Patent Application
20090291432
1. Field of the Invention
The present invention relates generally to a method for profiling an individual or group of individuals with respect to a neurological, psychiatric or psychological condition, phenotype or state, including a sub-threshold neurological, psychiatric or psychological condition, phenotype or state. More particularly, the present invention identifies genetic profiles associated with a neurological, psychiatric or psychological condition, phenotype or state which permit the development of agents useful in diagnosing the presence of a neurological, psychiatric or psychological condition, phenotype or state or a risk or likelihood of development of same. The present invention further contemplates methods for the treatment or prophylaxis of a neurological, psychiatric or psychological condition, phenotype or state in an individual, including implementing behavioral modification protocols, to ameliorate the risk of developing an adverse neurological, psychiatric or psychological condition, phenotype or state.
2. Description of the Prior Art
Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms a part of the common general knowledge in any country.
Psychological “disorders” are endemic in any society. Reference to “disorders” in this context means that an individual exhibits behavioral patterns which are inconsistent with society norms. Many psychological phenotypes have a physiological basis while others result from or are compounded by environmental conditioning. The difficulty for clinical psychiatrists, neurologists and psychologists is to diagnose a physiologically-based condition for which therapeutic intervention is possible.
One particularly complex psychological phenotype is schizophrenia. Schizophrenia is a common, chronic, disabling illness with an incidence of 15 new cases per 100,000 population per year (Kelly et al., Ir. J. Med. Sci. 172:37-40, 2003). Additionally, “unaffected” first degree relatives show both childhood (Niendam et. al., Am. J. Psychiatry. 160:2060-2062, 2003) and adulthood (MacDonald et al., Arch. Gen. Psychiatry. 60:57-65, 2003) deficits in cognitive functioning. Siblings of schizophrenic patients also exhibit an abnormal MRI response in the dorsolateral prefrontal cortex implicating inefficient information processing (Callicott et. al., Am. J. Psychiatry. 160:709-719, 2003). Furthermore, both schizophrenic subjects and their unaffected siblings show both reductions in hippocampal volume and hippocampal shape deformity (Tepest et al., Biol, Psychiatry. 54:1234-1240, 2003). Decreased temporoparietal P300 amplitude and increased frontal P300 amplitude are found in both schizophrenic patients and their siblings (Winterer et. al., Arch. Gen. Psychiatry. 60:1158-1167, 2003). Taken together, these findings indicate that the underlying pathophysiological state of schizophrenia is considerably more widespread in the general population than prevalence figures for schizophrenia would suggest and that a considerable genetic vulnerability for this disorder exists. Consequently, schizophrenia is a complex condition having a wide variety of manifestations some of which may have a pathophysiological origin whereas others may originate from environmental conditioning including substance abuse. This makes the treatment and diagnosis of schizophrenia difficult for clinicians.
The apparent high genetic risk for schizophrenia has led to considerable research efforts aimed at the identification of susceptibility genes. This has resulted in linkages or associations with regions 6p21-22, 1q21-22 and 13q32-34 with single studies reporting significance at P<0.05 (Owen et al., Mol. Psychiatry. 9:14-27, 2004) although a recent large multicenter linkage study of schizophrenia loci on chromosome 22q failed to find any evidence for linkage or association to schizophrenia (Mowry B J et. al., Mol. Psychiatry. 2004). Other regions that may be implicated include 8p21-22, 6q21-25, 5q21-q33, 10p15-p11 and 1q42 (Owen et al. 2004 supra). Despite this limited progress, the conclusive identification of specific molecular genetic etiological factors in the pathogenesis of schizophrenia has not occurred (Miyamoto et al., Mol. Intervent. 3:27-39, 2004).
Several lines of evidence have implicated the dopamine 2 receptor (DRD2) gene as a candidate gene for schizophrenia genetic susceptibility. For example, all anti-psychotic medications are either antagonists or partial agonists of DRD2. DRD2 receptor has been repeatedly demonstrated to be the primary site of action for these medications (Seeman and Kapur Proc. Natl. Acad. Sci. USA 97:7673-7675, 2000) indicating that schizophrenic symptoms are ameliorated by a reduction in DRD2 function. Additionally, recent evidence strongly suggests that schizophrenic patients have increased brain DRD2 density (Abi-Dargham et al., Proc. Natl. Acad. Sci. 97:8104-8109, 2000). However, the absence of a clear genetic link between the DRD2 and schizophrenia have hampered the development of appropriate therapeutic and diagnostic protocols.
The present invention now identifies a genetic link between DRD2 and a neurological, psychiatric or psychological condition, phenotype or state. In particular, a population of individuals having related pathopsychological symptoms and behavioral patterns are shown to exhibit a particular polymorphism with the genetic region encoding the DRD2 receptor. Even more particularly, the present invention identifies a polymorphism in or near the DRD2 genetic locus which is prevalent in individuals with schizophrenia, alcoholism or related neurological, psychiatric or psychological conditions including addictions including smoking and drug abuse.
Accordingly, the present invention contemplates a method for identifying a genetic profile associated with a neurological, psychiatric or psychological condition, phenotype or state including a sub-threshold neurological, psychiatric or psychological condition, phenotype or state in an individual or a group of individuals, said method comprising screening individuals for a polymorphism including a mutation in a genetic locus comprising the DRD2 gene, including its 5′ and 3′ terminal regions, promoter, introns and exons which has a statistically significant linkage or association to symptoms or behavior characterizing the neurological, psychiatric or psychological condition, phenotype or state or sub-threshold forms thereof.
Neurological, psychiatric or psychological conditions, phenotypes and states include, but are not limited to, Addiction, Alzheimer's Disease, Anxiety Disorders, Attention Deficit Hyperactivity Disorder (ADHD), Eating Disorders, Manic-Depressive Illness, Autism, Schizophrenia, Tourette's Syndrome, Obsessive Compulsive Disorder (OCD), Panic Disorder, Post Traumatic Stress Disorder (PTSD), Phobias, borderline personality disorder, bi-polar disorder, sleep disorders, Acute Stress Disorder, Adjustment Disorder, Agoraphobia Without History of Panic Disorder, Alcohol Dependence (Alcoholism), Amphetamine Dependence, Anorexia Nervosa, Antisocial Personality Disorder, Asperger's Disorder, Avoidant Personality Disorder, Brief Psychotic Disorder, Bulimia Nervosa, Cannabis Dependence, Cocaine Dependence, Conduct Disorder, Cyclothymic Disorder, Delirium, Delusional Disorder, Dementia Associated With Alcoholism, Dementia of the Alzheimer Type, Dependent Personality Disorder, Dysthymic Disorder, Generalized Anxiety Disorder, Hallucinogen Dependence, Histrionic Personality Disorder, Inhalant Dependence, Major Depressive Disorder, Manic Depression, Multi-Infarct Dementia, Narcissistic Personality Disorder, Nicotine Dependence, Opioid Dependence, Oppositional Defiant Disorder, Panic Disorder, Paranoid Personality Disorder, Parkinson's Disease, Phencyclidine Dependence, Schizoaffective Disorder, Schizoid Personality Disorder, Schizophreniform Disorder, Schizotypal Personality Disorder, Sedative Dependence, Separation Anxiety Disorder, Shared Psychotic Disorder, Smoking Dependence and Social Phobia. It should be noted, however, that a person considered not to suffer any symptom associated with the above disorders still falls within the scope of a “norma” or a non-symptomatic or non-pathogenic neurological, psychiatric or psychological condition, phenotype or state.
Schizophrenia and alcoholism are particularly exemplified herein associated with a polymorphism in the genetic locus comprising the DRD2 gene including its 5′ and 3′ terminal regions, promoter, introns and exons. A list of potential polymorphisms in the DRD2 gene are shown in Table 2.
In one embodiment polymorphism having a linkage or association to schizophrenia or alcoholism is a thymine (T) to cytosine (C) substitution at nucleotide position 957 (957C>T) using the numbering system from the cDNA sequence. This is represented as 957C>T. The nucleotide position is calculated using the cDNA sequence, wherein the numbering is calculated from the “A” of the AGT encoding the methionine being at position +1, encoding DRD2 (see SEQ ID NO:1). However, the present invention extends to any polymorphism or other mutation in the DRD2 genetic locus and which is linked to a neurological, psychiatric or psychological condition, phenotype or state such as schizophrenia or alcoholism.
The present invention enables clinicians to make a genetic-based diagnosis of a neurological, psychiatric or psychological condition, phenotype or state or a risk or likelihood that an individual will develop such a neurological, psychiatric or psychological condition, phenotype or state and can thereby implement treatment or preventative or symptom-ameliorating or controlling protocols including therapeutic intervention and/or behavioral modification protocols to reduce the adverse consequences of the neurological, psychiatric or psychological condition, phenotype or state.
In addition, the identification of a polymorphism including a mutation in the DRD2 genetic locus enables agents to be identified which mask the physiological impact or consequences of the genetic profile. For example, it is proposed that 957C>T results in decreased translation and stability of D2 mRNA. Consequently, agents which cause reduced levels of DRD2, such as DRD2 antagonists maybe useful in the treatment of schizophrenia or alcoholism or other neurological, psychiatric or psychological conditions, phenotypes or states.
The present invention further contemplates combinations of two or more polymorphisms such as 957C>T and Taq1A. The combinations may be at the same allele (i.e. haplotypes) or at different alleles.
Throughout the specification, unless the context requires otherwise, the word “comprise” or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:). The SEQ ID NOs: correspond numerically to the sequence identifiers <400>1, <400>2, etc. A sequence list is provided following the claims.
