WO1997034928A1

WO1997034928A1 - Method for assessing risk to develop psychotic disease

Info

Publication number: WO1997034928A1
Application number: PCT/GB1997/000681
Authority: WO
Inventors: David Andrew Collier; Maria Jesus Arranz
Original assignee: Smithkline Beecham Plc
Priority date: 1996-03-15
Filing date: 1997-03-12
Publication date: 1997-09-25
Also published as: AU1933697A

Abstract

A method for use in assessing whether a subject is likely to be at risk of developing a psychotic disorder, such as schizophrenia, bipolar altective disorder or unipolar depression, the method comprising testing for and detecting the presence or absence of DNA encoding the 10- and/or 12-repeat alleles of the VNTR on the hSERT gene in a biological sample obtained from said subject. The claimed invention also concerns a cell-line and a transgenic animal expressing the 10- and/or 12-repeat.

Description

METHOD FOR ASSESSING RISK TO DEVELOP PSYCHOTIC DISEASE

The present invention relates to a method of assessing the risk of developing a psychotic condition such as schizophrenia, bipolar affective disorder or unipolar depression.

Schizophrenia is a devastating psychiatric disease for which there is currently no cure, although advances are now being made in understanding its causes and controlling its symptoms. In general the age of onset is in late adolescence and it is a lifelong illness with a very poor prognosis. Subjects suffering from schizophrenia may exhibit positive symptoms, for example delusions and hallucinations, and /or negative symptoms such as withdrawal, isolation and demotivation leading ultimately to social decline and suicide. Early identification of subjects at risk of developing schizophrenia would enable early and if appropriate, prophylactic treatment to be given.

Bipolar affective disorder (which may also be referred to as bipolar depression or manic depression) is a condition which is episodic in nature and involves violent mood swings ranging from depressed mood to elation or mania.

Unipolar depression comprises a depressed mood state.

The human brain serotonin transporter (hSERT, 5-HTT) has been implicated in depression (MJ Owens and CB Nerneroff, Clin. Chem., Feb 1994, 40(2) 288-95, although it has also been reported that alterations in the primary structure of hSERT are not generally involved in the pathogenesis of unipolar depression and manic depressive illness (KP Lesch et al., Bio Psychiatry, 15 Feb 1995, 37(4) 215-23). Molecular cloning, expression and chromosomal location of the gene encoding hSERT are described by S. Rama oorthy et al (Proc. Natl. Acad. Sci. USA, March 1993, Vol 90, 2542-2546). Characterisation of this gene, including a tandem repeat DNA polymorphism (VNTR) has been reported by KP Lesch et al., (J. Neural Transm., 1994, 95(2) 157-62). This polymorphism is described by Ogilvie et al., 1996, The Lancet, Vol 347, 731-733, as a 16 or.17 bp repetitive element with the consensus sequence GGCTGYGACCY(R)GRRTG in which Y is C or T, R is A or G and (R) may be present or absent, located in intron 2; either 10 or 11 copies of the bp repeat were found in the majority of the population. It is now believed however that the repeat is present in either 10 or 12 copies (ie that the 11 -repeat described in the literature is actually a 12-repeat allele) and that there is also a rare 9-repeat allele which occurs in the population at low frequency.

It has now surprisingly been found that in a Caucasian population the 12-repeat allele occurs with a higher frequency in patients diagnosed as suffering from bipolar depression than in the general Caucasian population. Thus it is believed that possession of the 12-repeat allele is indicative of a higher than average probability of developing bipolar depression. It has also surprisingly been found that the 12-repeat allele occurs with a higher frequency in patients diagnosed as suffering from schizophrenia than in the general population. Thus it is believed that possession of the 12-repeat allele is indicative of a higher than average probability of developing schizophrenia, especially in a Caucasian population.

It is also believed that the 10-repeat allele occurs with a lower frequency in patients suffering from a psychotic disorder such as bipolar depression. The absence of the 10-repeat allele is therefore indicative of a higher than average probability of developing a psychotic disorder such as bipolar depression.

