CA2385194A1 - Method of diagnosis and treatment for affective disorders - Google Patents

Abstract

DNA sequences that are associated with affective disorders such as schizophrenia and method of screening and diagnoses thereof.

Description

4 This invention relates to the diagnosis and treatment for affective disorders, such as schizophrenia. This invention also relates to methods of screening for therapeutics for 6 affective disorders such as schizophrenia.

8 Common human neuropsychiatric and affective disorders are mufti-factorial or complex 9 in nature. To date, linkage studies have been relatively unsuccessful in identifying the genes for complex disorders such as bipolar disorder, schizophrenia, depression, anxiety-related traits and 11 autism with no loci having yet been unequivocally identified. Schizophrenia is one of the most 12 common serious psychiatric disorders characterised by profound disruption in cognition and 13 emotion, affecting the most fundamental human attributes: language, thought, perception, and 14 sense of self. The array of symptoms frequently includes hallucination and delusion. Studies of the prevalence of schizophrenia in the general population demonstrated that in general, 1-year 16 prevalence in adults between the ages of 18 to 54 is estimated to be 1.3 percent. Onset generally 17 occurs during young adulthood (mid-2()s for men, late-20s for women), although earlier and later 18 onsets do occur. It may be abrupt or gradual, but most people experience some early signs, such 19 as increasing social withdrawal, loss of interest, unusual behavior, or decreases in functioning prior to the beginning of active positive symptoms.
21 So far, the etiopathogenesis of schizophrenia has not yet been revealed.
The most 22 popular view points to the interaction of genetic factors and major environmental upheaval 23 during development of the brain. Family, twin, and adoption studies support the role of 24 I;enetic influences in schizophrenia (Kendler KS & Diehl SR, 1993 Schizophr Bull. 19:261-85; McGuffin P, Owen MJ & Farmex AE, 1995 Lancet 346(8976):678-82; Portin P &
26 .Alanen YO, 1997 Acta Psychiatr Scand. 95:73-80). Immediate biological relatives of 27 people with schizophrenia have about 10 times greater risk than that of the general 28 population. Given prevalence estimates, this translates into a 5 to 10 percent lifetime risk 29 for first-degree relatives (including children and siblings) and suggests a substantial genetic component to schizophrenia (Kety SS, 1987 J Psychiatry Res. 21:423-9;
Tsuang 1 MT, 1991 Br J Psychiatry. 158:165-70). What also bolsters a genetic role are findings that 2 the identical twin of a person with schizophrenia is at greater risk than a sibling or fraternal 3 twin, and that adoptive relatives do not share the increased risk of biological relatives.
4 However, in about 40 percent of identical twins in which one is diagnosed with schizophrenia, the other never meets the diagnostic criteria. The discordance among 6 identical twins clearly indicates that environmental factors likely also play a role.
7 However, despite the evidence for genetic vulnerability to schizophrenia, scientists have 8 not yet identified the genes responsible (Kendler KS & Diehl SR, 1993 Schizophr Bull.
9 19:261-85). The current consensus is that multiple genes are responsible (Kendler KS, MacLean CJ, O'Neill FA, Burke J, Murphy B, Duke F, Shinkwin R, Easter SM, Webb BT, I1 Zhang J, Walsh D & Straub RE, 1996 Am J Psychiatry. 153:1534-40; Kunugi H, Curtis D, 12 Vallada HP, Nanko S, Powell JF, Murray RM, McGuffin P, Owen MJ, Gill M &
Collier 13 I)A, 1996 Schizophr Res. 18;22:61-8; Portin P & Alanen YO, 1997 Acta Psychiatry Scand.
14 95:73-80; Straub RE, MacLean CJ, Martin RB, Ma Y, Myakishev MV, Harris-Kerr C, Webb BT, O'Neill FA, Walsh D & Kendler KS, 1998 Am J Med Genet. 10;81:296-301).
16 Excessive levels of the neurotransmitter dopamine have long been implicated in 17 schizophrenia, although it is unclear whether the excess is a primary cause of schizophrenia or a 18 result of a more fundamental dysfunction. More recent evidence implicates much greater 19 complexity in the dysregulation of dopamine and other neurotransmitter systems (Grace AA, 1991 Neuroscience 41:1-24; Grace, 1992 J Neural Transm Suppl. 36:91-131; Olie JP & Bayle 21 FJ, 1997 Encephale. 23 Spec No.2:2-9). Some of this research ties schizophrenia to certain 22 variations in dopamine receptors (Nakamura K, 1995 Seishin Shinkeigaku Zasshi. 97:529-50;
23 Serretti A, Macciardi F & Smeraldi E, 1998 Schizophr Res. 30;34:207-10), while other research 24 focuses on the serotonin system (Inayama Y, Yoneda H, Sakai T, Ishida T, Nonomura Y, Kono Y, 'Cakahata R, Koh J, Sakai J, Takai A, Inada Y & Asaba H, 1996 Am J Med Genet.
16;67:103-5).
26 Serotonin is a key neurotransmitter in the central nervous system, and dysregulation of 27 serotonergic pathways has been implicated in the pathogenesis of many complex psychiatric 28 diseases. Polymorphisms of many of the genes involved in serotonin biosynthesis, catabolism, 29 and response have been reported, suggesting that genetic variability may underlie the development of diseases such as schizophrenia, obsessive compulsive disorder, and suicide.

