# Patent

## Title
Genes associated with posttraumatic-stress disorder (PTSD)

## Abstract

The present invention relates to a method of identifying a predisposition for developing posttraumatic stress disorder (PTSD) in a subject comprising assessing in a sample obtained from said subject the expression level of one or more genes selected from the FK506 binding protein 5 (FKBP5) gene, the signal transducer and activator of transcription (STAT5B) gene and the nuclear factor I/A (NFIA) gene, wherein a decrease in the expression level of said one or more genes as compared to the expression level of the corresponding gene(s) of a control is indicative of a predisposition for developing PTSD. Further, the invention relates to a method of identifying a compound capable of preventing or treating PTSD or capable of serving as a lead compound for developing a compound capable of preventing or treating PTSD and also to a method of selecting a therapy to prevent or treat PTSD.

## Background
RELATED APPLICATION
This application claims the benefit of and priority to European application serial number 08016126.8 (EP) filed on 12 Sep. 2008 and International application serial number PCT/EP2009/061890 filed on 14 Sep. 2009 the contents of which are herein incorporated by reference in their entirety for any and all purposes.
SEQUENCE LISTING
The instant application contains a Sequence Listing that has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 2, 2009, is named VOS0001US.txt, and is 114,310 bytes in size.
The present invention relates to a method of identifying a predisposition for developing posttraumatic stress disorder (PTSD) in a subject comprising assessing in a sample obtained from said subject the expression level of one or more genes selected from the FK506 binding protein 5 (FKBP5) gene, the signal transducer and activator of transcription (STAT5B) gene and the nuclear factor I/A (NFIA) gene, wherein a decrease in the expression level of said one or more genes as compared to the expression level of the corresponding gene(s) of a control is indicative of a predisposition for developing PTSD. Further, the invention relates to a method of identifying a compound capable of preventing or treating PTSD or capable of serving as a lead compound for developing a compound capable of preventing or treating PTSD and also to a method of selecting a therapy to prevent or treat PTSD.
In this specification, a number of documents including patent applications and manufacturer's manuals are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.
The risk for the individual to get exposed by life-threatening events like natural disasters, accidents, attacks, assaults and other acts of violence, not to mention terrorist attacks, has considerably increased during the past decades. All these incidents can trigger the development of stress-related disorders. This includes the so called posttraumatic-stress disorder (PTSD), which is characterized by persistent and frightening re-experiencing of the traumatic event, accompanied by severely impairing sleep disturbances and avoidance behaviour. Besides PTSD, which is directly linked to a specific traumatic experience, also anxiety disorders and depression can be triggered by adverse events.
Posttraumatic stress disorder (PTSD) occurs in only a small proportion of those exposed to traumatic events. Several risk factors have been implicated in the development of PTSD. These include age at traumatization, gender, earlier childhood exposures (i.e., “pre-traumatic”) to adversity, personality characteristics, and familial psychopathology, including PTSD (Yehuda R, LeDoux J, Neuron 2007; 56: 19-32). There is evidence from twin studies that genetic factors contribute to the risk for PTSD (Stein et al., Am J Psychiatry 2002; 159: 1675-81; True et al., Arch Gen Psychiatry 1993; 50: 257-264; Xian et al., Drug Alcohol Depend 2000; 61: 95-102). However, susceptibility genes have not been confirmed in PTSD (Broekman et al., Neurosci Biobehav Rev 2007; 31: 348-62).
PTSD is on the molecular level characterized by a disturbed regulation of the primary stress-hormone system, the so-called HPA (hypothalamic-pituitary-adrenal) axis. The hypothalamic-pituitary-adrenal (HPA) axis is the major constituent of the neuroendocrine response to acute and chronic stress, resulting in the release of corticotropin releasing hormone (CRH) and vasopressin (AVP) from the parvocellular neurons of the hypothalamus into the portal vessels system to activate the synthesis and release of ACTH from the anterior pituitary. In turn, ACTH stimulates the adrenal cortex to synthesize and release glucocorticoids, in particular cortisol (de Kloet et al., Nat Rev Neurosci 2005; 6: 463-75). In PTSD, the fine-tuned regulation of this HPA axis is disturbed, which is indicated by reduced levels and an exaggerated responsiveness of ACTH and cortisol in these patients. Enhanced responsiveness of the glucocorticoid receptor (GR) appears to underlie these disturbances. These findings contrast with observations of greater cortisol levels and reduced GR responsiveness associated with major depression (Ribeiro et al., Am J Psychiatry 1993; 150: 1618-29; Holsboer F, Neuropsychopharmacology 2000; 23: 477-501).
Though initially interpreted as reflecting enduring consequences of trauma exposure, there has been reason to suspect that HPA axis alterations in PTSD reflect pre-traumatic risk factors. In several longitudinal studies, lower cortisol levels in the acute aftermath of trauma were associated with either the subsequent development of PTSD, or with the well-established risk factor of prior trauma exposure (e.g., childhood traumatisation) (Yehuda et al., Am J Psychiatry 2005; 162: 998-1000; Neylan et al., Psychoneuroendocrinology 2005; 30: 373-8; Oquendo et al., Neuropsychopharmacology 2003; 28: 591-8; King et al., Psychiatry Clin Neurosci 2001; 55: 71-4; Heim et al., Psychoneuroendocrinology 2000; 25: 1-35; Yehuda et al., Biol Psychiatry 1996; 40: 79-88; Lemieux A M, Coe C L, Psychosom Med 1995; 57: 105-15; Glover D A, Poland R E, Psychoneuroendocrinology 2002; 27: 805-19; Young E A, Breslau N, Arch Gen Psychiatry 2004; 61: 394-401; Pitman R K, Orr S P, Biol Psychiatry. 1990; 27: 245-7; Baker et al., Am J Psychiatry 2005; 162: 992-4). Recent studies of infant and adult children of parents with PTSD also support the idea that both low cortisol levels, and glucocorticoid responsiveness are risk factors for PTSD (Yehuda et al., J Clin Endocrinol Metab 2005; 90: 4115-8; Yehuda R, Bierer L M, Prog Brain Res 2008; 167: 121-35). These findings raised the possibility that reduced cortisol levels at the time of a traumatic exposure may compromise the inhibition of stress-induced biologic responses (e.g., during and following a traumatic event), resulting in prolonged physiological/emotional distress which would then facilitate the development of PTSD (Yehuda R, N Engl J Med 2002; 346: 108-14). Accordingly, the identification of biological processes that underpin enhanced GR responsiveness and/or lower cortisol levels are of particular interest. Indeed, it was recently observed that adults who experienced child abuse and have genetic variations in the FKBP5 gene (involved in the regulation of the GR) appear to be at greater risk of PTSD symptoms as adults (Binder et al., JAMA 2008; 299: 1291-305).
There have been two other studies examining gene expression following trauma exposure. In a study of persons exposed to severe trauma encountered in the emergency room who either did (n=8), or did not (n=6) meet criteria for PTSD at both a 1 month and 4 month follow-up, gene expression changes associated with several interesting molecular categories related to the stress response (Segman et al., Mol Psychiatry 2005; 10: 500-13, 425). Interestingly, the expression of FKBP5 was shown to be upregulated in this study. Given the proximity to trauma exposure, however, the findings may have reflected biological changes associated with recovering from the effects of trauma exposure, rather than with the development or persistence of chronic PTSD. A whole blood gene expression profiling study of 16 subjects (n=8 with PTSD) exposed to the Ramstein Airshow tragedy (16 years ago) attempted to examine processes associated with very chronic PTSD. This study used a special microarray chip modified to specifically detect genes associated with the immune and stress responses (Zieker et al., Mol Psychiatry 2007; 12: 116-8). Interestingly, little overlap in gene expression was reported in the two studies. Both studies reported on very few subjects, and neither study examined risk factors other than exposure to the focal trauma.
Even though effective psychopharmacological treatments and psychotherapeutic programs are available for PTSD, their efficiency is limited by the fact that they will not be initiated until the full-blown symptoms of the stress-related disorder-have occurred. At this stage of the disorder, the patients are already severely impaired, and it takes long time until full remission of the symptoms can be achieved.
The technical problem underlying the present invention was to identify improved means and methods for diagnosing PTSD.
The solution to this technical problem is achieved by providing the embodiments characterized in the claims.
Accordingly, the present invention relates in a first embodiment to a method of identifying a predisposition for developing posttraumatic stress disorder (PTSD) in a subject comprising assessing in a sample obtained from said subject the expression level of one or more genes selected from the FK506 binding protein 5 (FKBP5) gene, the signal transducer and activator of transcription (STAT5B) gene and the nuclear factor I/A (NFIA) gene, wherein a decrease in the expression level of said one or more genes as compared to the expression level of the corresponding gene(s) of a control is indicative of a predisposition for developing PTSD.
The term “predisposition for a disease” is established in the art and used herein analogously. The term “predisposition for developing posttraumatic stress disorder (PTSD)” as used in accordance with the present invention describes the status of a patient at risk to develop said disease once exposed to a trauma. A predisposition may be based on environmental, genetic and/or epigenetic factors. As used herein, the predisposition to develop PTSD is based on epigenetic factors affecting the expression of genes. Said predisposition to develop PTSD may be diagnosed according to the method of the invention upon exposure to a traumatic event as well as prior to exposure to said event.
“Posttraumatic stress disorder” as used herein relates to a condition characterized by the development of characteristic symptoms following exposure to a traumatic stressor such as direct personal experience of an event that involves actual or threatened death or serious injury, or other threat to one's physical integrity; or witnessing an event that involves death, injury, or a threat to the physical integrity of another person; or learning about unexpected or violent death, serious harm, or threat of death or injury experienced by a family member or other close associate. The person's response to the event generally involves intense fear, helplessness, or horror. In children, the response generally involves disorganized or agitated behavior. The characteristic symptoms resulting from the exposure to the extreme trauma include persistent reexperiencing of the traumatic event, persistent avoidance of stimuli associated with the trauma and numbing of general responsiveness, and persistent symptoms of increased arousal. In Posttraumatic Stress Disorder, the stressor is generally of extreme nature (c.f. above). In contrast, in Adjustment Disorder, the stressor can be of any severity. The diagnosis of Adjustment Disorder is appropriate both for situations in which the response to an extreme stressor does not meet the criteria for Posttraumatic Stress Disorder (or another specific mental disorder) and for situations in which the symptom pattern of Posttraumatic Stress Disorder occurs in response to a stressor that is not extreme (e.g., spouse leaving, being fired). Symptoms of avoidance, numbing, and increased arousal that are present before exposure to the stressor do not meet criteria for the diagnosis of Posttraumatic Stress Disorder and require consideration of other diagnoses (e.g., Brief Psychotic Disorder, Conversion Disorder, Major Depressive Disorder), these diagnoses should be given instead of, or in addition to, Posttraumatic Stress Disorder. Acute Stress Disorder is distinguished from Posttraumatic Stress Disorder because the symptom pattern in Acute Stress Disorder must occur within 4 weeks of the traumatic event and resolve within that 4-week period. If the symptoms persist for more than 1 month and meet criteria for Posttraumatic Stress Disorder, the diagnosis is changed from Acute Stress Disorder to Posttraumatic Stress Disorder. For example, severity of PTSD symptoms can be evaluated by using the Modified PTSD Symptom Scale (PSS) (Coffey et al., J Trauma Stress (1998), 11: 393-399; Falsetti et al., Behav Therapist (1993), 16: 161-162).
The term “expression level of one or more genes” as used in accordance with the method of the invention relates to the degree of gene expression of one or more genes. The term “gene expression” relates to a process by which information from a nucleic acid (e.g., DNA or RNA) sequence such as, e.g., a gene, is processed into a gene product (e.g., RNA or peptide/protein). Said process can be subdivided into a transcriptional and a translational process. Transcription describes in the context of gene expression the process of transcribing DNA into mRNA, whereas translation describes the process of translating mRNA into a peptide or protein. The term “peptide” as used herein describes a group of molecules consisting of up to 30 amino acids, whereas “proteins” consist of more than 30 amino acids. Peptides and proteins may further form dimers, trimers and higher oligomers, i.e. consisting of more than one molecule which may be identical or non-identical. The corresponding higher order structures are, consequently, termed homo- or heterodimers, homo- or heterotrimers etc. The terms “peptide” and “protein” (wherein “protein” is interchangeably used with “polypeptide”) also refer to naturally modified peptides/proteins wherein the modification is effected e.g. by glycosylation, acetylation, phosphorylation and the like. Such modifications are well-known in the art. In accordance with the invention, the expression level of one or more of the genes selected from FKBP5, STAT5B and NFIA and combinations thereof such as FKBP5, STAT5B and NFIA; FKBP5 and STAT5B; FKBP5 and NFIA; or STAT5B and NFIA can be assessed.
The assessment of “expression levels”, i.e. of the degree of expression, is the decisive factor in the process of diagnosing a risk to develop PTSD in accordance with method of the present invention. The term “level” as used in connection with gene expression in accordance with the present invention relates to the readout of any quantitative measure of gene expression. Said measure is dependent on, preferably at least roughly proportional to the number of mRNA molecules or peptide or protein molecules, hence allowing a quantitative assessment of gene expression. Depending on the means and methods employed for said quantitative measure the determined level of gene expression may be in the form of a variety of readout parameters, e.g., the intensity of a radioactivity signal (e.g., Northern blot with a radioactive probe), of a fluorescent signal (e.g., DNA microarray) or the mass-to-charge ratio (e.g., mass spectrometry). Means and methods that allow to quantitatively determine gene expression levels are well known in the art and non-limiting examples are further detailed herein below. The expression levels of genes generally and normally vary from subject to subject depending on age, sex and/or condition, inter alia, to a certain extent. Variations to be considered not normal, e.g. variations linked to a disease, have to be statistically significant when compared to a suitable control population.
Generally speaking, in healthy subjects expression levels are expressed at certain ratios contributing in concerted action to normal body functions. Disturbances, such as an upregulation and/or downregulation of one or more genes, in gene expression levels may lead to a medical condition if endogenous rescue mechanisms do not exist that can compensate for said disturbances. Several steps in the gene expression process may be modulated to regulate the expression level of a gene such as, e.g. the step of initiating the transcription process involving the amount of transcription factors, the presence of so called operons (e.g. lac operon) or enhancer/inhibitory sequences, the step of translation involving the half-life of the mRNA to be translated, the posttranslational modification of a protein having an effect on the half-life, the secretion, the folding of the latter. Gene regulation gives cells control over structure and function and is the basis for events like differentiation, morphogenesis or adaptability of a cell or a multicellular organism. In summary, the assessment of expression levels may prove in some cases to be a suitable tool for diagnosing or predicting a medical condition.
