WO2009036513A1

WO2009036513A1 - Diagnostic and therapeutic protocols

Info

Publication number: WO2009036513A1
Application number: PCT/AU2008/001395
Authority: WO
Inventors: Jeffrey Mark Craig; Richard Saffery; Craig Anthony Olsson
Original assignee: Murdoch Childrens Research Institute
Priority date: 2007-09-21
Filing date: 2008-09-19
Publication date: 2009-03-26

Abstract

The present invention relates generally to a method for profiling or stratifying an individual or group of individuals with respect to a mental health condition including a psychiatric, neurological or psychological condition, phenotype or state, said method comprising determining the presence or absence or extent of DNA methylation of the 5HTT promoter region and/or the level of expression of the 5HTT gene alone or in combination with the presence or absence or number of S-allelic forms of the 5HTT gene. Methods for the treatment or prophylaxis of a mental health condition including a psychiatric, neurological or psychological condition, phenotype or state are also contemplated together with therapeutic agents and protocols useful for same.

Description

DIAGNOSTIC AND THERAPEUTIC PROTOCOLS FIELD

[0001] The present invention relates generally to a method for profiling or stratifying an individual or group of individuals with respect to a mental health condition including a psychiatric, neurological or psychological condition, phenotype or state. Methods for the treatment or prophylaxis of a mental health condition including a psychiatric, neurological or psychological condition, phenotype or state are also contemplated together with therapeutic agents and protocols useful for same.

BACKGROUND

[0002] Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.

[0003] Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

[0004] Depression is a common health problem and a particular burden in young people because it interferes with normal development and is often associated with poor psychosocial and academic outcomes and an increased risk for other disorders (Birmaher et al, Child Adolesc Psychiatr Clin N Am 11:619-637, 2002). Most adults with recurrent depression have their initial depressive episodes as adolescents (Pine et al, Arch Gen Psychiatr 55:56-64, 1998). Persistent or recurrent depression incurs tremendous social and financial costs in the form of impaired relationships, lost productivity and lost wages (Pincus and Pettit, J Clin Psychiatry 62(6):5-9, 2001). Targeted interventions to reduce the burden of disease associated with chronic depression depend on accurate characterization of risk factors: environmental and biological. [0005] Serotonin (5HT) is a monoamine neurotransmitter that regulates a range of behaviors including mood, sleep, appetite and libido (Heils et al, J Neurochem 66:2621- 2624, 1996; Lesch et al, J New Transm 104:1259-1266, 1997). A class of antidepressant medications known as selective serotonin reuptake inhibitors (SSRIs) achieve their therapeutic effect by inhibiting the clearance of serotonin from the synaptic cleft (Montastruc et al, Therapie 52:143-149, 1997). One way in which SSRIs inhibit clearance is by blocking the action of a trans-membrane protein known as the serotonin transporter (5HTT or SLC6A4) [Vaswani et al, Prog Neuro-Psychopharmacol Biol Psychiatr 27:85- 102, 2003; Czlonkowska et al, Med Sci Monit P.-RA270-275, 2003]. 5HTT removes 5HT from the cleft through a process of 5HTT re-uptake into the pre-synaptic neuron (Ueno, J Med Invest 50(l-2):25-3l, 2003; Ramanoorthy et al, J Biol Chem 268:21626-21631, 1993). The fact that 5HT is known to play an important role in mood regulation and that SSRIs achieve important therapeutic gain through selective blocking of 5HTT, has led to considerable research interest in the role of natural variation within 5HTT in the aetiology of depression.

[0006] 5HTT (SLC6A4, MIM 182138) is located at 17qll.l-12 (Ueno, 2003 supra). A 44bp insertion/deletion polymorphism (5HTTLPR) that sits within the 5' regulatory region of 5HTT has been shown to play an important regulatory role in gene transcription (Lesch et al, Science 274:1527-1531, 1996; Bradley et al, American Journal Medical Genetics 136:58-61, 2005). Compared with the long variant (L-allele), the short variant (S-allele) results in lower 5HTT transcriptional activity (Heils et al, 1996 supra; Ramamoorthy et al, 1993 supra). The relationship between S-allele genotypes and common mental health problems such as depression and anxiety is unclear. For example, it is known that S-allele genotypes reduce risk of anxiety under conditions of insecure attachment (Olsson et al, MoI Psychiatr 10:868-876, 2005), while others have shown that S-allele genotypes increase risk of depressive symptoms, particularly under conditions of life stress (Caspi et al, Science 301:386-389, 2003; Kendler et al, Arch Gen Psychiatr 62(5):529-535, 2005). In some cases, both risk and protective effects have been reported within the one sample (Middeldorp et al, Behav Genet 37(2):294-301, 2007). [0007] In accordance with the present invention, a relationship is identified between 5HTTLPR genotypes and 5HTT and 5HTT promoter region DNA methylation in relation to mental health conditions including psychiatric, neurological and psychological disorders and conditions.

SUMMARY

[0008] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0009] Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO). The SEQ ED NOs correspond numerically to the sequence identifiers <400>l (SEQ ID NO:1), <400>2 (SEQ ID NO:2), etc. A summary of the sequence identifiers is provided in Table 1. A sequence listing is provided after the claims. Abbreviations used herein are defined in Table 2.

[0010] In accordance with the present invention, an individual is considered at risk of developing or may have a mental health condition including a psychiatric, neurological or psychological condition, phenotype or state when the individual carries a 5HTT promoter or 5HTT promoter region which has higher than low levels of DNA methylation, alone or in combination with an S-allelic form of the 5HTT gene or when the level of activity of 5HTT or 5HTT gene expression is reduced. Hence, an individual is considered at risk of developing or may have a mental health condition including a psychiatric, neurological or psychological condition, phenotype or state when the individual carries a 5HTT promoter or 5HTT promoter region which is more than low level DNA methylated, and the level of activity of 5HTT or 5HTT gene expression is reduced. Reference to "DNA methylated" or "DNA methylation" includes extent of methylation from low levels to high levels. A "low level" is generally considered within the bottom of the population distribution of 5HTT DNA methylation. The "bottom" includes the lower two tertiles of the population distribution. A "low level" is defined as less than 6% + 5% DNA methylation. A "high level" is greater than 6% ± 5%. [0011] A "5HTT promoter region" includes up to approximately 10kb up or down stream of the transcription initiation site of the 5HTT gene and further includes the 5HTT promoter.

[0012] Reference to reduced gene expression or activity includes a reduction in expression or activity due to histone modification as well as non-coding RNA binding.

[0013] Accordingly, one aspect of the present invention contemplates a method for identifying an epigenetic or epigenetic and genetic profile in a subject associated with a mental health condition including a psychiatric, neurological or psychological condition, phenotype or state, the method comprising determining the extent of DNA methylation of the 5HTT promoter or 5HTT promoter region and/or the level of expression of the 5HTT gene alone or in combination with an S-allelic form of the 5HTT gene wherein the presence of a DNA methylated 5HTT promoter or 5HTT promoter region or reduced level or activity of 5HTT with or without the S-allele is indicative of a subject being at risk of developing such a condition, phenotype or state.

[0014] For brevity, this aspect of the present invention may be stated as being a method for identifying an epigenetic profile in a subject associated with a mental health condition, said method comprising determining the presence or absence or extent of DNA methylation of the 5HTT promoter region and/or the level of expression of the 5HTT gene alone or in combination with the presence or absence or number of S-allelic forms of the 5HTT gene wherein a DNA methylated 5HTT promoter region or reduced level of activity of 5HTT with or without an S-allele is indicative of a subject being at risk of developing such a mental health condition.

[0015] Furthermore, the present invention contemplates a method for identifying an epigenetic and genetic profile in a subject associated with a mental health condition, said method comprising determining the presence or absence or extent of DNA methylation of the 5HTT promoter region and/or the level of expression of the 5HTT gene alone or in combination with the presence or absence or number of S-allelic forms of the 5HTT gene wherein a DNA methylated 5HTT promoter region or reduced level of activity of 5HTT with or without an S-allele is indicative of a subject being at risk of developing such a mental health condition.

[0016] Another aspect of the present invention provides a method for profiling or stratifying an individual or group of individuals for a level of risk in developing a mental health condition including a psychiatric, neurological or psychological condition, phenotype or state, the method comprising determining the extent of DNA methylation of the 5HTT promoter or 5HTT promoter region and/or level of 5HTT gene expression wherein:

(i) an individual with lower levels of a methylated 5HTT promoter or 5HTT promoter region DNA within the bottom of population distribution of 5HTT methylation is considered at a lower risk of developing the mental health condition; wherein the bottom of population distribution of 5HTT DNA methylation is defined as equal to or less than 6% plus or minus 5% DNA methylation;

(ii) an individual with a methylated 5HTT promoter or 5HTT promoter region DNA or reduced 5HTT gene expression alone or in combination with an S-allelic form of the 5HTT gene is more likely of developing the mental health condition compared to an individual in group (i); and

(iii) an individual with a highly methylated 5HTT promoter or 5HTT promoter region DNA or highly reduced 5HTT gene expression alone or in combination with multiple S- allelic forms of the 5HTT gene is more at risk of developing the mental health condition compared to an individual in group (ii).

[0017] Again for brevity, this aspect of the present invention provides a method for profiling or stratifying an individual or group of individuals for a level of risk in developing a mental health condition, said method comprising determining the presence or absence of or extent of DNA methylation of the 5HTT promoter region and/or level of 5HTT gene expression wherein:

(i) an individual without a methylated 5HTT promoter region DNA or having a low level DNA methylation is considered at a lower risk of developing said mental health condition;

(ii) an individual with a methylated 5HTT promoter region DNA or reduced 5HTT gene expression alone or in combination with an S-allelic form of the 5HTT gene is more likely of developing said mental health condition compared to an individual in group (i); and

(iii) an individual with a highly methylated 5HTT promoter region DNA or highly reduced 5HTT gene expression alone or in combination with an S-allelic form of the 5HTT gene is more at risk of developing said mental health condition compared to an individual in group (ii).

[0018] Another aspect of the present invention contemplates a method for diagnosing a mental health condition including a psychiatric, neurological or psychological condition, phenotype or state in an individual, said method comprising obtaining or extracting nucleic acid material from cells of the individual and screening for or otherwise detecting the extent of DNA methylation of the 5HTT promoter or 5HTT promoter region or level of 5HTT gene expression wherein an individual with a DNA methylated 5HTT promoter or 5HTT promoter region or reduced 5HTT gene expression has a statistically significant association with a particular mental health condition including a psychiatric, neurological or psychological condition, phenotype or state or a sub-threshold form thereof or that the individual is at risk of developing same when the individual has an S-allelic or multiple S- allelic forms of the 5HTT gene.

[0019] A further aspect of the present invention contemplates the a method for identifying an epigenetic or epi genetic and genetic profile in a subject associated with persisting depressive symptomatology (PDS), the method comprising determining the extent of DNA methylation of CpG dinucleotides in the 5HTT promoter or 5HTT promoter region and/or level of 5HTT gene expression wherein the presence of a DNA methylated 5HTT promoter or 5HTT promoter region or reduced 5HTT gene expression alone or in combination with one or more S-allelic forms of the 5HTT gene is indicative of a subject being at risk of developing PDS.

