA concentrations in setting up the pools.
- Genotyping individual samples provides higher sensitivity, reproducibility and accuracy and; is the preferred method used in the present invention.
- each individual is genotyped separately and simple gene counting is applied to determine the frequency of an allele of a biallelic marker or of a genotype in a given population.
- 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.
- single chromosomes can be studied independently, for example, by asymmetric PCR amplification (see Newton et al., 1989; Wu et al., 1989) or by isolation of single chromosome by limit dilution followed by PCR amplification (see Ruano et al., 1990). Further, a sample may be haplotyped for sufficiently close biallelic markers by double PCR amplification of specific alleles (Sarkar, G. and Sommer S.S., 1991). These approaches are not entirely satisfying either because of their technical complexify, the additional cost they entail, their lack of generalisation at a large scale, or the possible biases they introduce.
- an algorithm to infer the phase of PCR-amplified DNA genotypes introduced by Clark A.G. (1990) may be used. Briefly, the principle is to start filling a preliminary list of haplotypes present in the sample by examining unambiguous individuals, that is, the complete homozygotes and the single-site heterozygotes. Then other individuals in the same sample are screened for the possible occurrence of previously recognised haplotypes. For each positive identification, the complementary haplotype is added to the list of recognised haplotypes, until the phase information for all individuals is either resolved or identified as unresolved.
- This method assigns a single haplotype to each multiheterozygous individual, whereas several haplotypes are possible when there are more than one heterozygous site.
- a method based on an expectation-maximization (EM) algorithm (Dempster et al., J R. 1977) leading to maximum-likelihood estimates of haplotype frequencies under the assumption of Hardy- Weinberg proportions (random mating) is used (see Excoffier L. and Slatkin M., 1995).
- the EM algorithm is a generalised iterative maximum-likelihood approach to estimation that is useful when data are ambiguous and/or incomplete.
- the EM algorithm is used to resolve heterozygotes into haplotypes. Haplotype estimations are further described below under the heading "Statistical methods.). Any other method known in the art to determine or to estimate the frequency of a haplotype in a population may also be used. 2) Linkage Disequilibrium analysis
- Linkage disequilibrium is the non-random association of alleles at two or more loci and represents a powerful tool for mapping genes involved in disease traits (see Ajioka R.S. et al., 1997).
- Biallelic markers because they are densely spaced in the human genome and can be genotyped in more numerous numbers than other types of genetic markers (such as RFLP or VNTR markers), are particularly useful in genetic analysis based on linkage disequilibrium.
- the biallelic markers of the present invention may be used in any linkage disequilibrium analysis method known in the art.
- a disease mutation when first introduced into a population (by a new mutation or the immigration of a mutation carrier), it necessarily resides on a single chromosome and thus on a single "background” or “ancestral” haplotype of linked markers. Consequently, there is complete disequilibrium between these markers and the disease mutation: one finds the disease mutation only in the presence of a specific set of marker alleles. Through subsequent generations recombinations occur between the disease mutation and these marker polymorphisms, and the disequilibrium gradually dissipates. The pace of this dissipation is a function of the recombination frequency, so the markers closest to the disease gene will manifest higher levels of disequilibrium than those that are further away.
- 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".
- linkage disequilibrium the occurrence of pairs of specific alleles at different loci on the same chromosome is not random and the deviation from random is called linkage disequilibrium.
- Association studies focus on population frequencies and rely on the phenomenon of linkage disequilibrium. If a specific allele in a given gene is directly involved in causing a particular trait, its frequency will be statistically increased in an affected (trait positive) population, when compared to the frequency in a trait negative population or in a random control population. As a consequence of the existence of linkage disequilibrium, the frequency of all other alleles present in the haplotype carrying the trait-causing allele will also be increased in trait positive individuals compared to trait negative individuals or random controls.
- Case-control populations Inclusion criteria
- population-based association studies do not concern familial inheritance but compare the prevalence of a particular genetic marker, or a set of markers, in case-control populations. They are case-control studies based on comparison of unrelated case (affected or trait positive) individuals and unrelated control (unaffected or trait negative or random) individuals.
- the control group is composed of unaffected or trait negative individuals.
- the control group is ethnically matched to the case population.
- the control group is preferably matched to the case-population for the main known confusion factor for the trait under study (for example age-matched for an age-dependent trait).
- case population and “affected population” are used interchangeably.
- An important step in the dissection of complex traits using association studies is the choice of case-control populations (see Lander and Schork, 1994).
- a major step in the choice of case-control populations is the clinical definition of a given trait or phenotype. Any genetic trait may be analysed by the association method proposed here by carefully selecting the individuals to be included in the trait positive and trait negative phenofypic groups. Four criteria are often useful: clinical phenotype, age at onset, family history and severity.