In the present case, the expression level of the genes FKBP5, STAT5B and NFIA were surprisingly found to be disturbed, i.e. downregulated, in subjects having PTSD.
The skilled person is well aware of the molecular details involved in gene expression and there exists ample literature in said field (cf., e.g., Lodish et al., Molecular Cell Biology; W.H. Freeman & Co, New York; Alberts et al., Molecular Biology of the Cell, Garland Science, New York and London; G. M. Cooper, The Cell—A Molecular Approach, Sinauer Associates, Inc. Sunderland (Ma); Griffiths et al., Modern Genetic Analysis, W.H. Freeman, New York). Methods for “assessing” gene expression are also well-known to the person skilled in the art and part of the latter referenced textbooks.
The human FK506 binding protein 5 (FKBP5) gene located on chromosome 6 (6p21.3-p21.2) encodes the FK505 binding protein 5. This protein is a member of the immunophilin protein family, which play a role in basic cellular processes involving protein folding and trafficking. FK506-binding protein 5 is a so-called co-chaperone molecule interacting with the heat-shock protein 90 in an inactive glucocorticoid receptor complex. It is a negative regulator of glucocorticoid action by reducing the binding affinity of glucocorticoid receptors (Denny et al., Endocrinology 2000; 141: 4107-113). Preferably, the FKBP5 gene has a coding sequence as shown in SEQ ID NO: 1. However, as also variant or homologous alleles of the FKBP5 gene may exist the gene can also have a coding sequence being at least 95% identical to the sequence of SEQ ID NO:1 such as, being at least 96%, or preferably 97%, more preferred 98% and most preferred at least 99% identical.
The signal transducer and activator of transcription (STAT5B) gene located on chromosome 17 (17q11.2) encodes the STAT5B protein. This protein is a member of the STAT family of transcription factors and mediates the signal transduction triggered by various cell ligands, such as IL2, IL4, CSF1, and different growth hormones. This protein acts as a direct inhibitor of the nuclear translocation of activated glucocorticoid receptors (Goleva et al., J Immunol 2002; 169: 5934-40; Biola et al., Mol Endocrinol 2001; 15: 1062-76) thus reducing the effects of activated glucocorticoid receptors. Preferably, the STAT5B gene has a coding sequence as shown in SEQ ID NO: 2. However, as also variant or homologous alleles of the STAT5B gene may exist the gene can also have a coding sequence being at least 95% identical to the sequence of SEQ ID NO:2 such as, being at least 96%, or preferably 97%, more preferred 98% and most preferred at least 99% identical.
The nuclear factor I/A (NFIA) gene located on chromosome 1 (1p31.3-2) encodes the NFIA protein. This protein is a member of the Nuclear factor I (NFI) protein family, which are dimeric DNA-binding proteins with similar, and possibly identical, DNA-binding specificity. They function as cellular transcription factors and as replication factors for adenovirus DNA replication. The NFIA protein modulates the effects of activated glucocorticoid receptors at their target structures (Hebbar, Archer, J Biol Chem 2007; 282: 8284-91). Preferably, the NFIA gene has a coding sequence as shown in SEQ ID NO: 3. However, as also variant or homologous alleles of the NFIA gene may exist the gene can also have a coding sequence being at least 95% identical to the sequence of SEQ ID NO:3 such as, being at least 796%, or preferably 97%, more preferred 98% and most preferred at least 99% identical.
The designation of the genes when written out is interchangeable with the term in parentheses which is used as an abbreviation herein. For example, the FK506 binding protein 5 gene may also be described by using the term “FKBP5” or “FKBP5 gene”, the meaning is identical.
The term “sample” as used herein refers to a biological sample, such as, for example, cells, tissues (from any organ including post-mortem brain tissue), or fluids (including serum, whole blood, cerebrospinal fluid, lymph, saliva, milk, pus, urine, faeces), which has been isolated or obtained from an individual or from cell culture constituents of a cell culture comprising a subject's cells. Any tissue or liquid sample obtained from a patient and/or subject that comprises cells can be used for the assessment of expression levels of the one or more genes according to the method of the invention. It is well known in the art that nucleic acids such as, e.g., mRNA, and proteins of individuals can easily be obtained from blood samples. Thus, a preferred sample to assess gene expression levels in accordance with the method of the invention is blood. Methods for preparing the sample for nucleic acid and protein extraction are well known in the art, and can be carried out using commercially available kits such as, for example, the PAXgeneTM RNA Systems (Qiagen), RNeasyTM Kits (Qiagen) or protocols using TRIzol® reagent (Invitrogen).
In order to identify a predisposition for developing PTSD, the gene expression level(s) in a sample of a subject suspected to be predispositioned to develop PTSD are compared to the expression level(s) of the corresponding gene(s) in a control. Preferably a control is selected from i) a control subject that has been exposed to a trauma, but did not develop PTSD, ii) a group of subjects negative for PTSD and representative for the investigated population and iii) database entries. As mentioned above, a control subject has been exposed to a trauma, but did not develop PTSD. Preferred control subjects did not develop PTSD within 6 months from the exposure to trauma. Alternatively, the range of expression levels obtained from a group of subjects negative for PTSD and representative for the investigated population can be used as normal values. A “trauma” as referred to herein is the direct personal experience of an event that involves actual or threatened death or serious injury, or other threat to one's physical integrity; or witnessing an event that involves death, injury, or a threat to the physical integrity of another person; or learning about unexpected or violent death, serious harm, or threat of death or injury experienced by a family member or other close associate. Preferred is that a sample of a control subject is assessed more than once or that several samples of said control subject are obtained in order to increase the reliability of the data relating to the expression level. The data may further be pooled to calculate the mean or median and optionally the variance for each control subject. Furthermore preferred is that the expression level(s) are compared to the expression level(s) of corresponding gene(s) in samples of more than one control subject such as at least 2, 10, 20 or more preferred 50, and most preferred 100 and more control subjects. Furthermore also preferred is that the expression levels of the samples of the control subjects are pooled and the mean or median and optionally the variance is calculated. These values may, e.g., be deposited into a database as a standardized value for each gene and if required retrieved from a database, hence making the need to also experimentally assess the expression levels in a control sample every time the expression level in a patient sample is assessed dispensable. Accordingly a control may also be a database entry. Moreover, by using the variance of the expression level of the control sample, the statistical significance of deviations from the mean of controls in the sample to be assessed may be determined. Finally, and where deemed appropriate, age- or gender-specific controls may be used.
In accordance with the present invention, for the first time an association of gene expression and a predisposition to PTSD is demonstrated, wherein the genes are involved in the regulation of the HPA axis. In brief, 20 participants meeting criteria for lifetime PTSD, assessed five years after the attack on the World Trade Center (WTC), i.e. after “9/11”, and 20 participants matched with respect to severity of exposure to 9/11, age, gender, and race were recruited for the purpose of performing whole blood genome-wide expression analysis to identify altered gene activity patterns as risk factors for PTSD. Also, interactions between gene expression and PTSD severity were evaluated.
It could be demonstrated that the expression of genes encoding for FKBP5, STAT5B and NFIA proteins is differentially regulated in individuals diagnosed with PTSD in comparison to control individuals. Without being bound by a specific theory, it is considered that in response to stress, glucocorticoids are secreted from the cortex of the adrenal glands and subsequently exert diverse activities by binding at glucocorticoid receptors (GRs), which in turn are also the key regulatory element of the HPA axis. Briefly and in accordance with the description in the background section above, the hypothalamic-pituitary-adrenal (HPA) axis is involved in the neuroendocrine response to stress by triggering the release of corticotrophin releasing hormone (CRH) and vasopressin (AVP). Via a signal cascade involving, inter alia, ACTH glucocorticoids, in particular cortisol, are synthesized and released subsequently interacting with glucocorticoidreceptors (GRs). FKBP5 codes for a protein that modulates GR function by decreasing GR binding affinity for glucocorticoids, and thus reduces the sensitivity of this receptor. STAT5B and NFIA modulate the effects of GR by inhibiting their translocation to the target structure (STAT5B) or alter the effects of GR at their target structure (NFIA). In other words, FKBP5, STAT5B and NFIA are functionally related in that they modulate the activity of the HPA axis. Moreover, they act on a common pathway in that they control the sensitivity of the glucocorticoid receptors. On the molecular level, the reduced expression levels of FKBP5 and STAT5B lead to hypersensitive and hyperactive GR, which in turn results in a severe imbalance of the stress-hormone regulation. Reduced NFIA expression acts as a counterregulator of the increased GR activity by reducing GR binding at specific transcription factor sites. These findings match abnormal glucocorticoid levels frequently observed in PTSD. In this context, it is interesting to note that genetic polymorphisms in the FKBP5 gene, but not expression level alterations, have been previously identified as risk factors for the development of PTSD symptoms in children who were victims of severe accidents as well as in adults who were exposed to traumatic experiences at young age. Thus, with FKBP5, STAT5, and NFIA three genes directly involved in the actions of GR could be identified.
With the diagnostic method of the invention, an imminent risk for the development of PTSD can be identified, and medical interventions can be initiated at a very early stage of the disease process. Such a diagnostic method can be applied to individuals, who, for instance, just recently experienced a traumatic event like a natural disaster, an accident, attacks, assaults or other acts of violence. Individuals with a diminished expression level of these genes should receive immediate preventive medical attention to avoid the development of PTSD. Such a procedure will help to maintain health in many traumatized subjects who otherwise would be at risk to develop overt symptoms. Assessment of FKBP5, STAT5B, and/or NFIA gene expression levels will identify subjects who will benefit from preventive medical intervention, thus saving the health of the individual and reduce costs for the health-care system.
In a preferred embodiment of the method of the invention, additionally the expression level of one or both genes selected from the mannosidase, alpha, class 2C, member 1 (MAN2C1) gene; and DEAD box polypeptide 17 (DDX17) gene is assessed, wherein an increase in expression level said gene(s) as compared to the expression level of the corresponding gene(s) of a control is indicative of a predisposition for developing PTSD.
In a further preferred embodiment of the method of the invention, additionally the expression level of one or more further genes selected from the copine I (CPNE1) gene; tuberous sclerosis 1 (TSC1) gene; ADAM metallopeptidase domain 17 (ADAM17) gene; ring finger protein 181 (RNF181) gene; myosin IC (MYO1C) gene; MOB1, Mps One Binder kinase activator-like 1B (MOBKL1B) gene; glucosidase, beta, acid (GBA) gene; chromosome 2 open reading frame 34 (C2orf34) gene; transmembrane protein 167A (TMEM167A) gene; microtubule associated serine/threonine kinase family member 4 (MAST4) gene; N-acylsphingosine amidohydrolase (ASAH1) gene; CGG triplet repeat binding protein 1 (CGGBP1) gene; family with sequence similarity 175, member B (FAM175B) gene; bridging integrator 1 (BIN1) gene; kruppel-like factor 12 (KLF12) gene; methyltransferase like 7A (METTL7A) gene; transmembrane protein 49 (TMEM49) gene; ribosomal protein S6 kinase, 90 kDa, polypeptide (RPS6KA5) gene; and major histocompatibility complex, class II, DR beta (DR HLA-DRB1) gene; is assessed, wherein a decrease of the expression level of any one of said genes as compared to the expression level of the corresponding gene(s) of a control is indicative of a predisposition for developing PTSD.
Also in accordance with the invention, each of the above recited genes associated with the risk of developing PTSD is, when assessed on its own, i.e. without a second gene, suitable for predicting the risk of developing PTSD.
Preferably, the MAN2C1 gene has a coding sequence as shown in SEQ ID NO: 4; the DDX17 gene has a coding sequence as shown in SEQ ID NO: 5; the CPNE1 gene has a coding sequence as shown in SEQ ID NO: 6; the TSC1 gene has a coding sequence as shown in SEQ ID NO: 7; the ADAM17 gene has a coding sequence as shown in SEQ ID NO: 8; the RNF181 gene has a coding sequence as shown in SEQ ID NO: 9; the MYO1C gene has a coding sequence as shown in SEQ ID NO: 10; the MOBKL1B gene has a coding sequence as shown in SEQ ID NO: 11; the GBA gene has a coding sequence as shown in SEQ ID NO: 12; the C2orf34 gene has a coding sequence as shown in SEQ ID NO: 13; the TMEM167A gene has a coding sequence as shown in SEQ ID NO: 14; the MAST4 gene has a coding sequence as shown in SEQ ID NO: 15 or 16; the ASAH1 gene has a coding sequence as shown in SEQ ID NO: 17; the CGGBP1 gene has a coding sequence as shown in SEQ ID NO: 18, the FAM175B gene has a coding sequence as shown in SEQ ID NO: 19; the BIN1 gene has a coding sequence as shown in SEQ ID NO: 20; the KLF12 gene has a coding sequence as shown in SEQ ID NO: 21; the METTL7A gene has a coding sequence as shown in SEQ ID NO: 22; the TMEM49 gene has a coding sequence as shown in SEQ ID NO: 23; the RPS6KA5 gene has a coding sequence as shown in SEQ ID NO: 24; and the DR HLA-DRB1 gene has a coding sequence as shown in SEQ ID NO: 25. However, as also variant or homologous alleles of any of the above described genes may exist the latter gene(s) can also have a coding sequence being at least 95% identical to the sequence as depicted in the SEQ ID NO assigned to the gene such as, being at least 96%, or preferably 97%, more preferred 98% and most preferred at least 99% identical.
Besides assessing one or more of the genes selected from FKBP5, STAT5B and NFIA and combinations thereof in this embodiment it is preferred that the predictive power of the method of the invention is further increased by additionally assessing the expression levels of any one of the above-recited genes. Said genes have been demonstrated to also be significantly associated with the risk to develop PTSD and are hence suitable as molecular predictors of said risk to develop PTSD. In accordance with the method of the invention, any combination of genes may be chosen for identifying a predisposition to develop PTSD as long as at least one of FKBP5, STAT5B or NFIA is also assessed in accordance with the method of the invention. It is preferred that more than one additional gene is assessed, such as at least 2, 3, 4, 5, 6, 7, 8, more preferred at least 9, most preferred at least 10. Also, envisaged is the assessment of more than 10 additional genes such as at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or at least 21 genes. The selection of additional genes to be assessed is preferably based on the significance of association of the single genes as experimentally shown herein (cf. Table 1). A selection is advantageously based upon the reasoning that the lesser the association significance of a gene is, the more genes have to be assessed in order to be able to make a more substantiated diagnosis. The assessment of further genes that have been previously identified or will in the future be identified to be associated with PTSD which are not recited in this specification can be additionally assessed.
