Patent 6,844,432

The present invention relates to a method for diagnosing panic disorder, said method being based on the determination of the class of polymorphism of the short tandem repeat (STR) complex in the upstream region of human cholecystokinin gene. The polymorphic DNA comprises a DNA sequence, having a general formula (1): 5'(GGAA)_n1 X(GGAG)_n2 (GGAA)_n3 (GGGA)_n4 GAG(AGAC)_n5 Y(GGAA)_n6 3' (1) wherein X denotes a DNA sequence of SEQ ID NO: 1, Y denotes a DNA sequence of SEQ ID NO:2 and each of n1, n2, n3, n4, n5 and n6 denotes independently 0 or a positive integral number, whereby said DNA ranging from 363 to 399 base pairs in length. The invention also relates to an assay kit used for implementing said method.

Description of the Invention

RELATED APPLICATIONS

This application claims priority to Japanese Patent Application No. 2000-375090, filed on Dec. 8, 2000.

FIELD OF THE INVENTION

The present invention relates to novel polymorphisms, and more specifically, to polymorphisms of the short tandem repeat type in the 5'-upstream region of human cholecystokinin gene, and their use, i.e. a method and a kit for diagnosis of susceptibility to panic disorder.

BACKGROUND OF THE INVENTION

Panic disorder is a common and genetically complex mental illness, characterized by recurrent and unexpected panic attacks. It exhibits a lifetime prevalence rate of between 1.2/100 to 2.4/100 in the general population (Weissman, M. M. et al., Arch. Gen. Psychiatry, 1997; 54, 305-309). Family studies have consistently shown a higher prevalence ranging from between 7.7% to 20.5% in the first-degree relatives of probands. Twin studies have shown concordance rates of 25% for MZ twins and 10% for DZ twins (Skre, I. et al., Acta Psychiatr. Scand. 1993; 88, 85-92). These epidemiological studies suggest involvement of genetic factors in the development of this disease.

Cholecystokinin (CCK) is a neuropeptide as well as a gastrointestinal peptide hormone, and is reported to have the relation with pathogenesis of panic disorder and schizophrenia. The carboxy terminal tetrapeptide of CCK (CCK-4), expressed in the brain, is thought to act as a neurotransmitter and/or neuromodulator. It provokes panic attacks in subjects with panic disorder and normal controls (Bradwejn et al., 1990; Can. J. Psychiatry 35, 83-85; and de Montigny et al., 1989; Arch Gen Psychiatry 46, 511-517). Patients with panic disorder have a higher sensitivity to CCK-4 than controls (Bradwejn et al., 1991; Arch. Gen. Psychiatry 48, 603-610). Furthermore, this panicogenic effect of CCK-4 is inhibited by antagonists of CCK B receptor (Bradwejn et al., 1994; Arch. Gen. Psychiatry 51, 486-493) which constitutes a large proportion of the CCK receptors in the human brain (de Weerth et al., 1993; Biochem. Biophys. Res. Commun. 194, 811-818). These findings have intensified research into elucidating the role of CCK in panic disorder.

CCK-like peptides, mainly the carboxy terminal octapeptide of CCK (CCK-8), co-localize with dopamine in mesolimbic dopaminergic neurons (Hokfelt et al., 1980; Nature 285, 476-478). In rats, CCK B receptor antagonists are shown to decrease the number of spontaneously active midbrain dopamine neurons (Rasmussen et al., 1991; Eur. J. Pharmacol. 209, 135-138). In humans, CCK-8 is reported to have an anti-psychotic effect (Moroji et al., 1982; Arch. Gen. Psychiat. 39, 485-486), inferring a possible role in the pathology of schizophrenia.

Two groups simultaneously reported a single nucleotide polymorphism (SNP), -36C>T in the 5'-upstream region of the CCK gene. Wang et al., (1998) Am. J. Med. Genet. (Neuropsychiatr. Genet.) 81, 228-234, showed that the -36T allele was weakly associated with subjects manifesting panic attacks (P<0.05) but not with panic disorder. In a case-control study of schizophrenia, Bowen et al., (1998) Mol. Psychiatry 3, 67-71, found no association between this SNP and the disease.

