System and methods for determining propensity to post-traumatic stress disorder (PTSD)

Abstract

Accurate diagnosis and treatment planning for PTSD is difficult. The discovery of specific markers creates new opportunities for more accurate clinical assessments identifying groups that may experience better outcomes when exposed to an intervention. Embodiments include methods of determining a subject's susceptibility (i.e., risk) of developing PTSD after a traumatic event. An assay can detect specific markers (e.g., nucleotides or proteins) in a sample from a subject that is prone to PTSD or suspected of suffering from PTSD. A subject who is deemed at risk of PTSD or who is suffering from PTSD can be treated with, for example, psychotherapy (e.g., cognitive behavioral therapy and adjunct pharmacotherapy) and / or medications such as serotonin-specific reuptake inhibitor (SSRI's).

Description

SYSTEM AND METHODS FOR DETERMINING PROPENSITY TO POST-TRAUMATIC STRESS DISORDER (PTSD)RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application No. 63 / 617,766 filed on January 4, 2024, the contents of which are incorporated herein by reference.FIELD OF THE INVENTION

[0002] The invention relates generally to therapeutic diagnostics, more particularly, to systems and methods of diagnosing PTSD and / or determining a subject’s susceptibility to PTSD.BACKGROUND

[0003] Post-Traumatic Stress Disorder (PTSD) is a prevalent chronic psychiatric and emotional condition that may occur in people who have experienced or witnessed one or more traumatic events that were coupled with physical or emotional harm, in circumstances that may have been life-threatening to themselves or others. Individuals may have intense, disturbing thoughts and feelings related to their experience that persist long after such events. Examples vary widely and may include terrorist acts, combat and war, rape and / or sexual assault, natural disasters, serious accidents, as well as multi-generational trauma experienced by a specific cultural, racial, or ethnic group, such as the Holocaust, slavery, forced migrations, and colonization of indigenous tribes. This disorder can result in a profound disturbance to individual and family functioning, causing significant medical, financial, and social problems.

[0004] Treatments for PTSD include psychotherapy, in particular cognitive behavioral therapy and adjunct pharmacotherapy, primarily with the serotonin-specific reuptake inhibitor (SSRI’s). Many different pharmacological approaches have been investigated. One approach is based on the clinical hypothesis that Major Depressive Disorder (MDD) and PTSD may have much in common. For example, they have common riskfactors, have overlapping symptoms, and often occur together. Consequently, antidepressants, such as the SSRI drugs fluoxetine (Prozac™) and paroxetine (Paxil™), are widely considered effective at treating some symptoms of PTSD. Other commonly administered SSRI antidepressants have included venlafaxine (Effexor™), and sertraline (Zoloft™).

[0005] Despite decades of research and billions of dollars, much about PTSD remains unknown. There is presently no simple or accurate test for PTSD. Moreover, there is no method of early detection. The diagnosis of PTSD is established on the basis of clinical history and subjective mental status examination, using a clinically structured interview, symptom checklists, or patient self-reports. However, relying on these subjective tests makes it difficult to distinguish PTSD from other psychiatric disorders. This can lead to misdiagnosis and difficult treatment decisions as to both treatment interventions and a more definitive understanding of the etiology. The existing limitations of current clinical assessment would benefit substantially from a more objective means to enhance the ability to identify the PTSD patient, and therefore distinguish PTSD from other psychiatric disorders.

[0006] Biomarkers are a non-invasive and cost-effective means to aid in clinical management of patients, particularly in areas of disease detection, prognosis, monitoring and therapeutic stratification. For example, serum PSA is commonly used for prostate cancer screening in men over 50, but its usage remains controversial due to serum elevation in benign disease as well as prostate cancer. Nevertheless, PSA represents one of the most useful serological markers currently available. PSA is strongly expressed in only the prostate tissue of healthy men, with low levels in serum established by normal diffusion through various anatomical barriers. These anatomical barriers are disrupted upon development of prostate cancer, allowing increased amounts of PSA to enter circulation. Other common serological biomarkers include carcinoembryonic antigen (CEA) and carbohydrate antigen 19.9 (CA19.9) for gastrointestinal cancer, CEA, CYFRA 21-1 (cytokeratin 19 fragment), neuron-specific enolase (NSE), tissue polypeptide antigen (TPA), progastrin-releasing peptide (pro-GRP), and SCC antigen for lung cancer, CA 125 for ovarian cancer and prostatespecific antigen (PSA, also known as KLK3) in prostate cancer.