1 The serotonin transporter protein controls the concentration of serotonin in the synaptic 2 cleft by directing the sodium-dependent presynaptic reuptake of the neurotransmitter into 3 neurons. A reduced level of serotonerg:ic activity has been hypothesized to be important in the 4 aetiology of depressive disorders (Coppen A, 1967 Br J Psychiatry.
113(504):1237-64; Meltzer HfY, Lowy MT & Koenig JI, 1987 Adv Biochem Psychopharmacol. 43:165-82). Anti-6 depressants, such as selective serotonin reuptake inhibitors (SSRIs), elevate the synaptic 7 concentrations of serotonin by preventing its reuptake. The serotonergic system may also be 8 involved in schizophrenia since several atypical antipsychotics have recently been developed 9 which have lower affinities for D2 receptors than for serotonin receptor subtypes (Stockmeier ~;A, DiCarlo JJ, Zhang Y, Thompson ~ Meltzer HY, 1993 J Pharmacol Exp Ther.
266:1374-11 84). Moreover, serotonin receptor 5-H'r2A genotype influences clinical response to clozapine in 12 treatment-resistant schizophrenics (Arranz M, Collier D, Sodhi M, Ball D, Roberts G, Price J, 13 Sham P & Kerwin R 1995 Lancet 346:281-282). Antipsychotics such as clozapine act by 14 preventing the receptor binding of 5-HT. Finally, drugs such as cocaine, which are known to induce schizophrenia-like psychoses, act at the serotonin transporter.
Therefore, allelic variation 16 i:n the transporter gene may predispose to schizophrenia.
17 The human serotonin transporter gene or SERT, located on the human chromosome 18 17q11.2-12 (Ramamoorthy S, Bauman AL, Moore KR, Han H, Yang-Feng T, Chang AS, 19 (Janapathy V & Blakely RD, 1993 Proc Natl Acad Sci U S A. 15;90:2542-6), consists of 15 exons (Bradley CC & Blakely RD, 1997 J. Neurochem. 69, 1.356-1367). The expression of this 21 gene has been characterised most extensively in human brain, platelet and placenta. Alternative 22 splicing was observed in the human placental JAR cells (Bradley CC &
Blakely RD, 1997 J.
23 Neurochem. 69 1356-1357). The extra untranslated exon 1B (Fig 1) can be spliced into the 24 mRNA of the gene under some unknown situation. The dense existence of various transcriptional factor recognition sequences within the region upstream of exon 1B (Fig 1) 26 indicates that this region may have the function of regulating gene transcription or expression 27 (Bradley CC & Blakely RD, 1997 J. Neurochem. 69, 1356-1357).
28 Intron 2 contains several polymorphisms including a variable number tandem repeat 29 ( VNTR) composed of a 17 by unit repeat with 9, 10 and 12 copies in the second intron, a functional deletion/insertion locus at the regulatory region (5-HTT linked polymorphic region or 1 5-HTTLPR) as well as a PstI restriction fragment length polymorphism in the 3'-UTR. Several 2 positive associations between the SERT gene and anxiety-related traits (Lesch KP, Bengel D, 3 Heils A, Sabol SZ, Greenberg BD, Petri S, Benjamin J, Muller CR, Hamer DH &