In general, the polymorphism, such as SNPs, which exists frequently in the human genome, is thought to have a relationship with a specific disease or constitution. Therefore, it is desirable to find the polymorphism which is used for the diagnosis and treatment of these diseases and to provide high quality order-made medical care. However, in the prior art, no functional polymorphisms associated with psychiatric disease have been reported in the CCK gene.

SUMMARY OF THE DISCLOSURE

It is, therefore, the present invention is directed to providing a method for diagnosing panic disorder and related useful methods therefor, by analyzing the structure of the human CCK gene, which is suggested to have a relationship with a psychiatric disease.

The present invention is further directed to providing a kit or a reagent for implementing said methods.

Recognizing the importance of the above objects, the present inventors have investigated the CCK gene. These efforts led to the discovery of a novel short tandem repeat (STR) complex, located in the 5'-upstream region of the CCK gene, and this STR was found to be polymorphic with ten different allele lengths. Analysis of the DNAs of many subjects showed that the distribution of the class of polymorphism is significantly different between patients with panic disorder and controls, and that the identification of the class of the polymorphism is useful for the diagnosis of panic disorder. These findings have led to the following inventions.

According to a first aspect of the present invention, there is provided an isolated DNA which is derived from an upstream region of a human cholecystokinin gene, said DNA comprising a polymorphic DNA sequence having a general formula (1):

5'(GGAA)_n1 X(GGAG)_n2 (GGAA)_n3 (GGGA)_n4 GAG(AGAC)_n5 Y(GGAA)_n6 3' (1)

According to a second aspect of the present invention, there is provided a hybridization probe consisting essentially of said DNA as defined above.

According to a third aspect of the present invention, there is provided a method for determining a class of polymorphism of a short tandem repeat stretch in the 5'-upstream region of the human cholecystokinin gene, said method comprising:

a) obtaining a DNA sample from a subject;

b) amplifying the 5'-upstream region of the human cholecystokinin gene in the sample; and

c) identifying the class of polymorphism by comparing the amplified DNA with the isolated DNA of the first aspect of the invention.

In preferred embodiments of this aspect of the invention, the class of polymorphism is detected by at least a method selected from the group consisting of sequence determination, gel electrophoresis, southern blotting, restriction fragment length polymorphism (RFLP) method, single strand conformational polymorphism (SSCP) method and mass spectrometry.

According to a fourth aspect of the invention, there is provided an assay kit for determining a class of polymorphism of a short tandem repeat stretch in the 5'-upstream region of the human cholecystokinin gene, said kit comprising:

a) a sense primer consisting of continuous 10 to 30 nucleotide sequence of SEQ ID NO:3; and

b) an anti-sense primer consisting of continuous 10 to 30 nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:4.

According to a fifth aspect of the present invention, there is provided a method for diagnosing panic disorder, comprising:

a) obtaining from a subject a DNA sample comprising the short tandem repeat stretch in the 5'-upstream region of the human cholecystokinin gene; and

b) identifying a class of polymorphism present in the subject, wherein a specific class is an indication that said subject may suffer from panic disorder.

According to a sixth aspect of the present invention, there is provided a method for demonstrating a predisposition to panic disorder, comprising:

a) obtaining from a subject a DNA sample comprising the short tandem repeat stretch in the 5'-upstream region of the human cholecystokinin gene; and

b) identifying a class of polymorphism present in the subject, wherein a specific class is an indication of a predisposition to panic disorder in said subject.

According to a seventh aspect of the present invention, there is provided a method for predicting the efficacy of an anxiolytic drug for the treatment of panic disorders, comprising:

a) obtaining from a subject a DNA sample comprising the short tandem repeat stretch in the 5'-upstream region of the human cholecystokinin gene; and

b) identifying the class of polymorphism present in the subject, wherein a specific class is an indication that said anxiolytic drug may be effective for the treatment of panic disorders.