A summary of the sequence identifiers used throughout the subject specification is provided in Table 1.
The present invention is predicated in part on the identification of genetic profiles having a statistically significant association with a neurological, psychiatric or psychological condition, phenotype or state including a sub-threshold neurological, psychiatric or psychological condition, phenotype or state. By “genetic profiles” is meant that groups of individuals exhibiting a particular neurological, psychiatric or psychological condition, phenotype or state or sub-threshold forms thereof or who are at the risk of developing same exhibit a common polymorphism at or within the DRD2 genetic locus including its 5′ or 3′ terminal regions, promoter, exons or introns. The genetic profile may be a single polymorphism or multiple polymorphisms, that is two or more polymorphisms that are statistically significantly linked to a neurological, psychiatric or psychological condition, phenotype or state or sub-threshold forms thereof. Reference to a polymorphism in this context includes a mutation.
The singular forms “a”, “an”, and “the” include single and plural aspects unless the context clearly indicates otherwise. Thus, for example, reference to a “polymorphism” includes a single polymorphism, as well as two or more polymorphisms; reference to a psychological phenotype includes a single psychological phenotype, as well as two or more psychological phenotypes.
Accordingly, one aspect of the present invention contemplates a method for identifying a genetic profile associated with a neurological, psychiatric or psychological condition, phenotype or state including a sub-threshold neurological, psychiatric or psychological condition, phenotype or state in an individual or a group of individuals, said method comprising screening individuals for a polymorphism in a genetic locus comprising the DRD2 gene including its 5′ and 3′ terminal regions, promoter, introns and exons which has statistically significant linkage or association to symptoms or behavior characterizing the neurological, psychiatric or psychological condition, phenotype or state or sub-threshold forms thereof.
The genetic locus comprising the DRD2 gene may be referred to as the “DRD2 gene”, “DRD2 nucleic acid”, “DRD2 locus”, “DRD2 genetic locus” or “DRD2 polynucleotide”. Each refers to polynucleotides, all of which, are in the DRD2 region including its 5′ or 3′ terminal regions, promoter, introns or exons. Accordingly, the DRD2 locus is intended to include coding sequences, intervening sequences and regulatory elements controlling transcription and/or translation. The DRD2 genetic locus is intended to include all allelic variations of the DNA sequence on either or both chromosomes. Consequently, homozygous and heterozygous variations of the DRD2 locus are contemplated herein.
As indicated above, the DRD2 locus comprises different profiles for different neurological, psychiatric or psychological conditions, phenotypes or states or sub-threshold forms thereof. Such profiles include polymorphisms, although any nucleotide substitution, addition, deletion or insertion or other mutation in the DRD2 genetic locus is encompassed by the present invention when associated with a neurological, psychiatric or psychological condition, phenotype or state. Accordingly, the present invention extends to rare mutations which although not present in larger numbers of individuals in a population, when the mutation is present, it leads to a very high likelihood of development of a pathopsychological disorder.
The term “polymorphism” or “mutation” refers to a difference in a DNA or RNA sequence or sequences among individuals, groups or populations which give rise to a statistically significant phenotype or physiological condition. Examples of genetic polymorphisms include mutations that result by chance or are induced by external features. These polymorphisms or mutations may be indicative of a disease or disorder and may arise following a genetic disease, a chromosomal abnormality, a genetic predisposition, a viral infection, a fungal infection, a bacterial infection or a protist infection or following chemotherapy, radiation therapy or substance abuse including alcohol or drug abuse. The polymorphisms may also dictate or contribute to symptoms with a psychological phenotype. In a preferred aspect, the polymorphisms of the present invention are indicative of a neurological, psychiatric or psychological condition, phenotype or state or sub-threshold condition, phenotype or state thereof. As used herein, polymorphisms including mutations may refer to one or more changes in a DNA or RNA sequence which are present in a group of individuals having a particular neurological, psychiatric or psychological condition, phenotype or state or sub-threshold forms thereof or are at risk of developing same.
Examples of nucleotide changes contemplated herein include single nucleotide polymorphisms (SNPs), multiple nucleotide polymorphisms (MNPs), frame shift mutations, including insertions and deletions (also called deletion insertion polymorphisms or DIPS), nucleotide substitutions and nonsense mutations. Two or more polymorphisms may also be used either at the same allele (i.e. haplotypes) or at different alleles.
Examples of a neurological, psychiatric or psychological condition, phenotype or state contemplated by the present invention and which may be directly or indirectly linked to a genetic profile such as a polymorphism or mutation to the DRD2 genetic locus include but are not limited to Addiction, Alzheimer's Disease, Anxiety Disorders, Attention Deficit Hyperactivity Disorder (ADHD), Eating Disorders, Manic-Depressive Illness, Autism, Schizophrenia, Tourette's Syndrome, Obsessive Compulsive Disorder (OCD), Panic Disorder, Post Traumatic Stress Disorder (PTSD), Phobias, borderline personality disorder, bi-polar disorder, sleep disorders, Acute Stress Disorder, Adjustment Disorder, Agoraphobia Without History of Panic Disorder, Alcohol Dependence (Alcoholism), Amphetamine Dependence, Anorexia Nervosa, Antisocial Personality Disorder, Asperger's Disorder, Avoidant Personality Disorder, Brief Psychotic Disorder, Bulimia Nervosa, Cannabis Dependence, Cocaine Dependence, Conduct Disorder, Cyclothymic Disorder, Delirium, Delusional Disorder, Dementia Associated With Alcoholism, Dementia of the Alzheimer Type, Dependent Personality Disorder, Dysthymic Disorder, Generalized Anxiety Disorder, Hallucinogen Dependence, Histrionic Personality Disorder, Inhalant Dependence, Major Depressive Disorder, Manic Depression, Multi-Infarct Dementia, Narcissistic Personality Disorder, Nicotine Dependence, Opioid Dependence, Oppositional Defiant Disorder, Panic Disorder, Paranoid Personality Disorder, Parkinson's Disease, Phencyclidine Dependence, Schizoaffective Disorder, Schizoid Personality Disorder, Schizophreniform Disorder, Schizotypal Personality Disorder, Sedative Dependence, Separation Anxiety Disorder, Shared Psychotic Disorder and Social Phobia.
The most exemplified conditions herein up to the present time are schizophrenia and alcoholism. Reference herein to “schizophrenia” includes conditions which have symptoms similar to schizophrenia and hence are regard as schizophrenia-related conditions. Such symptoms of schizophrenia include behavioral and physiological conditions. Due to the composition of schizophrenia and related conditions, the ability to identify a genetic profile to assist in defining Schizophrenia is of significant importance. The present invention now provides this genetic profile.
Reference hereto to “alcoholism” refers to a disorder characterized by dependence on alcohol, repeated excessive use of alcoholic beverages, development of withdrawal symptoms on reducing or ceasing alcohol intake, morbidity that may include cirrhosis of the liver, and decreased ability to function socially and vocationally. It is characterized by a cluster of behavioral, cognitive, and physiological phenomena that develop after repeated substance misuse and that typically include a strong desire to take the alcohol, difficulties in controlling its use, persisting in its use despite harmful consequences, a higher priority given to its use than other activities and obligations, increased tolerance, and sometimes a physical withdrawal state.
Any number of methods may be used to calculate the statistical significance of a polymorphism and its association with a neurological, psychiatric or psychological condition. Particular statistical analysis methods which may be used are described in Fisher and vanBelle, “Biostatistics: A Methodology for the Health Sciences” Wiley-Intersciences (New York) 1993. This analysis may also include a regression calculation of which polymorphic sites in the DRD2 gene give the most significant contribution to the differences in phenotype. One regression model useful in the invention starts with a model of the form
r=r
0+(S×d)
where r is the response, r0 is a constant called the “intercept”, S is the slope and d is the dose. To determine the dose, the most-common and least common nucleotides at the polymorphic site are first defined. Then, for each individual in the trial population, one calculates a “dose” as the number of least-common nucleotides the individual has at the polymorphic site of interest. This value can be 0 (homozygous for the least-common nucleotide), 1 (heterozygous), or 2 (homozygous for the most common nucleotide). An individual's “response” is the value of the clinical measurement. Standard linear regression methods are then used to fit all the individuals' doses and responses to a single model (see e.g. Fisher and vanBelle, supra, Ch 9). The outputs of the regression calculation are the intercept r0, the slope S, and the variance (which measures how well the data fits this simple linear model). The Students t-test value and the level of significance can then be calculated for each of the polymorphic sites.
In relation to the genetic profile associated with schizophrenia, alcoholism or a related condition or other neurological, psychiatric or psychological condition, phenotype or state, the present invention encompasses any polymorphism or mutation within or proximal to the DRD2 genetic locus including its 5′ or 3′ terminal regions, promoter, introns and exons. Examples of possible polymorphisms or mutations are given in Table 2.
A particularly important polymorphism is at nucleotide position 957 (using cDNA nucleotide numbering). The majority of individuals have a T at position 957 (the “T allele”. However, a statistically significant number of individuals presenting with symptoms of schizophrenia, alcoholism, or other neurological, psychiatric or psychological conditions, phenotypes or states have an increase in the frequency of the C allele. This is referred to as a 957C>T polymorphism. Although not intending to limit the present invention to any one theory or mode of operation, a DRD2 genetic locus carrying 957C>T results in unstable D2 translation material and hence reduced levels of DRD2.