Murphy DL, 4 1996 Science. 274(5292):1527-31), major depression (Ogilvie AD, Battersby S, Bubb VJ, Fink C~, Harmar AJ, Goodwim GM & Smith CA, 1996 Lancet 347:731-733), and bipolar disorder 6 (Collier DA, Arrant MJ, Sham P, Battersby S, Vallada H, Gill P, Aitchison KJ, Sodhi M, Li T, 7 R,oberts GW, Smith B, Morton J, Murray RM, Smith D & Kirov G, 1996 Neuroreport 7:1675-8 1679) have been reported in Caucasian populations. However, other studies have failed to 9 replicate these findings {Esterling LE, Yoshikawa T, Turner G, Badner JA, Bengel D, Gershon )_?S, Berrettini WH & Detera-Wadleigh SD, 1998 Am J Med Genet 81:37-40;
Furlong RA, Ho L, 11 Walsh C, Rubinsztein JS, Jain S, Paykel ES, Easton DF & Rubinsztein DC, 1998 Am J Med I2 CJenet 81:58-63; Hoehe MR, Wendel B, Grunewald I, Chiaroni P, Levy N, Moms-Rosendahl D, 13 Macher JP, Sander T & Crocq MA 1998 Am J Med Genet 81:1-3). There is a possible trend in 14 schizophrenics showing enrichment for the 12-repeat allele in a population of UK (Collier DA, Arrant MJ, Sham P, Battersby S, Vallada H, Gill P, Aitchison KJ, Sodhi M, Li T, Roberts GW, 16 Smith B, Morton J, Murray RM, Smith D & Kirov G, 1996 Neuroreport 7:1675-1679); another 17 study using Transmitted Disequilibrium Test (TDT) analysis in 61 nuclear families from 18 Croatian population detected a transmission distortion for alleles of the intronic VNTR
19 polymorphism (chi2TDT max =14.33; P = 0.0002; corrected P value = 0.0003) resulting in more frequent than expected transmission of the 12 repeat allele {Hranilovic D, Schwab SG, Jernej B, 21 1{napp B, Albus M, Rietschel M, Kanyas K, Bornna,nn M, Lichtermann D, Maier W &
22 'JVildenauer DB, 2000 Mol Psychiatry. 5:91-5). Malhotra AK, Goldman D, Mazzanti C, Clifton 23 ,~, Breier A & Pickar D (1998 Mol Psychiatry 3:328-32) found that 5-HTTLPR
is associated 24 with psychosis in neuroleptic-free schizophrenia. Meanwhile, a significant decrease in the affinity of [3H]paroxetine binding to the hippocampal membrane from subjects who had 26 schizophrenia was also reported (Dean B, Hayes W, Opeskin K, Naylor L, Pavey G, Hill C, Keks 27 :~1 & Copolov DL, 1996 Behav Brain Res. 73:169-75). On the other hand, the serotonin 28 transporter may also be sensitive to the neuroleptic drugs. Available data show that neuroleptic 29 drugs can prominently change the transcription of SERT (Hernandez I &
Sokolov BP, 1997 Mol Psychiatry.2:57-64).