- the selection procedure for continuous or quantitative traits involves selecting individuals at opposite ends of the phenotype distribution of the trait under study, so as to include in these trait positive and trait negative populations individuals with non- overlapping phenotypes.
- case-control populations comprise phenotypically homogeneous populations.
- Trait positive and trait negative populations comprise phenotypically uniform populations of individuals representing each between 1 and 98%, preferably between 1 and 80%, more preferably between 1 and 50%, and more preferably between 1 and 30%, most preferably between 1 and 20% of the total population under study, and selected among individuals exhibiting non-overlapping phenotypes. The clearer the difference between the two trait phenotypes, the greater the probability of detecting an association with biallelic markers.
- 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.
- the methods of detecting an association between a haplotype and a phenotype of the invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination:
- said 13q31-q33-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID Nos 1 to 26,
- said 13q31-q33-related biallelic marker may be selected individually or in any combination from the biallelic markers described in Tables 6b and 6c; optionally, said control population may be a trait negative population, or a random population; optionally, said phenotype is a disease involving schizophrenia, a response to an agent acting on schizophrenia, or a side effects to an agent acting on schizophrenia.
- the mutant allele when a chromosome carrying a disease allele first appears in a population as a result of either mutation or migration, the mutant allele necessarily resides on a chromosome having a set of linked markers: the ancestral haplotype.
- This haplotype can be tracked through populations and its statistical association with a given trait can be analysed. Complementing single point (allelic) association studies with multi-point association studies also called haplotype studies increases the statistical power of association studies.
- haplotype association study allows one to define the frequency and the type of the ancestral carrier haplotype.
- a haplotype analysis is important in that it increases the statistical power of an analysis involving individual markers.
- 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.
- the biallelic markers of the present invention may also be used to identify patterns of biallelic markers associated with detectable traits resulting from polygenic interactions.
- the analysis of genetic interaction between alleles at unlinked loci requires individual genotyping using the techniques described herein.
- the analysis of allelic interaction among a selected set of biallelic markers with appropriate level of statistical significance can be considered as a haplotype analysis.
- Interaction analysis comprises stratifying the case-control populations with respect to a given haplotype for the first loci and performing a haplotype analysis with the second loci with each subpopulation.
- TDT tests for both linkage and association are not affected by population stratification. TDT requires data for affected individuals and their parents or data from unaffected sibs instead of from parents (see Spielmann S. et al., 1993; Schaid DJ. et al., 1996, Spielmann S. and Ewens W.J, 1998). Such combined tests generally reduce the false - positive errors produced by separate analyses. Statistical methods
- any method known in the art to test whether a trait and a genotype show a statistically significant correlation may be used.
- haplotype frequencies can be estimated from the multilocus genotypic data. Any method known to person skilled in the art can be used to estimate haplotype frequencies (see Lange K., 1997; Weir, B.S., 1996) Preferably, maximum-likelihood haplotype frequencies are computed using an Expectation- Maximization (EM) algorithm (see Dempster et al., 1977; Excoffier L. and Slatkin M., 1995).
- EM Expectation- Maximization
- This procedure is an iterative process aiming at obtaining maximum-likelihood estimates of haplotype frequencies from multi-locus genotype data when the gametic phase is unknown.
- Haplotype estimations are usually performed by applying the EM algorithm using for example the EM-HAPLO program (Hawley M.E. et al.,1994) or the Arlequin program (Schneider et al., 1997).
- the EM algorithm is a generalised iterative maximum likelihood approach to estimation and is briefly described below.
- phenotypes will refer to multi-locus genotypes with unknown phase.
- Genotypes will refer to known-phase multi-locus genotypes. Suppose a sample of N unrelated individuals typed for K markers.
- ⁇ t is an indicator variable which count the number of time haplotype t in genotype i. It takes the values of 0, 1 or 2.
- 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 :
- a aiaj V ⁇ 4 - V ( ⁇ 4 + ⁇ 3) ( ⁇ 4 + ⁇ 2), where :
- 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).
- This formula allows linkage disequilibrium between alleles to be estimated when only genotype, and not haplotype, data are available. Another means of calculating the linkage disequilibrium between markers is as follows.
- D aiaj pr(haplotype(a t ,aj)) - pr(a,- ).pr(aj ).
- 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).
- the p value related to a biallelic marker association is preferably about 1 x 10 "2 or less, more preferably about 1 x 10 "4 or less, for a single biallelic marker analysis and about 1 x 10 "3 or less, still more preferably 1 x 10 "6 or less and most preferably of about 1 x 10 *8 or less, for a haplotype analysis involving several markers.
- 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.