Accordingly, the present invention provides a genetic marker for a neurological, psychiatric or psychological condition, state or phenotype in an individual said genetic marker comprising a C at nucleotide position 957 wherein the presence of a 957C polymorphism is indicative of, or a predisposition of developing a neurological, psychiatric or psychological condition, phenotype or state selected from Addiction, Alzheimer's Disease, Anxiety Disorders, Attention Deficit Hyperactivity Disorder (ADHD), Eating Disorders, Manic-Depressive Illness, Autism, Schizophrenia, Tourette's Syndrome, Obsessive Compulsive Disorder (OCD), Panic Disorder, Post Traumatic Stress Disorder (PTSD), Phobias, borderline personality disorder, bi-polar disorder, sleep disorders, Acute Stress Disorder, Adjustment Disorder, Agoraphobia Without History of Panic Disorder, Alcohol Dependence (Alcoholism), Amphetamine Dependence, Anorexia Nervosa, Antisocial Personality Disorder, Asperger's Disorder, Avoidant Personality Disorder, Brief Psychotic Disorder, Bulimia Nervosa, Cannabis Dependence, Cocaine Dependence, Conduct Disorder, Cyclothymic Disorder, Delirium, Delusional Disorder, Dementia Associated With Alcoholism, Dementia of the Alzheimer Type, Dependent Personality Disorder, Dysthymic Disorder, Generalized Anxiety Disorder, Hallucinogen Dependence, Histrionic Personality Disorder, Inhalant Dependence, Major Depressive Disorder, Manic Depression, Multi-Infarct Dementia, Narcissistic Personality Disorder, Nicotine Dependence, Opioid Dependence, Oppositional Defiant Disorder, Panic Disorder, Paranoid Personality Disorder, Parkinson's Disease, Phencyclidine Dependence, Schizoaffective Disorder, Schizoid Personality Disorder, Schizophreniform Disorder, Schizotypal Personality Disorder, Sedative Dependence, Separation Anxiety Disorder, Shared Psychotic Disorder and Social Phobia.
Preferably, the neurological, psychiatric or psychological condition, phenotype or state is schizophrenia or alcoholism or a related condition.
In a most preferred embodiment, therefore, the present invention provides a genetic marker for a neurological, psychiatric or psychological condition, phenotype or state in an individual said genetic marker comprising a C at nucleotide position 957 wherein the presence of a 957C polymorphism is indicative of or a predisposition of developing schizophrenia or alcoholism or a related condition. The identification of such a marker allows for the diagnosis of a neurological, psychiatric or psychological condition, phenotype or state in an individual and the application of pharmacogenomics, or “personalized medicine,” which involves using genomic knowledge to tailor treatments that best suit the individual patient's needs.
Reference to nucleotide position “957” is based on the cDNA sequence (SEQ ID NO:1) or its corresponding location in the genomic sequence (SEQ ID NO:3). The numbering is calculated from the “A” in the AGT encoding the methionine or initiation codon and is designated as +1. The cDNA sequence carrying a 957C>T polymorphism is shown in SEQ ID NO:2. A range of potential polymorphisms is shown in Table 2.
In a related aspect, the present invention provides a method for detecting the presence of, or the propensity to develop a neurological, psychiatric or psychological condition phenotype or state or sub-threshold form thereof, wherein the condition, phenotype or state results from or is exacerbated by any insertion or deletion in the DRD2 genetic locus including its 5′ or 3′ terminal regions, promoter, exons or introns. Insertions or deletions may involve a single nucleotide or more than one such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 nucleotides within the region of interest.
In yet another aspect, the present invention provides a nonsense mutation which includes the introduction of a stop codon.
A neurological, psychiatric or psychological condition, phenotype or state or sub-threshold form thereof involving DRD2 or a risk of developing such a condition, phenotype or state may be ascertained by screening any tissue from an individual for genetic material carrying the DRD2 genetic locus for the presence of a polymorphism including a mutation which is associated with a particular neurological, psychiatric or psychological condition, phenotype or state or a sub-threshold form thereof or a pre-disposition for development of same. A 957C>T polymorphism on either or both alleles is one example of a genetic profile to be identified. Schizophrenia is an example of a particular neurological, psychiatric or psychological condition, phenotype or state. Most conveniently, blood is drawn and DNA extracted from the cells of the blood. In addition, prenatal diagnosis can be accomplished by testing fetal cells, placental cells or amniotic cells for a polymorphism in the DRD2 genetic locus.
Accordingly, another aspect of the present invention contemplates a method for diagnosing a neurological, psychiatric or psychological condition, phenotype or state in an individual, said method comprising obtaining or extracting DNA sample from cells of said individual and screening for or otherwise detecting the presence of a genetic profile in the DRD2 genetic locus including its 5′ or 3′ terminal region, promoter, intron or exons with a statistically significant association with a particular neurological, psychiatric or psychological condition, phenotype or state wherein the presence of that genetic profile is indicative of the neurological, psychiatric or psychological condition, phenotype or state or a sub-threshold form thereof or that the individual is at risk of developing same.
Generally, the genetic test is part of an overall diagnostic protocol involving psychological tests and certain behavioral analysis. Consequently, this aspect of the present invention may be considered as a confirmatory test or part of a series of tests in the final diagnosis of a neurological, psychiatric or psychological condition, phenotype or state.
Accordingly, another aspect of the present invention provides a diagnostic assay for a genetic profile predetermined to be associated with a particular neurological, psychiatric or psychological condition, phenotype or state said method comprising obtaining or extracting a DNA sample from cells of said individual and screening for or otherwise detecting the presence of a genetic profile in the DRD2 genetic locus including its 5′ or 3′ terminal region, promoter, intron or exons which has a statistically significant association with a particular neurological, psychiatric or psychological condition, phenotype or state wherein the presence of that genetic profile is indicative of the neurological, psychiatric or psychological condition, phenotype or state or a sub-threshold form thereof or that the individual is at risk of developing same.
As indicated above, the genetic profile is generally detecting a particular polymorphism or mutation within the DRD2 genetic locus or its 5′ or 3′ terminal regions, promoter, exons or introns. Any polymorphism or mutation such as those contemplated in Table 2 and which are found to be associated with a neurological, psychiatric or psychological condition, phenotype or state is encompassed by the present invention. In addition, examples of neurological, psychiatric or psychological conditions, phenotypes and states include but are not limited to Addiction, Alzheimer's Disease, Anxiety Disorders, Attention Deficit Hyperactivity Disorder (ADHD), Eating Disorders, Manic-Depressive Illness, Autism, Schizophrenia, Tourette's Syndrome, Obsessive Compulsive Disorder (OCD), Panic Disorder, Post Traumatic Stress Disorder (PTSD), Phobias, borderline personality disorder, bi-polar disorder, sleep disorders, Acute Stress Disorder, Adjustment Disorder, Agoraphobia Without History of Panic Disorder, Alcohol Dependence (Alcoholism), Amphetamine Dependence, Anorexia Nervosa, Antisocial Personality Disorder, Asperger's Disorder, Avoidant Personality Disorder, Brief Psychotic Disorder, Bulimia Nervosa, Cannabis Dependence, Cocaine Dependence, Conduct Disorder, Cyclothymic Disorder, Delirium, Delusional Disorder, Dementia Associated With Alcoholism, Dementia of the Alzheimer Type, Dependent Personality Disorder, Dysthymic Disorder, Generalized Anxiety Disorder, Hallucinogen Dependence, Histrionic Personality Disorder, Inhalant Dependence, Major Depressive Disorder, Manic Depression, Multi-Infarct Dementia, Narcissistic Personality Disorder, Nicotine Dependence, Opioid Dependence, Oppositional Defiant Disorder, Panic Disorder, Paranoid Personality Disorder, Parkinson's Disease, Phencyclidine Dependence, Schizoaffective Disorder, Schizoid Personality Disorder, Schizophreniform Disorder, Schizotypal Personality Disorder, Sedative Dependence, Separation Anxiety Disorder, Shared Psychotic Disorder and Social Phobia.
Schizophrenia and alcoholism are particularly contemplated by the present invention as is the 957C>T polymorphism.
Accordingly, in a preferred embodiment, the present invention is directed to a method for diagnosing a neurological, psychiatric or psychological condition, phenotype or state including schizophrenia in an individual or a risk of development of same, said method comprising obtaining or extracting a DNA sample from cells of said individual and screening for or otherwise detecting the presence of a polymorphism at cDNA nucleotide position number 957 wherein the presence of a C at position 957 is indicative of the individual having or at risk of developing an adverse neurological, psychiatric or psychological condition, phenotype or state selected from Addiction, Alzheimer's Disease, Anxiety Disorders, Attention Deficit Hyperactivity Disorder (ADHD), Eating Disorders, Manic-Depressive Illness, Autism, Schizophrenia, Tourette's Syndrome, Obsessive Compulsive Disorder (OCD), Panic Disorder, Post Traumatic Stress Disorder (PTSD), Phobias, borderline personality disorder, bi-polar disorder, sleep disorders, Acute Stress Disorder, Adjustment Disorder, Agoraphobia Without History of Panic Disorder, Alcohol Dependence (Alcoholism), Amphetamine Dependence, Anorexia Nervosa, Antisocial Personality Disorder, Asperger's Disorder, Avoidant Personality Disorder, Brief Psychotic Disorder, Bulimia Nervosa, Cannabis Dependence, Cocaine Dependence, Conduct Disorder, Cyclothymic Disorder, Delirium, Delusional Disorder, Dementia Associated With Alcoholism, Dementia of the Alzheimer Type, Dependent Personality Disorder, Dysthymic Disorder, Generalized Anxiety Disorder, Hallucinogen Dependence, Histrionic Personality Disorder, Inhalant Dependence, Major Depressive Disorder, Manic Depression, Multi-Infarct Dementia, Narcissistic Personality Disorder, Nicotine Dependence, Opioid Dependence, Oppositional Defiant Disorder, Panic Disorder, Paranoid Personality Disorder, Parkinson's Disease, Phencyclidine Dependence, Schizoaffective Disorder, Schizoid Personality Disorder, Schizophreniform Disorder, Schizotypal Personality Disorder, Sedative Dependence, Separation Anxiety Disorder, Shared Psychotic Disorder and Social Phobia.