[0020] Yet another aspect provides a method for profiling or stratifying an individual or group of individuals for a level of risk in developing PDS, the method comprising determining the and the extent of DNA methylation of the 5HTT promoter or 5HTT promoter region and/or level of 5HTT gene expression wherein:

(i) an individual with lower levels of a methylated 5HTT promoter or 5HTT promoter region DNA within the bottom of the population distribution of 5HTT methylation is considered at a lower risk of developing PDS; wherein the bottom of population distribution of 5HTT DNA methylation is defined as equal to or less than 6% plus or minus 5% DNA methylation;

(ii) an individual with a methylated 5HTT promoter or 5HTT promoter region DNA and/or reduced 5HTT gene expression alone or in combination with an S-allelic form of the 5HTT gene is more likely of developing PDS compared to an individual in group (i); and

(iii) an individual with a highly methylated 5HTT promoter or 5HTT promoter region DNA and/or highly reduced 5HTT gene expression alone or in combination with multiple S-allelic forms of the 5HTT gene is more at risk of developing PDS compared to an individual in group (ii).

[0021] A further aspect of the present invention contemplates the a method for identifying an epigenetic or epigenetic and genetic profile in a subject associated with depression including major depressive disorder, dysthymia and episodic depressive symptomatology, the method comprising determining the extent of DNA methylation of CpG dinucleotides in the 5HTT promoter or 5HTT promoter region and/or level of 5HTT gene expression wherein the presence of a DNA methylated 5HTT promoter or 5HTT promoter region or reduced 5HTT gene expression alone or in combination with one or more S-allelic forms of the 5HTT gene is indicative of a subject being at risk of developing depression including major depressive disorder, dysthymia and episodic depressive symptomatology.

[0022] Yet another aspect provides a method for profiling or stratifying an individual or group of individuals for a level of risk in developing depression including major depressive disorder, dysthymia and episodic depressive symptomatology, the method comprising determining the and the extent of DNA methylation of the 5HTT promoter or 5HTT promoter region and/or level of 5HTT gene expression wherein:

(i) an individual with lower levels of a methylated 5HTT promoter or 5HTT promoter region DNA within the bottom of the population distribution of 5HTT methylation is considered at a lower risk of developing depression including major depressive disorder, dysthymia and episodic depressive symptomatology; wherein the bottom of population distribution of 5HTT DNA methylation is defined as equal to or less than 6% plus or minus 5% DNA methylation;

(ii) an individual with a methylated 5HTT promoter or 5HTT promoter region DNA and/or reduced 5HTT gene expression alone or in combination with an S-allelic form of the 5HTT gene is more likely of developing depression including major depressive disorder, dysthymia and episodic depressive symptomatology compared to an individual in group (i); and

(iii) an individual with a highly methylated 5HTT promoter or 5HTT promoter region DNA and/or highly reduced 5HTT gene expression alone or in combination with multiple S-allelic forms of the 5HTT gene is more at risk of developing depression including major depressive disorder, dysthymia and episodic depressive symptomatology compared to an individual in group (ii). [0023] Even yet another aspect of the present invention contemplates a risk profile for a subject developing or being diagnosed with a mental health condition including a psychiatric, neurological or psychiatric condition, phenotype or state wherein the profile comprises a plurality of data comprising behavioral, environmental and social parameters in combination with one or more of the presence of DNA methylation of or extent of DNA methylation of the 5HTT promoter or 5HTT promoter region and/or reduced 5HTT gene expression.

[0024] Reference to a "mental health condition" includes a psychiatric, neurological or psychological condition, phenotype or state include drug addiction such as persisting depressive symptomatology (PDS), depression including major depressive disorder, dysthymia and episodic depressive symptomatology, addiction to prescribed licit and illicit substances, eating disorders including obesity, anorexia nervosa and bulimia, anxiety disorders including simple phobias, social phobias, generalized anxiety disorder, panic disorder (with and without agoraphobia) and OCD and bipolar disorder, schizophrenia, personality disorder including schizoid, narcissistic and paranoid personality features, acute stress disorder and adjustment disorder, schizoaffective disorder, schizoid personality disorder, schizophreniform disorder, schizotypal personality disorder. The present invention is particularly effective for diagnosing PDS and depression.

[0025] The identification of at risk individuals, especially at initial stages, enables early intervention with medical protocols at the medicament and/or psychology levels. The present invention contemplates, therefore, therapeutic and prophylactic protocols for individuals or groups of individuals at risk of developing a mental health condition including a psychiatric, neurological or psychological condition, phenotype or disorder, the protocol comprising identifying at risk individuals by determining the presence or absence of or extent of DNA methylation of the 5HTT promoter or 5HTT promoter region and/or level of expression of the 5HTT gene wherein the presence of a DNA methylated 5HTT promoter or 5HTT promoter region or reduced 5HTT gene expression alone or in combination with an S-allelic or multiple S-allelic forms of the 5HTT gene is indicative of a subject being at risk of developing such a mental health condition and then subjecting the individual to therapeutic intervention and/or monitoring, the intervention comprising the administration of medicaments and/or behavioral, social, environmental or psychological modification.

[0026] The therapeutic protocol may also involve stratifying or profiling individuals or groups of individuals by determining the presence or absence of or extent of DNA methylation of the 5HTT promoter or 5HTT promoter region and/or level of expression of the 5HTT gene wherein:

(i) an individual with lower levels of a methylated 5HTT promoter or 5HTT promoter region DNA within the bottom of the population distribution of 5HTT methylation is considered at a lower risk of developing a mental health condition; wherein the bottom of population distribution of 5HTT DNA methylation is defined as equal to or less than 6% plus or minus 5% DNA methylation;

(ii) an individual with a methylated 5HTT promoter or 5HTT promoter region DNA and/or exhibiting reduced 5HTT gene expression alone or in combination with an S-allelic form of the 5HTT gene is more likely of developing the mental health condition, phenotype or state compared to individuals in group (i); and

(iii) an individual with a highly methylated 5HTT promoter or 5HTT promoter region DNA and/or exhibiting reduced 5HTT gene expression alone or in combination with multiple S-allelic forms of the 5HTT gene is more at risk of developing the mental health condition, state or phenotype compared to the individuals in group (iii);

wherein, based on the level of risk, providing the individual or group of individuals with medicament and/or behavioral, environmental, social or psychological intervention.

[0027] The present invention further contemplates the use of a data set comprising information on the presence or extent of DNA methylation of the 5HTT promoter or 5HTT promoter region and/or levels of 5HTT or 5HTT gene expression alone or in combination with an S-allelic form of the 5HTT gene in the generation of a risk factor analysis of an individual developing a mental health condition including a psychiatric, neurological or psychological condition, phenotype or state.

[0028] The present invention further contemplates a method of treating a mental health condition including a psychiatric, neurological or psychological condition, phenotype or state in an individual the method comprising identifying an at risk individual and subjecting the individual to or medical intervention including gene therapy or psychological interventional to facilitate amelioration the symptoms and/or to prevent development of the symptoms.

[0029] Another aspect of the present invention provides a method for determining the likelihood of a subject responding favorably to a particular drug in the treatment of a mental health condition including a psychiatric, neurological or psychological condition, phenotype or state, the method comprising obtaining or extracting a nucleic acid sample from cells of said individual and screening for or otherwise detecting the presence or extent of DNA methylation of the 5HTT promoter or 5HTT promoter region and/or level of expression alone or in combination with an S-allelic form of the 5HTT gene wherein a drug which increases expression of the 5HTT gene is deemed more likely to be effective than a drug which inhibits 5HTT gene expression or which does not target 5HTT.

[0030] Still another aspect of the present invention provides a method for determining the likelihood of a subject responding favorably to an intervention in the treatment of a mental health condition including a psychiatric, neurological or psychological condition, phenotype or state, the intervention including behavioral, environmental, social or psychological intervention, the method comprising obtaining or extracting a nucleic acid sample from cells of said individual and screening for or otherwise detecting the presence or extent of DNA methylation of the 5HTT promoter or 5HTT promoter region and/or level of expression alone or in combination with the presence or absence of an S-allelic form of the 5HTT gene wherein an intervention which increases expression of the 5HTT gene is deemed more likely to be effective than an intervention which inhibits 5HTT gene expression.

TABLE l

Summary of sequence identifiers

TABLE 2 Abbreviations

BRIEF DESCRIPTION OF THE FIGURES

[0031] Some figures contain color representations or entities. Color photographs are available from the Patentee upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office.

[0032] Figure 1 is a schematic representation of 5HTT (SLC6A4) promoter region. The SLC6A4 gene is located on chromosome 17q and contains a small CpG island (green bar) associated with the gene promoter. Two overlapping methylation assays (Al and A2) spanning the SLC6A4 transcriptional start site (TS), exon 1 (El), parto of intron 1 (II) and the CpG island were performed in this study. A total of 86 CpG dinucleotides are present within the assayed regions and included in analysis. The 5HTTLPR polymorphism is upstream of this region and shows no overlap with the CpG dinucleotides.

[0033] Figure 2 is a tabular representation showing the relationships between residual number, sequonome output and analytic clusters for association analysis.

DETAILED DESCRIPTION

[0034] The singular forms "a", "an", and "the" include single and plural aspects unless the context clearly indicates otherwise. Thus, for example, reference to "a polymorphism" includes a single polymorphism, as well as two or more polymorphisms; reference to "an S-allele" includes a single S-allele or multiple S-alleles; reference to "the invention " includes single or multiple aspects of an invention; and so on.

[0035] The present invention identifies a relationship between the 5HTTLPR genotypes, DNA methylation of the 5HTT promoter or 5HTT promoter region, binding of non-coding RNA and/or histone modification and a mental health condition including psychiatric, neurological and psychological conditions, phenotypes and states. Such states include persisting depressive symptomatology or "PDS" and "depression" which encompasses major depressive disorder, dysthymia and episodic depressive symptomatology. The term "mental health" is to be read as including conditions, phenotypes and states encompassing a sub-threshold psychiatric, neurological and psychological condition, phenotype or state. A "condition, phenotype or state" also encompasses a "disorder". In accordance with the present invention, an individual or group of individuals is classified as "at risk" of developing symptomatic features of a mental health condition including a psychiatric, neurological or psychological condition, phenotype or state if he or she has a 5HTT promoter or 5HTT promoter region with DNA which is greater than lower methylated and/or have reduced expression of the 5HTT gene alone or in combination with an S-allele of the 5HTT gene. The term "at risk" includes the situation where the likelihood of developing the mental condition is environmentally dependent or associated. Reference to the "S-allele" includes S-allelic forms of the 5HTT gene as well as multiple S-allelic forms. An individual may be homozygous or heterozygous for the S-allele. A lower level of DNA methylation is in the bottom group of the population distribution of 5HTT gene methylation wherein the bottom group is equal to or less than 6% + 5% DNA methylation. The bottom group also includes the lower two tertiles of the population distribution. Reduced gene expression may also arise due to histone modification or non-coding RNA binding. [0036] Accordingly, one aspect of the present invention contemplates a method for identifying an epigenetic or epi genetic and genetic profile in a subject associated with a mental health condition including a psychiatric, neurological or psychological condition, phenotype or state, the method comprising determining the presence or absence of or extent of DNA methylation of the 5HTT promoter or 5HTT promoter region and/or the level of expression of the 5HTT gene alone or in combination with an S-allelic form of the

5HTT gene wherein the presence of a methylated 5HTT promoter or 5HTT promoter region DNA or reduced level or activity of 5HTT with or without the S-allele is indicative of a subject being at risk of developing such a mental health condition.