PREFERRED EMBODIMENT OF THE INVENTION

The genetic polymorphism of the present invention is found to be in the short tandem repeat (STR) stretch, located in the 5'-upstream region of the human CCK gene. The DNA sequence of this STR complex has been partially known as a draft sequence of the human genome project (GenBank Accession No. AC018358), however, it has not yet known if the STR is polymorphic, and the detailed structure of the class of polymorphism is initially disclosed by the present application. The DNA sample including this STR complex can be cloned easily from the human chromosome DNA by various methods known in the art. For example, it can be cloned from the gene library constructed by various vectors such as .lambda. phage, cosmid or plasmid, and preferably cloned by amplifying the 5'-upstream region of the human CCK gene with Polymerase Chain Reaction (PCR) as described in Examples of the present invention.

DNA amplification can be performed by any method known in the art, such as PCR technology. This technology relies on thermal strand separation followed by thermal dissociation. During this process, at least one primer per strand, cycling equipment, high reaction temperatures and specific thermostable enzymes are used (Saiki, et al., Science, 1985; 230, 1350-1354; Mullis and Faloona, Methods in Enzymology, 1987; 155, 335-351; U.S. Pat. Nos. 4,683,195 and 4,883,202). Alternatively, it is possible to amplify the DNA at a constant temperature (Nucleic Acids Sequence Based Amplification (NASBA) Kievits, T., et al., J. Virol Methods, 1991; 35, 273-286; and Malek, L. T., U.S. Pat. No. 5,130,238; and Strand Displacement Amplification (SDA), Walker, G. T. and Schram, J. L., European Patent Application Publication No. 0 500 224 A2; Walker, G. T., et al., Nuc. Acids Res., 1992; 20, 1691-1696; and the like). Any sequencing method known to a person skilled in the art may be employed. In particular, it is advantageous to use an automated DNA sequencer. The sequencing is preferably carried out with a double-stranded template by means of the chain-termination method using fluorescent primers. An appropriate kit for this purpose is provided from PE Applied Biosystems (PE Applied Biosystems, Norwalk, Conn., USA).

The STR complex of the present invention which is located in the upstream region of the human CCK gene, can be represented by the following general formula (1):

5'(GGAA)_n1 X(GGAG)_n2 (GGAA)_n3 (GGGA)_n4 GAG(AGAC)_n5 Y(GGAA)_n6 3' (1)

wherein X denotes a DNA sequence of SEQ ID NO: 1, Y denotes a DNA sequence of SEQ ID NO:2 and each of n1, n2, n3, n4, n5 and n6 denotes independently 0 or a positive integral number, whereby said DNA ranging from 363 to 399 base pairs in length. The typical structure of the polymorphic DNA of the present invention which has these 6 repeating units, is shown in FIG. 1, and the number of repeats is variable by changing the numbers of n1 to n6. Therefore, the total length of the repeated sequence which constitutes the polymorphic DNA of the present invention, is in the range of 363 to 399 bp. Even if the repeated sequences are the same length, their constitution may be different. The polymorphic DNA of the present invention comprises at least the above DNA sequence. In addition, this DNA may contain several nucleotide sequences at both of its ends, such as the primer sequences, which are used for amplifying the polymorphic DNA of the present invention and the like. The flanking DNA sequence is shown in SEQ ID NO:3 with respect to the 5'-upstream region, and SEQ ID NO:4 with respect to the 3'-downstream region of the polymorphic DNA.

The use of the polymorphism of the STR complex of the present invention is not limited to a specific embodiment, but is applicable for various embodiments such as diagnosis and treatment of panic disorder by using, for example, as a probe, i.e., susceptibility or predisposition to panic disorder, and selection of an anxiolytic drug for the treatment of panic disorders based on the above diagnosis. In view of the physiological functions of CCK, it is also possible to develop a novel drug for other diseases by studying the mechanism of gene expression of human CCK gene with said polymorphic DNA. These drugs may be useful for diagnosis and treatment of Parkinson's disease, obesity, bulimia and gastrointestinal diseases such as gallbladder and biliary tract disorder.