1 Liu et al. observed that the 12 repeat allele of intronic VNTR was significantly increased 2 in the schizophrenic cases from Shanghai (Liu W, Gu N, Feng G, Li S, Bai S, Zhang J, Shen T, 3 rue H, Breen G, St Clair D & He L, 1999 Pharmacognetics 9:491-5).
Fiskerstrand CE, Lovejoy 4 E,A & Quinn JP also reported (1999 FEES Lett 458:171-174) that the VNTR can act as a transcriptional regulator and potentially contribute to disease susceptibility.
6 Although many of the above-mentioned reports have shown some correlation between 7 BERT and schizophrenia, these genetic variations are not simple point mutations that can 8 conveniently be used for screening and diagnosis. It is therefore an object ofthe present 9 invention to provide further tools and methods for the diagnosis and treatment of affective disorders such as schizophrenia.

13 The present invention arose from the discovery of two single nucleotide polymorphisms I4 present in the promoter region of the human serotonin transporter gene.
These two nucleotide 1S sequences are associated with schizophrenia and may be a potential genetic factor linked to the 16 e;tiopathogenesis of schizophrenia.
17 Accordingly, one aspect of the present invention is to provide DNA
sequences that are 18 associated with affective disorders such as schizophrenia.
19 It is another aspect of the present invention to provide a method of screening and diagnosing affective disorders such as schizophrenia. The method includes obtaining human 21 DNA in suff dent quantity for sequence analysis, and identifying the DNA
sequence at position 22 1772 andlor position 1797 of the promoter region of the human serotonin transporter genes.
23 In yet another aspect of the present invention, recombinant DNA molecules are provided.
24 'These molecules contain a reporter gene and a section of the promoter region of the SERT gene that contains one or both of the above-identified SNPs and their flanking regions.
26 Yet a further aspect of the present invention is a method of drug screening using the 27 above-identified SNPs and their flanking regions by linking the SNP
polynucleotide to a genetic 28 vector and expressing such a recombinant genetic vector in a prokaryotic and eukaryotic system.
29 Another aspect of the present invention relates to an antisense oligonucleotide such as an RNA or an RNA analogue that is complementary to the flanking region of the SNP

1 polynucleotide. The antisense oligonucleotide may be used as a medicament for the treatment of 2 affective disorders such as schizophrenia. The method of treatment involves administering into a 3 human subject one or more antisense oligonucleotide in a therapeutically effective dosage to 4 modulate the expression of the SERT genes in said subject.
BRIEF DESCRIPTION OF THE FIGURES

6 Fig 1 is a nucleotide sequence of promoter 2 (underlined) and the Exon 1 B
of the human 7 BERT gene as reported by Bradley CC & Blakely RD, ( I 997 J. Neurochem.
69,1356-1367;

8 C~enbank Accession no. U79746). The two novel SNPs I 772T/A and I797T/C of the present 9 invention are identified by rectangular boxes with solid lines. Exon 1B is enclosed within the rectangle with dotted lines.
11 Fig 2 is the schematic diagram to show the relationship between the promoters and exons 12 (boxes) in the genomic nucleotide sequence (black line) of human serotonin transporter as 13 reported in the prior art. Totally, fifteen exons and two promoters are arrayed.
14 Fig 3 shows the data obtained from the sequencing gel of the flanking region around the two novel SNPs 1772T/A and I797T/C. Three genotypes A/A, T/A and T/T for 1772T/A and 16 '1/T, T/C and C/C for 1797T/C are shown.