The present invention therefore provides a method for use in assessing whether a subject is likely to be at risk of developing a psychotic disorder , the method comprising testing for and detecting the presence or absence of DNA encoding the 10- and/or 12- repeat alleles of the intron 2 VNTR of the hSERT gene in said subject. A positive result for the 12-repeat allele is indicative of a higher risk of developing a psychotic disorder. T e psychotic disorder may be for example schizophrenia, bipolar depression, unipolar disorder or schizoaffective disorder. Preferably the method is used in assessing the likelihood of developing schizophrenia or bipolar depression, most preferably bipolar depression.

In a further embodiment the present invention provides a method for assessing whether a subject is likely to be at risk of developing a psychotic disorder said method comprising the steps of:

(i) testing for the presence or absence of DNA encoding the 10- and/or 12-repeat alleles of the intron 2 VNTR of the hSERT gene in a sample containing DNA obtained from said subject, and

(ii) comparing the result with a pre-determined correlation between the distribution of the 10- and/or 12-repeat alleles of the intron 2 VNTR of the hSERT gene obtained for a population of subjects suffering from said condition and that obtained for a population not suffering from said condition.

The pre-determined correlation utilised in step (ii) may itself be obtained by the following series of steps: selecting a population or cohort of subjects diagnosed as suffering from a specified psychotic condition; taking from said cohort biological samples containing DNA and testing this for the presence or absence of the 10- and/or 12-repeat alleles of the intron 2 VNTR of the hSERT gene; making a comparison with the distribution of alleles in a control group of subjects, not suffering from said condition; performing a statistical analysis to determine if there is a statistically significant association between presence or absence of the 10- and/or 12-repeat alleles of the intron 2 VNTR of the hSERT gene and likelihood of developing said psychotic condition.

The present invention thus also provides a method for assessing whether a subject is likely to be at risk of developing a psychotic disorder, said method comprising the steps of: (i) correlating the distribution of the 10- and/or 12-repeat alleles of the intron 2

VNTR of the hSERT gene obtained for a population of subjects suffering from said condition and that obtained for a population not suffering from said condition;

(ii) testing for the presence or absence of DNA encoding the 10- and/or 12-repeat alleles of the intron 2 VNTR of the hSERT gene in a sample containing DNA obtained from said subject, and

(iii) comparing the result obtained in (ii) with the correlation between the distribution of the 10- and/or 12-repeat alleles of the intron 2 VNTR of the hSERT gene and the likelihood of developing said psychotic disorder obtained in (I).

Step (i) will itself comprise taking from the subjects biological samples containing DNA and testing this for the presence or absence of the alleles. It will be appreciated that where the correlation in (i) is known , eg as in the case of schizophrenia, bipolar depression and unipolar depression, the process comprises only steps (ii) and (iii).

In a further embodiment the invention also provides a method for generating a model to assess whether a subject is likely to develop a psychotic disorder which method comprises: (i) obtaining DNA samples from a cohort of patients diagnosed as suffering from a psychotic disorder

(ii) obtaining DNA samples from a control group of subjects diagnosed as not suffering from said psychotic disorder;

(iii) analysing the samples obtained in (i) and (ii) to identify whether they encode the 10- and/or 12-repeat alleles of the intron 2 VNTR of the hSERT gene

(iv) calculating the frequencies of these alleles in the samples from (i) and (ii) (v) comparing the frequencies of these alleles in the samples from (i) and (ii) (vi) performing statistical analysis on the results from (v) to generate a model for asssessing the risk of developing said psychotic disorder.

The invention also provides a method for assessing whether a given subject will be at risk of developing a psychotic disorder, which comprises comparing said subject's genotype with the model described above.

It will be appreciated that in any of the above methods, one or more of the steps may be effected by a computer-controlled system. Thus, for example, once the biological samples have been obtained, genotyping may be carried out by a computer controlled robotic system. A computer-controlled system may also be configured to make a comparison between the genotype obtained for a given individual and the 12- repeat allele, or with a predetermined correlation or model and further configured to give either a positive or negative readout depending on the outcome of the comparison. The present invention therefore extends to such computer-controlled ore computer- implemented methods. It will be appreciated by those skilled in the art that a diagnosis of schizophrenia can be made according to recognised criteria eg DSM-IIIR criteria.