[0007] Biomarkers are increasingly used to diagnose diseases promptly and accurately, and to identify individuals at high risk for certain conditions and tendencies even before clinical manifestations arise. There are presently no objective markers to validate the diagnosis or to serve as objective surrogate endpoints for therapy for PTSD. In the absence of clinically used markers for patients with PTSD, diagnosis has been dependent on subjective clinical assessments, or from asymptomatic diagnosis by a psychologist. Because PTSD can only be diagnosed through a personal interview of a patient, where the patient may be cognizant to give answers they know to be correct, the current methods leave it difficult to diagnose subjects suffering from these disorders. As a result, a majority of PTSD cases are often missed, misdiagnosed or left untreated in thousands of affected individuals. In some cases of these disorders being missed, the safety of the general public is also compromised.

[0008] There remains an unmet need for prognostic indicators that can aid in the detection of psychiatric disorders such as PTSD. In particular, there is a need for a process to objectively and reliably diagnose PTSD. Identifying individuals that are suffering from PTSD, along with those who are susceptible to PTSD, will allow early intervention and ensure proper treatment. Aspects of the present invention fulfill this need and provide further related advantages as described in the following summary.SUMMARY OF THE INVENTION

[0009] Aspects of the present disclosure teach certain benefits in construction and use which give rise to the exemplary advantages described below.

[0010] The present disclosure solves the problems described above by providing the ability to objectively diagnose PTSD, determine one’s susceptibility to PTSD and monitor the progress of patients with PTSD.

[0011] The methods and assays disclosed herein are directed to the examination of the amount of one or more biomarkers in a biological sample, wherein the determination of that amount is predictive or indicative of the risk of, presence and / or severity of PTSD. The disclosed methods and assays provide convenient, efficient, and potentially cost- effective means to obtain data and information useful in assessing appropriate or effective therapies for treating patients.

[0012] More specifically, embodiments include methods of detecting PTSD based on specific mRNAs and / or proteins that have altered expression levels. The Applicant has identified 11 genes that can be used as biomarkers to distinguish healthy individuals from individuals affected with PD. The use of biomarkers is non-invasive and more reliable than conventional methods. The biomarkers can predict if a subject is susceptible to PTSD. The biomarkers can also detect early-onset or asymptomatic PTSD. Embodiments also include methods of prognosis, patient monitoring and distinguishing between PTSD and ailments that present similar signs / symptoms (e.g., major depressive disorder).

[0013] In one embodiment, the method includes one or more mRNA biomarkers. In one embodiment, the method includes one or more protein biomarkers. In one embodiment, the method includes a combination of one or more mRNA biomarkers and one or more protein biomarkers.

[0014] Embodiments include the use of one or more of the following genes (or mRNAs) as biomarkers: ZDHHC14, PARK2, KAZN, TMEM51 -AS1 , ZNF813, LINC02335, MIR5007, TUC338, LINC02571 , HLA-B, SH3RF3 and PODXL.

[0015] Embodiments include the use of one or more of the following proteins as biomarkers: ZDHHC14, PARK2, KAZN, TMEM51 -AS1 , ZNF813, LINC02335, MIR5007, TUC338, LINC02571 , HLA-B, SH3RF3 and PODXL.

[0016] Embodiments include the use of one or more of the following genes (or mRNAs) as biomarkers: EXOC6, RAB27B, CCBL2, CDC42BPB, INPP4A, CTNND1 , TSFM, NCK1 , KHK, LMOD1 and PLCG1.

[0017] Embodiments include the use of one or more of the following proteins as biomarkers: EXOC6, RAB27B, CCBL2, CDC42BPB, INPP4A, CTNND1 , TSFM, NCK1 , KHK, LMOD1 and PLCG1.

[0018] Embodiments also include the use of the mRNAs and proteins identified herein as therapeutic targets for treating PTSD.

[0019] Additional embodiments include the use of one or more single nucleotide polymorphisms (“SNPs”) as biomarkers to diagnose PTSD, determine one’s susceptibility to PTSD and / or monitor the progress of patients with PTSD. In aspects, a polymorphism is identified in one or more of the following genes: SLC6A4, SLC6A3, DRD2, Glucocorticoid Receptor Gene, FKBP5, PACAP, PAC1 receptor, BDNF, GABAA receptor, RGS2, CCK or CNRI .

[0020] Embodiments also include a method of detecting PTSD or determining a prognosis of a subject with PTSD that includes steps of: a) measuring the expression level of at least one mRNA in a test sample of the subject, b) comparing the expression level of the mRNA in the test sample to a level in a base sample and c) detecting or determining the prognosis of PTSD based on altered expression the mRNA in the test sample. The method can be used with brain tissue, spinal fluid, blood, serum, saliva or other bodily fluids. The method can also compare levels of at least one protein between the base sample and the test sample. Further, conventional efforts and additional biomedical information can be considered. The method can also determine progression of PTSD and whether the subject is at risk of suffering from PTSD.