19 In the following example, schizophrenia is used as the specific embodiment to illustrate the utility and application of the present invention in the screening and treatment of affective 21 disorders.
22 The present invention relates to two single nucleotide polyrnorphisms (SNP) associated 23 ~Nith the etiopathogenesis of schizophrenia. Individuals with the altered nucleotide sequences or 24 t:he corresponding regions of the nucleotide sequences will be more susceptible to schizophrenia.
26 Nucleotide sequences associated with schizophrenia 27 This example describes the protocol to find and test these two single nucleotide 28 polymorphisms. In this example, PCR-amplified products are synthesized to encompass the 29 second promoter region of human serotonin transporter gene. A pair of primers specific to this promoter region that can be used for amplification are: the forward primer 5'-1 TTGGCCTTCAAGTTCGTCAGTTG-3' (positions 1389-1411 according to the sequence 2 numbering in Fig 1) and reverse primer 5'-CACTAGGGTTTGGCGTTTGCTG-3' (positions 3 1940-1961 according to sequence numbering in Fig 1 ) are used to amplify a 573 base pair long 4 DNA fragment.
The amplification reaction contains the following components: 5% Q-solution (Qiagen, 6 CimbH, Max-Voliner-Stra~ie, Germany), 50 mM KCI, 10 mM Tris-HCl (pH 8.0), 0.001% gelatin 7 (w/v), 2.5 mM MgCl2, 200 ~M dNTPs, L Opmol of each primer, 10 ng DNA and 1 U
Hotstar Taq 8 polymerase (Qiagen, GmbH, Max-Volmer-Stra~ie, Germany) in total volume of 151. PCR
9 amplification is performed in the thermal-cycler GeneAmp PCR System 9700 (PE
Applied Biosystems, Foster City, Calif, CA, USA) using 96-well microplates under the thermal cycle 11 conditions: 15 minutes at 95°C for denaturation, and 14 touch-down cycles for optimal DNA
12 amplification that entail denaturation at 95°C for 40 seconds, annealing for 60 seconds, and 13 polymerization at 72°C for 2 minutes. In these 14 cycles, the annealing temperature starts at 64°C
14 amd decreases by 0.5°C between each successive cycle. Then the DNA
fragment is amplified for ~ 30 cycles each entailing incubation at 94°C for 40 seconds, 57°C for 40 seconds, followed by at 16 7 2°C for 2 minutes. Finally, the PCR reaction is held at 72°C for 10 minutes.
17 The PCR products, purified using a commercial kit (Resin Wizard, Promega Corporation, 18 Madison, Wilsconsin, USA), are directly sequenced using the BigDye Terminator Cycle 19 Sequencing Ready Reaction Kit (PE Applied Biosystems) under the following conditions: 96°C
for 10 seconds followed by 30 cycles of 96°C for 10 seconds, SO°C for 5 seconds, and 60°C for 4 21 minutes.
22 One microliter of the sequencing reaction is loaded on the DNA Sequencer 377 (PE
23 .Applied Biosystems) and electrophoresed in 5% denaturing polyacrylamide gel at 3000 volts for 24 :i hours. The peak profiles of sequencing are visualized by inspection using the Polyphred software (Nickerson DA, Tobe VO & Taylor SL, 1997 Nucleic Acids Res.
15;25(14):2745-51).
26 To confirm the sequencing results, each PCR product is sequenced from both ends 27 using the same primers as those of the DNA amplification.
28 Referring to Figs 1 and 3, the two SNPs located in this promoter region are shown in 29 positions 1772 and 1797. 131 heathy controls and 134 schizophrenics that satisfy DSM III-R
criteria (American Psychiatric Association, I 980) were analysed in genotyping. Since the A/A

1 h~omozygotes for 1772T/A and C/C homozygotes for 1797T/C were very rare in our data (<5%), 2 the A/A and T/A samples were combined as allele A earners, and C/C and T/C
combined as 3 allele C carriers. The chi-square test for the genotypes between cases and controls shows a very 4 significant increase of homozygotes T /T earners in 1772T/A and homozygotes T/T earners in 1797T/C among the schizophrenics. 'the comparison of allele frequencies also gives a highly 6 significant difference between cases and controls.
7 Table 1 represents the frequency distribution of genotypes and alleles for 1772T/A and 8 1797T/C polymorphisms. The control is abbreviated as CON and schizophrenia is abbreviated as 9 SCH. Thep values from the chi-square test, odds ratios (OR) and 95%
confidence interval (95%CI) between 134 schizophrenics and 131 healthy controls are shown. Both the cases and 11 controls have come from Chinese Han population. Note that when calculating the p values, OR
12 and 95%CI for the genotypes, only the combined allele A carriers or combined allele C carriers 13 are considered. That is, for 1772T/A SNP, we compare the T/T carriers and combined allele A
14 carriers (T/A+A/A); for 1797T/C SNP, we compare the T/T carriers and combined allele C
carriers (T/C+C/C). The statistic software Epi Info version 6 is used for the significance test 16 (Dean AG et al, 1995 Epi Info, Version 6: A Word-Processing, Database, and Statistics Program 17 for Public Health on IBM-compatible Microcomputers. Centers for Disease Control and 18 Prevention, Atlanta, Georgia, U.S.A.).

1 Table 1 SNP Genotype CON SCH AlleleCON SCH

Total 131 134 Total 262 268 .