The same applies mutatis mutandis for other embodiments recited herein below.
In another preferred embodiment of the method of the invention, additionally the expression level of the MAN2C1 gene is assessed, wherein an increase of the expression level of the MAN2C1 gene as compared to the expression level of the corresponding gene of a control is indicative of a predisposition for developing PTSD.
In a further preferred embodiment of the method of the invention, additionally the expression level of one or more genes selected from CPNE1; ADAM17; and METTL7A is assessed, wherein a decrease of the expression level of any one of said genes as compared to the expression level of the corresponding gene(s) of a control is indicative of a predisposition for developing PTSD.
In another aspect of the studies performed by the present inventors they were able to establish a link between differential gene expression and symptom severity. Besides showing that differential expression of the genes identified herein above is associated with PTSD they could also demonstrate which of the identified differentially expressed genes correlates with the severity of PTSD. It was shown by regression analysis that the expression level of each of the genes MAN2C1, CPNE1, ADAM17 and METTL7A was significantly associated with PTSD symptom severity. In particular, an increased expression level of MAN2C1 or a decreased expression level of CPNE1, ADAM17 or METTL7A is indicative of the severity of the PTSD symptoms. Said association means that a weak dysregulation of expression is indicative of weak symptoms, while a strong dysregulation is indicative of strong symptoms. The severity of symptoms can be determined by standardized clinical rating procedures that are well-known in the art such as, e.g., the Clinician-Administered PTSD Scale (CAPS), which is a structured interview for assessing frequency and severity of the core and associated symptoms of PTSD using standard prompt questions and behaviourally-anchored rating scales (Blake D D et al. J Trauma Stress 1995; 8: 75-90). From the obtained responses, a general severity score can be calculated. Hence, when assessing the expression levels of the latter genes, the diagnostic conclusion may be twofold, i.e. (i) the patient is at risk for developing PTSD or not and (ii) if at risk the symptoms will be severe. The same applies—correspondingly adapted—to other embodiments recited herein below.
In a further embodiment the invention relates to a method of identifying a compound capable of preventing or treating PTSD or capable of serving as a lead compound for developing a compound capable of preventing or treating PTSD, the method comprising the steps of: (a) assessing the expression level of one or more genes selected from the FK506 binding protein 5 (FKBP5) gene, the signal transducer and activator of transcription (STAT5B) gene and the nuclear factor I/A (NFIA) gene in a cell contacted with a test compound or in a sample obtained from a subject and contacted with said test compound; and (b) assessing the expression level of said one or more gene(s) in a cell or a sample obtained from a subject, wherein said cell or sample was i) not contacted with said test compound; ii) contacted with a compound known to not affect the expression levels of said gene(s) of step (a), wherein an increase of the expression level of the gene(s) in step (a) as compared to step (b) i) or (b) ii) is indicative of said test compound being capable of preventing or treating PTSD or serving as a lead compound for developing a compound capable of preventing or treating PTSD; or iii) contacted with a compound known to enhance the expression level of said gene(s) of step (a), wherein an essentially equal level of expression or an increase of the expression level of the gene(s) in step (a) as compared to step (b) iii) is indicative of the compound being capable of preventing or treating PTSD or serving as a lead compound for developing a compound capable of preventing or treating PTSD.
Methods for the assessment of expression levels are well-known in the art and some are exemplarily described herein. The expression level of the one or more genes is to be assessed in a cell or in a sample obtained from a subject contacted with the test compound. Said cell may be part of a cell culture colony derived from an established cell line. Also, the cell may be part of a primary cell culture established from a sample of a subject contacted with the test compound. Suitable samples obtained from subjects for the assessment of expression levels are described herein elsewhere and may also be processed without prior steps, such as establishing a primary cell culture, in order to assess expression levels.
The term “compound” as used herein relates to a substance that may be solid, semisolid, semifluid, fluid or gaseous. Said compound may, however, also be comprised in a mixture, extract or composition.
The compound that is identified according to the method of the invention to be capable of preventing or treating PTSD or capable of serving as a lead compound for developing a compound capable of preventing or treating PTSD increases the gene expression levels of said one or more genes selected from FKBP5, STAT5B and NFIA which may, for example, be based on its inhibitory, promoting, agonistic or antagonistic activity in a direct or indirect interaction with the gene or any intermediate or final gene product(s). Said compound(s) may be chemically synthesized or produced via microbial fermentation but can also be comprised in, for example, samples, e.g., cell extracts from, e.g., plants, animals or microorganisms. Furthermore, said compound to be identified by the method of the invention may be known in the art but hitherto not known to be useful as a compound capable of preventing or treating PTSD.
The term “contacting a cell” with a compound in accordance with the method of the invention relates to the process of exposing the cell to a compound to be tested and allowing interaction of said compound with the cell. Depending on the potential mode of action of the compound to be tested the interaction may take place extracellularly and/or intracellularly resulting in the increase of the expression level of the genes FKBP5, STAT5B and/or NFIA—provided the compound is capable thereof. For example, a compound may bind to the cell surface and trigger a signal cascade resulting in increased expression of said target gene(s). However, preferably the compound is exposed to the cell under conditions allowing both extracellular interaction and the uptake into the cell, in particular the cytoplasma or nucleus, to exert its potential activity on regulatory events in the gene expression process of the target gene(s). The person skilled in the art is aware of conditions generally suitable for uptake of compounds such as, e.g., proteins or nucleic acid molecules, into cells and methods to enhance said uptake as regards rate and amount wherein said enhancement may include artificially modifying, e.g, proteins (see, for example, Patsch et Edenhofer, (2007), Handb. Exp. Pharmacol., 178, 203-232) or nucleic acids. Furthermore, he is also aware of cell lines naturally exhibiting the capacity of increased uptake capabilities relative to other cells. Such cells are, for example, cells like mucosal cells or intestinal cells. A number of mechanisms exist for the passage of various compounds across the plasma membrane, including passive diffusion, facilitated diffusion, and active transport systems. Passive diffusion of proteins through the bilayer lipid structure of the plasma membrane is a function of the size, lipid solubility, and charge of the protein molecule. A further uptake mechanism is endocytosis. Endocytosis is a process whereby cells absorb material from the outside by engulfing it with their cell membrane. Endocytosis works with macromolecules or particulate matter beyond a certain size threshold and also with fluids (pinocytosis). Correspondingly, the test compound should be contacted with the subject in a way that allows for the interaction of the test compound with cells that are part of the sample to be subsequently obtained from said subject and assessed in accordance with the method of the invention. For example, if blood is intended to be obtained as sample from the subject, intravenous administration of the test compound—if necessary as part of a therapeutically acceptable composition—will be suitable to allow for an interaction of cells of the sample with the test compound. Accordingly, said test compound may be, e.g., added to the culture medium or injected into a cell or administered to an individual prior to the assessment of the expression level in step (a). Moreover, the compound to be identified can be contained in libraries of small molecules, such as organic or inorganic small molecules which may be commercially available. In addition, libraries comprising antibodies or functional fragments or derivatives thereof (i.e. fragments or derivatives maintaining the binding specificity of the original antibody) may be used as a starting point in the identifying process. Suitable libraries are commercially available, for example from ChemBridge Corp., San Diego, USA. Also, libraries of aptamers such as peptide aptamers might be employed. The skilled person is of course free to use any other starting point of desired compounds for use in the method of the invention.
IF a composition containing (a) compound(s) is identified to be capable of preventing or treating PTSD or serving as a lead compound for developing a compound capable of preventing or treating PTSD in the method of the invention, then it is either possible to isolate the active compound(s) from the original composition identified as containing the compound(s) in question or one can further subdivide the original composition, for example, if it consists of a plurality of different test compounds, so as to reduce the number of different substances per sample and repeat the method with the subdivisions of the original composition. It can then be determined whether said subdivided composition or resulting compound displays the desired properties, for example, by the methods described herein or in the literature (“Cells: A laboratory manual”, v. 1-3, edited by Spector et al., Cold Spring Harbour Laboratory Press (1997); ISBN 10: 0879695218). Depending on the complexity of the compositions, the steps described above can be performed several times, preferably until the composition identified according to the method of the invention only comprises a limited number of or only one substance(s). Preferably said composition comprises substances of similar chemical and/or physical properties. The method of the present invention can be easily performed and the experimental setup without further ado designed by the person skilled in the art, for example, in accordance with other cell based screening assays described in the prior art. Such adaptation of the method of the invention is well within the skill of the person skilled in the art and can be performed without undue experimentation.
Compounds which can be tested in accordance with the present invention include peptides, proteins, nucleic acids, antibodies, small organic compounds, ligands, peptidomimetics, PNAs and the like. Said compounds may act as agonists or antagonists. Said compounds can also be functional derivatives or analogues of known drugs. Methods for the preparation of chemical derivatives and analogues are well known to those skilled in the art and are described in, for example, Beilstein, Handbook of Organic Chemistry, Springer edition New York Inc., 175 Fifth Avenue, New York, N.Y. 10010 U.S.A. and Organic Synthesis, Wiley, New York, USA. Also, peptide mimetics and/or computer-aided design of appropriate drug derivatives and analogues can be used.
Appropriate computer programs can be used for the identification of interactive sites of a compound putatively capable of preventing and/or treating PTSD by computer assisted searches for complementary structural motifs (Fassina, Immunomethods 5 (1994), 114-120). Further appropriate computer systems for the computer aided design of protein and peptides are described in the prior art, for example, in Berry, Biochem. Soc. Trans. 22 (1994), 1033-1036; Wodak, Ann. N. Y. Acad. Sci. 501 (1987), 1-13; Pabo, Biochemistry 25 (1986), 5987-5991. The results obtained from the above-described computer analysis can be used in combination with the method of the invention for, e.g., optimizing known inhibitors, analogs, antagonists or agonists. Appropriate peptidomimetics can also be identified by the synthesis of peptidomimetic combinatorial libraries through successive chemical modification and testing the resulting compounds, e.g., according to the methods described herein. Methods for the generation and use of peptidomimetic combinatorial libraries are described in the prior art, for example in Ostresh, Methods in Enzymology 267 (1996), 220-234 and Dorner, Bioorg. Med. Chem. 4 (1996), 709-715. Furthermore, the three-dimensional and/or crystallographic structure of said compounds can be used for the design of peptidomimetic drugs (Rose, Biochemistry 35 (1996), 12933-12944; Rutenber, Bioorg. Med. Chem. 4 (1996), 1545-1558). It is very well known how to obtain compounds to be tested in the method of the invention, e.g. by chemical or biochemical standard techniques. Thus, also comprised by the method of the invention are means of making or producing said compounds. In summary, the present invention provides a method for identifying compounds which can be used in specific doses for the prevention or treatment of PTSD.
Also, the method of the invention may be useful in identifying lead compounds. The term “lead compound” in accordance with the present invention refers to a compound discovered by the method of the invention which will be e.g. further optimized, in particular to be pharmaceutically more acceptable. The identified lead compounds may be optimized to arrive at a compound which may be, for example, used in a pharmaceutical composition for preventing or treating PTSD. Methods for the optimization of the pharmacological properties of compounds identified in screens, the lead compounds, are known in the art and comprise a method of modifying a compound identified as a lead compound to achieve: (i) modified site of action, spectrum of activity, organ specificity, and/or (ii) improved potency, and/or (iii) decreased toxicity (improved therapeutic index), and/or (iv) decreased side effects, and/or (v) modified onset of therapeutic action, duration of effect, and/or (vi) modified pharmacokinetic parameters (resorption, distribution, metabolism and excretion), and/or (vii) modified physico-chemical parameters (solubility, hygroscopicity, color, taste, odor, stability, state), and/or (viii) improved general specificity, organ/tissue specificity, and/or (ix) optimized application form and route by (i) esterification of carboxyl groups, or (ii) esterification of hydroxyl groups with carboxylic acids, or (iii) esterification of hydroxyl groups to, e.g. phosphates, pyrophosphates or sulfates or hemi-succinates, or (iv) formation of pharmaceutically acceptable salts, or (v) formation of pharmaceutically acceptable complexes, or (vi) synthesis of pharmacologically active polymers, or (vii) introduction of hydrophilic moieties, or (viii) introduction/exchange of substituents on aromates or side chains, change of substituent pattern, or (ix) modification by introduction of isosteric or bioisosteric moieties, or (x) synthesis of homologous compounds, or (xi) introduction of branched side chains, or (xii) conversion of alkyl substituents to cyclic analogues, or (xiii) derivatisation of hydroxyl group to ketales, acetales, or (xiv) N-acetylation to amides, phenylcarbamates, or (xv) synthesis of Mannich bases, imines, or (xvi) transformation of ketones or aldehydes to Schiff's bases, oximes, acetales, ketales, enolesters, oxazolidines, thiazolidines or combinations thereof.
The various steps recited above are generally known in the art. They include or rely on quantitative structure-activity relationship (QSAR) analyses (Kubinyi (1992) “Hausch-Analysis and Related Approaches”, VCH Verlag, Weinheim), combinatorial biochemistry, classical chemistry and others (see, for example, Holzgrabe and Bechtold (2000) Deutsche Apotheker Zeitung 140(8), 813).
