Methods and kits for determining susceptibility of a patient to bipolar disorders are described. The method comprises the steps of: (a) obtaining a sample from a patient; and (b) testing the sample for the presence of a polymorphism selected from (i) a 2 repeat allele in the variable number of tandem repeat (VNTR) region of the DRD4 gene and (ii) a 4 repeat allele in the VNTR region of the DRD4 gene, wherein the presence of the 2 repeat allele indicates that the patient is less susceptible to a bipolar disorder and the presence of the 4 repeat allele indicates that the patient is more susceptible to a bipolar disorder.

SUMMARY OF THE INVENTION

The present inventors have shown that there is an increased number of non-transmitted DRD4 2 repeat alleles and an increased number of transmissions for the 4 repeat alleles to bipolar patients when compared to what should be expected by chance. This effect was derived almost exclusively from the maternal meiosis. The excess of non-transmission of the 2 alleles to bipolar patients suggests that the 2 repeat allele may confer a protective role while the 4 repeat alleles may increase the risk in developing bipolar disorder when transmitted from the maternal side. Because of the occurrence in the human population of multiple sequences within the 48 bp repeat all the 2 repeat alleles that were informative from the maternal meioses were sequenced. No sequence variants for the 2 repeat allele were found.

The present invention therefore relates to a method of determining the susceptibility of a patient to a bipolar disorder comprising:

• (a) obtaining a sample from a patient; and
• (b) testing the sample for the presence of a polymorphism selected from (i) a 2 repeat allele in the variable number of tandem repeat (VNTR) region of the DRD4 gene and (ii) a 4 repeat allele in the VNTR region of the DRD4 gene, wherein the presence of the 2 repeat allele indicates that the patient is less susceptible to a bipolar disorder and the presence of the 4 repeat allele indicates that the patient is more susceptible to a bipolar disorder.

Preferably the polymorphisms are transmitted from the maternal side. Therefore the present invention also relates to a method of determining the susceptibility of a patient to a bipolar disorder as defined above, further comprising:

• (c) obtaining a sample from the patient's mother;
• (d) testing the mother's sample for the presence of a polymorphism selected from (i) a 2 repeat allele in the variable number of tandem repeat (VNTR) region of the DRD4 gene and (ii) a 4 repeat allele in the VNTR region of the DRD4 gene, wherein the presence of a 2 repeat allele from the mother in the sample from the patient indicates that the patient is less susceptible to a bipolar disorder and the presence of a 4 repeat allele from the mother in the sample from the patient indicates that the patient is more susceptible to a bipolar disorder.

In another embodiment of the invention, there are provided methods of detecting the presence of a polymorphism in the DRD4 gene associated with a bipolar disorder.

The present invention also provides a kit for determining susceptibility of a patient to a bipolar disorder, or for detecting the presence of a polymorphism associated with a bipolar disorder, comprising reagents necessary for determining the presence of a polymorphism selected from a 2 repeat allele in the VNTR region of the DRD4 gene and a 4 repeat allele in the VNTR region of the DRD4 gene and directions for its use.

Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

DETAILED DESCRIPTION OF THE INVENTION

The inconsistencies shown across the linkage studies that investigated the short arm of chromosome 11 in bipolar disorder do not exclude the possible small contribution of genes to disease (for e.g. DRD4 and TH) since linkage analysis has low power to detect genes with partial contribution to disease (Greenberg 1993). Association studies that investigated the role of DRD4 in bipolar disorder did not show evidence for association (De bruyn et al 1994; Serretti et al 1999; Lim et al 1994; Li et al 1999; Bocchefta et al 1999). In a similar fashion, the association studies of HUMTH01 in bipolar disorder produced positive and negative association and a meta-analysis of these studies showed no major effect attributable to the HUMTH01 in bipolar disorder (Reviewed in Furlong et al 1999). However, because the majority of studies that investigated DRD4 were case-controls and the only study conducted on families was in a small sample size (Bochetta et al 1999), the involvement of DRD4 in bipolar disorder cannot be ruled out. The case-control approach, in fact, is associated with the risk of producing spurious results because of the difference in allele frequencies across different populations (Kidd 1993). Limits of case-control studies are particularly evident when the candidate genes being studied have a demonstrated population-specific frequency for their alleles, as in the case of the DRD4 exon III VNTR (Chang et al 1999). Family-based association studies prevent the occurrence of false positive results derived from population stratification (Ott 1999), have good power to detect genes with partial effects as compared to linkage strategies, and allows the separate analysis of maternally and paternally transmitted alleles. Although the statistical power of family studies is lower when compared to case-control strategies (Rish 2000), maternal and paternal meiosis may help in understanding the presence of type I errors from true association (Petronis 2000). In the present study a family-based association study of the DRD4 48 bp repeats VNTR and the TH intron I tetranucleotide repeats VNTR allele was performed in a sample made up of subjects suffering from DSM IV bipolar disorder and their respective biological parents.