Similarly, a diagnosis of bipolar depression or unipolar depression can be made according to recognised criteria eg DSM-IV criteria .

The step of testing for and detecting the presence or absence of DNA encoding the 10- and or 12-repeat alleles may be carried out either directly or indirectly by any suitable means, such as by techniques well known in the art, and is preferably carried out ex vivo All generally involve the step of collecting a sample of biological material containing DNA from the subject, and then detecting which alleles the subject possesses from that sample. For example, the detecting step may be carried out by collecting a biological sample containing DNA from the subject, and then determining the presence or absence of DNA comprising a 10- and/or 12-repeat allele in the biological sample. Any biological sample which contains the DNA of that subject may be employed, including tissue samples and blood samples, with blood cells being a particularly convenient source. Determining the presence or absence of DNA comprising a 10- and/or 12- repeat allele may be carried out with an oligonucleotide probe labelled with a suitable detectable group; by means of an amplification reaction such as a polymerase chain reaction or ligase chain reaction, the product of which amplification reaction may then be detected with a labelled oligonucleotide probe or by direct staining of the product with any intercalating dye such as ethidium bromide after separation of the product on an agarose gel. Alternatively the allele may be detected by means of restriction nuclease digestion and electrophoretic separation to detect restriction fragment length polymorphism (RLFP). Further, the detecting step may include the step of detecting whether the subject is heterozygous or homozygous for the gene encoding a 10- and/or 12-repeat allele. Numerous different oligonucleotide probe assay formats are known which may be employed to carry out the present invention.

The present invention has utility in enabling improvements in the clinical management of patients suffering from a psychotic disorder, such as schizophrenia, bipolar depression or unipolar depression. By identifying in advance those who are likely to develop the disease it wil be more likely that clinical symptoms will also be identified at an earlier stage and hence treatment can be provided at an early juncture. Thus the invention provides direct benefits to the patient in terms of indicating the most appropriate therapy as early as possible in the treatment process and is of wider benefit in terms of health economics.

The present invention may also be of use to aid or confirm a diagnosis of a subject who appears to be suffering from a psychotic disorder.

In a further embodiment therefore the present invention provides a method of treating a psychotic disorder, said method comprising:

(i) testing for DNA encoding the 12-repeat allele of the intron 2 VNTR of the hSERT gene in a sample of biological material containing DNA obtained from a subject who appears to be suffering from a psychotic disorder, and (ii) if DNA encoding the 12-repeat allele is present in the sample, treating said subject with an antidepressant agent.

In a further embodiment the invention also provides an assay suitable for use in the method of the present invention, said assay comprising means for determining the presence or absence of DNA encoding the 10- and/or 12-repeat alleles of the hSERT gene in a biological sample. The present invention also provides a kit suitable for use in assessing the risk of a subject developing a psychotic disorder, said kit comprising:

(a) means for testing for the presence or absence of DNA encoding the 10- and/or 12-repeat alleles of the intron 2 VNTR of the hSERT gene in a sample of human DNA; (b) reagents for use in the detection process.

The means for testing preferably comprises a labelled probe or a restriction enzyme. The reagents may comprise for example diluents, wash solutions and control solutions.

In a further embodiment the present invention provides a cell line which expresses DNA encoding the 10- and/or 12-repeat alleles of the intron 2 VNTR of the hSERT gene. Such cell lines can be obtained using known recombinant techniques and methodology. In a yet further embodiment the present invention also provides a transgenic animal, in particular a transgenic mammal such as a mouse, which expresses the human hSERT gene containing the 10- and/or 12-repeat alleles. Such a transgenic animal may be used to screen for and identify novel antidepressant agents. Transgenic animals may be obtained using procedures which are standard in the field of genetic engineering.