- any marker in linkage disequilibrium with a first marker associated with a trait will be associated with the trait. Therefore, once an association has been demonstrated between a given biallelic marker and a trait, the discovery of additional biallelic markers associated with this trait is of great interest in order to increase the density of biallelic markers in this particular region. The causal gene or mutation will be found in the vicinity of the marker or set of markers showing the highest correlation with the trait.
- 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 present invention then also concerns biallelic markers and other polymo ⁇ hisms which are in linkage disequilibrium with the specific biallelic markers of the invention and which are expected to present similar characteristics in terms of their respective association with a given trait.
- the invnetion concerns biallelic markers which are in linkage disequilibrium with the specific biallelic markers.
- 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.
- the method used to detect such mutations generally comprises the following steps: (a) amplification of a region of the candidate DNA sequence comprising a biallelic marker or a group of biallelic markers associated with the trait from DNA samples of trait positive patients and trait negative controls; (b) sequencing of the amplified region; (c) comparison of DNA sequences from trait-positive patients and trait-negative controls; and (d) determination of mutations specific to trait-positive patients. Subcombinations which comprise steps (b) and (c) are specifically contemplated.
- candidate polymo ⁇ hisms be then verified by screening a larger population of cases and controls by means of any genotyping procedure such as those described herein, preferably using a microsequencing technique in an individual test format.
- Polymo ⁇ hisms are considered as candidate mutations when present in cases and controls at frequencies compatible with the expected association results.
- 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 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 any detectable trait, including predisposition to schizophrenia, age of onset of detectable symptoms, a beneficial response to or side effects related to treatment against schizophrenia.
- Such a diganosis can be useful in the monitoring, prognosis and/or prophylactic or curative therapy for schizophrenia.
- the diagnostic techniques of the present invention may employ a variety of methodologies to determine whether a test subject has a genotype associated with an increased risk of developing a detectable trait or whether the individual suffers from a detectable trait as a result of a particular mutation, including methods which enable the analysis of individual chromosomes for haplotyping, such as family studies, single sperm DNA analysis or somatic hybrids.
- the diagnostic techniques concern the detection of specific alleles present within the human chromosome 13q31-q33 region; optionally within the Region D subregion; and optionally within an sbgl, g34665, sbg2, g35017 or g35018 nucleic acid sequence.
- the invention concerns the detection of a nucleic acid comprising at least one of the nucleotide sequences of SEQ ID Nos. 1 to 26, 36 to 40 and 54 to 229 or a fragment thereof or a complementary sequence thereto including the polymo ⁇ hic base.
- nucleic acid sample from the individual and, determining, whether the nucleic acid sample contains at least one allele or at least one biallelic marker haplotype, indicative of a risk of developing the trait or indicative that the individual expresses the trait as a result of possessing a particular the human chromosome 13q3 l-q33 region, Region D, sbgl, g34665, sbg2, g35017 or g35018-related polymo ⁇ hism or mutation
- a nucleic acid sample is obtained from the individual and this sample is genotyped using methods described above in "Methods Of Genotyping DNA Samples For Biallelic markers.”
- the diagnostics may be based on a single biallelic marker or a on group of biallelic markers.
- a nucleic acid sample is obtained from the test subject and the biallelic marker pattern of one or more of the biallelic markers of the invention is determined.
- a PCR amplification is conducted on the nucleic acid sample to amplify regions in which polymorphisms associated with a detectable phenotype have been identified.
- the amplification products are sequenced to determine whether the individual possesses one or more human chromosome 13q3 l-q33 region, Region D, sbgl, g34665, sbg2, g35017 or g35018-related polymo ⁇ hisms associated with a detectable phenofype.
- the primers used to generate amplification products may comprise the primers listed in Table 6a.
- the nucleic acid sample is subjected to microsequencing reactions as described above to determine whether the individual possesses one or more human chromosome 13q31- q33 region-related polymo ⁇ hisms associated with a detectable phenotype resulting from a mutation or a polymorphism in the human chromosome 13q31-q33 region, Region D, sbgl, g34665, sbg2, g35017 or g35018-related biallelic marker.
- the primers used in the microsequencing reactions may include the primers listed in 6d.
- 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.
- An aspect of the present invention relates to the preparation of a medicament for the treatment of psychiatric disease, in particular schizophrenia and bipolar disorder.
- the present invention embodies medicaments acting on sbgl, g34665, sbg2, g35017 or g35018.
- medicaments of the invention act on sbgl , either directly or indirectly, by acting on the sbgl pathways.
- the medicaments may modulate, and more preferably decrease the level of sbgl activity which occurs in a cell or particular tissue, or increase or descrease the activity of the sbgl protein.
- 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.