The therapeutically useful compounds identified according to the method of the invention can be formulated and administered to a patient by methods well known in the art. Drugs or pro-drugs after their in vivo administration are metabolized in order to be eliminated either by excretion or by metabolism to one or more active or inactive metabolites (Meyer, J. Pharmacokinet. Biopharm. 24 (1996), 449-459). More specifically, a “prodrug” is a compound that is generally not biologically and/or pharmacologically active. After administration, the prodrug is activated, typically in vivo by enzymatic or hydrolytic cleavage and converted to a biologically and/or pharmacologically compound which has the intended medical effect. Prodrugs are typically formed by chemical modification of biologically and/or pharmacologically compounds. Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in Design of Prodrugs, ed. H. Bundgaard, Elsevier, 1985. Thus, rather than using the actual compound identified in accordance with the method of the present invention a corresponding formulation as a pro-drug can be used which is converted into its active in the patient. Precautionary measures that may be taken for the application of pro-drugs and drugs are described in the literature; see, for review, Ozama, J. Toxicol. Sci. 21 (1996), 323-329.
Preferably, said method is effected in high-throughput format. High-throughput assays, independently of being biochemical, cellular or other assays, generally may be performed in wells of microtiter plates, wherein each plate may contain 96, 384 or 1536 wells. Handling of the plates, including incubation at temperatures other than ambient temperature, and bringing into contact of test compounds with the assay mixture is preferably effected by one or more computer-controlled robotic systems including pipetting devices. In case large libraries of test compounds are to be screened and/or screening is to be effected within short time, mixtures of, for example 10, 20, 30, 40, 50 or 100 test compounds may be added to each well. In case a well exhibits biological activity, said mixture of test compounds may be de-convoluted to identify the one or more test compounds in said mixture giving rise to said activity.
The above definitions apply mutatis mutandis to the methods described in the following.
The method of the invention is based upon the presumption that an increase in expression levels of genes that are identified herein as being associated with the risk of developing PTSD and whose expression is downregulated in PTSD patients is suitable to prevent the onset of or to treat acute PTSD in a subject. The increase in expression levels is determined vis-à-vis the expression levels of a control sample or cell in step (b). Step (b) serving as control, it is self-evident that the control samples of step (b) i), ii) or iii) are not to be contacted with the test compound referred to in step (a). Said control sample or cell may be a sample or cell that has not been contacted with the compound to be tested. Additionally or alternatively the expression levels of the sample or cell may be compared to the expression levels of a sample or cell that has been contacted with a compound known to not affect the expression levels of the target genes. It is also envisaged that in addition to the comparison of the expression levels of the cells or the sample of step (a) against the one or both latter negative controls a comparison against a sample or cell contacted with a compound known to increase the expression level of the genes is effected. A corresponding method provides a qualitative assessment of the compound to be tested. It is alternatively possible to exclusively compare expression levels of the cell or sample of step (a) against the expression levels of a cell or sample that has been contacted with a compound known to increase the expression of the target genes, if it is desired to screen for compounds that have a similar or superior effect on the expression levels as the latter control. A corresponding experimental setup would hence allow identifying compounds that are similar or superior to existing compounds that are capable of increasing the expression levels of the target genes. In other words, corresponding compounds may be capable of effecting an essentially equal increase of the expression level or an higher increase of the expression levels as compared to the compound known to enhance the expression level of the target gene(s). The term “essentially equal” refers to an expression level that is at least 90% such as at least 91%, 92%, 93%, 94%, 95%, or preferably at least 96% or 97%, more preferred 98% or 99% and most preferred at least 100% as high as the expression level of the target gene(s) of the cell or sample that has been contacted with the compound known to enhance the expression level of said gene(s). Also, the expression level could be greater than 100% such as, e.g., at least 101%, 102%, 103%, 104%, 105%, or preferably at least 106% or 107%, more preferred at least 110% or 120% and most preferred at least 200% or more. Depending on the potency of the compound known to increase the expression level of the target genes, it may not be possible to further increase the expression levels and accordingly, suitable compounds to be identified by the method of the invention may in comparison only achieve a fraction of the increase in expression of the positive control. Hence, compounds that only result in expression level of the target gene(s) of at least 5% such as at least 10%, 20%, 30%, 40%, 50%, or preferably at least 60% or 70% and most preferred at least 80% or at least 89% as high as the expression level of the target gene(s) of the cell or sample that has been contacted with the compound known to enhance the expression level of said gene(s) can also be identified in accordance with the method of the invention. Accordingly, the identification of compounds that are capable of adjusting the expression level of the risk gene(s) as described herein towards the expression level of the corresponding gene(s) in a person not at risk for PTSD, preferably selectively and in a dose-dependent fashion, provides the means for a drug-based therapeutic intervention. The above applies mutatis mutandis to other embodiments recited herein.
In a preferred embodiment, the method of the invention further comprises synthesizing said compound being capable of preventing or treating PTSD or serving as a lead compound for developing a compound capable of preventing or treating PTSD.
As outlined above, it is well-known in the art how to obtain, produce and modulate the test compounds, e.g. by chemical or biochemical standard techniques. Thus, having identified a compound that is capable of preventing or treating PTSD or serving as a lead compound for developing a compound capable of preventing or treating PTSD the skilled person is in the position by employing said chemical or biochemical standard techniques to synthesize said identified compound in desired amounts.
In another preferred embodiment of the method of the invention, the method further comprises in step (a) additionally assessing the expression level of one or both further genes selected from MAN2C1 or DDX17 in a cell contacted with a test compound or in a sample obtained from a subject and contacted with said test compound; and in step (b) additionally assessing the expression level of MAN2C1 or DDX17 in a cell or a sample obtained from a subject, wherein said cell or sample was i) not contacted with the test compound; ii) contacted with a compound known to not affect the expression levels of MAN2C1 or DDX17 of step (a), wherein a decrease of the expression level of MAN2C1 or DDX17 in step (a) as compared to step (b) i) or (b) ii) is indicative of said test compound being capable or preventing or treating PTSD or serving as a lead compound for developing a compound capable of preventing or treating PTSD; or iii) contacted with a compound known to enhance the expression level of MAN2C1 or DDX17 of step (a), wherein an essentially equal level of expression or a decrease of the expression level of MAN2C1 or DDX17 in step (a) as compared to step (b) iii) is indicative of the compound being capable of preventing or treating PTSD or serving as a lead compound for developing a compound capable of preventing or treating PTSD.
In a different preferred embodiment of the method of the invention, the method further comprises in step (a) additionally assessing the expression level of one or more further genes selected from CPNE1, TSC1, ADAM17, RNF181, MYO1C, MOBKL1B, GBA, C2orf34, TMEM167A, MAST4, ASAH1, CGGBP1, FAM175B, BIN1, KLF12, METTL7A, TMEM49, RPS6KA5 and DR HLA-DRB1 in a cell contacted with a test compound or in a sample obtained from a subject and contacted with said test compound; and in step (b) additionally assessing the expression level of said one or more further genes selected from CPNE1, TSC1, ADAM17, RNF181, MYO1C, MOBKL1B, GBA, C2orf34, TMEM167A, MAST4, ASAH1, CGGBP1, FAM175B, BIN1, KLF12, METTL7A, TMEM49, RPS6KA5 and DR HLA-DRB1 in a cell or a sample obtained from a subject, wherein said cell or sample was i) not contacted with the test compound; ii) contacted with a compound known to not affect the expression levels of said one or more further genes selected from CPNE1, TSC1, ADAM17, RNF181, MYO1C, MOBKL1B, GBA, C2orf34, TMEM167A, MAST4, ASAH1, CGGBP1, FAM175B, BIN1, KLF12, METTL7A, TMEM49, RPS6KA5 and DR HLA-DRB1 of step (a), wherein an increase of the expression level of said one or more further genes selected from CPNE1, TSC1, ADAM17, RNF181, MYO1C, MOBKL1B, GBA, C2orf34, TMEM167A, MAST4, ASAH1, CGGBP1, FAM175B, BIN1, KLF12, METTL7A, TMEM49, RPS6KA5 and DR HLA-DRB1 in step (a) as compared to step (b) i) or (b) ii) is indicative of said test compound being capable or preventing or treating PTSD or serving as a lead compound for developing a compound capable of preventing or treating PTSD; or iii) contacted with a compound known to enhance the expression level of said one or more further genes selected from CPNE1, TSC1, ADAM17, RNF181, MYO1C, MOBKL1B, GBA, C2orf34, TMEM167A, MAST4, ASAH1, CGGBP1, FAM175B, BIN1, KLF12, METTL7A, TMEM49, RPS6KA5 and DR HLA-DRB1 of step (a), wherein an essentially equal level of expression or an increase of the expression level of said one or more further genes selected from CPNE1, TSC1, ADAM17, RNF181, MYO1C, MOBKL1B, GBA, C2orf34, TMEM167A, MAST4, ASAH1, CGGBP1, FAM175B, BIN1, KLF12, METTL7A, TMEM49, RPS6KA5 and DR HLA-DRB1 in step (a) as compared to step (b) iii) is indicative of the compound being capable of preventing or treating PTSD or serving as a lead compound for developing a compound capable of preventing or treating PTSD.
In step (b) ii) it is envisaged that a compound be used for contacting that is known not to affect the expression level(s) of any one of the additional gene(s) to be assessed in step (a). For example, if the expression level of KLF12 is additionally assessed, then the cell or sample of step (b) ii) was not contacted with a compound affecting expression of KLF12. The same applies mutatis mutandis to other embodiments herein.
As outlined supra, each of the recited genes is linked to the risk to develop PTSD also when assessed on its own, hence identifying a compound that alters the expression level of only one of said genes may be a suitable drug candidate.
In this embodiment, advantageously one or more of the above-recited genes is additionally assessed in order to identify a compound that alters the expression level of, e.g., more than one gene associated with the risk to develop PTSD or of one of said other (additional) gene(s) associated with the risk to develop PTSD, hence being a compound potentially more potent in preventing or treating PTSD or a more promising lead compound for developing a compound capable of preventing or treating PTSD.
In another preferred embodiment of the method of the invention, the method further comprises in step (a) additionally assessing the expression level of MAN2C1 in a cell contacted with a test compound or in a sample obtained from a subject and contacted with said test compound; and in step (b) additionally assessing the expression level of MAN2C1 in a cell or a sample obtained from a subject, wherein said cell or sample was i) not contacted with the test compound; ii) contacted with a compound known to not affect the expression levels of MAN2C1 of step (a), wherein a decrease of the expression level of MAN2C1 in step (a) as compared to step (b) i) or (b) ii) is indicative of said test compound being capable or preventing or treating PTSD or serving as a lead compound for developing a compound capable of preventing or treating PTSD; or iii) contacted with a compound known to decrease the expression level of MAN2C1 of step (a), wherein an essentially equal level of expression or a decrease of the expression level of MAN2C1 in step (a) as compared to step (b) iii) is indicative of the compound being capable of preventing or treating PTSD or serving as a lead compound for developing a compound capable of preventing or treating PTSD.
In a different preferred embodiment of the method of the invention, the method further comprises in step (a) additionally assessing the expression level of one or more further genes selected from CPNE1, ADAM17, and METTL7A in a cell contacted with a test compound or in a sample obtained from a subject and contacted with said test compound; and in step (b) additionally assessing the expression level of said one or more further genes selected from CPNE1, ADAM17, and METTL7A in a cell or a sample obtained from a subject, wherein said cell or sample was i) not contacted with the test compound; ii) contacted with a compound known to not affect the expression levels of said one or more further genes selected from CPNE1, ADAM17 and METTL7A of step (a), wherein an increase of the expression level of said one or more further genes selected from CPNE1, ADAM17 and METTL7A in step (a) as compared to step (b) i) or (b) ii) is indicative of said test compound being capable or preventing or treating PTSD or serving as a lead compound for developing a compound capable of preventing or treating PTSD; or iii) contacted with a compound known to enhance the expression level of said one or more further genes selected from CPNE1, ADAM17 and METTL7A of step (a), wherein an essentially equal level of expression or an increase of the expression level of said one or more further genes selected from CPNE1, ADAM17 and METTL7A in step (a) as compared to step (b) iii) is indicative of the compound being capable of preventing or treating PTSD or serving as a lead compound for developing a compound capable of preventing or treating PTSD.
Based on the foregoing, it is especially advantageous to identify compounds that are capable of adjusting the expression levels of MAN2C1, CPNE1, ADAM17 and METTL7A, i.e. decreasing the expression level of MAN2C1 and increasing the expression levels of CPNE1, ADAM17 and/or METTL7A, to a normal level when compared to controls as this may on the one hand prevent PTSD or on the other hand in a treatment regimen at least reduce the severity of symptoms in particularly severely affected PTSD patients. These results underscore the relevance of risk factors in association with gene expression in predicting PTSD severity, which may lead to new effective personalized treatment approaches, considering both genotype and biomarkers (Holsboer F. Challenges and opportunities for the development of personalized antidepressant medicines. Nat Rev Neurosci, in press 2008).
In a preferred embodiment of the method of the invention, the expression level of at least two of the genes selected from the FKBP5 gene, STAT5B gene and NFIA gene is assessed.
In order to increase the predictive power and reliability of the method of the invention the expression levels of at least two of the genes associated with PTSD are assessed. Accordingly, in a more preferred embodiment of the method, the expression levels of the FKBP5 gene and the STAT5B gene are assessed. Also preferred and in accordance with the foregoing is the assessment of the expression level of the FKBP5 gene and the NFIA gene or the STAT5B gene and the NFIA gene. Most preferred is that the expression level of all three genes is assessed.
In a preferred embodiment of the method of the invention, the sample is selected from blood, saliva and buccal smear.