[0021] Similarly, embodiments also include a method of detecting PTSD or determining a prognosis of a subject with PTSD that includes steps of: a) measuring the expressionlevel of at least one protein in a test sample of the subject, b) comparing the expression level of the protein in the test sample to a level in a base sample and c) detecting or determining the prognosis of PTSD based on altered expression the protein in the test sample. The method can be used with brain tissue, spinal fluid, blood, serum, saliva or other bodily fluids. The method can also compare levels of at least one mRNA between the base sample and the test sample. Further, conventional efforts (e.g., compiling a clinical history, mental status examination, clinically structured interview, symptom checklists and / or patient self-reports) and additional biomedical information can be considered. The method can also determine progression of PTSD and whether the subject is at risk of suffering from PTSD.

[0022] In aspects, the methods described herein include a treatment step. Treatment can include cognitive behavioral therapy and adjunct pharmacotherapy (e.g., selective serotonin reuptake inhibitors).

[0023] Embodiments include a method of determining if a subject has PTSD and / or assessing a subject for susceptibility to PTSD. The method can include steps of (a) obtaining a fluid sample from a subject, (b) applying the sample to a lateral flow immunoassay (LFA) strip, (c) analyzing the strip to identify causal biomarkers and calculate ratios and correlations between biomarker levels in the sample, (d) assessing the subject for susceptibility to PTSD, (e) determining that the subject is susceptible to PTSD, and (f) treating the subject for PTSD. The method can also include consideration of conventional efforts and additional biomedical information.

[0024] In aspects, the methods described herein use up converting phosphors (UCPs) are used in lateral flow immunoassays. In aspects, machine learning is used to identify causal biomarkers and calculating ratios and correlations between marker levels in a patient sample.

[0025] In aspects, a threshold value indicates that the subject suffers from PTSD or is susceptible to PTSD. In aspects, causal biomarkers include one or more of proteinsand mRNA, miRNA, siRNA, acetylation, proteomics, genomics, RANA, proteinomics, protein: protein interactions and protein: molecule interactions.

[0026] In aspects, the biomarkers include one or more of ZDHHC14, PARK2, KAZN, TMEM51 -AS1 , ZNF813, LINC02335, MIR5007, TUC338, LINC02571 , HLA-B, SH3RF3 and PODXL.

[0027] In aspects, the biomarkers include one or more of EXOC6, RAB27B, CCBL2, CDC42BPB, INPP4A, CTNND1 , TSFM, NCK1 , KHK, LMOD1 and PLCG1.

[0028] In aspects, the biomarkers described herein can be used to distinguish PTSD from another psychiatric disorder with similar signs / symptoms (e.g., major depressive disorder).

[0029] Embodiments also include a method of diagnosing PTSD disease or determining a prognosis of a subject with PTSD. The method can include steps of (a) measuring the expression level of at least one nucleic acid in a test sample from blood of the subject, (b) receiving the expression level of the at least one nucleic acid in the test sample by a computer and (c) comparing the expression level of the at least one nucleic acid in the test sample to a level in a base sample for the same at least one nucleic acid, and (d) receiving a result comparing the expression levels of the at least one nucleic acid in the test sample measured in (a) and the base sample measured in (c), (e) diagnosing or determining the prognosis of PTSD based on an altered or increased expression of the at least one nucleic acid in the test sample as compared to the base sample, and (f) treating the subject or administering a therapy to the subject for PTSD. In aspects, the treatment includes administering one or more drugs (e.g., Prazosin HCL, Sertraline HCL, Trazodone HCL, Clonazepam, Paroxetine and / or Venlafaxine.

[0030] Embodiments also include a diagnostic kit for diagnosing PTSD or determining a prognosis of a subject with PTSD. The kit can include a plurality of nucleic acid molecules, each nucleic acid molecule encoding a mRNA sequence. The nucleic acidmolecules identify variations in expression levels of one or more mRNAs in a sample from a test subject. The expression levels of one or more mRNAs can represent a nucleic acid expression fingerprint that is indicative for the presence of PTSD. In other embodiments, the kit utilizes an immune-affinity based assay to compare levels of protein to diagnose PTSD or determine a prognosis of a subject with PTSD.

[0031] Other features and advantages of aspects of the present invention will become apparent from the following more detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, the principles of aspects of the invention.Definitions

[0032] Reference in this specification to "one embodiment / aspect" or "an embodiment / aspect" means that a particular feature, structure, or characteristic described in connection with the embodiment / aspect is included in at least one embodiment / aspect of the disclosure. The use of the phrase "in one embodiment / aspect" or "in another embodiment / aspect" in various places in the specification are not necessarily all referring to the same embodiment / aspect, nor are separate or alternative embodiments / aspects mutually exclusive of other embodiments / aspects. Moreover, various features are described which may be exhibited by some embodiments / aspects and not by others. Similarly, various requirements are described which may be requirements for some embodiments / aspects but not other embodiments / aspects. Embodiment and aspect can in certain instances be used interchangeably.