[0037] Another aspect provides a method for identifying an epigenetic profile in a subject associated with a mental health condition, said method comprising determining the presence or absence or extent of DNA methylation of the 5HTT promoter region and/or the level of expression of the 5HTT gene alone or in combination with the presence or absence or number of S-allelic forms of the 5HTT gene wherein a DNA methylated 5HTT promoter region or reduced level of activity of 5HTT with or without an S-allele is indicative of a subject being at risk of developing such a mental health condition.

[0038] The method of the present invention extends to stratifying or profiling individuals for a mental health condition including a psychiatric, neurological or psychological condition, phenotype or state. The method is generally used in conjunction with behavioral tests and an understanding of environmental and social factors to which the individual(s) is/are exposed. Hence, an individual deemed "at risk" according to the method herein may not necessarily develop a condition, phenotype or state unless certain environmental or social factors are present.

[0039] Hence, the present invention identifies a link or relationship between the methylation of the 5HTT promoter or 5HTT promoter region DNA and/or levels or activity of 5HTT alone or in combination with an S-allelic form of the 5HTT gene and the presence of a mental health condition including a psychiatric, neurological or psychological condition, phenotype or state. The link or relationship comprises the combination of methylation including extent of methylation of the 5HTT promoter or 5HTT promoter region DNA and/or level of expression of the 5HTT gene with or without single or multiple S-allelic forms of the 5HTT gene. The link is stronger and the subject is more at risk the higher the level of 5HTT promoter or 5HTT promoter region DNA methylation and/or the lower the level of expression of the 5HTT gene alone or in combination with an S-allelic form of the 5HTT gene. A ranking of the level of risk enables stratification of individuals based on the presence of or extent of 5HTT promoter or 5HTT promoter region DNA methylation and/or 5HTT gene expression and the number of S-alleles. A "gene" in this context includes a gene having a promoter of operably linked thereto. A "5HTT promoter region" includes the 5HTT promoter as well as approximately 10kb up or downstream of the transcription initiation site of the 5HTT gene.

[0040] Another aspect of the present invention provides a method for profiling or stratifying an individual or group of individuals for a level of risk in developing a mental health condition including a psychiatric, neurological or psychological condition, phenotype or state, the method comprising determining the presence or absence of or extent of DNA methylation of the 5HTT promoter or 5HTT promoter region and/or level of 5HTT gene expression wherein:

(i) an individual with lower levels of a methylated 5HTT promoter or 5HTT promoter region DNA within the bottom of the population distribution of 5HTT methylation is considered at a lower risk of developing the mental health condition; wherein the bottom of population distribution of 5HTT DNA methylation is defined at equal to or less than 6% plus or minus 5% DNA methylation;

(ii) an individual with a methylated 5HTT promoter or 5HTT promoter region DNA or reduced 5HTT gene expression alone or in combination with an S-allelic form of DNA is more likely of developing the mental health condition compared to an individual in group (i); and (iii) an individual with highly a methylated 5HTT promoter or 5HTT promoter region DNA or highly reduced 5HTT gene expression is more at risk of developing the condition, state or phenotype compared to an individual in group (ii).

[0041] Put another way, the present invention provides a method for identifying an epigenetic and genetic profile in a subject associated with a mental health condition, said method comprising determining the presence or absence or extent of DNA methylation of the 5HTT promoter region and/or the level of expression of the 5HTT gene alone or in combination with the presence or absence or number of S-allelic forms of the 5HTT gene wherein a DNA methylated 5HTT promoter region or reduced level of activity of 5HTT with or without an S-allele is indicative of a subject being at risk of developing such a mental health condition.

[0042] Reference to the methylation of the 5HTT promoter or 5HTT promoter region DNA includes varying extents of DNA methylation from low to highly methylated DNA. hi one embodiment, the DNA methylation occurs in one or more CpG islands within the 5HTT promoter or 5HTT promoter region. A level of 6% or less includes 0, 1, 2, 3, 4, 5 and 6%, as well as fractions inbetween. A level of greater than 6% includes a fraction between 6 and 7 as well as 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 to 100%, as well as fractions inbetween.

[0043] A "mental health condition" covers psychiatric, neurological or psychological conditions, phenotypes and states such as, but are not limited to, persisting depressive symptomatology (PDS), depression including major depressive disorder, dysthymia and episodic depressive symptomatology, drug addiction including to prescribed licit and illicit substances, eating disorders including obesity, anorexia nervosa and bulimia, anxiety disorders including simple phobias, social phobias, generalized anxiety disorder, panic disorder (with and without agoraphobia) and OCD and bipolar disorder, schizophrenia, personality disorder including schizoid, narcissistic and paranoid personality features, acute stress disorder, adjustment disorder, schizoaffective disorder, schizoid personality disorder, schizophreniform disorder, schizotypal personality disorder.

[0044] Although not intending to limit the present invention to any one form of mental health condition including a psychiatric, neurological or psychological condition, phenotype or state, the method herein is particularly useful for identifying individuals at risk of developing PDS (persistence of depressive symptomatology) and/or depression.

[0045] Hence, a subject is at risk of having or developing PDS if they have a 5HTT promoter or 5HTT promoter region DNA which is methylated to any extent and/or if expression of the 5HTT gene is reduced alone or in combination with an S-allelic form of the 5HTT gene.

[0046] Accordingly, the present invention contemplates a method for identifying an epigenetic or epigenetic and genetic profile in a subject associated PDS, the method comprising determining the presence or absence of or extent of DNA methylation of the 5HTT promoter or 5HTT promoter region and/or level of 5HTT gene expression alone or in combination with an S-allelic form of the 5HTT gene wherein the presence of methylated 5HTT promoter DNA or 5HTT promoter region or reduced 5HTT gene expression with or without the S-allele is indicative of a subject being at risk of developing PDS.

[0047] Another aspect provides a method for profiling or stratifying an individual or group of individuals for a level of risk in developing PDS, the method comprising determining the presence or absence of or extent of DNA methylation of the 5HTT promoter or 5HTT promoter region and/or level of 5HTT gene expression wherein:

(i) an individual with lower levels of a methylated 5HTT promoter or 5HTT promoter region DNA within the bottom of the population distribution of 5HTT methylation is considered at a lower risk of developing PDS; (ii) an individual with a methylated 5HTT promoter or 5HTT promoter region DNA and/or reduced 5HTT gene expression alone or in combination with an S-allelic form of the 5HTT gene is more likely of developing PDS compared to an individual in group (i); and

(iii) an individual with highly a methylated 5HTT promoter or 5HTT promoter region DNA and/or highly reduced 5HTT gene expression alone or in combination with an S- allelic form of the 5HTT gene is more at risk of developing PDS compared to an individual in group (ii).

[0048] A further aspect of the present invention contemplates the a method for identifying an epi genetic or epigenetic and genetic profile in a subject associated with depression, the method comprising determining the extent of DNA methylation of CpG dinucleotides in the 5HTT promoter or 5HTT promoter region and/or level of 5HTT gene expression wherein the presence of a DNA methylated 5HTT promoter or 5HTT promoter region or reduced 5HTT gene expression alone or in combination with one or more S-allelic forms of the 5HTT gene is indicative of a subject being at risk of developing depression.

[0049] Yet another aspect provides a method for profiling or stratifying an individual or group of individuals for a level of risk in developing depression, the method comprising determining the and the extent of DNA methylation of the 5HTT promoter or 5HTT promoter region and/or level of 5HTT gene expression wherein:

(i) an individual with lower levels of a methylated 5HTT promoter or 5HTT promoter region DNA within the bottom of the population distribution of 5HTT methylation is considered at a lower risk of developing depression; wherein the bottom of population distribution of 5HTT DNA methylation is defined as equal to or less than 6% plus or minus 5% DNA methylation; (ii) an individual with a methylated 5HTT promoter or 5HTT promoter region DNA and/or reduced 5HTT gene expression alone or in combination with an S-allelic form of the 5HTT gene is more likely of developing PDS compared to an individual in group (i); and

(iii) an individual with a highly methylated 5HTT promoter or 5HTT promoter region DNA and/or highly reduced 5HTT gene expression alone or in combination with multiple S-allelic forms of the 5HTT gene is more at risk of developing depression compared to an individual in group (ii).

[0050] Reference to "depression" includes major depressive disorder, dysthymia and episodic depressive symptomatology. As indicated above, the diagnostic aspect of the present invention may also be conducted in conjunction with behavioral, social and environmental analyses.

[0051] Hence, the present invention contemplates a risk profile for a subject developing or being diagnosed with a mental health condition including a psychiatric, neurological or psychiatric condition, phenotype or state wherein the profile comprises a plurality of data comprising behavioral, environmental and social parameters in combination with one or more of the presence or extent of DNA methylation of the 5HTT promoter or 5HTT promoter region and/or reduced 5HTT gene expression alone or in combination with an S- allelic form of the 5HTT gene.

[0052] As indicated above, the 5HTT genetic locus and/or translational machinery may be subject to histone modification and/or non-coding RNA binding resulting in reduced expression.

[0053] Behavioral, environmental and social parameters include presence of peers and peer pressure, success or otherwise in social including family and work-related groups, living and working environments, levels of stress and in particular stress early on in life or during early adolescent age, anger management and exposure to anger or dangerous situations, expectations of others, levels of comfort in living and working and social situations, level of fiscal comfort, levels of social and work satisfaction, substance addiction and/or dependence phobias, amongst many other indicators.

[0054] The present invention is predicated in part on the 44 bp deletion/insertion polymorphism in the promoter region of the serotonin transporter gene, 5HTT: viz, 5HTTLPR S-allele or 5HTTLPR L-allele. Notwithstanding, however, all polymorphisms within the 5HTT promoter are encompassed by the present invention. Reference to a "polymorphism" includes, in one embodiment, a SNP; in another embodiment, a multiple nucleotide polymorphism (MNP); and in yet another embodiment, any nucleotide mutation such as an insertion, addition, substitution or deletion as well as rearrangements, microsatellites and variable repeats.

[0055] The term "polymorphism" or "mutation" or "epigenetic" or epigenetic and genetic profile" refers to a difference in a DNA or RNA sequence or sequences among individuals, groups or populations which give rise to a statistically significant neurological, psychiatric or physiological condition, phenotype or state. Examples of epigenetic or epigenetic and genetic polymorphisms include mutations or DNA methylations and/or binding of non- coding RNA and/or histone modifications which result by chance or are induced by external features. These polymorphisms or mutations may be indicative of a disease or disorder and may arise following a genetic disease, a chromosomal abnormality, a genetic predisposition, an inherited genetic condition, random mutation, a viral infection, a fungal infection, a bacterial infection or a protist infection or following chemotherapy, radiation therapy or substance abuse including alcohol or drug abuse. The polymorphisms may also dictate or contribute to symptoms with a psychological phenotype. In one aspect, the epigenetic or epigenetic and genetic profile comprises level or extent of DNA methylation of the 5HTT promoter or 5HTT promoter region and/or non-coding RNA binding and/or histone modification alone or in combination with an S-allelic form of the 5HTT gene which is indicative of a mental health condition including a psychiatric, neurological or psychological condition, phenotype or state or sub-threshold condition, phenotype or state thereof. As used herein, the epigenetic or epigenetic and genetic profile herein including mutations or DNA methylations and/or binding of non-coding RNA and/or histone modifications may refer to one or more changes in a DNA or RNA sequence which are present in a group of individuals having a particular mental health condition including a psychiatric, neurological or psychological condition, phenotype or state or sub-threshold forms thereof or are at risk of developing same. Non-coding RNA binding encompasses genetic signaling of spliced intronic or exonic RNA and generation of single or double stranded RNA entities including RNAi-like entities.