- Such medicaments may also increase or decrease the activity of a compound analogous to sbgl, a compound comprising an amino acid sequence having at least 25% homology to a sequence selected from the group consisting of SEQ ID NOs. 27 to 35, a compound comprising an amino acid sequence having at least 50% homology to a sequence selected from the group consisting of SEQ ID NOs. 27 to 35, and a compound comprising an amino acid sequence having at least 80% homology to a sequence selected from the group consisting of SEQ ID NOs. 27 to 35.
- Medicaments which increase or descrease the activity of these compounds in an individual may be used to ameliorate or prevent symptoms in individuals suffering from or predisposed to a psychiatric disease, as discussed above in the section entitled "indications”.
- sbgl activity may be increased or decreasing by the expression of the genes encoding the identified sbgl -modulating compounds using gene therapy. Examples of vectors and promoters suitable for use in gene therapy are described above. Sbgl activity may also be increased or decreased by preparing an antibody which binds to an sbgl peptide, an sbgl receptor or a protein related thereto, as well as fragments of these proteins. Such antibodies may modulate the interaction between sbgl and an sbgl receptor or a protein related thereto. Antibodies and methods of obtaining them are further described herein. As described above, the present invention provides cellular assays for identifying compounds for the treatment of psychiatric disease.
- the assays are based on detection of sbgl expression, measurement of sbgl protein activity, or based on the determination of other suitable schizophrenia, bipolar disorder or related psychiatric disease endpoints.
- Compounds for the treatment of psychiatric disease include derivative proteins or peptides which are capable of inhibiting the activity of a wild type sbgl protein, which may be identified by determining their ability to bind a wild type sbgl protein.
- Compounds also include antibodies, and small molecules and drugs which may be obtained using a variety of synthetic approaches familiar to those skilled in the art, including combinatorial chemistry based techniques.
- 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.
- sbgl, g34665, sbg2, g35017 or g35018 Individuals affected by or predisposed to schizophrenia and bipolar disorder may express abnormal levels of sbgl, g34665, sbg2, g35017 or g35018. Individuals having increased or decreased sbgl, g34665, sbg2, g35017 or g35018 activity in their plasma, body fluids, or body tissues may be at risk of devloping schizophrenia, bipolar disorder or a variety of potentially related psychiatric conditions.
- the present invention is a method for determining whether an individual is at risk of suffering from or is currently suffering from schizophrenia, bipolar disorder or other psychotic disorders, mood disorders, autism, substance dependence or alcoholism, mental retardation, or other psychiatric diseases including cognitive, anxiety, eating, impulse-control, and personality disorders, as defined with the Diagnosis and Statistical Manual of Mental Disorders fourth edition (DSM-IV) classification, comprising determining whether the individual has an abnormal level of sbgl activity in plasma, body fluids, or body tissues.
- the level of sbgl or analogous compounds in plasma, body fluids, or body tissues may be determined using a variety approaches. In particular, the level may be determined using ELISA, Western Blots, or protein electrophoresis.
- 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.
- the diagnostic techniques of the present invention may employ a variefy of methodologies to determine whether a test subject has a genotype associated with an increased risk of developing a detectable trait or whether the individual suffers from a detectable trait as a result of a particular mutation, including methods which enable the analysis of individual chromosomes for haplotyping, such as family studies, single sperm DNA analysis or somatic hybrids.
- the diagnostic techniques concern the detection of specific alleles present within the human chromosome 13q31-q33 region; optionally within the Region D subregion; and optionally within an sbgl, g34665, sbg2, g35017 or g35018 nucleic acid sequence. More particularly, the invention concerns the detection of a nucleic acid comprising at least one of the nucleotide sequences of SEQ ID Nos. 1 to 26, 36 to 40 and 54 to 229 or a fragment thereof or a complementary sequence thereto including the polymo ⁇ hic base.
- These methods involve obtaining a nucleic acid sample from the individual and, determining, whether the nucleic acid sample contains at least one allele or at least one biallelic marker haplotype, indicative of a risk of developing the trait or indicative that the individual expresses the trait as a result of possessing a particular the human chromosome 13q31-q33 region-related polymo ⁇ hism or mutation (trait-causing allele).
- a nucleic acid sample is obtained from the individual and this sample is genotyped using methods described above in "Methods Of
- the diagnostics may be based on a single biallelic marker or a on group of biallelic markers.
- a nucleic acid sample is obtained from the test subject and the biallelic marker pattern of one or more of a biallelic marker of the invention is determined.
- a PCR amplification is conducted on the nucleic acid sample to amplify regions in which polymo ⁇ hisms associated with a detectable phenotype have been identified.
- the amplification products are sequenced to determine whether the individual possesses one or more human chromosome 13q31-q33 region, Region D, sbgl, g34665, sbg2, g35017 or g35018-related polymo ⁇ hisms associated with a detectable phenotype.