[0033] The terms used in this specification generally have their ordinary meanings in the art, within the context of the disclosure, and in the specific context where each term is used. Certain terms that are used to describe the disclosure are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of the disclosure. It will be appreciated that the same thing can be said in more than one way.

[0034] Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein. Nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only, and is not intended to further limit the scope and meaning of the disclosure or of any exemplified term. Likewise, the disclosure is not limited to various embodiments given in this specification.

[0035] Without intent to further limit the scope of the disclosure, examples of instruments, apparatus, methods and their related results according to the embodiments of the present disclosure are given below. Note that titles or subtitles may be used in the examples for convenience of a reader, which in no way should limit the scope of the disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In the case of conflict, the present document, including definitions, will control.

[0036] The terms patient, individual or “subject” are used interchangeably herein, and is meant a mammalian subject to be treated, with human patients being preferred. In some cases, the processes of the invention find use in experimental animals, in veterinary application, and in the development of vertebrate models for disease, including, but not limited to, rodents including mice, rats, and hamsters; birds, fish reptiles, and primates.

[0037] The term “normal subject” refers to a subject that is not or has not suffered from neural injury manifest in psychiatric terms and does not have a history of past neural injuries or any psychiatric disorders.

[0038] The term “psychiatric disorder” refers to a mental disorder that interferes with the way a person behaves, interacts with others, and functions in daily life. TheDiagnostic and Statistical Manual (DSM) of Mental Disorders, published by the American Psychiatric Association, classifies psychiatric disorders such as PTSD, major depressive disorder (MDD), schizophrenia (SCZ) and borderline personality disorder (BPD).

[0039] The term “Traumatic Brain Injury” or “TBI” refers to a condition in which a traumatic blow to the head causes damage to the brain, often without penetrating the skull. Usually, the initial trauma can result in expanding hematoma, subarachnoid hemorrhage, cerebral edema, raised intracranial pressure (ICP), and cerebral hypoxia, which can, in turn, lead to severe secondary events due to low cerebral blood flow (CBF). Depending upon severity, TBI may also be classified as severe, mild or moderate.

[0040] The term “environmental factor” refers to exposures to substances (e.g., pesticides, industrial waste, etc.) where a subject lives or works. Common environmental factors include (a) chemicals (e.g., mold, pesticides, etc.), (b) air pollution, (c) climate change and natural disasters, (d) diseases caused by microbes, (e) lack of access to health care, (f) infrastructure issues, (g) poor water quality and (h) global environmental issues.

[0041] The term “polymorphism” refers to a the presence of two or more variant forms of a specific DNA sequence that can occur among different individuals or populations. The most common type of polymorphism involves variation at a single nucleotide (also called a single-nucleotide polymorphism, or SNP). Other polymorphisms can be much larger, involving longer stretches of DNA.

[0042] The term “personalized health care” or “precision medicine” refers to an emerging practice of medicine that uses an individual's genetic profile to guide decisions made in regard to the prevention, diagnosis and treatment of disease. The approach can also take into account other characteristics of an individual (e.g., biomarker levels, protein and molecular interactions, etc.). For the patient / consumer, personalized health care means being recognized as a unique individual based on their health, geneticmakeup, chemistry, history and circumstances, and receiving treatment tailored to one’s needs. This leads to better health outcomes in preventing, diagnosing, treating and managing health and disease.

[0043] The term “algorithm” refers to a procedure for solving a mathematical problem in a finite number of steps that frequently involves repetition of an operation.

[0044] The term “artificial intelligence” or “Al” refers to intelligence exhibited by machines, rather than humans. The term, as applied herein, refers to when a machine mimics "cognitive" functions that humans associate with other human minds, such as "learning" and "problem solving."

[0045] The term “big data” refers to large data sets that can be analyzed computationally to reveal patterns, trends, and associations, especially relating to human physiological responses, human behavior and interactions. This can include, for example, a databases of electronic health records from a hospital or clinic.

[0046] The term “classifier” refers to the mathematical function, implemented by a classification algorithm that maps input data to a category. An algorithm that implements classification, especially in a concrete implementation, is known as a classifier.

[0047] The term “computer learning” or “machine learning refers to an application of artificial intelligence (Al) that provides systems the ability to automatically learn and improve from experience without being explicitly programmed. Machine learning focuses on the development of computer programs that can access data and use it learn for themselves.