[0056] As indicated above, examples of a mental health condition including a psychiatric, neurological or psychological condition, phenotype or state contemplated by the present invention and which may be directly or indirectly linked to an epigenetic or epigenetic and genetic profile comprising 5HTT promoter or 5HTT promoter region DNA methylation alone or in combination with an S-allelic form of the 5HTT gene include but are not limited to PDS, depression including major depressive disorder, dysthymia and episodic depressive symptomatology, drug addiction including to prescribed licit and illicit substances, eating disorders including obesity, anorexia nervosa and bulimia, anxiety disorders including simple phobias, social phobias, generalized anxiety disorder, panic disorder (with and without agoraphobia) and OCD, and bipolar disorder, schizophrenia, personality disorder including schizoid, narcissistic and paranoid personality features, acute stress disorder, adjustment disorder, schizoaffective disorder, schizoid personality disorder, schizophreniform disorder and schizotypal personality disorder. The present invention is particularly effective for diagnosing PDS. PDS and depression are particular examples of a psychiatric, neurological or psychological condition, phenotype or state contemplated herein.

[0057] Any number of methods may be used to calculate the statistical significance of an association between the DNA methylation of the 5HTT promoter or 5HTT promoter region and the presence of an S-allele of the 5HTT gene with a psychiatric, neurological or psychological condition. Particular statistical analysis methods which may be used are described in Fisher and vanBelle, "Biostatistics: A Methodology for the Health Sciences" Wiley-Intersciences (New York) 1993. This analysis may also include a regression calculation of which combination of S-allele presence and number and 5HTT promoter or 5HTT promoter region DNA methylation gives the most significant contribution to the differences in phenotype. One regression model starts with a model of the form:

r= r_o+ (Sx d)

where r is the response, r$ is a constant called the "intercept", S is the slope and d is the dose. To determine the dose, the most-common and least common epigenetic or epigenetic and genetic profile of the 5HTT gene including its promoter or 5HTT promoter region are first defined. Then, for each individual in the trial population, one calculates a "dose" as the number of least-common profiles the individual has in the 5HTT gene region. This value can be 0 (homozygous for the least-common), 1 (heterozygous), or 2 (homozygous for the most common). An individual's "response" is the value of the clinical measurement. Standard linear regression methods are then used to fit all the individuals' doses and responses to a single model (see e.g. Fisher and vanBelle, supra, Ch 9). The outputs of the regression calculation are the intercept r₀, the slope S and the variance (which measures how well the data fit this linear model). The Students t-test value and the level of significance can then be calculated for each of the combinations of 5HTT promoter or 5HTT promoter region DNA methylation and presence of an S-allelic form of the 5HTT gene.

[0058] The present invention provides, therefore, an epigenetic or epigenetic and genetic marker for a mental health condition including a psychiatric, neurological or psychological condition, state or phenotype in an individual wherein the epigenetic or epigenetic and genetic marker is the presence or absence or extent of DNA methylation of 5HTT promoter or 5HTT promoter region and the presence of single or multiple S-alleles of the 5HTT gene.

[0059] Genetic assays are conveniently conducted in or on nucleic acid material (e.g. DNA or niRNA) extracted from cells obtained in blood or other cell-containing fluid drawn from a subject. Hence, a test is conducted in a sample which may be referred to as a biological sample and includes any form of tissue or fluid or biopsy sample from a subject such as body fluid, buccal swabs, nasal epithelia, brain biopsy, blood, plasma, serum, spinal fluid, excretia (such as urine and faeces) and lymph fluid.

[0060] In addition, prenatal diagnosis can be accomplished by testing fetal cells, placental cells or amniotic cells for the 5HTT epigenetic or epigenetic and genetic profile. The "5HTT epigenetic or epigenetic and genetic profile" includes extent of DNA methylation of the 5HTT promoter or 5HTT promoter region and the presence and number of the S- allele of the 5HTT gene. The 5HTT promoter region includes the 5HTT promoter.

[0061] Accordingly, another aspect of the present invention contemplates a method for diagnosing a mental health condition including a psychiatric, neurological or psychological condition, phenotype or state in an individual, the method comprising obtaining or extracting nucleic acid material from cells of the individual and screening for or otherwise detecting the presence or absence or extent of DNA methylation of the 5HTT promoter or 5HTT promoter region or level of 5HTT gene expression alone or in combination with an S-allelic form of the 5HTT gene wherein 5HTT promoter or 5HTT promoter region DNA methylation or reduced 5HTT gene expression with or without the S-allelic form of the 5HTT gene has a statistically significant association with a particular mental health condition including a psychiatric, neurological or psychological condition, phenotype or state or a sub-threshold form thereof or that the individual is at risk of developing same.

[0062] Detecting 5HTT promoter or 5HTT promoter region DNA methylation may be done directly or indirectly via levels of expression of the 5HTT gene. Low level of expression is an indication of potential methylation.

[0063] Generally, the genetic test is part of an overall diagnostic protocol involving clinical assessment and diagnostic tools such as pencil-and-paper tests. Consequently, this aspect of the present invention may be considered as a confirmatory test or part of a series of tests in the final diagnosis of a mental health condition including a psychiatric, neurological or psychological condition, phenotype or state. [0064] The methods and assays of the present invention are further directed to detecting the epigenetic or epigenetic and genetic profile around the 5HTT genetic region in an individual associated with "normal" behavior, hi other words, an individual which may be at risk such as through his or her genetic lines or because of substance abuse or who has behavioral tendencies which suggest a particular mental health condition including a psychiatric, neurological or psychological condition, phenotype or state can be screened for the presence or absence of extent of 5HTT promoter or 5HTT promoter region DNA methylation wherein a 5HTT promoter or 5HTT promoter region which is DNA methylated alone or with an S-allelic form of the 5HTT gene is at least suggestive of an epigenetic and or genetic basis for any symptoms associated with the mental health condition including a psychiatric, neurological or psychological condition, phenotype or state for which the individual first presented to a clinician.

[0065] A "mental health condition" includes a psychiatric, neurological or psychological condition, phenotype or state which causes an adverse condition or may represent "normal" behavior. The latter constitutes behavior consistent with societal "norms".

[0066] Reference herein to an "individual" includes a human which may also be considered a subject, patient, host, recipient or target.

[0067] The present invention enables, therefore, a stratification of individuals based on an epigenetic or epigenetic and genetic profile. The stratification or profiling enables early diagnosis, conformation of a clinical diagnosis, treatment monitoring and treatment selection for a mental health condition including a psychiatric, neurological or psychological conditions phenotype or state.

[0068] There are many methods which may be used to detect a DNA sequence profile or

DNA 'methylation profiles. Direct DNA sequencing, either manual sequencing or automated fluorescent sequencing can detect sequence variation including the S-allele or

L-allele. Another approach is the single-stranded conformation polymorphism assay (SSCP) (Orita, et al, Proc. Natl. Acad. ScL USA. 86:2766-2770, 1989). This method can be optimized to detect most DNA sequence variation. The reduced detection sensitivity is a disadvantage, but the increased throughput possible with SSCP makes it a viable alternative to direct sequencing for mutation detection. The fragments which have shifted mobility on SSCP gels are then sequenced to determine the exact nature of the DNA sequence variation. Other approaches based on the detection of mismatches between the two complementary DNA strands include clamped denaturing gel electrophoresis (CDGE) (Sheffield et al, Proc. Natl. Acad. ScL USA 86:232-236, 1989), heteroduplex analysis (HA) (White et al, Genomics 72:301-306, 1992) and chemical mismatch cleavage (CMC) (Grompe et al, Proc. Natl. Acad. ScL USA 55:5855-5892, 1989). None of the methods described above detects large deletions, duplications or insertions, nor will they detect a mutation in a regulatory region or a gene. Other methods which would detect these classes of mutations include a protein truncation assay or the asymmetric assay. A review of available methods of detecting DNA sequence variation can be found in Kwok, Curr Issues MoI. Biol. 5(2):A3-60, 2003; Twyman and Primrose, Pharmacogenomics. 4(1):67- 79, 2003; Edwards and Bartlett, Methods MoI. Biol. 226:287-294, 2003 and Brennan, Am. J. Pharmacogenomics. l(4):395-302, 2001. Once a mutation is known, an allele-specific detection approach such as allele-specific oligonucleotide (ASO) hybridization can be utilized to rapidly screen large numbers of other samples for that same mutation. Such a technique can utilize probes which are labeled with gold nanoparticles or any other reporter molecule to yield a visual color result (Elghanian et al, Science 277:1078-1081, 1997).

[0069] A rapid preliminary analysis to detect S-allelic or L-allelic DNA sequences can be performed by looking at a series of Southern blots of DNA cut with one or more restriction enzymes, preferably with a large number of restriction enzymes. Each blot contains a series of normal individuals and a series of individuals having neurologic or neuropsychiatric diseases or disorders or any other neurological, psychiatric or psychological condition, phenotype or state. Southern blots displaying hybridizing fragments (differing in length from control DNA when probed with sequences near or to the genetic locus being tested) indicate a possible mutation or polymorphism. If restriction enzymes which produce very large restriction fragments are used, then pulsed field gel electrophoresis (PFGE) is employed. Alternatively, the desired region of the genetic locus being tested can be amplified, the resulting amplified products can be cut with a restriction enzyme and the size of fragments produced for the different polymorphisms can be determined.

[0070] Other methods for confirming the presence of a susceptibility allele include denaturing gradient gel electrophoresis [DGGE] (Wartell et al, Nucl. Acids Res. 18:2699- 2705, 1990; Sheffield et al, 1989 supra), RNase protection assays (Finkelstein et al, Genomics 7:167-172, 1990; Kinszler et al, Science 257:1366-1370, 1991), the use of proteins which recognize nucleotide mismatches, such as the E. coli mutS protein (Modrich^rc. Rev. Genet. 25:229-253, 1991), allele-specific PCR (Ruano and Kidd, Nucl. Acids Res. 77:8392, 1989) and PCR amplification of the site of the deletion or addition in 5HTTLPR followed by digestion using a restriction endonuclease that cuts or fails to cut when the variant allele is present.

[0071] Additionally, real-time PCR such as the allele specific kinetic real-time PCR assay can be used or allele specific real-time TaqMan probes.

[0072] For allele-specific PCR, primers are used which hybridize at their 3' ends to a particular target genetic locus or mutation. If the particular polymorphism or mutation is not present, an amplification product is not observed. Amplification Refractory Mutation System (ARMS) can also be used, as disclosed in European Patent Application Publication No. 0332435. Insertions and deletions of genes can also be detected by cloning, sequencing and amplification, hi addition, restriction fragment length polymorphism (RFLP) probes for the gene or surrounding marker genes can be used to score alteration of an allele or an insertion in a polymorphic fragment. Such a method is particularly useful for screening relatives of an affected individual for the presence of an allele found in that individual. Other techniques for detecting insertions and deletions as known in the art can be used. [0073] In SSCP, DGGE and the RNase protection assay, an electrophoretic band appears which is absent if the polymorphism or mutation is not present. SSCP detects a band which migrates differentially because the sequence change causes a difference in single-strand, intramolecular base pairing. RNase protection involves cleavage of the mutant polynucleotide into two or more smaller fragments. DGGE detects differences in migration rates of mutant sequences compared to wild-type sequences, using a denaturing gradient gel. In an allele-specific oligonucleotide assay, an oligonucleotide is designed which detects a specific sequence and the assay is performed by detecting the presence or absence of a hybridization signal, In the mutS assay, the protein binds only to sequences that contain a nucleotide mismatch in a heteroduplex between mutant and wild-type sequences.