The method and assay of the present invention are further directed to detecting the form of the polymorphism in an individual associated with “normal” behavior. In other words, an individual which may be at risk such as through his or her genetic lines or because of substance abuse or who has behavioral tendencies which suggest a particular neurological, psychiatric or psychological condition, phenotype or state can be screened for the presence of a T at cDNA nucleotide number 957 wherein the presence of a T is at least suggestive of a non-genetic basis for any symptoms associated with the neurological, psychiatric or psychological condition, phenotype or state for which the individual first presented to a clinician.
Consequently, a “neurological, psychiatric or psychological condition, phenotype or state” may be an adverse condition or may represent “normal” behavior. The latter constitutes behavior consistent with societal “norms”.
Reference herein to an “individual” includes a human which may also be considered a subject, patient, host, recipient or target.
There are many methods which may be used to detect a DNA sequence profile. Direct DNA sequencing, either manual sequencing or automated fluorescent sequencing can detect sequence variation including a polymorphism or mutation. Another approach is the single-stranded conformation polymorphism assay (SSCP) (Orita, et al., Proc. Natl. Acad. Sci. USA. 86:2766-2770, 1989). This method does not detect all sequence changes, especially if the DNA fragment size is greater than 200 bp, but can be optimized to detect most DNA sequence variation. The reduced detection sensitivity is a disadvantage, but the increased throughput possible with SSCP makes it an attractive, viable alternative to direct sequencing for mutation detection. The fragments which have shifted mobility on SSCP gels are then sequenced to determine the exact nature of the DNA sequence variation. Other approaches based on the detection of mismatches between the two complementary DNA strands include clamped denaturing gel electrophoresis (CDGE) (Sheffield et al. Proc. Natl. Acad. Sci. USA 86:232-236, 1989), heteroduplex analysis (HA) (White et al. Genomics 12:301-306, 1992) and chemical mismatch cleavage (CMC) (Grompe et al. Proc. Natl. Acad. Sci. USA 86:5855-5892, 1989). None of the methods described above detects large deletions, duplications or insertions, nor will they detect a mutation in a regulatory region or a gene. Other methods which would detect these classes of mutations include a protein truncation assay or the asymmetric assay. A review of currently available methods of detecting DNA sequence variation can be found in Kwok (Curr Issues Mol. Biol. 5(2):43-60, 20030, Twyman and Primrose (Pharmacogenomics. 4(1):67-79, 2003), Edwards and Bartlett (Methods mol. Biol. 226:287-294, 2003) and Brennan (Am. J. Pharmacogenomics. 1(4):395-302, 2001). Once a mutation is known, an allele-specific detection approach such as allele-specific oligonucleotide (ASO) hybridization can be utilized to rapidly screen large numbers of other samples for that same mutation. Such a technique can utilize probes which are labelled with gold nanoparticles or any other reporter molecule to yield a visual color result (Elghanian et al. Science 277:1078-1081, 1997).
A rapid preliminary analysis to detect polymorphisms in DNA sequences can be performed by looking at a series of Southern blots of DNA cut with one or more restriction enzymes, preferably with a large number of restriction enzymes. Each blot contains a series of normal individuals and a series of individuals having neurologic or neuropsychiatric diseases or disorders or any other neurological, psychiatric or psychological condition, phenotype or state. Southern blots displaying hybridizing fragments (differing in length from control DNA when probed with sequences near or including the DRD2 genetic locus) indicate a possible mutation or polymorphism. If restriction enzymes which produce very large restriction fragments are used, then pulsed field gel electrophoresis (PFGE) is employed. Alternatively, the desired region of the DRD2 locus can be amplified, the resulting amplified products can be cut with a restriction enzyme and the size of fragments produced for the different polymorphisms can be determined.
Detection of point mutations may be accomplished by molecular cloning of the DRD2 alleles and sequencing the alleles using techniques well known in the art. Also, the gene or portions of the gene may be amplified, e.g., by PCR or other amplification technique, and the amplified gene or amplified portions of the gene may be sequenced.
Methods for a more complete, yet still indirect, test for confirming the presence of a susceptibility allele include: 1) single-stranded conformation analysis (SSCP) (Orita et al., 1989: supra); 2) denaturing gradient gel electrophoresis (DGGE) (Wartell et al. Nucl. Acids Res. 18:2699-2705, 1990; Sheffield et al. 1989 supra); 3) RNase protection assays (Finkelstein et al. Genomics 7:167-172, 1990; Kinszler et al. Science 251:1366-1370, 1991); 4) allele-specific oligonucleotides [ASOs] (Conner et al. Proc. Natl. Acad. Sci. USA 80:278-282, 1983); 5) the use of proteins which recognize nucleotide mismatches, such as the E. coli mutS protein (Modrich Ann. Rev. Genet. 25:229-253, 1991); 6) allele-specific PCR (Ruano and Kidd Nucl. Acids Res. 17:8392, 1989); and 7) PCR amplification of the site of the polymorphism followed by digestion using a restriction endonuclease that cuts or fails to cut when the variant allele is present.
Additionally, you could add real-time PCR such as the allele specific kinetic real-time PCR assay can be used or allele specific real-time TaqMan probes.
For allele-specific PCR, primers are used which hybridize at their 3′ ends to a particular DRD2 genetic locus polymorphism or mutation. If the particular polymorphism or mutation is not present, an amplification product is not observed. Amplification Refractory Mutation System (ARMS) can also be used, as disclosed in European Patent Application Publication No. 0332435. Insertions and deletions of genes can also be detected by cloning, sequencing and amplification. In addition, restriction fragment length polymorphism (RFLP) probes for the gene or surrounding marker genes can be used to score alteration of an allele or an insertion in a polymorphic fragment. Such a method is particularly useful for screening relatives of an affected individual for the presence of the mutation found in that individual. Other techniques for detecting insertions and deletions as known in the art can be used.
In SSCP, DGGE and the RNase protection assay, a electrophoretic band appears which is absent if the polymorphism or mutation is not present. SSCP detects a band which migrates differentially because the sequence change causes a difference in single-strand, intramolecular base pairing. RNase protection involves cleavage of the mutant polynucleotide into two or more smaller fragments. DGGE detects differences in migration rates of mutant sequences compared to wild-type sequences, using a denaturing gradient gel. In an allele-specific oligonucleotide assay, an oligonucleotide is designed which detects a specific sequence, and the assay is performed by detecting the presence or absence of a hybridization signal. In the mutS assay, the protein binds only to sequences that contain a nucleotide mismatch in a heteroduplex between mutant and wild-type sequences.
Mismatches, according to the present invention, are hybridized nucleic acid duplexes in which the two strands are not 100% complementary. Lack of total homology may be due to deletions, insertions, inversions or substitutions. Mismatch detection can be used to detect point mutations in the gene or in its mRNA product. While these techniques are less sensitive than sequencing, they are simpler to perform on a large number of samples. An example of a mismatch cleavage technique is the RNase protection method. In the practice of the present invention, the method involves the use of a labelled riboprobe which is complementary to the human wild-type DRD2 genetic locus. The riboprobe and either mRNA or DNA isolated from the person are annealed (hybridized) together and subsequently digested with the enzyme RNase A which is able to detect some mismatches in a duplex RNA structure. If a mismatch is detected by RNase A, it cleaves at the site of the mismatch. Thus, when the annealed RNA preparation is separated on an electrophoretic gel matrix, if a mismatch has been detected and cleaved by RNase A, an RNA product will be seen which is smaller than the full length duplex RNA for the riboprobe and the mRNA or DNA. The riboprobe need not be the full length of the mRNA or gene but can be a segment of either. If the riboprobe comprises only a segment of the mRNA or gene, it will be desirable to use a number of these probes to screen the whole mRNA sequence for mismatches.
In similar fashion, DNA probes can be used to detect mismatches, through enzymatic or chemical cleavage (see, for example, Cotton et al. Proc. Natl. Acad. Sci. USA 87:4033-40371988; Shenk et al. Proc. Natl. Acad. Sci. USA 72:989-993, 1975; Novack et al. Proc. Natl. Acad. Sci. USA 83:586-590, 1986). Alternatively, mismatches can be detected by shifts in the electrophoretic mobility of mismatched duplexes relative to matched duplexes (see, for example, Cariello Am. J. Human Genetics 42:726-734, 1988). With either riboprobes or DNA probes, the cellular mRNA or DNA which might contain a mutation can be amplified using PCR (see below) before hybridization. Changes in DNA of the DRD2 genetic locus can also be detected using Southern blot hybridization, especially if the changes are gross rearrangements, such as deletions and insertions.