[0048] The term “transfer learning” or “deep transfer learning” is a type of machine learning that can leverage existing, generalizable knowledge from other related tasks to enable learning of a separate task with a small set of data. The data sets available totrain models for in silico drug discovery efforts are often small. Indeed, the sparse availability of labeled data is a major barrier to artificial-intelligence-assisted drug discovery. One solution to this problem is to develop algorithms that can cope with relatively heterogeneous and scarce data. Deep transfer learning is the most commonly used type of transfer learning in the field of drug discovery.

[0049] The term “Markov model” refers to a stochastic model used to model pseudo- randomly changing systems. It is assumed that future states depend only on the current state, not on the events that occurred before it (that is, it assumes the Markov property). Generally, this assumption enables reasoning and computation with the model that would otherwise be intractable. For this reason, in the fields of predictive modelling and probabilistic forecasting, it is desirable for a given model to exhibit the Markov property.

[0050] The term “omics” refers to utilizing multiple molecular disciplines that involve the characterization of global sets of biological molecules such as DNAs, RNAs, proteins, and metabolites. For example, genomics investigates thousands of DNA sequences, transcriptom ics investigates all or many gene transcripts, proteomics investigates large numbers of proteins, and metabolomics investigates large sets of metabolites. Omic data can include genomic data, transcriptom ics, proteomics, epigenomics and metabolomics.

[0051] The term “historical data” refers to data (e.g., physiological measurements and actions of individuals) recorded and / or stored in a database that can be accessed for analysis and / or comparison. The data can be raw (e.g., sensor data) or processed (e.g. fused data). Historical data can include (a) data compiled from groups / populations of individuals and (b) data compiled from an individual person. For example, data can be recorded from healthy people and people with known ailments. An analysis of the data can indicate variations in physiological measurements that can be correlated with ailments. Likewise, data from an individual person can be recorded and stored. Thiscan allow the system to identify patterns, variations and / or aberrations in activity for that particular person.

[0052] As used herein, "additional biomedical information" refers to one or more evaluations of an individual, other than using any of the biomarkers described herein, that are associated with health and / or susceptibility to PTSD. Accordingly, "additional biomedical information" includes any of the following: physical descriptors of an individual, the height, weight and / or BMI of an individual, the gender of an individual, the ethnicity of an individual, family history, smoking history, occupational history, etc. Additional biomedical information can be obtained from an individual using routine techniques known in the art, such as from the individual themselves by use of a routine patient questionnaire or health history questionnaire, etc., or from a medical practitioner, etc. Testing of biomarker levels in combination with an evaluation of any additional biomedical information may, for example, improve sensitivity, specificity, and / or AUC for predicting vulnerability to PTSD (or other related issues) as compared to biomarker testing alone or evaluating any particular item of additional biomedical information alone.

[0053] The term “comorbid disorders” refers to additional diseases or conditions that a subject has at the same time as a primary health concern. Conditions described as comorbidities are often chronic or long-term conditions.

[0054] The term “drug addiction” refers to a complex set of behaviors typically associated with misuse of certain drugs, developing over time and with higher drug dosages. Addiction includes psychological compulsion, to the extent that the affected person persists in actions leading to dangerous or unhealthy outcomes. Opioid addiction includes insufflation or injection, rather than taking opioids orally as prescribed for medical reasons.

[0055] The term “formulation” as used herein refers to the antibodies disclosed herein and excipients combined together which can be administered and has the ability to bindto the corresponding receptors and initiate a signal transduction pathway resulting in the desired activity. The formulation can optionally comprise other agents.

[0056] The term "administration" refers to the introduction of an amount of a predetermined substance into a patient by a certain suitable method. The composition disclosed herein may be administered via any of the common routes, as long as it is able to reach a desired tissue, for example, but is not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, or intrarectal administration. However, since peptides are digested upon oral administration, active ingredients of a composition for oral administration should be coated or formulated for protection against degradation in the stomach.

[0057] The term “medicament,” “active agent” or “active ingredient” refers to a substance, compound, or molecule, which is biologically active or otherwise, induces a biological or physiological effect on a subject to which it is administered to. In other words, “active agent” or “active ingredient” refers to a component or components of a composition to which the whole or part of the effect of the composition is attributed. An active agent can be a primary active agent, or in other words, the component(s) of a composition to which the whole or part of the effect of the composition is attributed. An active agent can be a secondary agent, or in other words, the component(s) of a composition to which an additional part and / or other effect of the composition is attributed.

[0058] The term “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, in a sterile composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo. In one aspect, the pharmaceutical composition is substantially free of endotoxins or is non-toxic to recipients at the dosage or concentration employed.