[0074] Mismatches, according to the present invention, are hybridized nucleic acid duplexes in which the two strands are not 100% complementary. Lack of total homology may be due to deletions, insertions, inversions or substitutions. Mismatch detection can be used to detect point mutations in the gene or in its mRNA product. While these techniques are less sensitive than sequencing, they are simpler to perform on a large number of samples. An example of a mismatch cleavage technique is the RNase protection method. In the practice of the present invention, the method involves the use of a labeled riboprobe which is complementary to the 5HTT genetic locus. The riboprobe and either mRNA or DNA isolated from the person are annealed (hybridized) together and subsequently digested with the enzyme RNase A which is able to detect some mismatches in a duplex RNA structure. If a mismatch is detected by RNase A, it cleaves at the site of the mismatch. Thus, when the annealed RNA preparation is separated on an electrophoretic gel matrix, if a mismatch has been detected and cleaved by RNase A, an RNA product will be seen which is smaller than the full length duplex RNA for the riboprobe and the mRNA or DNA. The riboprobe need not be the full length of the mRNA or gene but can be a segment of either. If the riboprobe comprises only a segment of the mRNA or gene, it will be desirable to use a number of these probes to screen the whole mRNA sequence for mismatches. [0075] In similar fashion, DNA probes can be used to detect mismatches, through enzymatic or chemical cleavage (see, for example, Cotton et al, Proc. Natl. Acad. Sci. USA 57:4033-4037,1988; Shenk et al, Proc. Natl. Acad. Sci. USA 72:989-993, 1975; Novack et al, Proc. Natl. Acad. Sci. USA 53:586-590, 1986). Alternatively, mismatches can be detected by shifts in the electrophoretic mobility of mismatched duplexes relative to matched duplexes (see, for example, Cariello Am. J. Human Genetics 42:126-734, 1988). With either riboprobes or DNA probes, the cellular mRNA or DNA which might contain a mutation can be amplified using PCR (see below) before hybridization. Changes in DNA of the associated genetic polymorphisms or genetic loci can also be detected using Southern blot hybridization, especially if the changes are gross rearrangements, such as deletions and insertions.

[0076] Alteration of mRNA expression from the 5HTT genetic locus can be detected by any techniques known in the art. These include Northern blot analysis, PCR amplification and RNase protection. Diminished mRNA expression can indicate methylated gene promoter . Alteration of expression can also be detected by screening for alteration of wild-type protein. For example, monoclonal antibodies immunoreactive with the serotonin transporter can be used to screen a tissue. Lack of cognate antigen or a reduction in the levels of antigen would indicate less of expression and possibly methylation. Such immunological assays can be done in any convenient formats known in the art. These include Western blots, immunohistochemical assays and ELISA assays. Any means for detecting an altered 5HTT protein can be used to detect alteration of the "wild-type" protein. Functional assays, such as protein binding determinations, can be used. In addition, assays can be used which detect the protein function. Reduced expression or levels of 5HTT may be indicative of methylation of the promoter or coding regions.

[0077] Primer pairs disclosed herein are useful for determination of the nucleotide sequence of the 5HTTLPR genetic locus using PCR. The pairs of single-stranded DNA primers can be annealed to sequences within or surrounding the gene in order to prime amplifying DNA synthesis of the gene itself. A complete set of these primers allows synthesis of all of the nucleotides of the gene coding sequences, i.e. the exons. The set of primers preferably allows synthesis of both intron and exon sequences. Allele-specific primers can also be used. Such primers anneal only to particular polymorphic or mutant alleles and thus will only amplify a product in the presence of the polymorphic or mutant allele as a template.

[0078] In order to facilitate subsequent cloning of amplified sequences, primers may have restriction enzyme site sequences appended to their 5' ends. Thus, all nucleotides of the primers are derived from the 5HTT genetic locus, except for the few nucleotides necessary to form a restriction enzyme site. Such enzymes and sites are well known in the art. The primers themselves can be synthesized using techniques which are well known in the art. Generally, the primers can be made using oligonucleotide synthesizing machines which are commercially available.

[0079] The nucleic acid probes provided by the present invention are useful for a number of purposes. They can be used in Southern blot hybridization to genomic /RNA as DNA and in the RNase protection method for detecting point mutations already discussed above. The probes can be used to detect PCR amplification products. They may also be used to detect mismatches in the target genes or mRNA using other techniques.

[0080] The present invention identifies the presence of a genetic profile of the 5HTT genetic locus associated with a mental health condition including a psychiatric, neurological or psychological condition, phenotype or state or a sub-threshold form thereof or an individual of risk of developing same. In order to detect a target genes or mutation, a biological sample is prepared and analyzed for a particular type of allele. As indicated above, the biological sample includes brain biopsy, nasal epithelia and buccal swab.

[0081] As used herein, the phrase "amplifying" refers to increasing the content of a specific genetic region of interest within a sample. The amplification of the genetic region of interest may be performed using any method of amplification known to those of skill in the relevant art. In a preferred aspect, the present method for detecting a polymorphism utilizes PCR as the amplification step. [0082] PCR amplification utilizes primers to amplify a genetic region of interest. Reference herein to a "primer" is not to be taken as any limitation to structure, size or function. Reference to primers herein, includes reference to a sequence of deoxyribonucleotides comprising at least three nucleotides. Generally, the primers comprises from about three to about 100 nucleotides, preferably from about five to about 50 nucleotides and even more preferably from about 10 to about 25 nucleotides such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 nucleotides. The primers of the present invention may be synthetically produced by, for example, the stepwise addition of nucleotides or may be fragments, parts or portions or extension products of other nucleic acid molecules. The term "primer" is used in its most general sense to include any length of nucleotides which, when used for amplification purposes, can provide free 3' hydroxyl group for the initiation of DNA synthesis by a DNA polymerase. DNA synthesis results in the extension of the primer to produce a primer extension product complementary to the nucleic acid strand to which the primer has annealed or hybridized.

[0083] In a particular embodiment, one of the at least two primers is involved in an amplification reaction to amplify a target sequence. If this primer is also labeled with a reporter molecule, the amplification reaction will result in the incorporation of any of the label into the amplified product. The terms "amplification product" and "amplicon" may be used interchangeably.

[0084] The primers and the amplicons of the present invention may also be modified in a manner which provides either a detectable signal or aids in the purification of the amplified product. [0085] A range of labels providing a detectable signal may be employed. The label may be associated with a primer or amplicon or it may be attached to an intermediate which subsequently binds to the primer or amplicon. The label may be selected from a group including a chromogen, a catalyst, an enzyme, a fluorophore, a luminescent molecule, a chemiluminescent molecule, a lanthanide ion such as Europium (Eu³ ), a radioisotope and a direct visual label. In the case of a direct visual label, use may be made of a colloidal metallic or non-metallic particular, a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like. A large number of enzymes suitable for use as labels is disclosed in U.S. Patent Nos. 4,366,241, 4,843,000 and 4,849,338. Suitable enzyme labels useful in the present invention include alkaline phosphatase, horseradish peroxidase, luciferase, β- galactosidase, glucose oxidase, lysozyme, malate dehydrogenase and the like. The enzyme label may be used alone or in combination with a second enzyme which is in solution. Alternatively, a fluorophore which may be used as a suitable label in accordance with the present invention includes, but is not limited to, fluorescein-isothiocyanate (FITC) and the fluorochrome is selected from FITC, cyanine-2, Cyanine-3, Cyanine-3.5, Cyanine-5, Cyanine-7, fluorescein, Texas red, rhodamine, lissamine and phycoerythrin.

[0086] Examples of fluorophores are provided in Table 3.

TABLE 3

Ex: Peak excitation wavelength (nm) 2 Em: Peak emission wavelength (nm)

[0087] In order to aid in the purification of an amplicon, the primers or amplicons may additionally be incorporated on a bead. The beads used in the methods of the present invention may either be magnetic beads or beads coated with streptavidin.

[0088] The extension of the hybridized primer to produce an extension product is included herein by the term amplification. Amplification generally occurs in cycles of denaturation followed by primer hybridization and extension. The present invention encompasses form about one cycle to about 120 cycles, preferably from about two to about 70 cycles, more preferably from about five to about 40 cycles, including 10, 15, 20, 25 and 30 cycles, and even more preferably, 35 cycles such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 61, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120 cycles.

[0089] In order for the primers used in the methods of the present invention to anneal to a nucleic acid molecule containing the gene of interest, a suitable annealing temperature must be determined. Determination of an annealing temperature is based primarily on the genetic make-up of the primer, i.e. the number of A, T, C and Gs, and the length of the primer. Annealing temperatures contemplated by the methods of the present invention are from about 40°C to about 80°C, preferably from about 5O⁰C to about 7O⁰C, and more preferably about 65°C such as 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80⁰C. [0090] The PCR amplifications performed in the methods of the present invention include the use of MgCl₂ in the optimization of the PCR amplification conditions. The present invention encompasses MgCl₂ concentrations for about 0.1 to about 10 mM, preferably from 0.5 to about 5 mM, and even more preferably 2.5 mM such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mM.

[0091] Methylation sensitive enzymes are a useful way of screening for DNA methylation of the 5HTT promoter or 5HTT promoter region. There are a range of assays which can be employed to assess DNA methylation of the 5HTT promoter or 5HTT promoter region. DNA methylation is generally but not excessively considered to be within CpG dinucleotides. Approximately 86 CpG dinucleotidesare present in the 5HTT promoter. The present invention extends to DNA methylation of from one CpG dinculeotides to all 86 CpG dinucleotides including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 or 86 CpG dinucleotides. DNA methylation is linked to reduced expression. Hence, the present invention extends to the combination of level of expression of the 5HTT gene and presence and/or number of S-alleles of 5HTTLPR. Reduced 5HTT gene expression alone or in combination with an allelic form of the 5HTT gene is indicative of an individual at risk of developing a mental health condition including a psychiatric, neurological or psychological condition, phenotype or state.

[0092] In order to detect the presence of DNA methylation of the 5HTT promoter or 5HTT promoter region and the S-allele or L-allele of 5HTTLPR predisposing an individual to a mental health condition including a psychiatric, neurological or psychological condition, phenotype or state or sub-threshold form thereof or a risk of developing same, a biological sample such as blood, buccal swab, brain biopsy or nasal epithelia is obtained and analyzed for the extent of DNA methylation of the 5HTT promoter or 5HTT promoter region or level of expression of the 5HTT gene and the presence or absence of an S-allele. Results of these tests and interpretive information are returned to the health care provider for communication to the tested individual, Such diagnoses may be performed by diagnostic laboratories, or, alternatively, diagnostic kits are manufactured and sold to health care providers or to private individuals for self-diagnosis. Suitable diagnostic techniques include those described herein as well as those described in U.S. Pat. Nos. 5,837,492; 5,800,998 and 5,891,628.