- the primers used to generate amplification products may comprise the primers listed in Table 6a.
- the nucleic acid sample is subjected to microsequencing reactions as described above to determine whether the individual possesses one or more human chromosome 13q31- q33 region, Region D, sbgl, g34665, sbg2, g35017 or g35018-related polymo ⁇ hisms associated with a detectable phenotype resulting from a mutation or a polymo ⁇ hism in the human chromosome 13q31-q33 region.
- the primers used in the microsequencing reactions may include the primers listed in Table 6d.
- 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 6b.
- 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. 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.
- the invention concerns a method for the treatment of schizophrenia or bipolar disorder, or a related disorder comprising the following steps: - selecting an individual whose DNA comprises alleles of a biallelic marker or of a group of biallelic markers of the human chromosome 13q31-q33 region, preferably Region D-related markers, and more preferably sbgl, g34665, sbg2, g35017 or g35018-related markers associated with schizophrenia or bipolar disorder;
- Another embodiment of the present invention comprises a method for the treatment of schizophrenia or bipolar disorder comprising the following steps:
- the present invention concerns a method for the treatment of schizophrenia or bipolar disorder comprising the following steps:
- the present invention also concerns a method for the treatment of schizophrenia or bipolar disorder comprising the following steps:
- the invention also concerns a method for the treatment of schizophrenia or bipolar disorder in a selected population of individuals.
- the method comprises:
- a "positive response" to a medicament can be defined as comprising a reduction of the symptoms related to the disease.
- a "negative response" to a medicament can be defined as comprising either a > lack of positive response to the medicament which does not lead to a symptom reduction or which leads to a side-effect observed following administration of the medicament.
- the invention also relates to a method of determining whether a subject is likely to respond positively to treatment with a medicament.
- the method comprises identifying a first population of individuals who respond positively to said medicament and a second population of individuals who respond negatively to said medicament.
- One or more biallelic markers is identified in the first population which is associated with a positive response to said medicament or one or more biallelic markers is identified in the second population which is associated with a negative response to said medicament.
- the biallelic markers may be identified using the techniques described herein.
- a DNA sample is then obtained from the subject to be tested.
- the DNA sample is analyzed to determine whether it comprises alleles of one or more biallelic markers associated with a positive response to treatment with the medicament and/or alleles of one or more biallelic markers associated with a negative response to treatment with the medicament.
- the medicament may be administered to the subject in a clinical trial if the DNA sample contains alleles of one or more biallelic markers associated with a positive response to treatment with the medicament and/or if the DNA sample lacks alleles of one or more biallelic markers associated with a negative response to treatment with the medicament.
- the medicament is a drug acting against schizophrenia or bipolar disorder.
- the evaluation of drug efficacy may be conducted in a population of individuals likely to respond favorably to the medicament.
- Another aspect of the invention is a method of using a medicament comprising obtaining a DNA sample from a subject, determining whether the DNA sample contains alleles of one or more biallelic markers associated with a positive response to the medicament and/or whether the DNA sample contains alleles of one or more biallelic markers associated with a negative response to the medicament, and administering the medicament to the subject if the DNA sample contains alleles of one or more biallelic markers associated with a positive response to the medicament and/or if the DNA sample lacks alleles of one or more biallelic markers associated with a negative response to the medicament.
- the invention also concerns a method for the clinical testing of a medicament, preferably a medicament acting against schizophrenia or or bipolar disorder or symptoms thereof.
- the method comprises the following steps:
- a medicament preferably a medicament susceptible of acting against schizophrenia or or bipolar disorder or symptoms thereof to a heterogeneous population of individuals
- said biallelic marker may optionally comprise:
- biallelic marker selected from the group consisting of biallelic markers Al to A489;
- a biallelic marker selected from the group consisting of biallelic markers 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 A295, A303 to A307, A330, A334 to A335 and A346 to 357; (c) a biallelic marker selected from the group consisting of biallelic markers Al to A69,
- 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 g35018-related markers Al to A39;
- a biallelic marker selected from the group consisting of A239, A227, A 198, A228,
- a biallelic marker selected from the group consisting of A48, A60, A61, A62, A65, ' A70, A75, A76, A80, A 107, A 108, A 198, A218, A221, A223, A227, A228, A239, A285, A286, A287, A288, A290, A292, A293, A295.A299 and A304;
- a biallelic marker selected from the group consisting of A304, A307, A305, A298,
- 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.
- Such methods are deemed to be extremely useful to increase the benefit/risk ratio resulting from the administration of medicaments which may cause undesirable side effects and/or be inefficacious to a portion of the patient population to which it is normally administered.