DNA sequences of the DRD2 gene which have been amplified by use of PCR may also be screened using allele-specific probes. These probes are nucleic acid oligomers, each of which contains a region of the gene sequence harboring a known mutation. For example, one oligomer may be from about 3 to about 100 nucleotides in length such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100. An oligomer of about 20 nucleotides in length is particularly convenient. These oligomers correspond to a portion of the gene sequence. By use of a battery of such allele-specific probes, PCR amplification products can be screened to identify the presence of a previously identified mutation in the gene. Hybridization of allele-specific probes with amplified DRD2 genetic sequences can be performed, for example, on a nylon filter. Hybridization to a particular probe under high stringency hybridization conditions indicates the presence of the same mutation in the tissue as in the allele-specific probe.
Once the site containing the polymorphisms has been amplified, the SNPs can also be detected by primer extension. Here a primer is annealed immediately adjacent to the variant site, and the 5′ end is extended a single base pair by incubation with di-deoxytrinucleotides. Whether the extended base was a A, T, G or C can then be determined by mass spectrometry (MALDI-TOF) or fluorescent flow cytometric analysis (Taylor et al. Biotechniques 30:661-669, 2001) or other techniques.
Nucleic acid analysis via microchip technology is also applicable to the present invention. In this technique, thousands of distinct oligonucleotide probes are built up in an array on a silicon chip. Nucleic acids to be analyzed are fluorescently labeled and hybridized to the probes on the chip. It is also possible to study nucleic acid-protein interactions using these nucleic acid microchips. Using this technique, one can determine the presence of mutations or even sequence the nucleic acid being analyzed or one can measure expression levels of a gene of interest. The method is one of parallel processing of many, including thousands, of probes at once and can tremendously increase the rate of analysis.
The most definitive test for mutations in the DRD2 genetic locus is to directly compare genomic DRD2 sequences from patients with those from a control population. Alternatively, one can sequence mRNA after amplification, e.g., by PCR, thereby eliminating the necessity of determining the exon structure of the candidate gene.
Mutations falling outside the coding region of DRD2 can be detected by examining the non-coding regions, such as introns and regulatory sequences near or within the genes. An early indication that mutations in non-coding regions are important may come from Northern blot experiments that reveal messenger RNA molecules of abnormal size or abundance in patients as compared to those of control individuals.
Alteration of mRNA expression from the DRD2 genetic locus can be detected by any techniques known in the art. These include Northern blot analysis, PCR amplification and RNase protection. Diminished mRNA expression indicates an alteration of the wild-type gene. Alteration of wild-type genes can also be detected by screening for alteration of wild-type protein. For example, monoclonal antibodies immunoreactive with DRD2 can be used to screen a tissue. Lack of cognate antigen or a reduction in the levels of antigen would indicate a mutation. Antibodies specific for products of mutant alleles could also be used to detect mutant gene product. Such immunological assays can be done in any convenient formats known in the art. These include Western blots, immunohistochemical assays and ELISA assays. Any means for detecting an altered protein can be used to detect alteration of the wild-type DRD2 gene. Functional assays, such as protein binding determinations, can be used. In addition, assays can be used which detect DRD2 biochemical function. Finding a mutant DRD2 gene product indicates alteration of a wild-type DRD2 gene.
A mutant DRD2 gene or corresponding gene products can also be detected in other human body samples which contain DNA, such as serum, stool, urine and sputum. The same techniques discussed above for detection of mutant genes or gene products in tissues can be applied to other body samples. By screening such body samples, an early diagnosis can be achieved for subjects at risk of developing a particular neurological, psychiatric or psychological condition, phenotype or state or sub-threshold forms thereof.
The primer pairs of the present invention are useful for determination of the nucleotide sequence of a particular DRD2 allele using PCR. The pairs of single-stranded DNA primers can be annealed to sequences within or surrounding the gene in order to prime amplifying DNA synthesis of the gene itself. A complete set of these primers allows synthesis of all of the nucleotides of the gene coding sequences, i.e., the exons. The set of primers preferably allows synthesis of both intron and exon sequences. Allele-specific primers can also be used. Such primers anneal only to particular DRD2 polymorphic or mutant alleles, and thus will only amplify a product in the presence of the polymorphic or mutant allele as a template.
In order to facilitate subsequent cloning of amplified sequences, primers may have restriction enzyme site sequences appended to their 5′ ends. Thus, all nucleotides of the primers are derived from the gene sequence or sequences adjacent the gene, except for the few nucleotides necessary to form a restriction enzyme site. Such enzymes and sites are well known in the art. The primers themselves can be synthesized using techniques which are well known in the art. Generally, the primers can be made using oligonucleotide synthesizing machines which are commercially available. Given the sequence of each gene and polymorphisms described herein, design of particular primers is well within the skill of the art. The present invention adds to this by presenting data on the intron/exon boundaries thereby allowing one to design primers to amplify and sequence all of the exonic regions completely.
The nucleic acid probes provided by the present invention are useful for a number of purposes. They can be used in Southern blot hybridization to genomic DNA and in the RNase protection method for detecting point mutations already discussed above. The probes can be used to detect PCR amplification products. They may also be used to detect mismatches with the DRD2 gene or mRNA using other techniques.
The present invention identifies the presence of an altered (or a mutant) DRD2 genetic locus associated with a neurological, psychiatric or psychological condition, phenotype or state, including schizophrenia or a sub-threshold form thereof or an individual of risk of developing same. In order to detect a DRD2 gene polymorphism or mutation, a biological sample is prepared and analyzed for a difference between the sequence of the allele being analyzed and the sequence of the “wild-type” allele. In this context, a “wild-type” allele includes the nucleotide at a given position most commonly represented in the population and for which there is not direct evidence for these individuals having the neurological, psychiatric or psychological condition, phenotype or state under investigation. Polymorphic or mutant alleles can be initially identified by any of the techniques described above. The polymorphic or mutant alleles may then be sequenced to identify the specific polymorphism or mutation of the particular allele. Alternatively, polymorphic or mutant alleles can be initially identified by identifying polymorphic or mutant (altered) proteins, using conventional techniques. The polymorphisms or mutations, especially those statistically associated with a neurological, psychiatric or psychological condition, phenotype or state or a sub-threshold form thereof are then used for the diagnostic and prognostic methods of the present invention.
As used herein, the phrase “amplifying” refers to increasing the content of a specific genetic region of interest within a sample. The amplification of the genetic region of interest may be performed using any method of amplification known to those of skill in the relevant art. In a preferred aspect, the present method for detecting a polymorphism utilizes PCR as the amplification step.
PCR amplification utilizes primers to amplify a genetic region of interest. Reference herein to a “primer” is not to be taken as any limitation to structure, size or function. Reference to primers herein, includes reference to a sequence of deoxyribonucleotides comprising at least 3 nucleotides. Generally, the primers comprises from about 3 to about 100 nucleotides, preferably from about 5 to about 50 nucleotides and even more preferably from about 10 to about 25 nucleotides such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 nucleotides. The primers of the present invention may be synthetically produced by, for example, the stepwise addition of nucleotides or may be fragments, parts or portions or extension products of other nucleic acid molecules. The term “primer” is used in its most general sense to include any length of nucleotides which, when used for amplification purposes, can provide free 3′ hydroxyl group for the initiation of DNA synthesis by a DNA polymerase. DNA synthesis results in the extension of the primer to produce a primer extension product complementary to the nucleic acid strand to which the primer has annealed or hybridized.
Accordingly, the present invention extends to an isolated oligonucleotide which comprises from about 3 to about 100 consecutive nucleotides from the DRD2 genetic locus and which encompass at least one polymorphism or mutation associated with or otherwise likely to be found in individuals with a particular neurological, psychiatric or psychological condition, phenotype or state such as those selected from normal behavior, Addiction, Alzheimer's Disease, Anxiety Disorders, Attention Deficit Hyperactivity Disorder (ADHD), Eating Disorders, Manic-Depressive Illness, Autism, Schizophrenia, Tourette's Syndrome, Obsessive Compulsive Disorder (OCD), Panic Disorder, Post Traumatic Stress Disorder (PTSD), Phobias, borderline personality disorder, bi-polar disorder, sleep disorders, Acute Stress Disorder, Adjustment Disorder, Agoraphobia Without History of Panic Disorder, Alcohol Dependence (Alcoholism), Amphetamine Dependence, Anorexia Nervosa, Antisocial Personality Disorder, Asperger's Disorder, Avoidant Personality Disorder, Brief Psychotic Disorder, Bulimia Nervosa, Cannabis Dependence, Cocaine Dependence, Conduct Disorder, Cyclothymic Disorder, Delirium, Delusional Disorder, Dementia Associated With Alcoholism, Dementia of the Alzheimer Type, Dependent Personality Disorder, Dysthymic Disorder, Generalized Anxiety Disorder, Hallucinogen Dependence, Histrionic Personality Disorder, Inhalant Dependence, Major Depressive Disorder, Manic Depression, Multi-Infarct Dementia, Narcissistic Personality Disorder, Nicotine Dependence, Opioid Dependence, Oppositional Defiant Disorder, Panic Disorder, Paranoid Personality Disorder, Parkinson's Disease, Phencyclidine Dependence, Schizoaffective Disorder, Schizoid Personality Disorder, Schizophreniform Disorder, Schizotypal Personality Disorder, Sedative Dependence, Separation Anxiety Disorder, Shared Psychotic Disorder and Social Phobia.
Schizophrenia and alcoholism are considered as particular examples of a neurological, psychiatric or psychological condition, phenotype or state. Insofar as the oligonucleotide primers seek to identify a polymorphism at position 957 (using cDNA numbering) of the DRD2 gene, then the preferred oligonucleotides are defined by SEQ ID NO:4 (C957) or SEQ ID NO:5 (T957). A convenient reverse primer includes SEQ ID NO:6.