Total 131 134 Total 262 268 P Value -_ 0.02 0.02 0.03 0.04 OR 2.08 1.79 1.89 1.5 95% CI 1.06-4.14 1.05-3.04 I.O1-3.56 1.00-2.42 3 Transcriptional element analysis using the software TRANSFAC (Wingender E, Chen X, 4 Hehl R, Karas H, Liebich I, Matys V, Meinhardt T, Pru~i M, Reuter I &
Schacherer F, 2000 S Nucleic Acids Res. 28,316-319) shows that the surrounding region of the 6 polymorphism is a potential recognition site of the myeloid zinc finger gene 1 protein, the 7 t~anscriptional factor MZF-1. MZF-1 is a transcription factor expressed in hematopoietic 8 progenitor cells committed to myeloid differentiation. It belongs to a family of zinc finger DNA-9 binding proteins, many of which have been demonstrated to have roles in regulating transcription during development. A consensus sequence for binding to zinc fingers 1-4 of the MZF-1 binding 1 I site is shown in column 5 of Table 2, derived from the bound oligonucleotide sequences 12 indicated in columns 1-4. As seen in column 6 of Table 2, the 1772-A SNP
allele and its flanking 13 sequence correspond closely to the MZF-1 binding site. Thus a change from the 1772-A allele to 1 the 1772-T allele could cause an important departure from the MZF-1 binding consensus 2 sequence, and sharply diminish effectiveness of the 1765-1772 sequence toward binding to 3 MZF-1. As shown in column 5 of Table 2, the bottom consensus base position is overwhelming 4 by A among the oligonucleotides bound to MZF-1 zinc forgers 1-4, rarely C or G; in no instance is this position a T. For ease of description, the 1765 to 1772 sequence of the promoter region of 6 the SERT gene may be referred to as the 1772 MZF-like sequence.

8 7.'able 2. Comparison of Consensus Sequence for Binding to MZF-1 and the 1772 SNP and 9 H'lanking Sequence in SERT Promoter Region.
Base Consensus 1772 SNP and Corresponding Frequencies*

Sequence for Flanking Position A C G T Binding to Sequence in Zinc (nucleotides SERT
Fingers 1-4 1765-1772 Promoter of Re ion 11 'kat different base positions among oligonucleotides observed to bind to zinc fingers 1-4 of MZF-12 1; these frequencies give rise to the consensus binding sequence shown in volumn 5 (Morns JF, 13 '.Hromas R & Rauscher FJ, Mol. Cell Biol. 1994, 14;1786-1795).

1 Table 3. Consensus Sequence for Binding to NKX-2.5 and 1797 SNP and Flanking 2 Sequence in SERT Promoter Region.
Ba se Consensus Core1797 SNP and Corresponding Frequencies*

Sequence for Flanking Position A C G T Binding to Sequence in the NKX- (nucleotides SERT
2.5 1790-1797 Promoter __ Re ion 4 ~'at different base positions among oligonucleotides observed to bind to NKX-2.5; these S frequencies give rise to consensus binding sequence shown in volumn 5. (Chen CY & Schowartz 6 ltJ, J. Biol. Chem, 1995, 270:15628-15633).
7 Table 3 shows the consensus gene sequence for binding the mouse NKX-2.5 protein 8 encoded by the marine nkx-2.5 homeobox gene, and its relationship with the flanking region of 9 the 1797 SNP. For the ease of discussion, this region (nucleotides 1790 to 1797 of the promoter region of the SERT gene) may be referred to as the 1797 NKX-like sequence. The reported 11 NKX-2.5 consensus sequence is directly adjacent to the 1797 SNP site.
Although the human 12 protein corresponding to the mouse NKX-2.5 DNA binding protein has not yet been identified, it 13 would not be unexpected that the flanking region of the 1797 SNP might also closely match the 14 binding sequence for the human counterpart of NKX-2.5.
16 Method of diagnosing and screening for affective disorders such as schizophrenia 17 From the information disclosed above, it becomes possible to screen for individuals with 18 risk or susceptibility to affective disorders such as schizophrenia by analyzing the promoter 1 rc;gion of the SERT gene to identify the allele or genotype (or both) of the subject at position 2 1772 or 1797 (or both). The method used may involve common techniques such as PCR, under 3 the same conditions and using the same primers as described in the previous section, followed by 4 TINA sequence analysis. Other primers upstream and downstream of the 1772 or 1797 positions may also be used and may be determined by one skilled in the art without undue 6 experimentation. If the base T is found in position 1772 or 1797, the patient may be at a higher 7 risk for developing schizophrenia than if A or C is found in position 1772 or 1797 respectively.