[0059] The terms “treating,” “treatment” and the like are used herein, without limitation, to mean obtaining a desired pharmacologic and / or physiologic effect. The effect maybe prophylactic in terms of completely or partially preventing a disorder or sign or symptom thereof, and / or may be therapeutic in terms of amelioration of the symptoms of the disease or infection, or a partial or complete cure for a disorder and / or adverse effect attributable to the disorder. In regard to PTSD, treatments can include psychotherapy, medications or a combination of both. Common treatments for PTSD include, for example: Trauma-focused psychotherapy (e.g., Cognitive Processing Therapy (CPT), Prolonged Exposure (PE) and Eye Movement Desensitization and Reprocessing (EMDR)), Brief Eclectic Psychotherapy as well as medications. Some common medications for PTSD include Prazosin HCL, Sertraline HCL, Trazodone HCL, and Clonazepam. The National Center for PTSD recommends Sertraline (Zoloft™), Paroxetine (Paxil™), and Venlafaxine (Effexor®).

[0060] The term “prognosis” refers to the likely outcome or course of a disease and / or the chance of recovery or recurrence. As used herein, it refers to the probable outcome of PTSD, including whether a subject will respond to treatment or mitigation efforts and / or the likelihood that the PTSD will progress. This is in contrast to a “diagnosis” which refers to identifying an ailment or disease, usually from examining a subject.

[0061] The term “health evaluation” or “health assessment” refers to a plan of care that identifies the specific needs of a person and how those needs will be addressed by the healthcare system or healthcare provider. Conventionally, a health assessment follows an evaluation of a subject’s health status by performing a physical exam after taking a health history.

[0062] The term “single nucleotide polymorphisms” or “SNPs” refers to the most common type of genetic variation among people. Each SNP represents a difference in a single nucleotide. For example, a SNP may replace the nucleotide cytosine (C) with the nucleotide thymine (T) in a certain stretch of DNA. SNPs occur normally throughout a person’s DNA. They occur almost once in every 1 ,000 nucleotides on average, which means there are roughly 4 to 5 million SNPs in a person's genome. Most commonly, SNPs are found in the DNA between genes. They can act as biological markers to helplocate genes that are associated with disease (e.g., PTSD). When SNPs occur within a gene or in a regulatory region near a gene, they may play a more direct role in disease by affecting the gene’s function. Most SNPs have no effect on health or development. Some of these genetic differences, however, have proven to be important in the study of human health. SNPs can help predict an individual’s response to certain drugs, susceptibility to environmental factors such as toxins, and risk of developing diseases. SNPs can also be used to track the inheritance of disease-associated genetic variants within families.

[0063] The term “cis-regulatory elements” refers to regions of non-coding DNA (e.g., promoters, enhancers, and silencers) that regulate the transcription of nearby genes. In contrast, “trans-regulatory factors” regulate (or modify) the expression of distant genes by combining with their target sequences

[0064] The term "biomarker" refers generally to a DNA, RNA, protein, carbohydrate, or glycolipid-based molecular marker, the expression or presence of which in a sample can be detected by standard methods (or methods disclosed herein) and is predictive or prognostic of the effective responsiveness or sensitivity of a mammalians subject with an ailment. Biomarkers may be present in a test sample but absent in a control sample, absent in a test sample but present in a control sample, or the amount or of biomarker can differ between a test sample and a control sample. For example, protein biomarkers can be present in such a sample, but not in a control sample, or certain biomarkers are seropositive in the sample, but seronegative in a control sample. Also, expression of such a biomarker may be determined to be higher than that observed from a control sample. The terms "marker" and "biomarker" are used herein interchangeably.

[0065] The amount of the biomarker can be measured in a test sample and compared to the “normal control level,” utilizing techniques such as reference limits, discrimination limits, or risk defining thresholds to define cutoff points and abnormal values for an ailment. The normal control level means the level of one or more biomarkers orcombined biomarker indices typically found in a subject not suffering from the ailment. Such normal control level and cutoff points can vary based on whether a biomarker is used alone or in a formula combining with other biomarkers into an index. Alternatively, the normal control level can be a database of biomarker patterns from previously tested subjects who did not experience the ailment over a clinically relevant time.

[0066] After selection of a set of biomarkers, well-known techniques such as crosscorrelation, Principal Components Analysis (PCA), factor rotation, Logistic Regression (LogReg), Linear Discriminant Analysis (LDA), Eigengene Linear Discriminant Analysis (ELDA), Support Vector Machines (SVM), Random Forest (RF), Recursive Partitioning Tree (RPART), related decision tree classification techniques, Shrunken Centroids (SC), StepAIC, Kth-Nearest Neighbor, Boosting, Decision Trees, Neural Networks, Bayesian Networks, Support Vector Machines, and Hidden Markov Models, Linear Regression or classification algorithms, Nonlinear Regression or classification algorithms, analysis of variants (ANOVA), hierarchical analysis or clustering algorithms; hierarchical algorithms using decision trees; kernel based machine algorithms such as kernel partial least squares algorithms, kernel matching pursuit algorithms, kernel Fisher's discriminate analysis algorithms, or kernel principal components analysis algorithms, or other mathematical and statistical methods can be used to develop a formula for calculation of a risk score. A selected population of individuals is used, where historical information is available regarding the values of biomarkers in the population and their clinical outcomes. To calculate a risk score for a given individual, biomarker values are obtained from one or more samples collected from the individual and used as input data.