However, the present invention extends to any oligomeric which encompasses a polymorphism within the DRD2 genetic locus.
Examples of these from the DRD2 cDNA include the following or their complementary forms:
Accordingly, the present invention extends to an isolated oligonucleotide from DRD2 cDNA when encompassing a polymorphism or mutation associated with a neurological, psychiatric or psychological conditions, phenotype or state selected from SEQ ID NO: 8 through SEQ ID NO:2616.
There are 20 mer oligonucleotides and as indicated above, the present invention extends to oligonucleotides from 3 to 100 nucleotides in length. In fact, the full length cDNA may also be employed. For example, SEQ ID NO:2 provides the full cDNA sequence of DRD2 cDNA comprising a C at position 957.
In a preferred embodiment, one of the at least two primers is involved in an amplification reaction to amplify a target sequence. If this primer is also labeled with a reporter molecule, the amplification reaction will result in the incorporation of any of the label into the amplified product. The terms “amplification product” and “amplicon” may be used interchangeably.
The primers and the amplicons of the present invention may also be modified in a manner which provides either a detectable signal or aids in the purification of the amplified product.
A range of labels providing a detectable signal may be employed. The label may be associated with a primer or amplicon or it may be attached to an intermediate which subsequently binds to the primer or amplicon. The label may be selected from a group including a chromogen, a catalyst, an enzyme, a fluorophore, a luminescent molecule, a chemiluminescent molecule, a lanthanide ion such as Europium (Eu34), a radioisotope and a direct visual label. In the case of a direct visual label, use may be made of a colloidal metallic or non-metallic particular, a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like. A large number of enzymes suitable for use as labels is disclosed in U.S. Pat. Nos. 4,366,241, 4,843,000 and 4,849,338. Suitable enzyme labels useful in the present invention include alkaline phosphatase, horseradish peroxidase, luciferase, p-galactosidase, glucose oxidase, lysozyme, malate dehydrogenase and the like. The enzyme label may be used alone or in combination with a second enzyme which is in solution. Alternatively, a fluorophore which may be used as a suitable label in accordance with the present invention includes, but is not limited to, fluorescein-isothiocyanate (FITC), and the fluorochrome is selected from FITC, cyanine-2, Cyanine-3, Cyanine-3.5, Cyanine-5, Cyanine-7, fluorescein, Texas red, rhodamine, lissamine and phycoerythrin.
Examples of fluorophores are provided in Table 3.
1Ex: Peak excitation wavelength (nm)
2Em: Peak emission wavelength (nm)
In order to aid in the purification of an amplicon, the primers or amplicons may additionally incorporate a bead. The beads used in the methods of the present invention may either be magnetic beads or beads coated with streptavidin.
The extension of the hybridized primer to produce an extension product is included herein by the term amplification. Amplification generally occurs in cycles of denaturation followed by primer hybridization and extension. The present invention encompasses form about 1 cycle to about 120 cycles, preferably from about 2 to about 70 cycles, more preferably from about 5 to about 40 cycles, including 10, 15, 20, 25 and 30 cycles, and even more preferably, 35 cycles such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120 cycles.
In order for the primers used in the methods of the present invention to anneal to a nucleic acid molecule containing the gene of interest, a suitable annealing temperature must be determined. Determination of an annealing temperatures is based primarily on the genetic make-up of the primer, i.e. the number of A, T, C and Gs, and the length of the primer. Annealing temperatures contemplated by the methods of the present invention are from about 40° C. to about 80° C., preferably from about 50° C. to about 70° C., and more preferably about 65° C. such as 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80° C.
The PCR amplifications performed in the methods of the present invention include the use of MgCl2 in the optimization of the PCR amplification conditions. The present invention encompasses MgCl2 concentrations for about 0.1 to about 10 mM, preferably from 0.5 to about 5 mM, and even more preferably 2.5 mM such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mM.
Polymorphisms of the present invention may be detected due to the presence of a base mis-match in the heteroduplexes formed following PCR amplification. A base mis-match occurs when two nucleotide sequences are aligned with substantial complementarity but at least one base aligns to a base which would result in an “abnormal” binding pair. An abnormal binding pair occurs when thymine (T) were to bind to a base other than adenine (A), if A were to bind to a base other than T, if guanine (G) were to bind to a base other than cytosine (C) or if C was to bind to a base other than G.
In order to detect the presence of a DRD2 allele predisposing an individual to an inability to overcome a neurological, psychiatric or psychological condition, phenotype or state or sub-threshold form thereof or a risk of developing same, a biological sample such as blood is obtained and analyzed for the presence or absence of one or more susceptibility alleles of the DRD2 genetic locus identified as being statistically significantly associated with the neurological, psychiatric or psychological condition, phenotype or state of interest of DRD2. Results of these tests and interpretive information are returned to the health care provider for communication to the tested individual. Such diagnoses may be performed by diagnostic laboratories, or, alternatively, diagnostic kits are manufactured and sold to health care providers or to private individuals for self-diagnosis. Suitable diagnostic techniques include those described herein as well as those described in U.S. Pat. Nos. 5,837,492; 5,800,998 and 5,891,628.
According to the present invention, a method is also provided for supplying wild-type DRD2 function to a cell which carries a DRD2 allele mutant or polymorphism. Supplying such a function should allow normal functioning of the recipient cells. The wild-type gene or a part of the gene may be introduced into the cell in a vector such that the gene remains extrachromosomal. In such a situation, the gene will be expressed by the cell from the extrachromosomal location. More preferred is the situation where the wild-type gene or a part thereof is introduced into the mutant cell in such a way that it recombines with the endogenous mutant gene present in the cell. Such recombination requires a double recombination event which results in the correction of the gene mutation. Vectors for introduction of genes both for recombination and for extrachromosomal maintenance are known in the art, and any suitable vector may be used. Methods for introducing DNA into cells such as electroporation, calcium phosphate co-precipitation and viral transduction are known in the art, and the choice of method is within the competence of the practitioner. Conventional methods are employed, including those described in U.S. Pat. Nos. 5,837,492; 5,800,998 and 5,891,628.
The identification of the association between the DRD2 gene polymorphism/mutations and a psychological phenotype or sub-threshold psychological phenotype permits the early presymptomatic screening of individuals to identify those at risk for developing a neurological, psychiatric or psychological condition, phenotype or state or sub-threshold neurological, psychiatric or psychological condition, phenotype or state such as schizophrenia or to identify the cause of such disorders or the risk that any individual will develop same. To identify such individuals, the alleles are screened as described herein or using conventional techniques, including but not limited to, one of the following methods: fluorescent in situ hybridization (FISH), direct DNA sequencing, PFGE analysis, Southern blot analysis, single stranded conformation analysis (SSCP), linkage analysis, RNase protection assay, allele-specific oligonucleotide (ASO), dot blot analysis and PCR-SSCP analysis. Also useful is the recently developed technique of DNA microchip technology. Such techniques are described in U.S. Pat. Nos. 5,837,492; 5,800,998 and 5,891,628, each incorporated herein by reference.
Genetic testing enables practitioners to identify individuals at risk for certain behavioral states including substance addition or an inability to overcome a neurological, psychiatric or psychological condition, phenotype or state or a sub-threshold form thereof after initial treatment. For particular at risk couples, embryos or fetuses may be tested after conception to determine the genetic likelihood of the offspring being pre-disposed to the neurological, psychiatric or psychological condition, phenotype or state. Certain behavioral or therapeutic protocols may then be introduced from birth or early childhood to reduce the risk of the neurological, psychiatric or psychological condition, phenotype or state developing. Presymptomatic diagnosis will enable better treatment of these disorders, including the use of existing medical therapies. Genetic testing will also enable practitioners to identify individuals having diagnosed disorders (or in an at risk group) which have polymorphism identified in the DRD2 genetic locus. Genotyping of such individuals will be useful for (a) identifying neurological, psychiatric or psychological condition, phenotype or state or a sub-threshold form thereof that will respond to drugs affecting DRD2 activity, (b) identifying a neurological, psychiatric or psychological condition, phenotype or state or sub-threshold neurological, psychiatric or psychological condition, phenotype or state that in an individual which respond well to placebos versus those that respond better to active drugs and (c) guide new drug discovery and testing. Further, the present invention provides a method for screening drug candidates to identify molecules useful for treating neurological, psychiatric or psychological conditions, phenotypes or states involving the DRD2 gene or its expressive product. Drug screening is performed by comparing the activity of native genes and those described herein in the presence and absence of potential drugs. In particular, these drugs may have the affect of masking a polymorphism or mutation or may bind to a particular polymorphism or mutation enabling it to be used as a diagnostic agent. The terms “drug”, “agent”, “therapeutic molecule”, “prophylactic molecule”, “medicament”, “candidate molecule” or “active ingredient” may be used interchangeable in describing this aspect of the present invention.
The goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g., agonists, antagonists, inhibitors) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, e.g., enhance or interfere with the function of a polypeptide in vivo or which are specific for a targetable (e.g. a polymorphism) and hence is a useful diagnostic. Several approaches for use in rational drug design include analysis of three-dimensional structure, alanine scans, molecular modeling and use of anti-id antibodies. These techniques are well known to those skilled in the art, including those described in U.S. Pat. Nos. 5,837,492; 5,800,998 and 5,891,628.