The method for determining a class of polymorphism of the STR complex of the invention is carried out on a DNA sample from the subject. The DNA sample to be tested can be obtained from cells which have been withdrawn from the patient. These cells are preferably blood cells (for example mononucleated cells), which are easily obtained by the simple withdrawal of blood. Other cell types, such as fibroblasts, epithelial cells, keratinocytes, etc., can also be employed.

With regard to the method for determining a class of polymorphism of the STR complex, it can be used to compare the DNA sample from the subject, with the polymorphic DNA comprising the DNA sequence represented by said general formula (1) ranging from 363 to 399 base pairs in length. The useable techniques for comparing the extracted DNAs are direct sequencing, gel electrophoresis subsequent to southern hybridization, DNA amplification by using PCR method subsequent to gel electrophoresis, southern hybridization, the SSCP and RFLP techniques, and the like. A combination of more than two of the above methods are also appropriate. Gel separation techniques have the advantage of making it possible to discriminate between different alleles in terms of their size without it being necessary to sequence the DNA fragments. This technique is based on the migration, under denaturing conditions, of the denatured DNA fragments in a preferably polyacrylamide gel. As a similar technique, capillary electrophoresis and mass spectrometry can determine the DNA size. The bands can be visualized by any technique known to the skilled person, with the technique being based, for example, on the following: using labeled primers (fluorescence or radioactivity); introducing .alpha.-³² PdCTP into the tested fragments; visualizing with ethidium bromide; or by means of hybridization (blotting) with a radiolabelled probe.

The single strand conformation polymorphism (SSCP) detection technique is also a method involving separation on an acrylamide gel, but under non-denaturing conditions. It is performed preferably with capillary electrophoresis equipment. This technique makes it possible to discriminate between different DNA fragments in terms of their conformation.

Alternatively, the DNA chip method can be employed (Barinaga M., Science, 1991; 253, 1489; Bains, W., Bio/Technology, 1992; 10, 757-758; Wang et al., Science, 1998; 280, 1077-1082). These methods usually attach specific DNA sequences to very small specific areas of a solid support, such as micro-wells of a DNA chip. Each class of polymorphic DNA of the present invention can be used for the DNA chip when they are hybridized with the amplified DNA fragment of the subject, and then detected by the pattern of hybridization. Consequently, the comparison for determining the class of polymorphisms of the STR complex of the present invention means to identify a class of polymorphisms based on their nucleotide sequence, length of DNA fragments, and DNA three dimensional structure, or their sequence homology between the sample DNA and the probe DNA.

In one embodiment of the present invention, the STR complex region of the human CCK gene is amplified, and said amplified DNA is compared, as described above, to determine the class of polymorphism of the STR complex. With regard to the method of DNA amplification, the PCR method is commonly used, but it is also possible to amplify the DNA at a constant temperature. The primers which are used for the aforementioned DNA amplification can be synthesized by any technique known to a person skilled in the art, based on the unique sequence flanking to the STR complex region. The nucleotide sequence shown in SEQ ID NO:3 is the sequence of the 5'-upstream region of the STR complex. A single-stranded primer can be synthesized by selecting any continuous 10 to 30 base sequence from the sequence of SEQ ID NO:3. The nucleotide sequence shown in SEQ ID NO:4 is the sequence of the 3'-downstream region of the STR complex. Another single-stranded reverse primer can be synthesized by selecting any continuous 10 to 30 base sequence from the complement sequence of SEQ ID NO:4. The length of these oligonucleotide primers are commonly in the range of 10 to 30 nucleotides in length, preferably in the range of 18 to 25 nucleotides in length, and more preferably each primer is 21 nucleotides in length, shown as SEQ ID NO:5 and SEQ ID NO:6. Each of these lengths can be used in the present invention.

The reaction mixture for amplifying the DNA comprises 4 deoxynucleotide phosphates (dATP, dGTP, dCTP, dTTP) and heat stable DNA polymerase (such as Taq polymerase), which are all known to the skilled person in the art.