- selection tests are carried out to determine whether the DNA of this individual comprises alleles of a biallelic marker or of a group of biallelic markers associated with a positive response to treatment or with a negative response to treatment which may include either side effects or unresponsiveness.
- the selection of the patient to be treated using the method of the present invention can be carried out through the detection methods described above.
- the individuals which are to be selected are preferably those whose DNA does not comprise alleles of a biallelic marker or of a group of biallelic markers associated with a negative response to treatment.
- the knowledge of an individual's genetic predisposition to unresponsiveness or side effects to particular medicaments allows the clinician to direct treatment toward appropriate drugs against schizophrenia or bipolar disorder or symptoms thereof.
- the clinician can select appropriate treatment for which negative response, particularly side effects, has not been reported or has been reported only marginally for the patient.
- the biallelic markers of the invention have demonstrated an association with schizophrenia and bipolar disorders.
- the present invention also comprises any of the prevention, diagnostic, prognosis and treatment methods described herein using the biallelic markers of the invention in methods of preventing, diagnosing, managing and treating related disorders, particularly related CNS disorders.
- related disorders may comprise psychotic disorders, mood disorders, autism, substance dependence and alcoholism, mental retardation, and other psychiatric diseases including cognitive, anxiety, eating, impulse-control, and personality disorders, as defined with the Diagnosis and Statistical Manual of Mental Disorders fourth edition (DSM-IV) classification".
- vector is used herein to designate either a circular or a linear DNA or RNA molecule, which is either double-stranded or single-stranded, and which comprise at least one polynucleotide of interest that is sought to be transferred in a cell host or in a unicellular or multicellular host organism.
- the present invention encompasses a family of recombinant vectors that comprise a polynucleotide derived from an sbgl, g34665, sbg2, g35017 or g35018 nucleic acid sequence. Consequently, the present invention further comprises recombinant vectors comprising:
- sbgl genomic DNA or cDNAs comprised in the nucleic acids of any of nucleotide positions 215819 to 215941, 215819 to 215975, 216661 to 216952, 216661 to 217061, 217027 to 217061, 229647 to 229742, 230408 to 230721, 231272 to 231412, 231787 to 231880, 231870 to 231879, 234174 to 234321, 237406 to 237428, 239719 to 239807, 239719 to 239853, 240528 to 240569, 240528 to 240596, 240528 to 240617, 240528 to 240644, 240528 to 240824, 240528 to 240994, 240528 to 241685 and 240800 to 240993 of SEQ ID No. 1, SEQ ID Nos 2 to 26 and primate sbgl DNAs of SEQ ID Nos 54 to 111 , and the complements thereof;
- g34665 genomic DNA or cDNAs comprised in the nucleic acids of any of nucleotide positions 292653 to 292841, 295555 to 296047 and 295580 to 296047 of SEQ ID No. 1 , and the complements thereof;
- sbg2 genomic DNA or cDNAs comprised in the nucleic acids of any of nucleotide positions 201 188 to 201234, 214676 to 214793, 215702 to 215746 and 216836 to 216915 of SEQ ID No. 1 , and the complements thereof;
- 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.
- a recombinant vector of the invention may comprise any of the polynucleotides described herein, as well as any sbgl, g34665, sbg2, g35017 or g35018 primer or probe as defined above.
- a recombinant vector of the invention is used to amplify the inserted polynucleotide derived from an sbgl, g34665, sbg2, g35017 or g35018 genomic sequence or cDNA of the invention in a suitable cell host, this polynucleotide being amplified at every time that the recombinant vector replicates.
- 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.
- Expression requires that appropriate signals are provided in the vectors, said signals including various regulatory elements, such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells.
- signals including various regulatory elements, such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells.
- Dominant drug selection markers for establishing permanent, stable cell clones expressing the products are generally included in the expression vectors of the invention, as they are elements that link expression of the drug selection markers to expression of the polypeptide.
- the present invention relates to expression vectors which include nucleic acids encoding an sbgl, g34665, sbg2, g35017 or g35018 protein or variants or fragments thereof, under the control of a regulatory sequence of the respective sbgl, g34665, sbg2, g35017 or g35018 regulatory polynucleotides, or alternatively under the control of an exogenous regulatory sequence.
- the invention also pertains to a recombinant expression vector useful for the expression of a sbgl, g34665, sbg2, g35017 or g35018 cDNA sequence.
- 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.
- a recombinant vector according to the invention comprises, but is not limited to, a YAC (Yeast Artificial Chromosome), a BAC (Bacterial Artificial Chromosome), a phage, a phagemid, a cosmid, a plasmid or even a linear DNA molecule which may comprise a chromosomal, non-chromosomal, semi-synthetic and synthetic DNA.