A substance identified as a modulator of polypeptide function may be peptide or non-peptide in nature. Non-peptide “small molecules” are often preferred for many in vivo pharmaceutical uses. Accordingly, a mimetic or mimic of the substance (particularly if a peptide) may be designed for pharmaceutical use.
The designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a “lead” compound. This approach might be desirable where the active compound is difficult or expensive to synthesize or where it is unsuitable for a particular method of administration, e.g., pure peptides are unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal. Mimetic design, synthesis and testing are generally used to avoid randomly screening large numbers of molecules for a target property.
Once the pharmacophore has been found, its structure is modelled according to its physical properties, e.g., stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g., spectroscopic techniques, x-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modeling process. A template molecule is then selected, onto which chemical groups that mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted thereon can be conveniently selected so that the mimetic is easy to synthesize, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound. Alternatively, where the mimetic is peptide-based, further stability can be achieved by cyclizing the peptide, increasing its rigidity. The mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent it is exhibited. Further optimization or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.
Briefly, a method of screening for a substance which modulates activity of a polypeptide may include contacting one or more test substances with the polypeptide in a suitable reaction medium, testing the activity of the treated polypeptide and comparing that activity with the activity of the polypeptide in comparable reaction medium untreated with the test substance or substances. A difference in activity between the treated and untreated polypeptides is indicative of a modulating effect of the relevant test substance or substances.
Following identification of a substance which modulates or affects DRD2 activity, the substance may be further investigated. Furthermore, it may be manufactured and/or used in preparation, i.e., a manufacture or formulation, or a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals directly or via gene therapy.
The DRD2 polypeptides, antibodies, peptides and nucleic acids of the present invention can be formulated in pharmaceutical compositions, which are prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington's Pharmaceutical Sciences, 18th Ed. 1990, Mack Publishing Co., Easton, Pa. The composition may contain the active agent or pharmaceutically acceptable salts of the active agent. These compositions may comprise, in addition to one of the active substances, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., intravenous, oral, intrathecal, epineural or parenteral.
The present invention provides information necessary for physicians to select drugs for use in the treatment of a neurological, psychiatric or psychological condition, phenotype or state or a sub-threshold form thereof. With the identification that polymorphisms within the DRD2 gene are associated with a neurological, psychiatric or psychological condition, phenotype or state including a sub-threshold form thereof, such as schizophrenia, antipsychotic medications, such as partial agonists or antagonists of DRD2 can be selected for the treatment of such conditions.
The present invention further contemplates a method of treating a neurological, psychiatric or psychological condition, phenotype or state in an individual the method comprising identifying a polymorphism in the DRD2 genetic locus with the neurological, psychiatric or psychological condition, phenotype or state and subjecting the individual to gene therapy to alter the gene or genetic sequence having a different polymorphism or to treat the defect caused by the polymorphism or to subject the individual to behavioral modification protocols to help ameliorate the symptoms.
Using the compositions of the present invention, gene therapy may be recommended when a particular polymorphism conferring, for example, a disease condition or a propensity for development of neurological, psychiatric or psychological condition, phenotype or state is identified in an embryo. Genetically modified stem cells may then be used to alter the genotype of the developing cells. Where an embryo has developed into a fetus or for post-natal subjects, localized gene therapy may still be accomplished. Alternatively, a compound may be identified which effectively masks a particular undesired polymorphic variant or which influences the expression of a more desired phenotype. For example, one polymorphic variant of a receptor may result in an instability of the mRNA transition product.
Accordingly, the present invention also provides genetic test kits which allow the rapid screening of a DRD2 polymorphism or polymorphisms within a test sample or multiple test samples. The kits of the present invention comprise one or more sets of primers, as described herein, which are specific for the amplification of a genetic region of interest. In addition, the genetic testing kits of the present invention provide a PCR mix, comprising MgCl2. In a preferred aspect, the MgCl2 is provided at a concentration of 2.5 mM. Additionally, the genetic test kits of the present invention provide instructions for using the primers of the present invention to obtain the desired duplexes, as well as instructions as to the analysis of the duplexes using d-HPLC.
The present invention is further described with reference to the following non-limiting Examples.
One hundred and fifty three unrelated Caucasian patients (133 males, 20 females) attending various psychiatric units for the treatment of their schizophrenia were recruited for the study. Patients were being treated at the Fortitude Valley Community Mental Health Centre, the Royal Brisbane Mental Health Unit and the Park Psychiatric Hospital. Inclusion criteria were being between 18 and 65 years of age and having a DSM IV diagnosis of schizophrenia. In this particular study potential participants were excluded if they had Schizoaffective Disorder, Bipolar Disorder, Dementia, Organic Brain Syndrome or Major Depressive Disorder with Delusions. The study further included One hundred and forty eight controls (41 females, 107 males). A 10 mL blood sample was drawn from each subject for DNA extraction. All participants provided informed consent and were able to terminate participation at any time without prejudice. Institutional ethics approval was obtained from the university, clinics and hospitals involved.
DNA was extracted from leucocytes using standard techniques and subsequently used as a template for determination of 957C>T genotypes. Genotyping was performed by real-time PCR using the Applied Biosystems 7000 sequence detection system (Applied Biosystems, Foster City, Calif., USA). Sequence specific primers were designed for the C allele (5′-ATGGTCTCCACAGCACTCTC-3′ SEQ ID NO:4), the T allele (5′-ATGGTCTCCACAGCACTCTT-3′ SEQ ID NO:5) and a common reverse primer (5′-CATTGGGCATGGTCTGGATC-3′ SEQ ID NO:6). A total of 5-10 ng of genomic DNA was amplified in 1×SYBR green PCR master mix (Applied Biosystems) containing 0.4 μM of allele specific forward primer and 0.4 μM of common reverse primer in a 25 μl volume. Amplification conditions were: 50° C. for 2 min, 95° C. for 10 min, followed by 40 cycles of 95° C. for 15 s and 60° C. for 1 min. A cycle time (Ct) value was obtained by setting the threshold during geometric phase of amplification and scored relative to the ΔCt generated between the matched and mismatched primer pairs.
Information coded from interview proformas was entered into a computer data base. Chi-square test were employed to compare differences in non continuous variables between 957C>T genotype groups. A p-value of ≦0.05 was considered to be statistically significant.
To evaluate the frequency of 957C and 957T in patients with schizophrenia and alcoholism and in controls, 153 patients meeting DSM-1V criteria for schizophrenia, 132 severely alcoholic subjects and 148 general population controls were genotyped for the 957C>T polymorphism.
The observed allele frequency and genotype frequencies of the 957C>T polymorphism in control, schizophrenic and alcoholic individuals revealed a significant increase in the frequency of the 957C allele in both schizophrenia (Table 4) and alcoholism (Table 5) compared to the controls. The genotype frequencies in the schizophrenic and alcoholic groups also differed significantly from expected values compared to controls although it is interesting to note that the heterozygote frequency is approximately the same in the two groups. The schizophrenic, alcoholic and control groups appeared to be in Hardy-Weinberg equilibrium based on the respective allele frequency of each group.
Using standard population genetics calculations and assuming a prevalence of schizophrenia of 1% in the general population CC alleles account for 25% of the heritability of schizophrenia.
One hundred and sixty unrelated Caucasian patients (137 males, 23 females) attending various psychiatric units for the treatment of their schizophrenia were recruited for the study. Patients were being treated at the Fortitude Valley Community Mental Health Centre, the Royal Brisbane Mental Health Unit and the Park Psychiatric Hospital, Brisbane, Australia. Inclusion criteria were being between 18 and 65 years of age and having a DSM IV diagnosis of schizophrenia. In this particular study potential participants were excluded if they had Schizoaffective Disorder, Bipolar Disorder, Dementia, Organic Brain Syndrome or Major Depressive Disorder with Delusions. The study further included two hundred and twenty nine controls (134 males, 95 females). A 10 mL blood sample was drawn from each subject for DNA extraction.
One hundred and ten unrelated Caucasian patients (110 males, 0 females) attending Greenslopes Private Hospital, Brisbane, Australia, were collected. Inclusion criteria were being between 18 and 65 years of age and having a DSM IV diagnosis of Post Traumatic Stress Disorder. The study further included two hundred and twenty nine controls (134 males, 95 females). A 10 mL blood sample was drawn from each subject for DNA extraction.
Two hundred and thirty two unrelated Caucasian patients (151 Males, 81 females) attending treatment at The Royal Brisbane Hospital, Brisbane, Australia, were collected for analysis. Inclusion criteria were being between 18 and 65 years of age and having a DSM IV diagnosis of alcohol dependence. The study further included two hundred and twenty nine controls (134 males, 95 females). A 10 mL blood sample was drawn from each subject for DNA extraction.
One hundred and fifty unrelated Caucasian patients (76 Males, 74 females) attending The Royal Brisbane Hospital were collected. Inclusion criteria were being between 18 and 65 years of age and having a DSM IV diagnosis of nicotine dependence. The study further included two hundred and twenty nine controls (134 males, 95 females). A 10 mL blood sample was drawn from each subject for DNA extraction.
One hundred and eighteen unrelated Caucasian patients (69 Males, 49 females) attending treatment at The Royal Brisbane Hospital were collected for analysis. Inclusion criteria were being between 18 and 65 years of age and having a DSM IV diagnosis of opioid dependence. The study further included two hundred and twenty nine controls (134 males, 95 females). A 10 mL blood sample was drawn from each subject for DNA extraction.