[0093] Genetic testing enables practitioners to identify or stratify individuals at risk for certain behavioral states including substance addition or an inability to overcome a mental health condition including a psychiatric, neurological or psychological condition, phenotype or state or a sub-threshold form thereof after initial treatment. For particular at risk couples, embryos or fetuses may be tested after conception to determine the epigenetic or epigenetic and genetic likelihood of the offspring being pre-disposed to the neurological, psychiatric or psychological condition, phenotype or state. Certain behavioral or therapeutic protocols may then be introduced from birth or early childhood to reduce the risk of the mental health condition including a psychiatric, neurological or psychological condition, phenotype or state developing. Presymptomatic diagnosis will enable better treatment of these disorders, including the use of existing medical therapies. Genetic testing will also enable practitioners to identify individuals having diagnosed disorders (or in an at risk group) which have an at risk 5HTT epigenetic or epigenetic and genetic profile. Genotyping of such individuals is useful for (a) identifying a mental health condition including a psychiatric, neurological or psychological condition, phenotype or state or a sub-threshold form thereof in a subject which will respond to drugs affecting gene product activity; (b) identifying a mental health condition including a psychiatric, neurological or psychological condition, phenotype or state or sub-threshold neurological, psychiatric or psychological condition, phenotype or state in a subject which will respond to specific medications or medication types with fewer adverse effects; (c) identifying a mental health condition including a psychiatric, neurological or psychological condition, phenotype or state or sub-threshold neurological, psychiatric or psychological condition, phenotype or state in a subject which will respond to behavioral, social, environmental and/or counselling; and (d) guide new drug discovery and testing. "Respond" means, in one embodiment, "respond well", i.e. ameliorating one or more symptoms of the mental health condition.

[0094] Hence, the identification of at risk individuals, especially at initial stages, enables early intervention with medical protocols at the medicament and/or psychology levels. The present invention contemplates, therefore, therapeutic and prophylactic protocols for individuals or groups of individuals at risk of developing a mental health condition including a psychiatric, neurological or psychological condition, phenotype or disorder said protocol comprising identifying at risk individuals by determining the presence or absence or extent of DNA methylation of the 5HTT promoter or 5HTT promoter region and/or level of expression of the 5HTT gene alone or in combination with an S-allelic form of the 5HTT gene wherein the presence of a DNA methylated 5HTT promoter or 5HTT promoter region or reduced 5HTT gene expression with or without the S-allele is indicative of a subject being at risk of developing such a mental health condition and then subjecting the individual to therapeutic intervention and/or monitoring, the intervention comprising the administration of medicaments and/or behavioral, social, environmental or psychological modification. The behavioral, social, environmental and psychological modification includes removing an individual from a stressful environment and/or counselling an individual who has had a traumatic experience.

[0095] The therapeutic protocol may also involve stratifying or profiling individuals or groups of individuals by determining the presence or absence of or extent of DNA methylation of the 5HTT promoter or 5HTT promoter region and/or level of expression of the 5HTT gene wherein:

(i) an individual without a DNA methylated 5HTT promoter or 5HTT promoter region and is considered at a lower risk of developing the condition, phenotype or state;

(ii) an individual with a methylated 5HTT promoter or 5HTT promoter region DNA and/or exhibiting reduced 5HTT gene expression alone or in combination with an S-allelic form of the 5HTT gene is more likely of developing the mental health condition compared to individuals in group (i); and

(iii) an individual with a highly methylated 5HTT promoter or 5HTT promoter region DNA and/or exhibiting reduced 5HTT gene expression alone or in combination with multiple S-allelic forms is more at risk of developing the condition, state or phenotype compared to the individuals in group (iii);

[0096] The therapeutic protocol of the present invention encompasses a method of treatment or prophylaxis of a mental health condition including a psychiatric, neurological or psychological condition, phenotype or disorder. The diagnostic aspect allows the targeted implementation of treatment and preventative interventions including medication and behavioral therapy to reduce the adverse consequences of the mental health condition including a psychiatric, neurological or psychological condition, phenotype or state. This type of therapeutic or prophylactic intervention encompasses personalized medicine and pharmaco genomic analysis and screening.

[0097] Further, the present invention provides a method for screening drug candidates to identify molecules useful for treating mental health condition including a psychiatric, neurological or psychological conditions, phenotypes or states involving the 5HTT genetic locus expression of this locus. Drug screening can be performed by comparing the activity of the serotonin transporter in the presence and absence of potential drugs. In particular, these drugs may have the affect of masking the DNA methylation of the 5HTT promoter or 5HTT promoter region or de-methylating the promoter DNA or 5HTT promoter region or otherwise elevating expression of the 5HTT gene. The terms "drug", "agent", "therapeutic molecule", "prophylactic molecule", "medicament", "candidate molecule" or "active ingredient" may be used interchangeable in describing this aspect of the present invention. [0098] The goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g. agonists, antagonists, inhibitors) in order to fashion drugs which are, for example, more active or stable forms of 5HTT, or which, e.g. enhance or interfere with the function of 5HTT in vivo. Several approaches for use in rational drug design include analysis of three- dimensional structure, alanine scans, molecular modeling and use of anti-idiotypic antibodies. These techniques are well known to those skilled in the art, including those described in U.S. Pat. Nos. 5,837,492; 5,800,998 and 5,891,628.

[0099] A substance identified as a modulator of 5HTT function may be peptide or non- peptide in nature. Non-peptide "small molecules" are often preferred for many in vivo pharmaceutical uses. Accordingly, a mimetic or mimic of the substance (particularly if a peptide) may be designed for pharmaceutical use.

[0100] The designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a "lead" compound. This approach might be desirable where the active compound is difficult or expensive to synthesize or where it is unsuitable for a particular method of administration, e.g, pure peptides are unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal, Mimetic design, synthesis and testing are generally used to avoid randomly screening large numbers of molecules for a target property.

[0101] Once the pharmacophore has been found, its structure is modeled according to its physical properties, e.g, stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g, spectroscopic techniques, x-ray diffraction data and NMR.

Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modeling process. A template molecule is then selected, onto which chemical groups that mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted thereon can be conveniently selected so that the mimetic is easy to synthesize, is likely to be pharmacologically acceptable and does not degrade in vivo, while retaining the biological activity of the lead compound. Alternatively, where the mimetic is peptide-based, further stability can be achieved by cyclizing the peptide, increasing its rigidity. The mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent it is exhibited. Further optimization or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.

[0102] Briefly, a method of screening for a substance which modulates activity of 5HTT may include contacting one or more test substances with 5HTT in a suitable reaction medium, testing the activity of 5HTT and comparing that activity with the activity of

5HTT in comparable reaction medium untreated with the test substance or substances. A difference in activity between the treated and untreated 5HTT is indicative of a modulating effect of the relevant test substance or substances. Alternatively, de-methylating agents may be sought.

[0103] Following identification of a substance which modulates or affects of 5HTT activity, the substance may be further investigated. Furthermore, it may be manufactured and/or used in preparation, i.e, a manufacture or formulation, or a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals directly or via gene therapy.

[0104] Potential therapeutic agents can be formulated in pharmaceutical compositions, which are prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington's Pharmaceutical Sciences, 18th Ed. 1990, Mack Publishing Co., Easton, Pa. The composition may contain the active agent or pharmaceutically acceptable salts of the active agent. These compositions may comprise, in addition to an active substance, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g. intravenous, oral, intrathecal, epineural, sub-coetaneous, or parenteral as well as via a patch.

[0105] The present invention provides information necessary for medical practitioners to select drugs for use in the treatment of a neurological, psychiatric or psychological condition, phenotype or state or a sub-threshold form thereof. With the identification of a genetic profile of an at risk individual, antipsychotic mediation medications can be selected for use in combination with 5HTT modulators.

[0106] The present invention further contemplates a method of treating a mental health condition including a psychiatric, neurological or psychological condition, phenotype or state in an individual the method comprising identifying an at risk individual and subjecting the individual to gene therapy or medical or psychological interventional to facilitate amelioration the symptoms and/or to prevent development of the symptoms.

[0107] Another aspect of the present invention provides a method for determining the likelihood of a subject responding favorably to a particular drug in the treatment of a mental health condition including a psychiatric, neurological or psychological condition, phenotype or state said method comprising obtaining or extracting a nucleic acid sample from cells of said individual and screening for or otherwise detecting the presence or extent of DNA methylation of the 5HTT promoter or 5HTT promoter region and/or level of expression alone or in combination with an S-allelic form of the 5HTT gene wherein a drug which increases expression of the 5HTT gene is deemed more likely to be effective than a drug which inhibits 5HTT gene expression or which does not target 5HTT.

[0108] Still another aspect of the present invention provides a method for determining the likelihood of a subject responding favorably to an intervention in the treatment of a mental health condition, the intervention include behavioral, environmental, social or psychological intervention said method comprising obtaining or extracting a nucleic acid sample from cells of said individual and screening for or otherwise detecting the presence or extent of DNA methylation of the 5HTT promoter region and/or level of expression alone or in combination with the presence or absence of an S-allelic form of the 5HTT gene wherein an intervention which increases expression of the 5HTT gene is deemed more likely to be effective than an intervention which inhibits 5HTT gene expression.

[0109] Gene therapy may be recommended when a particular genetic profile around the 5HTT genetic locus is identified conferring, for example, a propensity for development of mental health condition including a psychiatric, neurological or psychological condition, phenotype or state is identified in an embryo. Genetically modified stem cells may then be used to alter the genotype of the developing cells. Where an embryo has developed into a fetus or for post-natal subjects, localized gene therapy may still be accomplished. Alternatively, a compound may be identified which effectively masks a particular undesired genetic profile.

[0110] Accordingly, the present invention also provides genetic test kits which allow the rapid screening of a particular epigenetic or epigenetic and genetic profile surrounding the

5HTT genetic locus within a test sample or multiple test samples. The kits of the present invention comprise one or more sets of primers, as described herein, which are specific for the amplification of the 5HTT genetic region of interest and/or level of 5HTT gene transcript. In addition, or alternatively, the genetic test kit may comprise antibodies to 5HTT. Furthermore, the genetic test kits of the present invention provide instructions for using the primers or antibodies.

[0111] Therapeutic kits are also contemplated by the present invention. For example, the kit may comprise a diagnostic or polymorphism detection component and a selection of therapeutics, the choice of use of which is dependent on the outcome of the diagnostic assay.

[0112] The present invention is further described with reference to the following non- limiting Examples. Procedures and methods used in these Examples are provided herein below. Procedure and sample

[0113] The Victorian Adolescent Health Cohort is a representative sample of young Australians living in the state of Victoria, Australia, who have been followed across eight waves since 1992. The first six waves were conducted at 6 month intervals (1992-1996) when participants were at secondary school. The remaining two waves were post secondary school when participants were aged 20-years (wave 7) and 24-years (wave 8), in 1998 and 2002, respectively. The study has an average response rate of 80% (min 75%; max 86%).

[0114] At each wave, extensive data have been collected on key adolescent and young adult mental and behavioral health issues including psychiatric morbidity, drug use (tobacco, alcohol and illicit drug use), eating disorders and obesity, anti-social behavior, personality and attachment style. The most recent assessment (wave 8) had an exclusive focus on substance use behavior and involved collection of cheek swab samples from 963 consenting participants.

Phenotype definitions

[0115] Mental health was assessed within the ICDlO framework using the Clinical

Interview Schedule (Revised) [CIS-R] (Lewis et al, Psychol Med iS(3j:737-745, 1988). Persistence of depressive symptomatology (PDS) was calculated as the number of waves (1 to 7) that a young person scored 11 or more on the CIS-R: a level at which a general practitioner would become concerned. If the PDS score was greater than five, participants were categorized as cases. If the PDS score was zero, participants were categorized as controls.