In a preferred embodiment of the present invention, the length of the amplified DNA is dependent on the class of the polymorphism sought to be identified. As is explained in detail hereafter, e.g., in example 3, the length of the upstream region of the STR complex is measured by gel electrophoresis. Any suitable Gel for electrophoresis, such as polyacrylamide, can be selected. In the case of polyacrylamide, the gel can be used in the range of 3% to 8% by concentration, preferably 4% to 5% by concentration of acrylamide. Capillary electrophoresis apparatus equipped with high performance polymer can also be used. It is a commonly used method for a person skilled in the art to determined the molecular weight of DNA fragments precisely by using suitable molecular weight markers.

The method of the present invention can be carried out by determining one of the alleles or genotype consisting of both alleles of the individual at the STR complex. The alleles are divided into three classes according to their length: Short (S) (363-375 base pairs), Medium (M) (379-383 base pairs) and Long (L) (387-399 base pairs). The polymorphic length of the alleles are related to a possible susceptibility to panic disorder. As shown in Table 2, described in detail in Example 4, the distribution of the M/M genotype are 17% in controls, but 34% in panic disorder which is twice that of the control. Thus, the individual of M/M genotype may have higher risk of panic disorder than those of other genotypes.

The accuracy of the diagnostic method of the present invention can be improved by combining it with any other method of diagnosis, including diagnoses of genetic element and serum protein. For example, other SNP markers are combined. As is described in detail in the Examples, the distribution of the haplotype, having both the M class of the STR complex and the two SNPs of the upstream of CCK gene (-188G and -36T), of panic disorder patients is significantly higher than that of the control. These results suggest that the combination of the diagnostic method of the present invention with that of other suitable polymorphisms improves the accuracy of the diagnosis of panic disorder.

According to a further aspect of the present invention, there is provided a method of order-made therapy, comprising the analysis of a predisposition for patients of panic disorder. Although the panic disorder is a complex illness associated with various causes, drugs such as antidepressants or benzodiazepines are mainly used for the treatment of the disease. However, there are many patients for whom these drugs have no therapeutic effect. The method of the present invention provides a novel and promising treatment to control the function of CCK in these patients.

An assay kit for determining a class of polymorphism of the STR complex of the present invention comprises oligonucleotide primers consisting of the unique sequence in the flanking regions of the STR complex. The nucleotide sequence shown in SEQ ID NO:3 is the sequence of the 5'-upstream region of the STR complex. A single-stranded primer can be synthesized by selecting any continuous 10 to 30 base sequence from the sequence of SEQ ID NO:3. The nucleotide sequence shown in SEQ ID NO:4 is the sequence of the 3'-downstream region of the STR complex. Another single-stranded reverse primer can be synthesized by selecting any continuous 10 to 30 base sequence from the complement sequence of SEQ ID NO:4. The length of these oligonucleotide primers are commonly in the range of 10 to 30 nucleotide length, preferably in the range of 18 to 25 nucleotides in length, and more preferably each 21 nucleotide length shown as SEQ ID NO:5 and SEQ ID NO:6 can be used. In addition to these primers, the assay kit comprises 4 deoxynucleotide phosphates (dATP, dGTP, dCTP, dTTP) and an effective amount of a nucleic acid producing catalyst. A number of biological enzymes are known in the art which are useful as polymerizing agents. These include, but are not limited to E. coli DNA polymerase I, Klenow fragment, bacteriophage T7 RNA polymerase, reverse transcriptase, and polymerases derived from thermophilic bacteria, such as Thermus aquaticus. The latter polymerases are known for their high temperature stability, and include, for example, the Taq DNA polymerase I. Other enzymes such as Ribonuclease H can be included in the assay kit for regenerating the template DNA.

Claim 1 of 6 Claims

What is claimed is:

1. An isolated DNA which is derived from the upstream region of the human cholecystokinin gene, said DNA consisting of a polymorphic DNA sequence, having a general formula (1):

5'(GGAA)_n1 X(GGAG)_n2 (GGAA)_n3 (GGGA)_n4 GAG(AGAC)_n5 Y(GGAA)_n6 3' (1)

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