A preferred sample to be used in the method of the invention is blood (cf. also supra) due to the ease of accessibility and clinical routine of blood collection as well as the standardized protocols to extract and purify nucleic acids and/or proteins from blood constituents. Preferred in accordance with the method of the invention are peripheral blood mononuclear cells as blood constituent that is to be further processed for extraction of nucleic acids and/or proteins. Suitable methods for extraction and purification of nucleic acids and/or proteins form blood are well-known to the person skilled in the art and have been described herein above. Also preferred as sample in accordance with the method of the invention are saliva and buccal smear samples that are equally conveniently accessible for collection, however, without having to injure a subject in order to get to the sample as compared to blood collection. Preferred are buccal mucosa epithelial cells that can be isolated from buccal swabs for further processing to extract and purify nucleic acids and/or proteins in order to determine expression levels of genes associated with the risk to develop PTSD.
In a further preferred embodiment of the method of the invention, said test compound is selected from a small molecule, an antisense nucleic acid molecule, a siRNA, a shRNA, a miRNA, a ribozyme, a peptide aptamer, a nucleic acid based aptamer, an antibody or a combination thereof.
The term “small chemical molecule” as used herein may describe, for example, a small organic molecule. Organic molecules relate or belong to the class of chemical compounds having a carbon basis, the carbon atoms linked together by carbon-carbon bonds. The original definition of the term organic related to the source of chemical compounds, with organic compounds being those carbon-containing compounds obtained from plant or animal or microbial sources, whereas inorganic compounds were obtained from mineral sources. Organic compounds can be natural or synthetic. Alternatively the compound may be an inorganic compound. Inorganic compounds are derived from mineral sources and include all compounds without carbon atoms (except carbon dioxide, carbon monoxide and carbonates). Preferably, the small molecule has a molecular weight of less than about 2000 amu, or less than about 1000 amu such as 500 amu, and even less than about 250 amu. The size of a small molecule can be determined by methods well-known in the art, e.g., mass spectrometry. Small molecules may be designed, for example, in silico based on the crystal structure of potential drug targets, where sites presumably responsible for the biological activity and involved in the regulation of expression of genes identified herein, can be identified and verified in in vivo assays such as in vivo HTS (high-throughput screening) assays.
The term “antisense nucleic acid molecule” is known in the art and refers to a nucleic acid which is complementary to a target nucleic acid. An antisense molecule according to the invention is capable of interacting with, more specifically hybridizing with the target nucleic acid. By formation of the hybrid, transcription of the target gene(s) and/or translation of the target mRNA is reduced or blocked. Preferably, the nucleic acid molecule is a antisense RNA molecule. Standard methods relating to antisense technology have been described (see, e.g., Melani et al., Cancer Res. (1991) 51:2897-2901).
For therapeutic uses, the RNA inactivation by antisense molecules or by ribozymes (cf. infra) appears to be implementable. Both classes of compounds can be synthesized chemically or produced in conjunction with a promoter by biological expression in vitro or even in vivo.
Small interfering RNAs (siRNA), sometimes known as short interfering RNAs or silencing RNAs, are a class of 18 to 30, preferably 20 to 25, most preferred 21 to 23 or 21 nucleotide-long double-stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway where the siRNA interferes with the expression of a specific gene. In addition to their role in the RNAi pathway, siRNAs also act in RNAi-related pathways, e.g. as an antiviral mechanism or in shaping the chromatin structure of a genome.
Said siRNA molecule or a metabolic processing product thereof is capable of mediating target-specific nucleic acid modifications, particularly RNA interference and/or DNA methylation. Preferably at least one RNA strand has a 5′- and/or 3′-overhang. Preferably, one end of the double-strand has a 3′-overhang from 1-5 nucleotides, more preferably from 1-3 nucleotides and most preferably 2 nucleotides. The other end may be blunt-ended or has up to 6 nucleotides 3′-overhang. In general, any RNA molecule suitable to act as siRNA is envisioned in the present invention.
Preferred siRNAs have a well defined structure: a short double-strand of RNA (dsRNA) with 2-nt 3′ overhangs on either end. Each strand has a 5′ phosphate group and a 3′ hydroxyl (—OH) group. As regards naturally occurring siRNAs, this structure is the result of processing by dicer, an enzyme that converts either long dsRNAs or small hairpin RNAs into siRNAs. SiRNAs can also be exogenously (artificially) introduced into cells to bring about the specific knockdown of a gene of interest. Essentially any gene of which the sequence is known can thus be targeted based on sequence complementarity with an appropriately tailored siRNA.
The most efficient silencing was so far obtained with siRNA duplexes composed of 21-nt sense and 21-nt antisense strands, paired in a manner to have a 2-nt 3′-overhang. The sequence of the 2-nt 3′ overhang makes a small contribution to the specificity of target recognition restricted to the unpaired nucleotide adjacent to the first base pair (Elbashir et al., EMBO J 2001, 20(23):6877-6888). 2′-deoxynucleotides in the 3′ overhangs are as efficient as ribonucleotides, but are often cheaper to synthesize and probably more nuclease resistant.
A short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. shRNA uses a vector introduced into cells and utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs which match the siRNA that is bound to it.
Si/shRNAs to be used in the method of the present invention are preferably chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. Suppliers of RNA synthesis reagents are Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part of Perbio Science, Rockford, Ill., USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland, Mass., USA), and Cruachem (Glasgow, UK). Most conveniently, siRNAs are obtained from commercial RNA oligo synthesis suppliers, which sell RNA-synthesis products of different quality and costs. In general, the RNAs applicable in the present invention are conventionally synthesized and are readily provided in a quality suitable for RNAi.
Further molecules effecting RNAi include, for example, microRNAs (miRNA). Said RNA species are single-stranded RNA molecules which as endogenous RNA molecules regulate gene expression. Upon binding to a complementary mRNA transcript triggers the degradation of said mRNA transcript through a process similar to RNA interference. Accordingly, miRNAs may be employed to directly or indirectly regulate the expression of genes associated with PTSD.
A “ribozyme” (from ribonucleic acid enzyme, also called RNA enzyme or catalytic RNA) is an RNA molecule that catalyzes a variety of reactions. Many natural ribozymes catalyze either their own cleavage or the cleavage of other RNAs, but they have also been found to catalyze the aminotransferase activity of the ribosome.
Examples of well-characterized small self-cleaving RNAs are the hammerhead, hairpin, hepatitis delta virus, and in vitro-selected lead-dependent ribozymes. The organization of these small catalysts is contrasted to that of larger ribozymes, such as the group I intron.
The principle of catalytic self-cleavage has become well established in the last 10 years. The hammerhead ribozymes are characterized best among the RNA molecules with ribozyme activity. Since it was shown that hammerhead structures can be integrated into heterologous RNA sequences and that ribozyme activity can thereby be transferred to these molecules, it appears that catalytic antisense sequences for almost any target sequence can be created, provided the target sequence contains a potential matching cleavage site.
The basic principle of constructing hammerhead ribozymes is as follows: An interesting region of the RNA, which contains the GUC (or CUC) triplet, is selected. Two oligonucleotide strands, each with 6 to 8 nucleotides, are taken and the catalytic hammerhead sequence is inserted between them. Molecules of this type were synthesized for numerous target sequences. They showed catalytic activity in vitro and in some cases also in vivo. The best results are usually obtained with short ribozymes and target sequences.
Aptamers are oligonucleic acid or peptide molecules that bind a specific target molecule. Aptamers are usually created by selecting them from a large random sequence pool, but natural aptamers also exist in riboswitches. Aptamers can be used for both basic research and clinical purposes as macromolecular drugs. Further, they can be combined with ribozymes to self-cleave in the presence of their target molecule.
More specifically, aptamers can be classified as DNA or RNA aptamers or peptide aptamers. Whereas the former consist of (usually short) strands of oligonucleotides, the latter consist of a short variable peptide domain, attached at both ends to a protein scaffold. Nucleic acid aptamers are nucleic acid species that may be engineered through repeated rounds of in vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms.
Peptide aptamers are proteins that are designed to interfere with other protein interactions inside cells. They consist of a variable peptide loop attached at both ends to a protein scaffold. This double structural constraint greatly increases the binding affinity of the peptide aptamer to levels comparable to an antibody's (nanomolar range). The variable loop length is typically comprised of 10 to 20 amino acids, and the scaffold may be any protein which have good solubility properties. Currently, the bacterial protein Thioredoxin-A is the most used scaffold protein, the variable loop being inserted within the reducing active site, which is a -Cys-Gly-Pro-Cys- loop in the wild protein, the two cysteins lateral chains being able to form a disulfide bridge. Peptide aptamer selection can be made using different systems, but the most used is currently the yeast two-hybrid system.
Aptamers offer the utility for biotechnological and therapeutic applications as they offer molecular recognition properties that rival those of the commonly used biomolecules, in particular antibodies. In addition to their discriminate recognition, aptamers offer advantages over antibodies as they can be engineered completely in a test tube, are readily produced by chemical synthesis, possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications.
Non-modified aptamers are cleared rapidly from the bloodstream, with a half-life of minutes to hours, mainly due to nuclease degradation and clearance from the body by the kidneys, a result of the aptamer's inherently low molecular weight. Unmodified aptamer applications currently focus on treating transient conditions such as blood clotting, or treating organs such as the eye where local delivery is possible. This rapid clearance can be an advantage in applications such as in vivo diagnostic imaging. Several modifications, such as 2′-fluorine-substituted pyrimidines, polyethylene glycol (PEG) linkage, etc. are available to scientists with which the half-life of aptamers easily can be increased to the day or even week time scale.
A recent development is the combination of an aptamer recognizing a small compound with a hammerhead ribozyme. The conformational change induced in the aptamer upon binding the target molecule, is supposed to regulate the catalytic function of the ribozyme.
The term “antibody” as used herein can be, for example, relate to polyclonal or monoclonal antibodies. The term “antibody” also comprises derivatives or fragments thereof with retained binding specificity. Techniques for the production of antibodies are well known in the art and described, e.g. in Harlow and Lane “Antibodies, A Laboratory Manual”, Cold Spring Harbor Laboratory Press, 1988 and Harlow and Lane “Using Antibodies: A Laboratory Manual” Cold Spring Harbor Laboratory Press, 1999. The antibodies can be used in accordance with the method of the invention to interfere with the regulation of expression of any gene associated with a risk to develop PTSD described herein. Also, antibodies may be useful, for example, for immunoprecipitation, affinity purification and immunolocalization of the gene products, i.e. the proteins, of the genes associated with a risk to develop PTSD as well as for the monitoring of the presence and amount of such proteins, for example, in cultures of eukaryotic cells or organisms.
An antibody to be used in accordance with the invention also includes embodiments such as chimeric (human constant domain, non-human variable domain), single chain and humanized (human antibody with the exception of non-human CDRs) antibodies, as well as antibody fragments, like, inter alia, Fab or Fab′ fragments. Antibody fragments or derivatives further comprise Fd, F(ab′)2, Fv or scFv fragments; see, for example, Harlow and Lane (1988) and (1999), loc. cit. Various procedures are known in the art and may be used for the production of such antibodies and/or fragments. For example, the (antibody) derivatives can be produced by peptidomimetics. Further, techniques described for the production of single chain antibodies (see, inter alia, U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies specific for any target epitope/s. Also, transgenic animals or plants (see, e.g., U.S. Pat. No. 6,080,560) may be used to express (humanized) antibodies specific for any target epitope/s. For the preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples for such techniques include the hybridoma technique (Köhler and Milstein, Nature 256 (1975), 495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor, Immunology Today 4 (1983), 72) and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), 77-96). Surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies which bind to an epitope of an polypeptide of the invention (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). It is also envisaged in the context of this invention that the term “antibody” comprises antibody constructs which may be expressed in cells, e.g. antibody constructs which may be transfected and/or transduced via, amongst others, viruses or plasmid vectors.
The antibody to be used in the method of the invention is capable to specifically bind/interact with a target epitope. The term “specifically binding/interacting with” as used in accordance with the present invention means that the antibody does not or essentially does not cross-react with an epitope of similar structure. Cross-reactivity of a panel of antibodies under investigation may be tested, for example, by assessing binding of said panel of antibodies under conventional conditions to the epitope of interest as well as to a number of more or less (structurally and/or functionally) closely related epitopes. Only those antibodies that bind to the epitope of interest in its relevant context (e.g. a specific motif in the structure of a protein) but do not or do not essentially bind to any of the other epitope are considered specific for the epitope of interest and thus to be antibodies in accordance with this invention. Corresponding methods are described e.g. in Harlow and Lane, 1988 and 1999, loc cit.
With regard to genes whose expression levels are upregulated when indicative for a risk to develop PTSD it is possible to decrease expression levels by using, e.g., the above described antisense nucleic acid molecule, the RNA molecules or antibodies. Said compounds may be designed by methods well-known in the art to interact target-specifically with either mRNA or protein molecules and thereby reduce the expression level. For example, RNA molecules suitable for RNA interference can be designed leading to a reduction of mRNA molecules and hence of the expression level of the target gene(s). However, it may also be possible to indirectly reduce the expression level of said gene(s) by using any of the above-mentioned compounds by reducing and/or inhibiting, e.g., protein or mRNA molecules that are involved in the regulation of expression of said target gene(s). Also target gene(s) whose expression is decreased when indicative for a risk to develop PTSD may directly or indirectly be targeted by the above compounds resulting in an increase of expression levels. For example, said target gene(s) may be targeted indirectly by increasing the amount of protein or mRNA molecules that are involved in the regulation of expression of said gene(s) such as, e.g., transcription factors or transcription enhancing factors.
In preferred embodiments of the method of the invention, the expression level is assessed on the transcriptional level or on the translational level.