[0116] Selecting from the extremes of the distribution for depression identifies a clinically meaningful outcome (chronic or recurrent depression), increases the likelihood that a case is a true positive and maximizes statistical power by maximizing the contrast between groups. Selecting from the extreme of the depression liability spectrum also selects for comorbid anxiety. All depression cases met criteria for clinically significant general anxiety on at least one occasion. High liability for depression in this sample, therefore, identifies subjects with a history of both depression and anxiety.

Epigenetic and Genetic analysis [0117] The 799bp CpG island 3 ' of the SLC6A4 promoter was quantitatively analyzed for DNA methylation using Sequenom Mass ARRAY EpiTYPing. Two amplicons were designed to selectively amplify bisulfite converted DNA with the following primers:

Fl 5'-TATTGTTAGGTTTTAGGAAGAAAGAGAGAG (SEQ ID NO: 1) and Rl 5'-CCCTCACATAATCTAATCTCTAAATAACC (SEQ ID NO:2); F2 5'-TTTAGAGATTAGATTATGTGAGGGTT (SEQ ID NO:3) and R2 5'-AAACTCTCCCTTTACATAAAACCACCTAC (SEQ ID NO:4).

[0118] The length of DNA assayed extended slightly beyond the CpG island, encompassing a total of 86 CpG residues. There were no CpG residues in the overlapping portion of the two amplicons and subsequently the CpG residues were labeled 1 to 86, 5' to

3' (Figure 1). Methylation analysis involved a non-random cleavage of nucleic acid into fragments often encompassing more than one CpG dinucleotide residue. Each cleavage fragment yielded a single methylation level and was therefore considered as a single data point - labeled a CpG unit. The 86 CpG residues were assayed as 36 CpG units, labeled consecutively from 1-36 (Figure 2). For data analytic purposes, the 36 CpG units were clustered based on geographic proximity. The classification of the 86 residues into 36

CpG units and then 6 analytic clusters is presented in Figure 2. Clusters 1-3 had missing methylation data on 5/150 participants. Clusters 4-6 had missing methylation data on 24/150 participants.

Analysis

[0119] AU analyses were conducted using STATA8 (StataCorp, STATA. Release 8.0 ed.

Stata Corporation: College Station, TX, 2003). The absence of αpriori data on differences in 5HTT promoter methylation levels between depressed and non-depressed groups made planning a nested case-control study difficult. Power calculations were based on a substantial difference between groups, on the assumption that a substantial difference in methylation would more likely translate into a clinical effect. Assuming a difference in methylation status of 35% or greater (15% cases cf. 50% controls), it was determined that at least 20 cases and 100 controls would be needed to achieve power and precision of 80% and 5%, respectively. The final analysis was based on 25 cases and 125 controls.

[0120] Analyses were conducted separately for seven different classifications of 5HTT promoter methylation. The first classification was based on the mean methylation level (density) of the promoter-associated CpG island, then separately for methylation levels within the analytic clusters of CpG units determined by geographic proximity (Figure 2). Examination of methylation density distributions for the entire sample indicated that an effective way of capturing variation was to tertile the methylation distribution.

[0121] Logistic regression was used to examine association between (1) the 5HTTLPR and methylation textiles; and (2) methylation tertiles and adolescent depression status (as per the case-control design of the study). Staged adjustment for functional polymorphisms in related neuro-behavioral pathways (MAOA, COMT, DRD4) was conducted to test the robustness of association. Interaction was examined using the likelihood ratio test.

Further investigation of interaction was done by comparing those with at least one copy of the S-allele carriers and in the top tertile of 5HTT promoter methylation (LS/SS +

HighMeth) with the remaining sample. Association between the 5HTTLPR and methylation status was confirmed using analysis of variance based on the log transformation of the continuous methylation distributions for each cluster. All analyses were adjusted for sex. EXAMPLE 1 Methylation densities

[0122] Table 4 presents 5HTT promoter methylation (with 95% confidence intervals) for the entire CpG island and the 6 analytic clusters defined within the island.

TABLE 4

Percentage 5HTT methylation for complete CpG island and six clusters within the island

Cluster n % 95% CI

All 145 6.3 (5.8- 6.8)

1 145 4.2 (3.7- 4.6)

2 145 5.6 (5.3- 6.0)

3 145 4.0 (3.6- 4.5)

4 126 . 4.3 (3.8- 4.9)

5 126 8.7 (7.8-9.6)

6 126 8.0 (6.1- 9.9)

EXAMPLE 2 Association between 5HTTLPR and 5HTT methylation

[0123] Based on data from the entire 5HTT promoter region, it was found that the odds of being in the top tertile of 5HTT promoter methylation were notably reduced among carriers of S-allele genotypes (two-fold reductions per allele). On stratification by analytic cluster, it was observed that this association was being driven by effects within cluster 5 only (see Table 5).

TABLE 5

Association between increasing copies of the 5HTTLPR short allele and 5HTT promoter methylation density

5HTT 5HTTLPR

Cluster Methylation L/L L/S s/s OR¹ 95% CI P

All low 27 46 24 1.0 high 21 21 6 0.56 (0.34- 0.93) 0.026

1 low 32 51 24 1.0 high 16 16 6 0.69 (0.41- 1.2) 0.166

2 low 32 45 20 1.0 high 16 22 10 0.99 (0.61- 1.6) 0.966

3 low 39 51 25 1.0 high 9 16 5 1.0 (0.57- 1.7) 0.988

4 low 30 35 21 1.0 high 13 21 6 0.86 (0.51- 1.5) 0.582

5 low 22 40 22 1.0 high 21 16 5 0.47 (0.27- 0.82) 0.008

6 low 26 37 21 1.0 high 17 19 6 0.65 (0.38- 1.1) 0.111 [0124] Stratification by 5HTTLPR genotype showed that the odds of being in the top tertile of 5HTT promoter methylation decreased with additional copies of the S-allele in a dose dependent manner. Association remained after adjusting for background functional mutations in related pathways (see Table 6). There was association between COMT Met¹⁵⁸ allele and 5HTT promoter methylation.

TABLE 6

Association between 5HTTLPR and 5HTT promoter methylation density at cluster 5, with and without adjustment for selected functional loci in related pathways

5HTT Methylation (Cluster 5)

Model 1 Model 2

OR 95% CI P OR 95% CI P

5HTTLPR LL 1.0 1.00

LS 0.43 (0.19 -0.99) 0.048 0.38 (0.15 -0.95) 0.039

SS 0.24 (0.08 -0.75) 0.014 0.28 (0.08 -0.93) 0.038

COMT 0.49 (0.27 -0.90) 0.021

MAOA 1.2 (0.51 -3) 0.642

DRD4 1.2 (0.47 -3) 0,730

EXAMPLE 3 Association with persistent depressive symptoms during adolescence

[0125] No univariate association was observed between 5HTTLPR genotypes and PDS (OR = 1.3, 95% CI = 0.70-2.4, p = 0.396). However, the odds of reporting PDS during adolescence were substantially increased (~four-fold) among those with the highest level of 5HTT promoter methylation within analytic cluster 5 only (see Table 7). Association was not observed for any other cluster, or for the entire region.

TABLE 7

Association between 5HTT promoter methylation density and persistent depressive symptomatology

5HTT Depressive symptoms

Cluster Methylation none >5wvs OR 95% CI P

_ low 86 37 1.0 high 11 11 2.2 (0.86- 5.8) 0.100

1 low 88 35 1.0 high 19 3 0.4 (0.10- 1.3) 0.126

2 low 82 41 1.0 high 15 7 0.83 (0.30- 2.3) 0.723

3 low 98 25 1.0 high 17 5 1.5 (0.48- 4.9) 0.467

4 low 76 30 1.0 high 10 10 2.4 (0.87- 6.7) 0.092

5 low 75 31 1.0 high 9 11 3.8 (1.3- 11) 0.013

6 low 74 32 1.0 high 10 10 1.8 (0.66- 5.0) 0.246 [0126] When 5HTTLPR and 5HTT promoter methylation variables were modeled simultaneously: (1) the strength of association between 5HTT promoter methylation and PDS increased from four-fold to six-fold; and (2) an association between S-allele genotypes and PDS became apparent (two-fold increase per S-allele) [see Table 8], Furthermore, there was evidence of interaction between S-allele genotypes and 5HTT promoter methylation (likelihood ratio test, tf = 4.4, p = 0.035). However, stratified sample sizes were small, preventing adjustment for related genetic exposures and necessitating cautious interpretation of results.

TABLE 8

Association between 5HTTLPR and depressive symptoms stratified by low and high C5 methylation density, with and without adjustment for selected functional loci in related pathways

>5 wvs Depressive Symptomatology

Model 1 Model 2 Model 3

OR 95% CI P OR 95% CI p OR 95% CI P

5HTTLPR 1.3 (0.7 -2.5) 0.396 2.3 (1.0 -5.2) 0.040 2.5 (1.1 -5.9) 0.031

C5 Methylation 6.3 (1.8 -21) 0.003 5.7 (1.6 -20) 0.007

COMT 0.66 (0.30 -1.5) 0.300

MAOA 0.68 (0.21 -2.2) 0.526

DRD4 2.8 (0.79 -9.8) 0.110

[0127] To explore interaction effects within the limits of the sample size, some cells were strategically collapsed to create a composite 5HTTLPR/5HTT promoter methylation exposure group (see Table 9). There was no elevation in PDS among carriers of S-allele genotypes with the lowest levels of 5HTT promoter methylation (bottom two tertiles). However, PDS was substantially elevated (-seven-fold) among carriers of S-allele genotypes with the highest levels of 5HTT promoter methylation (top tertile). These data suggest that relationships between 5HTTLPR and 5HTT promoter methylation may not be linear. TABLE 9

Association between carriers of a combined risk factor (S-allele genotypes+High C5 methylation) and depressive symptoms, with and without adjustment for selected functional loci in related pathways

>5 wvs Depressive Symptomatology

Model 1

5HTTLPR C5 methylattion OR 95% CI P

LL Low 1.0

LS or SS Low 1.2 (0.1 -15) 0.872

LL High 1.0

LS or SS High 7.2 (2.3 -23) 0.001

Note: Estimates adjusted for sex, COMT, MAOA, DRD4

EXAMPLE 4

Association between 5HTTLPR and 5HTT methylation on depressive symptoms

[0128] The data herein demonstrate an association between 5HTTLPR allelic variation and 5HTT promoter methylation levels. Additional copies of the S-allele are associated with reduced 5HTT promoter methylation. This suggests a negative feedback loop in which the transcription limiting effects of the 5HTT S-allele maybe offset by transcription enhancing effects involving reductions in 5HTT promoter methylation.

[0129] No association was found between 5HTTLPR S-allele genotypes and depression in a univariate model. When methylation was taken into account the odds of observing persistent depressive symptoms doubled with additional copies of the S-allele. More detailed investigations showed that the effect of S-allele genotypes was no longer evident among those with low 5HTT promoter methylation (i.e, those with least compromised 5HTT transcription), but intensified among those who had high 5HTT promoter methylation (i.e, those with most compromised 5HTT transcription). Thus, it is proposed that the relationship between epigenetic and genetic processes in creating risk for depressive symptoms is better described by a quadratic (U-shaped) function than a simple linear function.