9 Method of screening for therapeutic drugs for the treatment of affective disorders such as schizophrenia 11 As discussed above, the flanking region of 1772 SNP is a putative binding site for MZF-12 1. The flanking region of 1797 SNP is a putative binding site for NKX-2.5.
Thus, if these 13 flanking regions are cloned into a genetic vector linked to or proximal to a reporter gene, the 14 expression of the recombinant vector may be used to screen for drugs that modulate the activity of the relevant flanking region. The reporter gene may be any gene that encodes a protein 16 product that can serve as a marker for the detection of gene expression.
Many such reporter 17 genes are known and available to those of ordinary skill in the art, such as ~3-galactosidase ([3-18 gal), chloramphenicol acetyltranferase (CAT), luciferase (LUC) and alkaline phosphatase (AP).
19 'Che reporter gene together with the promoter region of interest can then be inserted into an appropriate expression vector using techniques that are known in the art.
Examples of 21 <:xpression vectors include pGL-2-Basic Plasmid (Promega, Madison, Wisconsin). The 22 recombinant plasmid construct can then be transfected into a cell line that is used for the 23 screening of the therapeutic drugs. The cell line is preferably of human origin and more 24 preferably of neuronal origin. Transfection techniques known in the art may be used such as the use of Lipofectin agent (Life Technologies Inc.).
26 For screening of drugs, the test molecule can be administered into the cell culture 27 containing the transfected cell line and the expression of the reporter genes monitored. The level 28 of gene expression of the reported gene may be determined using a technique that is appropriate 29 for the particular reporter gene. For example, luciferase activity may be detected using luminometric methods with luciferin as the enzyme substrate. Other detection methods for 1 specific reporter genes are within the knowledge of one of ordinary skill in the art and may be 2 found in standard laboratory manuals such as Sambrook J, Fritsch EF &
Maniatis T, Molecular 3 C.'loning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, New 4 Fork, 1989.
Using the above-described screening methods, the effect of the 1772 and 1797 single 6 nucleotide polymorphism on gene expression can be analyzed. For example, a higher expression 7 of the 17727 allele as compared to the 1772A allele would show that increased SERT gene 8 expression may be one of the causes for increased risk for schizophrenia.
Accordingly, a 9 compound added to the culture medium containing the transfected cells having the 17727 allele that brings about reduced expression of the reporter genes would indicate that the compound 11 could modulate expression of the SERT gene. If such modulation results in gene expression 12 levels that are comparable to the normal expression levels found with the 1772A allele, the 13 compound would be identified as a useful candidate drug for the treatment of affective disorders 14 such as schizophrenia.
The reverse would be true for a case in which the 17727 allele is found to cause a 16 reduced expression of the reporter gene in transfected cells compared to the 1772A allele. In 17 such a case, a compound added to the test medium that results in a higher expression of the 18 reporter gene for the 17727 allele would be a candidate compound potentially useful for the 19 treatment of affective disorders such as schizophrenia.
21 Antisense oligonucleotides and methods of treating affective disorders 22 As mentioned above, the 17727 and 17977 SNP mutations may cause the up-regulation 23 or down-regulation of the SERT gene. A method of testing the effect of these mutations has 24 been described above. If these mutations cause an up-regulation of the SERT
gene, and are associated with affective disorders such as schizophrenia, it then becomes possible to treat these 26 disorders by suppressing SERT gene expression using antisense technology.
This method 27 :involves the use of antisense oligonucleotides which have sequences that are complementary to 28 'the regulatory sequences of the SERT gene. These regulatory sequences include the 1772 MZF-29 like sequence, the 1797 NKX-like sequence and the complementary sequences thereof.