As previously outlined herein supra, the expression of a gene is a process that can be subdivided into two distinct processes characterised by their end products. Without wishing to be bound by a specific scientific theory but to provide a simplistic view of a complicated process, the process of transcription can be said to result in the generation of mRNA, which in the second process, i.e. translation, is used as a starting point for the production of a peptide or protein. Accordingly, it is possible to determine the expression level of a gene not only on the transcriptional level, but also on the translational level. Nevertheless, a variety of methods have been developed and successfully employed to determine translational gene activity, which are well-known in the art (cf., e.g., Kingsmore S F, Nat Rev Drug Discovery 2006; 5: 310-20; Turck C W et al (in press), Proteomic strategies for biomarker discovery—from differential expression to isoforms to pathways. In: Turck C W (ed) Biomarkers for psychiatric disorders. Springer, Berlin Heidelberg New York.), all of which generally can be employed in accordance with the invention.
In a more preferred embodiment, the expression on the transcriptional level is assessed using Northern blotting, reverse transcriptase polymerase chain reaction-based methods, microarray or tag-based methods.
“Northern blotting” is a technique used to study gene expression and is based upon the electrophoretic separation on either agarose or denaturing polyacrylamide gels, wherein the latter gels are preferentially used for smaller fragments of RNA, and the subsequent detection of the target RNA with a hybridisation probe. The probe may be made from DNA or RNA. Also, a “reverse Northern blot” may be performed in order to assess gene expression levels. In this assay, the substrate nucleic acid which is affixed to a membrane is a collection of isolated DNA fragments and the probe is RNA extracted from a sample and e.g. radioactively labeled. The basic principle and technique of Northern blotting is well-known in the art and the skilled person knows how to adapt said technique to his specific experimental needs, particularly how to design suitable probes.
DNA microarrays are devices for high-throughput assays used to simultaneously assay a variety of target genes and capture the status quo of a sample with respect to, e.g., gene expression or genotype. The use of microarrays has dramatically increased in the last 10 years and the use as well as the design of microarray studies can be considered common general knowledge in the technical field of the invention, i.e. medical genetics. Generally, it consists of an arrayed series of a specified number of microscopic spots, also called features, where DNA oligonucleotides are immobilized each containing picomoles of a specific DNA sequence. This can be a short section of a gene or other DNA element that are used as probes to hybridize a cDNA or cRNA sample (called target) under high-stringency conditions. Probe-target hybridization is usually detected and quantified by fluorescence-based detection of fluorophore-labeled targets to determine relative abundance of nucleic acid sequences in the target.
In standard microarrays, the probes may be attached to a solid surface by a covalent bond to a chemical matrix (via epoxy-silane, amino-silane, lysine, polyacrylamide or others). The solid surface can be glass or a silicon chip, in which case they are commonly known as gene chip or colloquially “Affy chip” when an Affymetrix chip is used. Other microarray platforms, such as Illumina, use microscopic beads, instead of a large solid support. DNA arrays are different from other types of microarray only in that they either measure DNA or use DNA as part of its detection system.
DNA microarrays can be used to measure changes in expression levels of the genes associated with a risk to develop PTSD in accordance with the method of the invention. Factors to be considered when conducting microarray experiments are the experimental design and the methods of analyzing the data, which are mostly effected using computer-aided analyzing programs, all of which belong to the common general knowledge in the technical field of the invention (cf. e.g., Bakay M et al., BMC Bioinformatics 2002, 3: 4; Gentleman R et al. (2005). Bioinformatics and computational biology solutions using R and bioconductor. Springer: New York; Heber S. & Sick B., Omics 2006; 10: 358-68).
The reverse transcriptase polymerase chain reaction-based methods to be used in accordance with the method of the invention are based on the principle of initially converting in a first step RNA obtained from a sample into DNA. Briefly, an mRNA template, dNTPs and reverse transcriptase are combined with a DNA primer in a reverse transcriptase buffer to produce complementary DNA (cDNA). In a second step, the generated cDNA is subjected to PCR that amplifies the target cDNA. The resulting DNA amplicons may be employed in further assays such as, e.g., a Northern blot. A preferred reverse transcriptase polymerase chain reaction-based method is the so called (quantitative) real-time PCR which is used to simultaneously amplify and quantify a target DNA molecule. The general principles of PCR as well as the numerous established variations based on said principle are well-known in the art and described in a variety of textbooks as well as scientific articles (cf., e.g., Saiki R K et al., Science 1988; 239: 487-91; Mullis K B et al. (1994). PCR: Polymerase Chain Reaction. Birkhauser: Boston). Briefly, PCR is performed on an automated cycler device, which can heat and cool containers with the reaction mixture in a very short time. The PCR, generally, consists of many repetitions of a cycle which consists of: (a) a denaturing step, which melts both strands of a DNA molecule and terminates all previous enzymatic reactions; (b) an annealing step, which is aimed at allowing the primers to anneal specifically to the melted strands of the DNA molecule; and (c) an extension step, which elongates the annealed primers by using the information provided by the template strand. Generally, PCR can be performed for example in a 50 μl reaction mixture containing 5 μl of 10×PCR buffer with 1.5 mM MgCl2, 200 μM of each deoxynucleoside triphosphate, 0.5 μl of each primer (10 μM), about 10 to 100 ng of template DNA and 1 to 2.5 units of Taq Polymerase. The primers for the amplification may be labeled or be unlabeled. DNA amplification can be performed, e.g., with a model 2400 thermal cycler (Applied Biosystems, Foster City, Calif.): 2 min at 94° C., followed by 30 to 40 cycles consisting of annealing (e. g. 30 s at 50° C.), extension (e. g. 1 min at 72° C., depending on the length of DNA template and the enzyme used), denaturing (e. g. 10 s at 94° C.) and a final annealing step at 55° C. for 1 min as well as a final extension step at 72° C. for 5 min. Suitable polymerases for use with a DNA template include, for example, E. coli DNA polymerase I or its Klenow fragment, T4 DNA polymerase, Tth polymerase, Taq polymerase, a heat-stable DNA polymerase isolated from Thermus aquaticus Vent, Amplitaq, Pfu and KOD, some of which may exhibit proof-reading function and/or different temperature optima. The person skilled in the art knows how to optimize PCR conditions for the amplification of specific nucleic acid molecules with primers of different length and/or composition or to scale down or increase the volume of the reaction mix.
The exponential amplification via reverse transcription polymerase chain reaction provides for a highly sensitive technique, where a very low copy number of RNA molecules can be detected. Reverse transcription polymerase chain reaction is widely used in the diagnosis of genetic diseases—applied to the method of the invention for the semiquantitative determination of the abundance of specific different RNA molecules within a cell or tissue as a measure of gene expression.
Tag-based methods refer to methods relying on the extraction of a short sequence (tag) that is unique for a specific transcript. These tags are ligated together, cloned and sequenced. The advantage of tag-based methods over microarray methods is their potential to evaluate expression of known as well as unknown genes. The disadvantages are the higher efforts and the qualitative nature of the standard procedure. Most prominent example for tag-based gene expression methods is the Serial Analysis of Gene Expression (SAGE), which is meanwhile a standard method in the science and introduced in many review articles and text books (Velculescu et al., Science 1995; 270: 484-7; Nielsen K L (2008) Serial Analysis of Gene Expression (SAGE); Methods and protocols. Humana Press: Totowa, N.J.).
Further developments of the SAGE technique resulted in so called Digital Gene Expression methods with an improved detection for low-abundance transcripts (Kin et al., Science 2007; 316: 1481-4).
The detection of proteins to determine the expression levels of genes associated with a risk to develop PTSD in accordance with the invention may be performed using a variety of methods suitable to quantify protein amounts.
In a more preferred embodiment of the method of the invention, the expression on the translational level is assayed using antibody arrays or mass spectrometry assays.
Antibody arrays facilitate the simultaneous detection of multiple proteins and can be designed as antibody microarrays to allow for a high-throughput setup. For example, antibodies directed to the proteins of the genes associated with a risk to develop PTSD may be arranged to form an antibody array that can be used as a test system in a clinical setting as well as private practice for the diagnosis of a risk to develop PTSD.
Mass spectrometry is an analytical technique that identifies the chemical composition of a compound or sample on the basis of the mass-to-charge ratio of charged particles. The method employs chemical fragmentation of a sample into charged particles (ions) and measurements of two properties, charge and mass, of the resulting particles, the ratio of which is deduced by passing the particles through electric and magnetic fields in a mass spectrometer. The design of a mass spectrometer has three essential modules: an ion source, which transforms the molecules in a sample into ionized fragments; a mass analyzer, which sorts the ions by their masses by applying electric and magnetic fields; and a detector, which measures the value of some indicator quantity and thus provides data for calculating the abundances each ion fragment present. Besides allowing a quantitative analysis of a sample in accordance with the method of the invention, the technique also other uses, such as identifying unknown compounds, determining the isotopic composition of elements in a compound, determining the structure of a compound by observing its fragmentation, studying the fundamentals of gas phase ion chemistry (the chemistry of ions and neutrals in vacuum), and determining other physical, chemical, or biological properties of compounds. Mass spectrometry assays for quantitative analysis of samples described herein are well-known in the art. For example, the Absolute QUAntification (AQUA) mass spectrometry method using stable isotope labelled internal standard peptides is a frequently applied technique to determine precisely quantitative protein expression levels from cell lysates (Kirkpatrick D S et al., Methods 2005; 35: 265-43).
A further embodiment of the invention relates to a method of selecting a therapy to prevent or treat PTSD comprising the steps of: (a) identifying a predisposition for developing posttraumatic stress disorder according to the method of the invention; and (b) selecting a therapy based on the results obtained in the preceding step.
As is evident to the person skilled in the art, the knowledge deduced from the present invention can now be used to exactly and reliably characterize the expression profile of a subject as far as it is relevant in the identification of a predisposition to develop PTSD. Advantageously, PTSD can be predicted or diagnosed and preventive or therapeutical measures can be applied accordingly. Moreover in accordance with the foregoing, in cases where a given therapy proves to be not effective, a suitable individual therapy can be designed based on the knowledge of the individual gene expression profile of a subject with respect to the genes associated with a risk to develop PTSD and new and/or improved therapeutics can be identified, for example, by the method of the invention, and/or developed as has been discussed supra.
A variety of therapies exist to treat PTSD. Generally, physicians choose from psychotherapeutic interventions alone or in combination with psychopharmacological drugs from the type of a selective serotonin reuptake inhibitor (SSRI) or of a benzodiazepine (BZD) for severe forms of the disorder. While psychotherapeutic interventions are costly in terms of money and time, SSRIs and BZD have also impediments, especially with respect to their side-effects. SSRIs can induce a number of medical problems related to the peripheral effects of these drugs. This includes nausea, stomach upset, muscle pain, tachycardia or heart arrhythmia, diminished sexual interest and/or performance, and sleep disturbances. BZD are usually medically well tolerated, but can produce psychomotor side effects and memory disturbances, as well as and foremost can induce tolerance, withdrawal symptoms, and dependence. If an ongoing development of PTSD can be diagnosed prior to the development of full symptoms, the outcome of the disorder would be more favorable (Breslau N. J Clin Psychiatry 2001; 62 Suppl 17: 55-9), especially, as patients with a predominant avoidance behavior, which are less likely to seek medical treatment, are at greater risk to develop a chronic course of the disorders (Perkonigg et al., Am J Psychiatry 2005; 162: 1320-27). As is evident from the above, once diagnosed with a predisposition to develop PTSD a subject may upon exposure to a traumatic event or prior thereto be treated depending on his/her individual genetic make up and accordingly, psychotherapeutic and/or psychopharmacological drugs may be selected by the physician as therapeutical intervention. If the development of severe symptoms can be predicted according to the method of the invention, even a suitable psychopharmacological drug may be selected as therapy such as, e.g. a SSRI, which can be given at a lower dosage than it can be expected for the case of the development of severe symptoms, potentially also saving the subject from side effects.
As outlined above, a reliable method to diagnose a predisposition to develop PTSD has not been available prior to the present invention. Hence, an early diagnosis of a predisposition potentially demands for a different therapy than treating acute PTSD symptoms. Further, depending on the severity of the symptoms it may also be necessary to adjust a therapy accordingly.
In conclusion, due to the invention it is possible to select a suitable therapy such as, e.g., a drug and/or psychotherapy, and a suitable dosage regimen having overall a more beneficial effect than therapeutic approaches without having regard to the individual gene expression profile of the genes associated with a risk to develop PTSD. The effects of therapies, e.g., pharmacologic effects of drugs, can be determined by methods well-known in the art and include, for example, in vitro methods or collecting data relating to disease symptoms in a patient or group of patients.
In accordance with the above, in a preferred embodiment, the method of the invention comprises prior to step (b) a further step (a′) of applying and monitoring a therapy.
The step of selecting a therapy may comprise the additional step (a′) of applying and monitoring a therapy and based on its outcome select a therapy. For example, subjects that are diagnosed to be at risk for developing PTSD and put on a prophylactic therapy can be monitored and, if necessary, their therapy subsequently be adjusted or changed depending on whether PTSD symptoms occur and to which extent or not. Also, a therapy may be applied to a patient having acute PTSD and routinely the effect of the therapy is monitored and recorded. The recorded data provides the basis for the assessment whether the therapy applied is beneficial to the patient or not. Based on said assessment the person skilled in the art, in this case likely a clinician, will be able to adjust the currently applied therapy, e.g., by increasing/decreasing dosage regimen or dosage amount of the therapy, or decide to completely switch to another therapy.
In accordance with the foregoing, the therapy comprises in another preferred embodiment administration of psychopharmacological medication and/or psychotherapy.