[0130] Greater understanding of epigenetic programming is important for at least two reasons. First, epigenetic programming is a result of environmental exposures that are potentially modifiable in prevention settings. Second epigenetic programming is reversible offering the promise of more effective medication treatments. The early (infant) emotional environment may be important (Weaver et al, Nature Neuroscience 7(8): 847-854, 2004). To the extent to which 5HTT promoter methylation is subject to change across the life course, environmental exposures across other watershed periods in human development, including puberty and menopause, may also represent critical windows within which methylation levels may be subject to changes and re-set. [0131] Associations between 5HTTLPR S-alleles and 5HTT promoter methylation have been previously reported although the findings have been inconsistent. One explanation of the conflicting observations is methodological: study design and phenotype definition being important reasons for non-replication (Olsson et al, 2007 supra).

[0132] Methylation of CpG islands at gene promoters is thought to assist in gene silencing by blocking the binding of transcriptional activators and/or by recruiting a complex of silencing factors to the promoter (Craig, Bioassays 27:17-28, 2005). However, little is known about factors controlling 5HTT promoter methylation, or the consequences of 5HTT promoter methylation.

[0133] Brain pathways involved in mood and cognition - such as the serotinergic system - are arguably systems with tight homeostatic limits requiring sensitive regulation. One form of regulation is via epigenetic involving methylation of CpG islands.

[0134] The present invention identifies the relationship between 5HTTLPR genotypes and 5HTT promoter methylation and the relationship of each to persistent depression.

[0135] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. BIBLIOGRAPHY

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Claims

CLAIMS:

1. A method for identifying an epigenetic profile in a subject associated with a mental health condition, said method comprising determining the presence or absence or extent of DNA methylation of the 5HTT promoter region and/or the level of expression of the 5HTT gene alone or in combination with the presence or absence or number of S-allelic forms of the 5HTT gene wherein a DNA methylated 5HTT promoter region or reduced level of activity of 5HTT with or without an S-allele is indicative of a subject being at risk of developing such a mental health condition.

2. A method for identifying an epigenetic and genetic profile in a subject associated with a mental health condition, said method comprising determining the presence or absence or extent of DNA methylation of the 5HTT promoter region and/or the level of expression of the 5HTT gene alone or in combination with the presence or absence or number of S-allelic forms of the 5HTT gene wherein a DNA methylated 5HTT promoter region or reduced level of activity of 5HTT with or without an S-allele is indicative of a subject being at risk of developing such a mental health condition.

3. The method of Claim 1 or 2 wherein the mental health condition is a psychiatric, neurological or psychological condition phenotype or state.

4. The method of any one of Claims 1 to 3 wherein the 5HTT promoter region is the 5HTT promoter.

5. The method of any one of Claims 1 to 3 wherein the 5HTT promoter region is a region spanning up to 10kb up or downstream of the transcription initiation site of the 5HTT gene.

6. The method of any one of Claims 1 to 5 wherein the extent of DNA methylation of the 5HTT promoter region is greater than lower level methylation when considered in the bottom of population distribution of 5HTT methylation wherein the bottom group is equal to or less than about 6% + 5% DNA methylation.

7. A method for profiling or stratifying an individual or group of individuals for a level of risk in developing a mental health condition, said method comprising determining the presence or absence of or extent of DNA methylation of the 5HTT promoter region and/or level of 5HTT gene expression wherein:

8. The method of Claim 7 wherein the mental health condition is a psychiatric, neurological or psychological condition phenotype or state.

9. The method of Claim 7 or wherein the 5HTT promoter region is the 5HTT promoter.

10. The method of Claim 7 or 8 wherein the 5HTT promoter region is a region spanning up to 10kb up or downstream of the transcription initiation site of the 5HTT gene.

11. The method of any one of Claims 1 to 10 wherein the mental health condition is selected from the list consisting of persistent depressive disorder (PDS), depression including major depressive disorder, dysthymia and episodic depressive symptomatology, drug addiction including to prescribed licit and illicit substances, eating disorders including obesity, anorexia nervosa and bulimia, anxiety disorders including simple phobias, social phobias, generalized anxiety disorder, panic disorder (with and without agoraphobia) and OCD and bipolar disorder, schizophrenia, personality disorder including schizoid, narcissistic and paranoid personality features, acute stress disorder, adjustment disorder, schizoaffective disorder, schizoid personality disorder, schizophreniform disorder and schizotypal personality disorder.

12. The method of Claim 11 wherein the mental health condition is persistent depression symptomatology (PDS).

13. The method of Claim 11 wherein the mental health condition is depression.

14. The method of any one of Claims 1 to 13 wherein an at risk individual comprises a DNA methylated 5HTT promoter and one or more S-alleles of 5HTTLPR.

15. The method of Claim 14 wherein the 5HTT promoter DNA is methylated at from about 1 to 86 CpG sites.

16. The method of Claim 1 or 2 or 7 wherein the individual is an adolescent.

17. A therapeutic protocol comprising determining the presence or absence or extent of DNA methylation of the 5HTT promoter region and/or level of expression of the 5HTT gene wherein:

(i) an individual with lower levels of a methylated 5HTT promoter region DNA within the bottom of the population distribution of 5HTT methylation is considered at a lower risk of mental health condition; wherein the bottom of population distribution of 5HTT DNA methylation is defined at equal to or less than 6% plus or minus 5% DNA methylation;

(ii) an individual with a methylated 5HTT promoter region DNA and/or exhibiting reduced 5HTT gene expression alone or in combination with an S-allelic form of the 5HTT gene is more likely of developing said mental health condition compared to individuals in group (i); and

(iii) an individual with a highly methylated 5HTT promoter region DNA and/or exhibiting reduced 5HTT gene expression alone or in combination with multiple S-allelic forms of the 5HTT gene is more at risk of developing said mental health condition compared to the individuals in group (iii);

18. The protocol of Claim 17 wherein the mental health condition is a psychiatric, neurological or psychological condition phenotype or state.

19. The protocol of Claim 17 or 18 wherein the 5HTT promoter region is the 5HTT promoter.

20. The protocol of Claim 17 or 18 wherein the 5HTT promoter region is a region spanning up to 10kb up or downstream of the transcription initiation site of the 5HTT gene.

21. The protocol of Claim 17 or 18 or 19 or 20 wherein the mental health condition is selected from the list consisting of PDS, depression including major depressive disorder, dysthymia and episodic depressive disorder, drug addiction including to prescribed licit and illicit substances, eating disorders including obesity, anorexia nervosa and bulimia, anxiety disorders including simple phobias, social phobias, generalized anxiety disorder, panic disorder (with and without agoraphobia) and OCD, bipolar disorder, schizophrenia, personality disorder including schizoid, narcissistic and paranoid personality features, acute stress disorder, adjustment disorder, schizoaffective disorder, schizoid personality disorder, schizophreniform disorder and schizotypal personality disorder.

22. The protocol of Claim 21 wherein the mental health condition is PDS.

23. The protocol of Claim 21 wherein the mental health condition is depression.

24. . The protocol of any one of Claims 17 to 23 wherein an at risk individual comprises a DNA methylated 5HTT promoter and one or more S-alleles of 5HTTLPR.

25. The protocol of Claim 24 wherein the 5HTT promoter is DNA methylated at from about 1 to 86 CpG sites.

26. Use of a data set comprising information on the presence or extent of DNA methylation of the 5HTT promoter region and/or levels of 5HTT or 5HTT gene expression and the presence or absence of an S-allelic form of the 5HTT gene in the generation of a risk factor analysis of an individual developing a mental health condition.

27. A therapeutic and prophylactic protocol for individuals or groups of individuals at risk of developing a mental health condition, said protocol comprising identifying at risk individuals by determining the presence or absence or extent of DNA methylation of the 5HTT promoter region and/or level of expression of the 5HTT gene alone or in combination with an S-allelic form of the 5HTT gene wherein a DNA methylated 5HTT promoter region or reduced 5HTT gene expression with or without an S-allele is indicative of a subject being at risk of developing such a mental health condition and then subjecting the individual to therapeutic intervention and/or monitoring, said intervention comprising the administration of medicaments and/or behavioral, social, environmental or psychological modification.

28. A therapeutic protocol comprising stratifying or profiling individuals or groups of individuals by determining the presence or absence of or extent of DNA methylation of the 5HTT promoter region and/or level of expression of the 5HTT gene wherein:

(i) an individual without a DNA methylated 5HTT promoter region or having a low level DNA methylation is considered at a lower risk of developing the mental health condition;

(ii) an individual with a methylated 5HTT promoter region DNA and/or exhibiting reduced 5HTT gene expression alone or in combination with an S-allelic form of the 5HTT gene is more likely of developing the mental health condition compared to individuals in group (i); and

(iii) an individual with a highly methylated 5HTT promoter DNA and/or exhibiting reduced 5HTT gene expression alone or in combination with multiple S-allelic forms of the 5HTT gene is more at risk of developing the condition, state or phenotype compared to the individuals in group (iii);

29. The protocol of Claim 27 or 28 wherein the mental health condition is a psychiatric, neurological or psychological condition phenotype or state.

30. The protocol of Claim 27 or 28 or 29 wherein the 5HTT promoter region is the 5HTT promoter.

31. The protocol of Claim 27 or 28 or 29 wherein the 5HTT promoter region is a region spanning up to 10kb up or downstream of the transcription initiation site of the 5HTT promoter.

32. The protocol of Claim 30 or 31 wherein the extent of DNA methylation of the 5HTT promoter region is greater than lower level methylation when considered in the bottom of population distribution of 5HTT methylation wherein the bottom group is equal to or less than about 6% + 5% DNA methylation.

33. A method of treating a mental health condition in an individual, the method comprising identifying an at risk individual and subjecting the individual to gene therapy or medical or psychological interventional to facilitate amelioration the symptoms and/or to prevent development of the symptoms.

34. A method for determining the likelihood of a subject responding favorably to a particular drug in the treatment of a mental health condition said method comprising obtaining or extracting a nucleic acid sample from cells of said individual and screening for or otherwise detecting the presence or extent of DNA methylation of the 5HTT promoter region and/or level of expression alone or in combination with the presence or absence of an S-allelic form of the 5HTT gene wherein a drug which increases expression of the 5HTT gene is deemed more likely to be effective than a drug which inhibits 5HTT gene expression or which does not target 5HTT.

35. A method for determining the likelihood of a subject responding favorably to an intervention in the treatment of a mental health condition, the intervention including behavioral, environmental, social or psychological intervention, said method comprising obtaining or extracting a nucleic acid sample from cells of said individual and screening for or otherwise detecting the presence or extent of DNA methylation of the 5HTT promoter region and/or level of expression alone or in combination with the presence or absence of an S-allelic form of the 5HTT gene wherein an intervention which increases expression of the 5HTT gene is deemed more likely to be effective than an intervention which inhibits 5HTT gene expression.

36. The method of Claim 34 or 35 wherein the mental health condition is a psychiatric, neurological or psychological condition phenotype or state.

37. The method of any one of Claims 34 to 36 wherein the 5HTT promoter region is the 5HTT promoter.

38. The method of any one of Claims 34 to 36 wherein the 5HTT promoter region is a region spanning up to 1 Okb up or downstream of the transcription initiation site of the 5HTT gene.

39. A method according to any one of the preceding claims wherein reduced expression of 5HTT genetic locus is due to histone modification and/or non-coding RNA binding.