DNA was extracted from leucocytes using standard techniques and subsequently used as a template for determination of 957C>T genotypes. Genotyping was performed by real-time PCR using the Applied Biosystems 7000 sequence detection system (Applied Biosystems, Foster City, Calif., USA). Sequence specific primers were designed for the C allele (5′-ATGGTCTCCACAGCACTCTC-3′ SEQ ID NO:4), the T allele (5′-ATGGTCTCCACAGCACTCTT-3′ SEQ ID NO:5) and a common reverse primer (5′-CATTGGGCATGGTCTGGATC-3′ SEQ ID NO:6). A total of 5-10 ng of genomic DNA was amplified in 1×SYBR green PCR master mix (Applied Biosystems) containing 0.4 μM of allele specific forward primer and 0.4 μM of common reverse primer in a 25 μl volume. Amplification conditions were: 50° C. for 2 min, 95° C. for 10 min, followed by 40 cycles of 95° C. for 15 s and 60° C. for 1 min. A cycle time (Ct) value was obtained by setting the threshold during geometric phase of amplification and scored relative to the ΔCt generated between the matched and mismatched primer pairs.
DNA was extracted from leucocytes using standard techniques and subsequently used as a template for determination of Taq1 A (C<T) genotypes. Genotyping was performed by PCR-restriction fragment length polymorphism (RFLP) of amplified PCR fragments. The genomic sequence of 501 bp of the 3′-flanking region of DRD2 was amplified by PCR with the primer pair, forward primer (5′-GCACGTGCCACCATACCC-3′ SEQ ID NO:2630) and a reverse primer (5′-TGCAGAGCAGTCAGGCTG-3′ SEQ ID NO:2631). A total of 5-10 ng of genomic DNA was amplified in a PCR master mix containing 0.2 μM of forward primer and 0.2 μM of reverse primer, 1×PCR buffer, 1.5 mM MgCl2, 200 μM dNTPs, H20 and 1 unit of Platinum Taq DNA Polymerase (Invitrogen) in a 25 μl volume. Amplification conditions were: Step 1: 94° C. for 4 min, Step 2: 94° C. for 30 s, Step 3: 68° C. for 30 s, Step 4: 72° C. for 30 s, Steps 2-4 were repeated by 40 cycles followed by 72° C. for 3 min. Amplified PCR fragments were digested with Taqa I (5′ . . . T▾CGA . . . 3′) restriction enzyme (New England Biolabs) and digested fragments were visualised via 2% agarose gel electrophoresis and ethidium bromide staining.
-141delC
DNA was extracted from leucocytes using standard techniques and subsequently used as a template for determination of -141C Ins/Del genotypes. Genotyping was performed by RFLP of amplified PCR fragments. The genomic sequence of 284 bp of the 5′-flanking region and 274 bp of exon 1 of DRD2 was amplified by PCR with the primer pair, forward primer (5′-ACTGGCGAGCAGACGGTGAGGACCC-3′ SEQ ID NO:2632) and a reverse primer (5′-TGCGCGCGTGAGGCTGCCGGTTCGG-3′ SEQ ID NO:2633). A total of 5-10 ng of genomic DNA was amplified in a PCR master mix containing 0.2 μM of forward primer and 0.2 μM of reverse primer, 1×PCR buffer, 1.5 mM MgCl2, 200 μM dNTPs, H20, 2× enhancer solution (Invitrogen) and 1 unit of Platinum Taq DNA Polymerase (Invitrogen) in a 25 μl volume. Amplification conditions were: Step 1: 95° C. for 3 min, Step 2: 95° C. for 30 s, Step 3: 68° C. for 30 s, Step 4: 72° C. for 30 s, Steps 2-4 were repeated by 40 cycles followed by 72° C. for 2 min. Amplified PCR fragments were digested with BstN1 (5′ . . . CC▾(A/T)GG . . . 3′) restriction enzyme (New England Biolabs) and digested fragments were visualised via 2% w/v agarose gel electrophoresis and ethidium bromide staining.
Information coded from interview proformas was entered into a computer data base. Chi-square test were employed to compare differences in non continuous variables between 957C>T genotype groups. A p-value of ≦0.05 was considered to be statistically significant. Two stringent statistical tests were applied to test the null hypothesis that there was no difference between the case and the control groups: Compare 2 Version 1.25 was used for the comparison of two independent groups or samples. Fisher's exact P and Chi square for a 2 by 2 contingency table (allele frequencies) and a 2 by 3 table (for genotype frequencies). A further test (chi-squared for goodness of fit) was applied to determine whether the case population mimicked the control population based on the control allele frequencies. Raw genotype data for each individual for the control and test population were entered into the PHASE v2.1.1 program to generate predicted haplotype numbers and haplotype frequencies. Haplotype numbers generated were analyzed using the Compare 2 Version 1.25 using a 2 by k table to generate a likelihood-ratio Chi square and odds ratios.
The analysis on the 957CT polymorphism was extended to schizophrenia by expanding the number of schizophrenic patients and the number of control subjects. This result confirms and strengthens the conclusions from the study in Example 2, i.e. there is a strong association between the DRD2 957C allele and schizophrenia (see Tables 7 to 10).
This study was further extended by including the analysis of 2 further polymorphisms to include a total of 3 polymorphisms (957C>T, Taq 1A and -141delC). These 3 polymorphisms were analyzed in the control and schizophrenia patients and these were also analyzed in a several other patient groups including, post traumatic stress disorder (PTSD), alcohol dependence, nicotine dependence and opiate dependence. First, the patient groups and the control group were tested to see if they were in Hardy-Weinberg equilibrium (Table 6). All groups were in equilibrium for each of the 3 SNPs except the -141delC polymorphism in the alcohol dependence group. The genetic association of each of the 3 polymorphisms was tested in each of the patient groups by analysis of the genotype (Table 7) and allele (Table 9) frequencies. The 957C allele of the 957C>T polymorphism was significantly associated with schizophrenia, PTSD, alcohol dependence and nicotine dependence but there was no association with opiate dependence (when analyzed by goodness-of-fit to the control population and a 2×2 contingency Table 8).
The genotype frequencies in the schizophrenia, PTSD, alcohol dependence and nicotine dependence groups also differed significantly from expected values compared to controls although it is interesting to note that the heterozygote frequencies are approximately the same in the patient groups compared to the control group (Table 9).
In order to fully explore the presence of alleles that are associated with disease the data for the individual SNP genotypes was analyzed to generate haplotypes for the 3 SNPs in the control group and each of the patient groups using the PHASE program. The haplotypes were generated for all 3 SNPs at once, to give a total of 8 possible haplotypes or 2 at a time in 3 possible combinations to give a total of 4 possible haplotypes (Table 11).
The association of the haplotypes with disease was tested by performing a 2×8 comparison table, for the 3 SNP haplotypes or a 2×4 table for the 2 SNP haplotypes. For the 3 SNP haplotypes the overall likelihood ratio chi-square gave a significant P value for schizophrenia, PTSD, alcohol dependence and nicotine dependence but there appeared to be no association with opiate dependence. For those diseases that showed a haplotype association, in each case the 122 haplotype was about 2 to 4.5 times more likely to be found with disease than the 111 or 112 haplotypes, respectively. In addition, the 211 haplotype showed a tendency to be found with disease although this was only significant for Alcohol dependence where it was approximately 10 times more likely to be found with disease than the 122 “disease haplotype” and about 40 times more likely to be found with disease than the 112 “healthy haplotype”.
Legend for the Data Presented Below
For the -141delC/957C>T 2 SNP haplotypes the overall likelihood ratio chi-square gave a significant or nearly significant P value for schizophrenia, PTSD, alcohol dependence and nicotine dependence but there appeared to be no association with opiate dependence (Table 13). However, none of the individual haplotypes showed a significant association with disease relative to the -141delC, C/957C>T, C haplotype.
The following data are a combination of 2 SNPs of the choices shown above
For the -141 delC/Taq1A 2 SNP haplotypes the overall likelihood ratio chi-square was not significantly more likely to be found with disease for any of the diseases studied. The 957C>T/Taq1A 2 SNP haplotypes showed the strongest association with disease. The overall likelihood ratio chi-square values gave strongly significant P values for schizophrenia, alcohol dependence and nicotine dependence, and nearly significant P values for PTSD but there appeared to be no association with opiate dependence. Most of the 957C>T/Taq1A individual haplotypes also showed very strong and significant association relative to the 22 (957C>T, C/Taq1A, A1) “disease haplotype”. In particular, the 22 haplotype was 2.5 to 4 times more likely to be found with disease than the 12 (957C>T, T/Taq1A, A1) haplotype for all disease groups except opiate dependence.
In conclusion, it is clear that the 957C>T polymorphism is very strongly associated with 4 disease groups, schizophrenia, PTSD, alcohol dependence and nicotine dependence. The Taq1A polymorphism is showing the same pattern of association although the strength of the association is weaker, especially for alcohol dependence and PTSD but the -141delC polymorphism is not showing any association with any of the disease groups, possibly because the polymorphism has a low allele frequency. The use of haplotypes containing 2 or 3 SNPs greatly increases the power to detect alleles that are associated with disease. For example the 122 and the 221 haplotypes (-141delC, CC/957C>T, C/Taq1A, A1 and -141delC, C/957C>T, C/Taq1A, A2; respectively) appear to be strongly associated with disease relative to the 111 and 112 haplotypes (-141delC, CC/957C>T, T/Taq1A, A2 and -141delC, CC/957C>T, T/Taq1A, A1; respectively).
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2004902919 | Jun 2004 | AU | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/AU2005/000775 | 6/1/2005 | WO | 00 | 8/14/2008 |