The results of the present study showed an increased number of non-transmitted DRD4 2 repeat alleles and an increased number of transmissions for the 4 repeat alleles when compared to what should be expected by chance. This effect was derived almost exclusively from the maternal meiosis. The excess of non-transmission of the 2 alleles to bipolar patients is suggesting that the 2 repeat allele may confer a protective role while the 4 repeats alleles may increase the risk in developing bipolar disorder, when transmitted from the maternal side. Furthermore, the effect of the maternal meioses in the biased transmission of the 4-repeat allele was stronger when the families with a maternal history of bipolar disorder were considered (for the 4 repeat allele 10 transmissions and 2 non transmissions were observed; chi-square=5.33, 1 df, p=0.02).

The present invention therefore relates to a method of determining the susceptibility of a patient to a bipolar disorder comprising:

• (a) obtaining a sample from a patient; and
• (b) testing the sample for the presence of a polymorphism selected from (i) a 2 repeat allele in the variable number of tandem repeat (VNTR) region of the DRD4 gene and (ii) a 4 repeat allele in the VNTR region of the DRD4 gene, wherein the presence of the 2 repeat allele indicates that the patient is less susceptible to a bipolar disorder and the presence of the 4 repeat allele indicates that the patient is more susceptible to a bipolar disorder.

Preferably the polymorphism is transmitted from the maternal side. Therefore the present invention further relates to a method of determining the susceptibility of a patient to a bipolar disorder as defined above, further comprising:

• (c) obtaining a sample from the patient's mother;
• (d) testing the mother's sample for the presence of a polymorphism selected from (i) a 2 repeat allele in the variable number of tandem repeat (VNTR) region of the DRD4 gene and (ii) a 4 repeat allele in the VNTR region of the DRD4 gene, wherein the presence of a 2 repeat allele from the mother in the sample from the patient indicates that the patient is less susceptible to a bipolar disorder and the presence of a 4 repeat allele from the mother in the sample from the patient indicates that the patient is more susceptible to a bipolar disorder.

The term "repeat unit in the VNTR region of the DRD4 gene" means a 48 bp unit found in the variable number of tandem repeat (VNTR) region in the third exon of the dopamine D4 receptor (DRD4). In the 2-allele polymorphism, 2 of the 48 bp units are present and in the 4-allele polymorphism, 4 of the 48 bp units are present.

The term "bipolar disorder" refers to any type of bipolar disorder, including, but limited to, Bipolar I, Bipolar II, and Schizoaffective Bipolar-type Disorder.

The sample obtained from the patient can be any biological sample containing nucleic acids including, but not limited to, blood, urine, skin, hair, sperm, buccal mucosa as well as tissue samples and fractions of any of the foregoing.

The sample may be tested for the presence of a polymorphism in the DRD4 gene using a variety of techniques known in the art. Generally, nucleic acids are obtained from the sample and amplified using the Polymerase Chain Reaction (PCR) using primers to the appropriate region of the DRD4 gene. For example when assaying for polymorphisms in the VNTR region, primers to the VNTR are used (Lichter et al. 1993). The PCR products can be subjected to any method that would allow one to identify the presence of a polymorphism. In one embodiment, the PCR products may be subjected to an electrophoretic assay (such as gel electrophoresis or capillary electrophoresis) to determine the relative size of the PCR product. For example, the size of the PCR product can be determined by comparing its migration on an electrophoresis gel with a 50 bp ladder. Once the size has been determined in this manner, it can be compared with the predicted size of the repeat alleles to confirm its identity. In another embodiment, the PCR products may be probed with a fluorescently-labeled nucleic acid sequence specific for a region in the polymorphism. In a further embodiment, the PCR products may be sequenced using techniques known in the art including commercially available sequencing kits to determine if the polymorphism is present in the sample. Other sequencing technologies such as Denaturing High Pressure Liquid Chromatography or mass spectroscopy may also be employed. In yet another embodiment, detection of polymorphism can be performed by using restriction enzymes or Single Stranded Conformation Polymorphism (SSCP) techniques. In addition, methods for high throughput detection of nucleotide polymorphisms may be used such as DNA chip technology. Combinations of any of the above methods may be used.

