Note: Descriptions are shown in the official language in which they were submitted.
CA 02385194 2002-05-07
1 METHOD OF DIAGNOSIS AND TREATMENT FOR
2 AFFECTIVE DISORDERS
3 FIELD OF INVENTION
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.
7 DESCRIPTION OF RELATED ART
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
CA 02385194 2002-05-07
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.
2
CA 02385194 2002-05-07
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
3
CA 02385194 2002-05-07
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).
4
CA 02385194 2002-05-07
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.
11
12 SUMMARY OF THE INVENTION
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
5
CA 02385194 2002-05-07
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.
17
18 DETAILED DESCRIPTION OF THE INVENTION
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'-
6
CA 02385194 2002-05-07
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
7
CA 02385194 2002-05-07
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.).
19
8
CA 02385194 2002-05-07
1 Table 1
SNP Genotype CON SCH AlleleCON SCH
T/T 99 116
T 229 249
T/A 31 17
1772T/A
A 33 19
A/A 1 1
Total 131 134 Total 262 268
T/T 71 91
.
T 197 221
T/C 55 39
1797T/C
C 65 47
C/C 5 4
Total 131 134 Total 262 268
1772T/A 1797T/C 1772T/A 1797T/C
P Value -_
0.02 0.02 0.03 0.04
OR 2.08 1.79 1.89 1.5
5
95% CI 1.06-4.14 1.05-3.04 I.O1-3.56 1.00-2.42
2
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
1772T/A
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
9
CA 02385194 2002-05-07
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.
7
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
MZF-1
8 3 6 3 N C 1765
4 2 11 3 G C 1766
3 3 5 9 N T 1767
0 0 20 0 G G 1768
1 0 18 1 G G 1769
0 0 19 1 G G 1770
0 0 20 0 G G 1771
18 1 1 0 A T-A 1772
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).
14
CA 02385194 2002-05-07
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
1 0 1 5 T A 1790
0 4 0 3 Y C 1791
7 0 0 0 A A 1792
7 0 0 0 A A 1793
0 0 7 0 G G 1974
0 0 0 7 T T 1795
1 0 6 0 G G 1796
0 1 1 3 T-C 1797
3
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
11
CA 02385194 2002-05-07
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.
8
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
12
CA 02385194 2002-05-07
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.
13
CA 02385194 2002-05-07
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.
13
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.
14
CA 02385194 2002-05-07
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.