1 Examples of preferred antisense oligonucleotides include those that have modified 2 backbones or non-natural internucleoside linkages, for example phosphorothioates and 3 phosphotriesters as known in the art. The antisense oligonucleotides according to the present 4 invention also include "prodrugs" that are therapeutic agents prepared in an inactive form and administered to the body. Upon entry into the body, they are converted into an active form 6 through the action of endogenous enzymes or other chemical conditions.
Examples of such 7 prodrug oligonucleotides include SATE [S-acyl-2-thioethyl-phosphate]
derivatives according to 8 the method disclosed in WO 9324510 to Gosselin G & Imbach JL, published 9 December 1993 9 and incorporated herein by reference.
For use as therapeutic or prophylactic medicament, the antisense oligonucleotides may be 11 formulated in a pharmaceutically suitable carrier and administered to the body subcutaneously, 12 orally or using other parenteral routes.

14 Summary Of The Sequences Sequence ID No.l is the nucleotide sequence containing the 1772T SNP and flanking 16 bases.
17 Sequence ID No.2 is the nucleotide sequence containing the 1772A SNP and flanking 18 gases.
19 Sequence ID No.3 is the nucleotide sequence containing the 1797T SNP and flanking bases.
21 Sequence ID No.4 is the nucleotide sequence containing the 1797C SNP and flanking 22 bases.
23 Sequence ID No.S is the nucleotide sequence containing the 1772T SNP and 7 bases 24 upstream therefrom.
Sequence ID No.6 is the nucleotide sequence containing the 1772A SNP and 7 bases 26 upstream therefrom.
27 Sequence ID No.7 is the nucleotide sequence containing the 1797T SNP and 7 bases 28 upstream therefrom 29 Sequence ID No.8 is the nucleotide sequence containing the 1797C SNP and 7 bases upstream therefrom.

2 Although the invention has been described with reference to certain specific embodiments, various modifications thereof will be apparent to those skilled in the art without 4 departing from the spirit and scope of the invention as outlined in the claims appended hereto.

Claims (8)

1. An isolated polynucleotide comprising a member selected from the group consisting of:
sequence ID No.2, sequence ID No.4, sequence ID No.6 and sequence ID No.8.

2. A method of screening for or diagnosing affective disorders comprising identifying the DNA sequence at position 1772 of the promoter region of the human serotonin transporter gene.

3. A method of screening for or diagnosing affective disorders comprising identifying the DNA sequence at position 1797 of the promoter region of the human serotoin transporter genes.

4. A method according to Claim 2 wherein said method further comprises identifying the DNA sequence at position 1797 of the said promoter region.

5. A recombinant DNA molecule comprising a reporter gene operably linked to a promoter, said promoters comprising a member selected from the group consisting of sequence ID No.1, sequence ID No.3, sequence ID No.5 sequence ID No.7, sequence ID No.2, sequence ID No.4, sequence ID No.6 and sequence ID No.8.

6. A method of drug screening comprising:
- tranfecting a plasmid into a prokaryotic or eukaryotic expression system, said plasmid comprising a reporter gene operably linked to a promoter comprising a member selected from the group consisting of sequence ID No. 1, sequence ID No.2, sequence ID No.3, sequence ID No.4, sequence ID No.5, sequence ID No.6, sequence ID No.7 and sequence ID No.8;

- expressing and culturing said plasmid in said expression system;

- adding a drug to be screened into said culture; and - analysizing the gene expression of said reported gene.

7, An antisense oligonucleotide comprising a sequence complementary to a polynucleotide comprising a member selected from the group consisting of sequence No, 1, sequence No.2, sequence No.3, sequence No.4, sequence No.5, sequence No.6, sequence ID No.7 and sequence ID No.8.

8. A method of treating affective disorders comprising administering to a human an antisense oligonucleotide at a therapeutically effective dosage, said antisense oligonucleotide having a sequence complementary to a polynucleotide comprising a member selected from the group consisting of sequence ID No.1, sequence ID No.2, sequence ID No.3, sequence ID No.4, sequence ID No.5, sequence ID No.6, sequence ID No.7 and sequence ID No.8.