Without wishing to be bound by theory it is believed that the hSERT VNTR 12- repeat may not itself confer susceptibility to a psychotic disorder, but rather that it may be a genetic marker for another polymoφhism with which it is in linkage disequilibrium, said other polymoφhism being responsible for conferring susceptibility to a psychotic disorder. Therefore, any of the methods described herein which involve testing for the presence of hSERT 10- or 12-alleles may also extend to testing for the presence of a polymorphism in linkage disequilibrium with any of said alleles.

EXAMPLES

Example 1 - Bipolar Depression

Method Genotyping for VNTR polymorphism was carried out using blood samples obtained from 191 Caucasian individuals diagnosed as suffering from bipolar depression (DSM IV). Genotyping was also carried out on a control group of 187 Caucasian subjects not diagnosed as suffering from bipolar depression.

Detection of VNTR polymorphism by PCR

The method used to detect the polymoφhism of the VNTR in the serotonin transporter hSERT (5-HTT) was based on the method of Lesch et al. (J Neural Transm. 1994 95(2) 157-62) with the following modification: the oligonucleotide primers 5' gtcagtatcaacaggctgcgag 3' and 5' tgttcctagtcttacgccagtg 3' were used. The size of the DNA PCR products after the use of these oligonucleotide primers was determined by a standard method ie electrophoresis in an agarose gel next to DNA size standards and then staining of the DNA with ethidium bromide and visualisation of the DNA under an ultraviolet li *geh* t.

Data

Allele Controls Bipolar patients 12 repeats 202 259 10 repeats 169 120 9 repeats 3 3 total alleles 324 382

Statistical analysis:

Chi-square of bipolar patients versus controls (allele - wise) = 15.27 p = 0.00048 Chi-square of bipolar patients versus controls (genotype - wise) = 14.27 p = 0.002

Example 2 - Schizophrenia

Method

Genotyping for VNTR polymorphism was carried out using blood samples obtained from individuals diagnosed as suffering from schizophrenia (DSM IIIR) and undergoing

- 7 - SUBST1TUTE SHEET (RULE 26) treatment with clozapine. Genotyping was also carried out on a control group of patients not suffering from schizophrenia.

Detection of VNTR polymorphism by PCR

The method used to detect the polymorphism of the VNTR in the serotonin transporter hSERT (5-HTT) was based on the method of Lesch et al. (J Neural Transm. 1994 95(2) 157-62) with the following modification: the oligonucleotide primers 5' gtcagtatcacaggctgcgag 3' and 5' tgttcctagtcttacgccagtg 3' were used. The size of the DNA PCR products after the use of these oligonucleotide primers was determined by a standard method ie electrophoresis in an agarose gel next to DNA size standards and then staining of the DNA with ethidium bromide and visualisation of the DNA under an ultraviolet light.

Data

Allele Controls Clozapine treated schizophrenics

12 repeats 202 162 10 repeats 169 94 9 repeats 3 2 total alleles 374 258

Statistical analysis:

Chi-square of clozapine treated schizophrenics versus controls = 4.26 p = 0.08

Example 3 (Bipolar and unipolar depression)

Method

Genotyping for VNTR polymorphism was carried out using blood samples obtained from individuals diagnosed as suffering from unipolar and/or bipolar depression (DSM IV). Genotyping was also carried out on a control group of patients not suffering from unipolar and/or bipolar depression.

Detection of VNTR polymorphism by PCR

The method used to detect the polymorphism of the VNTR in the serotonin transporter hSERT (5-HTT) was based on the method of Lesch et al. (J Neural Transm. 1994 95(2) 157-62) with the following modification: the oligonucleotide primers 5' gtcagtatcacaggctgcgag 3' and 5' tgttcctagtcttacgccagtg 3' were used. The size of the DNA PCR products after the use of these oligonucleotide primers was determined by a standard method ie electrophoresis in an agarose gel next to DNA size standards and then staining of the DNA with ethidium bromide and visualisation of the DNA under an ultraviolet lisht.