[0067] Tests to measure biomarkers and biomarker panels can be implemented on a variety of diagnostic test systems. Diagnostic test systems are apparatuses that typically include means for obtaining test results from biological samples. Examples of such means include modules that automate the testing (e.g., biochemical, immunological, nucleic acid detection assays). Some diagnostic test systems are designed to handle multiple biological samples and can be programmed to run thesame or different tests on each sample. Diagnostic test systems typically include means for collecting, storing and / or tracking test results for each sample, usually in a data structure or database. Examples include well-known physical and electronic data storage devices (e.g., hard drives, flash memory, magnetic tape, paper printouts). It is also typical for diagnostic test systems to include means for reporting test results. Examples of reporting means include visible display, a link to a data structure or database, or a printer. The reporting means can be a data link to send test results to an external device, such as a data structure, data base, visual display, or printer.

[0068] The term "detecting" or "determining" with respect to a biomarker value includes the use of both the instrument required to observe and record a signal corresponding to a biomarker value and the material / s required to generate that signal. In various embodiments, the biomarker value is detected using any suitable method, including fluorescence, chemiluminescence, surface plasmon resonance, surface acoustic waves, mass spectrometry, infrared spectroscopy, Raman spectroscopy, atomic force microscopy, scanning tunneling microscopy, electrochemical detection methods, nuclear magnetic resonance, quantum dots, and the like.

[0069] The term “physiological event” refers to a response or reaction of the body to a stimulus. Most are automatic / instinctive physiological responses. The healthy state of the body depends upon the integrity of various organ systems. The organ systems in the body function in a particular manner constantly. The mechanisms, by which the organ systems of the body function, can be referred to as “physiological mechanisms.” Physiological mechanisms explain any health-related events or outcomes.Physiological mechanisms can be altered voluntarily. For example, exercise causes alteration in the cardiac physiology of resting state.

[0070] The term “immunoassay” refers to a biochemical test that measures the presence or concentration of a substance in a sample, such as a biological sample. It is common to use the reaction of an antibody to its cognate antigen, for example the specific binding of an antibody to a protein. Both the presence of antigen and theamount of antigen present can be measured. The presence and amount (i.e. , abundance) of the protein can be determined or measured. Measuring the quantity of antigen (such as a biomarker) can be achieved by a variety of methods. A common method is to label either the antigen or antibody with a detectable label (e.g., a fluorescent tag, enzymatic linkage or radioactive isotope).

[0071] The term “lateral flow assay,” “lateral flow immunoassay” or “LFA” refers to a diagnostic device used to confirm the presence or absence of a target analyte. LFA- based tests often use a paper-based platform for the detection and quantification of analytes, where the sample is placed on a test device and the results are displayed within 5 - 30 minutes. LFA-based tests are widely used in hospitals and clinical laboratories for the qualitative and quantitative detection of specific antigens and antibodies, as well as products of gene amplification. The principle behind the LFA is relatively simple. A liquid sample (or its extract) containing the analyte of interest moves without the assistance of external forces (capillary action) through various zones of polymeric strips, on which molecules that can interact with the analyte are attached. A typical lateral flow test strip has overlapping membranes that are mounted on a backing card.

[0072] The term “target analyte” or “analyte” refers to a molecule, compound or particle to be detected. Target analytes bind to binding ligands (both capture and soluble binding ligands), as is more fully described below. In some embodiments, the target analyte is a protein such as a biomarker as described herein.

[0073] The term “substrate” or “solid support” refers to a material that can be modified to contain discrete individual sites appropriate of the attachment or association of capture ligands. Suitable substrates include metal surfaces such as gold, electrodes as defined below, glass and modified or functionalized glass, fiberglass, resins, silica or silica-based materials, carbon, metals, inorganic glasses and other polymers.