The above definitions and descriptions apply to all embodiments of the present invention.

The method of the invention may also be used to detect a polymorphism in the DRD4 gene associated with a bipolar disorder. Accordingly, in a further embodiment, the present invention relates to a method of detecting the presence of a polymorphism in the DRD4 gene associated with a bipolar disorder in a patient comprising:

• (a) obtaining a sample from a patient; and
• (b) testing the sample for the presence of a polymorphism selected from (i) a 2 repeat allele in the variable number of tandem repeat (VNTR) region of the DRD4 gene and (ii) a 4 repeat allele in the VNTR region of the DRD4 gene, wherein the presence of the 2 repeat allele or the presence of the 4 repeat allele indicates the presence of a polymorphism in the DRD4 gene associated with bipolar disorder in the patient.

Preferably the polymorphism is transmitted from the maternal side. Therefore the present invention also relates to a method of detecting the presence of a polymorphism in the DRD4 gene associated with a bipolar disorder in a patient as defined above, further comprising:

• (c) obtaining a sample from the patient's mother;
• (d) testing the mother's sample for the presence of a polymorphism selected from (i) a 2 repeat allele in the variable number of tandem repeat (VNTR) region of the DRD4 gene and (ii) a 4 repeat allele in the VNTR region of the DRD4 gene, wherein the presence of a 2 repeat allele from the mother in the sample from the patient or the presence of a 4 repeat allele from the mother in the sample from the patient indicates the presence of a polymorphism in the DRD4 gene associated with a bipolar disorder in the patient.

The invention also includes kits for use in the above methods for detecting the presence of the polymorphism in the VNTR region of the DRD4 gene. Accordingly, the present invention provides a kit for determining the susceptibility of a patient to a bipolar disorder, or for determining the presence of a polymorphism in the DRD4 gene associated with a bipolar disorder, comprising reagents necessary for determining the presence of a polymorphism selected from (i) a 2 repeat allele in the VNTR region of the DRD4 gene and (ii) a 4 repeat allele in the VNTR region of the DRD4 gene and directions for its use.

The reagents useful in the kit can be determined by one of skill in the art and can include primers to the appropriate regions of the DRD4 gene in order to amplify nucleic acids from a test sample using PCR. The kit may further include nucleic acid probes useful in determining the presence of the VNTR variant. The kit may also include electrophoretic markers such as a 50 bp ladder. Other components of the kit can include nucleotides, enzymes and buffers useful in a method of the invention. As an example, a kit of the invention may include primers for amplifying the region surrounding the VNTR variant, DNA polymerase, each of dATP, dTTP, dCTP and dGTP, 7-deaza-dGTP, 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl₂ and 5% DMSO. The kit will also include detailed instructions for carrying out the method for detecting the presence of the VNTR variant of the DRD4 gene.
 

Claim 1 of 10 Claims

1. A method of determining the susceptibility of a patient to a bipolar disorder comprising:

(a) obtaining a sample from a patient; and

(b) testing the sample for the presence of a polymorphism selected from (i) a 2 repeat allele in the variable number of tandem repeat (VNTR) region of the DRD4 gene and (ii) a 4 repeat allele in the VNTR region of the DRD4 gene, wherein the VNTR region is a 48bp unit found in the third exon of the DRD4 gene and wherein the presence of the 2 repeat allele indicates that the patient is less susceptible to a bipolar disorder and the presence of the 4 repeat allele indicates that the patient is more susceptible to a bipolar disorder.

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