Data

Allele Controls Bipolar patients Unipolar patients 12 repeats 202 259 96 10 repeats 169 120 76 9 repeats 3 3 0 total alleles 374 382 172

Statistical analysis:

Chi-square of unipolar patients versus controls = 1.48 p = 0.47 Chi-square of bipolar patients versus controls = 17.07 p = 0.00019 Chi-square of bipolar patients versus unipolar patients = 9.48 p = 0.0087

Claims

Claims:

1. A method for use in assessing whether a subject is likely to be at risk of developing a psychotic disorder, the method comprising testing for and detecting the presence or absence of DNA encoding the 10- and/or 12-repeat alleles of the intron 2 VNTR of the hSERT gene in a biological sample obtained from said subject.

2. A method for assessing whether a subject is likely to be at risk of developing a psychotic disorder said method comprising the steps of: (i) testing for the presence or absence of DNA encoding the 10- and/or 12-repeat alleles of the intron 2 VNTR of the hSERT gene in a sample containing DNA obtained from said subject, and

3. A method for assessing whether a subject is likely to be at risk of developing a psychotic disorder, said method comprising the steps of: (i) correlating the distribution of the 10- and/or 12-repeat alleles of the intron 2

VNTR of the hSERT gene obtained for a population of subjects suffering from said condition and that obtained for a population not suffering from said condition; (ii) testing for the presence or absence of DNA encoding the 10- and/or 12-repeat alleles of the intron 2 VNTR of the hSERT gene in a sample containing DNA obtained from said subject, and

4. A method for generating a model to assess whether a subject is likely to develop a psychotic disorder which method comprises:

(i) obtaining DNA samples from a cohort of patients diagnosed as suffering from a psychotic disorder;

(ii) obtaining DNA samples from a control group of subjects diagnosed as not suffering from a psychotic disorder; (iii) analysing the samples obtained in (i) and (ii) to identify whether they encode the 10- and/or 12-repeat alleles of the intron 2 VNTR of the hSERT gene; (iv) calculating the frequencies of these alleles in the samples from (i) and (ii); (v) comparing the frequencies of these alleles in the samples from (i) and (ii); (vi) performing statistical analysis on the results from (v) to generate a model for asssessing the risk of developing a psychotic disorder.

5. A method for assessing whether a given subject will be at risk of developing a psychotic disorder, which comprises comparing said subject's genotype with a model as claimed in claim 4.

6. A method according to any of claims 1 to 5 wherein a positive result for the 12-repeat allele is indicative of a higher risk of developing a psychotic disorder.

7. A method of treating a psychotic disorder, said method comprising:

(i) testing for DNA encoding the 12-repeat allele of the intron 2 VNTR of the hSERT gene in a sample of biological material containing DNA obtained from a subject who appears to be suffering from a psychotic disorder, and (ii) if DNA encoding the 12-repeat allele is present in the sample, treating said subject with a neuroleptic agent.

8. A method according to any of claims 1 to 7 wherein the psychotic disorder is schizophrenia.

9. A method according to any of claims 1 to 7 wherein the psychotic disorder is bipolar affective disorder.

10. A method according to any of claims 1 to 9 wherein at least one step is computer-controlled.

11. An assay suitable for use in a method according to any of claims 1 to 10, said assay comprising means for determining the presence or absence of DNA encoding the 10- and/or 12-repeat alleles of the intron 2 VNTR of the hSERT gene in a biological sample.

12. A kit suitable for use in assessing the risk of a subject developing a psychotic disorder, said kit comprising: (a) means for testing for the presence or absence of DNA encoding the 10- and/or 12-repeat alleles of the intron 2 VNTR of the hSERT gene in a sample of human DNA;

(b) reagents for use in the detection process.

13. A cell line which expresses DNA encoding the 10- and/or 12-repeat alleles of the intron 2 VNTR of the hSERT gene.

14. A transgenic animal which expresses the human hSERT gene containing the 10- and/or 12-repeat alleles of the intron 2 VNTR.