- a recombinant vector can comprise a transcriptional unit comprising an assembly of:
- Enhancers are cis-acting elements of DNA, usually from about 10 to 300 bp in length that act on the promoter to increase the transcription.
- Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell.
- a recombinant protein when expressed without a leader or transport sequence, it may include a N- terminal residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.
- 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.
- an sbgl, g34665, sbg2, g35017 or g35018 polypeptide or fragments or variants thereof may be useful in order to correct a genetic defect related to the expression of the native gene in a host organism or to the production of a biologically inactive sbgl, g34665, sbg2, g35017 or g35018 protein.
- the present invention also comprises recombinant expression vectors mainly designed for the in vivo production of the sbgl, g34665, sbg2, g35017 or g35018 polypeptide by the introduction of the appropriate genetic material in the organism of the patient to be treated.
- said genetic material comprises at least one nucleotide sequence selected from the group of nucleotide posittion ranges consisting of:
- sbgl genomic DNA or cDNAs comprised in the nucleic acids of any of nucleotide positions 215819 to 215941, 215819 to 215975, 216661 to 216952, 216661 to 217061, 217027 to 217061, 229647 to 229742, 230408 to 230721, 231272 to 231412, 231787 to 231880,
- g34665 genomic DNA or cDNAs comprised in the nucleic acids of any of nucleotide positions 292653 to 292841, 295555 to 296047 and 295580 to 296047 of SEQ ID No. 1, and the complements thereof;
- sbg2 genomic DNA or cDNAs comprised in the nucleic acids of any of nucleotide positions 201188 to 201234, 214676 to 214793, 215702 to 215746 and 216836 to 216915 of
- 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
- 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.
- a suitable promoter may be heterologous with respect to the nucleic acid for which it controls the expression or alternatively can be endogenous to the native polynucleotide containing the coding sequence to be expressed. Additionally, the promoter is generally heterologous with respect to the recombinant vector sequences within which the construct promoter/coding sequence has been inserted.
- 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.
- Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-L. Selection of a convenient vector and promoter is well within the level of ordinary skill in the art. The choice of a promoter is well within the ability of a person skilled in the field of genetic engineering. For example, one may refer to the book of Sambrook et al.(1989) or also to the procedures described by Fuller et al.(1996).
- 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.
- sbgl genomic DNA or cDNAs comprised in the nucleic acids of any of nucleotide positions 215819 to 215941, 215819 to 215975, 216661 to 216952, 216661 to 217061, 217027 to 217061, 229647 to 229742, 230408 to 230721, 231272 to 231412, 231787 to 231880, 231870 to 231879, 234174 to 234321, 237406 to 237428, 239719 to 239807, 239719 to 239853, 240528 to 240569, 240528 to 240596, 240528 to 240617, 240528 to 240644, 240528 to 240824, 240528 to 240994, 240528 to 241685 and 240800 to 240993 of SEQ ID No. 1, SEQ ID Nos 2 to 26 and primate sbgl DNAs or SEQ ID Nos. 54 to 111, and the complements thereof;
- g34665 genomic DNA or cDNAs comprised in the nucleic acids of any of nucleotide positions 292653 to 292841, 295555 to 296047 and 295580 to 296047 of SEQ ID No. 1, and the complements thereof;
- sbg2 genomic DNA or cDNAs comprised in the nucleic acids of any of nucleotide positions 201188 to 201234, 214676 to 214793, 215702 to 215746 and 216836 to 216915 of SEQ ID No. 1 , and the complements thereof;
- 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.
- selectable Markers Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct.
- the selectable marker genes for selection of transformed host cells are preferably dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, TRP1 for S. cerevisiae or tetracycline, rifampicin or ampicillin resistance in E. coli, or levan saccharase for mycobacteria, this latter marker being a negative selection marker.
- 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).
- Suitable vectors are known to those of skill in the art, and commercially available, such as the following bacterial vectors: pQE70, pQE60, pQE-9 (Qiagen), pbs, pDIO, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia); pQE-30 (QIAexpress).
- Bacteriophage vectors The PI bacteriophage vector may contain large inserts ranging from about 80 to about
- 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.
- PI clone comprising an sbgl polynucleotide sequence in a transgenic animal, typically in transgenic mice
- it is desirable to remove vector sequences from the PI DNA fragment for example by cleaving the PI DNA at rare-cutting sites within the PI polylinker (S ⁇ l, Notl or Sail).
- the PI insert is then purified from vector sequences on a pulsed-field agarose gel, using methods similar using methods similar to those originally reported for the isolation of DNA from YACs (Schedl et al., 1993a; Peterson et al., 1993, ).