The figures show:
FIG. 1:
Gene activity of FKBP5, STAT5B, and NFIA involved in the fine-regulation of the HPA axis as well as of 21 further genes was profoundly altered in survivors of the New York WTC terrorist attacks who developed PTSD. Effect sizes were beyond the limits of a large effect according to common statistical interpretation guidelines (dashed lines).



The examples illustrate the invention:
EXAMPLE 1
Participants and Clinical Evaluation
A random sample of Caucasians who met criteria for PTSD in at least two out of four waves following 9/11 was invited by mail to participate in this study. After 20 participants were successfully recruited, a random sample of participants who had not met criteria for PTSD at any time were invited to participate, selected to match the PTSD participants in severity of trauma exposure (i.e., high direct vs. low and/or indirect exposure), age, and gender. All participants were Caucasians according to their responses in a self-report questionnaire asking for nationality, first language and ethnicity of the participant and all 4 grandparents. The study was approved by the Mount Sinai School of Medicine and the New York Academy of Medicine. All participants provided written, informed consent, and then further screened to determine eligibility. Participants were not invited to participate further if they had psychotic or bipolar illness, alcohol or substance dependence, or major medical, endocrinological, or neurological illness, confirmed by medical examination. No participant was in active treatment at the time of the study, and none were taking antidepressants.
Diagnostic evaluation at the time of the blood draw (wave 5) was performed by trained psychologists with established interrater reliability on the Clinician Administered PTSD scale (CAPS) (see, Blake et al., J Trauma Stress 1995; 8: 75-90) and the Structured Clinical Interview for the DSM-IV (SCID) (see, Spitzer R L, Gibbon M., Williams J B W. Structured Clinical Interview for DSM-IV Axis 1 Disorders (SCID). New York State Psychiatric Institute, Biometrics Research: New York, 1995). These scales, respectively, determined the presence of PTSD and confirmed the absence of other psychiatric disorders. To supplement information already collected in previous waves, information about other lifetime traumatic life events was obtained using the Trauma History Questionnaire (Green B L, Trauma history questionnaire. In Stamm B. (ed) Measurement of Stress, Trauma, and Adaptation. Sidran Press: Lutherville, Md., 1996, pp 366-369). Participants also completed the Childhood Trauma Questionnaire (Bernstein et al., Child Abuse Negl 2003; 27: 169-190).
EXAMPLE 2
Blood Drawing and Processing
Fasting blood samples were obtained by routine venipuncture between 08:00-09:00 h. Plasma samples were frozen for the subsequent determination of cortisol by radioimmunoassay (RIA). The intra-assay and inter-assay coefficients of variation for this method was 4.0 and 6.8%.
For RNA, blood was processed using the Paxgene RNA stabilization system, which prevents degradation of many short-lived RNA transcripts and prevent further transcription and metabolic activity from altering the composition of the sample.24 In addition, the samples were subjected to the globin mRNA reduction method as this greatly improves the data quality of stabilized RNA samples hybridized to microarrays. Gene expression studies were carried out using an Affymetrix Human Genome U133 Plus 2.0 Array containing 47 000 transcripts, using standard methods.
Quantitative polymerase chain reaction (qPCR) was performed to validate FKBP5 gene expression. For this procedure, total RNA prepared as above was used to generate cDNA using SuperScript II Reverse Transcriptase (Invitrogen, CA) according to the manufacturer's instructions. Gene expression probes and primers for FKBP5 were designed based on the NCBI sequence (NM—004117.2) using the ProbeFinder v2.41 software (Roche, http://www.universalprobelibrary.com). Four different exon junctions including exon 4 to 5 (4&5), exon 7 to 8 (7&8), exon 8 to 9 (8&9) and exon 11 to 12 (11&12) were quantitatively amplified. qPCR was performed on an ABI PrismTM 7900HT sequence detection system (Applied Biosystems, CA). Each sample was analyzed in quadruplicate in a reaction including 25 ng cDNA, 200 nM of each primer, 100 nM UPL probe, and 1× Platinum Quantitative PCR SuperMix-Uracil-N-Glycosylase (UDG) with ROX (Invitrogen, CA). The values were evaluated using the Sequence Detection Software v2.2.1 (Applied Biosystems, CA).
To test whether the observed FKBP5 expression differences might be partially determined by genotypic variation, we genotyped four single nucleotide polymorphisms (SNPs) in the FKBP5 gene region (rs3800737, rs9296158, rs1360780, rs9470080), which were previously reported as associated with PTSD risk or PTSD related symptoms (Koenen et al., Mol Psychiatry 2005; 10: 1058-9; Binder et al., JAMA 2008; 299: 1291-305). Genotyping was performed with a Roche LightCycler 480 System using allele-specific hybridization probes obtained from Metabion International AG (Martinsried, Germany); sequences are available upon request. None of the SNPs showed significant deviation from Hardy-Weinberg Equilibrium (p>0.09); genotypes could be determined with a call rate of greater than 97%. Linkage disequilibrium (LD) structure was evaluated with HAPLOVIEW, version 4.0, (http://www.broad.mit.edu/mpg/haploview/) revealing r2 between 0.77 and 0.95, which agrees with previous reports about the LD structure of this gene (Binder et al., Nat Genet 2004; 36: 1319-25; Binder et al., JAMA 2008; 299: 1291-305).
EXAMPLE 3
Statistical Analysis
Groups were compared on demographic, clinical measures and biological measures using t-tests, or chi-square tests as appropriate.
To analyze the microarray data, RNA expression was compared between cases and controls using dChip 2007 (build date Sep. 5, 2007). Invariant Set Normalization was carried out with all 40 arrays and model-based expression was evaluated using PM-MM probe data. Parameters were chosen using empirically derived false discovery rates (FDR). The use of t-test P-value for identifying differentially expressed genes showed a U-shaped curve in these analyses, with a minimum FDR at P=0.01. Other parameters showed increased FDR with more restrictive filtering. Differentially expressed genes were therefore first identified using P-values of 0.01 or lower as the criterion. As an example, using these parameters and comparing 20 controls and 20 cases with lifetime PTSD led to the identification of genes with an empirical median FDR of 16% and an empirical 90% FDR of 59% (from 200 permutations). Subsequently, the large proportion of these genes where the absolute expression differences were ≦50 were identified as very low-expressing genes and removed from further analyses.
To determine how the above noted gene expression changes might differentiate between persons with and without PTSD, the data were subjected to linear discriminate analysis (LDA), as implemented in dChip.
Data analysis for qPCR was performed using qBase. Reference genes chosen from GAPDH, ACTB, B2M and RPLPO were included based on the minimal coefficient of variation. A normal control sample was run in the each plate as a normalization control set to a value of 1. ANOVA was used to compare the expression levels between PTSD and controls.
Stepwise regression analyses were used to predict the contribution of individual genes to PTSD severity. For genes associated with FKBP5 additional regression analyses were performed to predict the contribution of trauma related variables and plasma cortisol levels to gene expression.







TABLE 1







Gene expression of 24 genes was distinctly altered in survivors


of the Sep. 11, 2001, terrorist attacks on the New York World


Trade Center, who developed PTSD compared to survivors


without PTSD. Among these genes, FKBP5, STAT5B, and


NFIA form a network related to the regulatory function of the


glucocorticoid receptor, which is the main regulatory


element of the primary stress-hormone system.












Fold-




Transcript
change1
p















FKBP5 (transcript a, b)
0.79/0.78
<0.0087



STAT5B
0.77
0.0068



NFIA
0.87
0.0058



MAN2C1
1.33
0.0009



DDX17
1.32
0.0073



CPNE1
0.87
0.0081



TSC1
0.86
0.0080



ADAM17
0.85
0.0090



RNF181
0.84
0.0078



MYO1C
0.84
0.0055



MOBKL1B
0.83
0.0071



GBA
0.81
0.0032



C2orf34
0.80
0.0043



TMEM167A
0.80
0.0099



MAST4
0.77
0.0027



ASAH1
0.77
0.0014



CGGBP1
0.76
0.0055



FAM175B
0.76
0.0059



BIN1
0.75
0.0024



KLF12
0.75
0.0027



METTL7A
0.73
0.0068



TMEM49
0.71
0.0080



RPS6KA5
0.71
0.0011



DR HLA-DRB1
0.24
0.0092







1of gene expression in PTSD compared with trauma exposed controls.

## Claims

1. A method for diagnosing and treating a human subject with a predisposition to post-traumatic stress disorder (PTSD), comprising:
i) detecting in a blood sample obtained from the human subject a test expression level of FK506 binding protein (FKBP5) gene mRNA;
ii) comparing the test expression level to a mean expression level of FK506 binding protein gene mRNA in control blood samples from a population not predisposed to PTSD and detecting that the test expression level is decreased compared to the mean expression level;
iii) correlating the detected test expression level with the presence of a predisposition to PTSD in the subject; and
iv) applying to the subject a therapy to treat or prevent PTSD selected from psychopharmacological medication and/or psychotherapy.

2. The method of claim 1, further comprising detecting in a sample from said subject at least a second test expression level of a mRNA product of a gene selected from the group of genes consisting of: a signal transducer and activator of transcription (STAT5B) gene, a nuclear factor 1A (NF1A) gene, a mannosidase-alpha class 2C, member 1 (MAN2C1) gene; a DEAD box polypeptide 17 (DDX17) gene, a copine I (CPNE1) gene, a tuberous sclerosis 1 (TSC1) gene, an ADAM metallopeptidase domain 17 (ADAM17) gene, a ring finger protein 181 (RNF181) gene, a myosin IC (MYO1C) gene, a Mps One Binder kinase activator-like 1B (MOBKL1B) gene, a glucosidase-beta acid (GBA) gene, a chromosome 2 open reading frame 34 (C2orf34) gene, a transmembrane protein 167A (TMEM167A) gene, a microtubule associated serine/threonine kinase family member 4 (MAST4) gene, a N-acylsphingosine amidohydrolase (ASAH1) gene, a CGG triplet repeat binding protein 1 (CGGBP1) gene, a family with sequence similarity 175, member B (FAM175B) gene, a bridging integrator 1(BIN1) gene, a kruppel-like factor 12 (KLF12) gene, a methyltransferase like 7A (METTL7A) gene, a transmembrane protein 49 (TMEM49) gene, a ribosomal protein S6 kinase, 90 kDa polypeptide (RPS6KA5) gene, and a major histocompatibility complex, class II, DR beta (DR HLA-DRB1) gene.

3. The method of claim 1, wherein the test expression level of FK506 binding protein gene mRNA is about 79% or less of the mean expression level of FK506 binding protein gene mRNA in control blood samples from a population not predisposed to PTSD.

4. The method of claim 1, in which the expression level of mRNA is assessed using a nucleic acid amplification method that utilizes a primer that is specific for binding to a nucleotide sequence present in the mRNA and that is labeled so as to be detectable, or in which the expression level of mRNA is assessed using a method of binding mRNA that is labeled for detection to nucleic acids immobilized on a substrate.

5. The method of claim 4, in which the label is a fluorescent label or a radioactive label.

6. The method of claim 4, in which the FK506 bindin protein gene mRNA includes an exon junction in the FKBP5 gene.

7. The method of claim 6, in which the exon junction is one or more selected from the group consisting of the exon junction joining exons 4 and 5, the exon junction joining exons 7 and 8, the exon junction joining exons 8 and 9, the exon junction joining exons 11 and 12.

8. The method of claim 2, in which the expression level of said FK506 binding protein mRNA is less than the expression level of said FK506 binding protein mRNA in a population not predisposed to PTSD, and the expression level of the mRNA of the MAN2C1 gene or the product of the DDX17 gene is assessed and said expression level of the mRNA of the MAN2C1 gene or the mRNA of the DDX17 gene is greater than the mean expression level of said MAN2C1 or DDX17 mRNA in control samples from a population not predisposed to PTSD.

9. The method of claim 8, in which the expression level of the mRNA of the MAN2C1 gene or the mRNA of the DDX17 gene is assessed and said expression level of the mRNA of the MAN2C1 gene or of the DX17 gene is about 1.3times the mean expression level of said MAN2C1 or DDX17 mRNA in control samples from a population not predisposed to PTSD.

10. The method of claim 2, in which the expression levels of at least a FK506binding protein gene, a STAT5 gene and the expression level of mRNA of all three of said FK506 binding protein, STAT5 and NFIA genes are decreased compared to the mean expression level of said FK506 binding protein, STATS and NFIA mRNAs in control samples from a population not predisposed to PTSD.

11. The method of claim 1, further comprising assessment in said sample of the expression level of at least a second mRNA analyte species of a gene selected from the group of genes consisting of a CPNE1 gene, a TSC1 gene, a ADAM17 gene, a RNF181 gene, a MYO1C gene, a MOBKL1B gene, a GBA gene, a C2orf34 gene or a TMEM167A gene, and the expression level of said FK506 binding protein is about 79% compared to the mean expression level of said FK506 binding protein mRNA in control samples from a population not predisposed to PTSD, and the expression level of said second mRNA analyte species is from 80% to 87% compared to the mean expression level of said second mRNA analyte species in control samples from a population not predisposed to PTSD.

12. The method of claim 1, further comprising assessment in said sample of the expression level of at least a second mRNA analyte species of at least one gene selected from the group consisting of a MAST4 gene, an ASAH1 gene, a CGGBP1 gene, a FAM175B gene, a BIN1 gene, a KLF12 gene, a METTL7A gene, a TMEM49 gene and a PRS6KA5 gene is also assessed, and the mRNA expression level of said FK506 binding protein is about 79% compared to the mean mRNA expression level of said FK506 binding protein product in control samples from a population not predisposed to PTSD, and the expression level of second mRNA analyte is from 71% to 77 compared to the mean expression level of said second mRNA analyte in control samples from a population not predisposed to PTSD.

13. The method of claim 2, in which the FKBP5 gene has a coding sequence as shown in SEQ ID NO: 1, the STAT5B gene has a coding sequence as shown in SEQ ID NO: 2, the NF1A gene has a coding sequence as shown in SEQ ID NO: 3, the MAN2C1 gene has a coding sequence as shown in SEQ ID NO: 4; the DDX17 gene has a coding sequence as shown in SEQ ID NO: 5; the CPNE1 gene has a coding sequence as shown in SEQ ID NO: 6; the TSC1 gene has a coding sequence as shown in SEQ ID NO: 7; the ADAM17 gene has a coding sequence as shown in SEQ ID NO: 8; the RNF181 gene has a coding sequence as shown in SEQ ID NO: 9; the MYO1C gene has a coding sequence as shown in SEQ ID NO: 10; the MOBKL1B gene has a coding sequence as shown in SEQ ID NO: 11; the GBA gene has a coding sequence as shown in SEQ ID NO: 12; the C2orf34 gene has a coding sequence as shown in SEQ ID NO: 13; the TMEM167A gene has a coding sequence as shown in SEQ ID NO: 14; the MAST4 gene has a coding sequence as shown in SEQ ID NO: 15 or 16; the ASAH1 gene has a coding sequence as shown in SEQ ID NO: 17; the CGGBP1 gene has a coding sequence as shown in SEQ ID NO: 18, the FAM175B gene has a coding sequence as shown in SEQ ID NO: 19; the BIN1 gene has a coding sequence as shown in SEQ ID NO: 20; the KLF12 gene has a coding sequence as shown in SEQ ID NO: 21; the METTL7A gene has a coding sequence as shown in SEQ ID NO: 22; the TMEM49 gene has a coding sequence as shown in SEQ ID NO: 23; the RPS6KA5 gene has a coding sequence as shown in SEQ ID NO: 24; and the DR HLA-DRB1 gene has a coding sequence as shown in SEQ ID NO: 25, or said gene has a coding sequence that is at least 95% identical to said SEQ ID NO.

14. The method of claim 13, in which said gene has a coding sequence that is at least 98% identical to said SEQ ID NO.

15. The method of claim 1, in which the subject is treated by administration of a benzodiazepine compound or one or more selective serotonin reuptake inhibitors.