- the resulting purified insert DNA can be concentrated, if necessary, on a Millipore
- 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
- a suitable vector for the expression of an sbgl polypeptide encoded by polynucleotides of SEQ ID No. 1 or fragments or variants thereof is a baculovirus vector that can be propagated in insect cells and in insect cell lines.
- a specific suitable host vector system is the pVL1392/1393 baculovirus transfer vector (Pharmingen) that is used to transfect the SF9 cell line (ATCC N°CRL 171 1) which is derived from Spodoptera frugiperda.
- Suitable vectors for the expression of the sbgl polypeptide encoded by the SEQ ID No. 1 or fragments or variants thereof in a baculovirus expression system include those described by Chai et al.(1993), Vlasak et al.(1983) and Lenhard et al.(1996).
- 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).
- Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery systems of choice for the transfer of exogenous polynucleotides in vivo, particularly to mammals, including humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host.
- 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).
- AAV adeno-associated virus
- the adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a he ⁇ es virus, as a helper virus for efficient replication and a productive life cycle (Muzyczka et al., 1992). It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (Flotte et al., 1992; Samulski et al., 1989; McLaughlin et al., 1989).
- AAV adeno-associated virus
- 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.
Abstract
Description
Claims
Priority Applications (4)
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EP00914325A EP1165836A2 (en) | 1999-03-30 | 2000-03-30 | Schizophrenia associated genes, proteins and biallelic markers |
CA002361408A CA2361408A1 (en) | 1999-03-30 | 2000-03-30 | Schizophrenia associated genes, proteins and biallelic markers |
AU35719/00A AU778868B2 (en) | 1999-03-30 | 2000-03-30 | Schizophrenia associated genes, proteins and biallelic markers |
JP2000608788A JP2002539845A (en) | 1999-03-30 | 2000-03-30 | Schizophrenia-related genes, proteins and biallelic markers |
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US12690399P | 1999-03-30 | 1999-03-30 | |
US60/126,903 | 1999-03-30 | ||
US13206599P | 1999-04-30 | 1999-04-30 | |
US13197199P | 1999-04-30 | 1999-04-30 | |
US60/131,971 | 1999-04-30 | ||
US60/132,065 | 1999-04-30 | ||
US14392899P | 1999-07-14 | 1999-07-14 | |
US60/143,928 | 1999-07-14 | ||
US14591599P | 1999-07-27 | 1999-07-27 | |
US60/145,915 | 1999-07-27 | ||
US14645399P | 1999-07-29 | 1999-07-29 | |
US14645299P | 1999-07-29 | 1999-07-29 | |
US60/146,453 | 1999-07-29 | ||
US60/146,452 | 1999-07-29 | ||
US16228899P | 1999-10-28 | 1999-10-28 | |
US60/162,288 | 1999-10-28 |
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JP (1) | JP2002539845A (en) |
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WO2002066672A2 (en) * | 2001-01-16 | 2002-08-29 | Genset S.A. | Treatment of cns disorders using d-amino acid oxidase and d-aspartate oxidase antagonists |
WO2003050303A2 (en) * | 2001-12-12 | 2003-06-19 | Genset S.A. | Biallelic markers of d-amino acid oxidase and uses thereof |
WO2003087408A2 (en) * | 2002-04-05 | 2003-10-23 | University Court Of The University Of Edinburgh | Schizophrenia associated genes |
EP1470245A2 (en) * | 2001-04-24 | 2004-10-27 | PHARMACIA & UPJOHN COMPANY | Single nucleotide polymorphisms diagnostic for schizophrenia |
EP1495041A1 (en) * | 2002-02-20 | 2005-01-12 | Sirna Therapeutics, Inc. | RNA INTERFERENCE MEDIATED INHIBITION OF G72 AND D-AMINO ACID OXIDASE (DAAO) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA) |
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EP1573039A2 (en) * | 2002-11-01 | 2005-09-14 | The Board of Trustees of The Leland Stanford Junior University | Compositions and methods for diagnosing and treating mood disorders |
EA008252B1 (en) * | 2000-12-05 | 2007-04-27 | Женсет | Antibody or antigene binding domain specifically associated with a polypeptide of potential-dependent voltage-gated ion channel |
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- 2000-03-30 JP JP2000608788A patent/JP2002539845A/en not_active Withdrawn
- 2000-03-30 WO PCT/IB2000/000435 patent/WO2000058510A2/en active Search and Examination
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EP1470245A4 (en) * | 2001-04-24 | 2005-08-31 | Pharmacia & Upjohn Co Llc | Single nucleotide polymorphisms diagnostic for schizophrenia |
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AU3571900A (en) | 2000-10-16 |
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WO2000058510A3 (en) | 2001-08-09 |
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EP1165836A2 (en) | 2002-01-02 |
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