[0074] The term “up-converting nano-phosphors” or “upconverting materials” refers to compounds that emit light at a wavelength that is shorter than the wavelength of light they have been photoexcited with which give them applications in biomedical imaging. The so-called anti-Stokes shift in these materials limits the autofluorescence of nearby molecules within a sample. Compared with gold nanoparticles, a lateral flow test using upconverting phosphor nanoparticles (UCNPs) is more sensitive (approximately tenfold) and robust, due to the unique feature of using the lower energy 980 nm infrared light (excitation light) to generate higher energy visual light (emission light). This light process is called “up-conversion,” which does not occur in nature. Thus, UCNPs as a reporter label do not generate background fluorescence (autofluorescence) compared with conventional fluorescent labels, such as fluorescently labeled nanoparticles and quantum dots. Moreover, UCNPs do not fade, allowing the lateral flow strips based on UCNPs to be stored in the long term. More importantly, the lateral flow test based on UCNPs has no interference from red blood cell hemolysis that is a problem sometimes encountered in lateral flow test based on colloidal gold-labeled using blood.

[0075] The term “omics” refers to comprehensive approaches for analysis of complete genetic or molecular profiles of humans and other organisms. Since the process of mapping and sequencing the human genome began, new technologies have made it possible to obtain a huge number of molecular measurements within a tissue or cell. These technologies can be applied to a biological system of interest to obtain a snapshot of the underlying biology at a resolution that has never before been possible. Broadly speaking, the scientific fields associated with measuring such biological molecules in a high-throughput way are called “omics.”

[0076] The term “treatment regime refers” to a treatment plan that specifies the dosage, the schedule, and the duration of treatment for a medicament (e.g., an opioid agonist). A “standard regime” refers to one that would be proposed by a competent medical professional (e.g., a licensed physician) pursuant to current guidelines, laws, regulations and safety protocols for a particular substance.

[0077] The term “risk mitigation strategies” refers to efforts that can be employed to help prevent or reduce a patient from suffering from PTSD. These can include, for example, medication, counseling, cognitive therapy, exercise and lifestyle changes.

[0078] The term “cognitive behavior therapy” or “CBT” refers to a type of psychotherapy. It may help you to change unhelpful or unhealthy ways of thinking, feeling and behaving. CBT uses practical self-help strategies. These are designed to immediately improve your quality of life. CBT can be an effective way to treat depression and anxiety.

[0079] As applicable, the terms "about" or "generally", as used herein in the specification and appended claims, and unless otherwise indicated, means a margin of + / - 20%. Also, as applicable, the term "substantially" as used herein in the specification and appended claims, unless otherwise indicated, means a margin of + / - 10%. It is to be appreciated that not all uses of the above terms are quantifiable such that the referenced ranges can be applied.

[0080] Many known and useful compounds and the like can be found in Remington’s Pharmaceutical Sciences (13thEd), Mack Publishing Company, Easton, PA — a standard reference for various types of administration.DETAILED DESCRIPTION

[0081] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the subject technology as claimed. Additional features and advantages of the subject technology are set forth in the description below, and in part will be apparent from the description, or may be learned by practice of the subject technology. The advantages of the subject technology will be realized and attained by the structure particularly pointed out in the written description and claims hereof.

[0082] Recent studies have demonstrated the presence of mRNAs / proteins in saliva, circulating blood or other sample (e.g., cerebrospinal fluid) and their potential for use as biomarkers in the diagnosis of various diseases. Applicants present the use of specific biomarkers for detecting Post-Traumatic Stress Disorder (PTSD). Specifically, the Applicant has recognized mRNA and protein expression profiling in subjects with PTSD.

[0083] Specific mRNAs and proteins or “biomarkers” are aberrantly expressed in tissues affected by PTSD as compared to healthy tissue. The biomarkers provide diagnostic methods for diagnosing / predicting PTSD and / or prognosticating the effectiveness of treatment. Moreover, biomarker expression can provide insights into cellular processes involved in the progression of PTSD. Thus, the biomarkers can be used as targets to prevent and / or treat PTSD and related symptoms.

[0084] Accordingly, the invention is based on the finding that PTSD can be reliably identified based on particular biomarker expression profiles with high sensitivity and specificity. The expression of biomarkers typically includes both up- and down- regulated levels of mRNAs / proteins. An analysis of mRNA / protein expression biomarkers allows for creation of a “fingerprint” by analyzing mRNA / protein expression patterns in diseased and healthy subjects. Thereafter, individual mRNA / protein expression levels can be used for the detection of PTSD at early stages of the disease (e.g., when asymptomatic). It is also possible to monitor progression of PTSD and predict if a subject is susceptible to PTSD.

[0085] Methods for detecting biomarkers to be assessed include protocols that examine the presence and / or expression of a desired nucleic acid. Tissue or cell samples can be conveniently assayed for, e.g., genetic- marker mRNAs or DNAs using Northern, dot-blot, or polymerase chain reaction (PCR) analysis, array hybridization, RNase protection assay, or using DNA SNP chip microarrays, which are commercially available, including DNA microarray snapshots. For example, real-time PCR (RT-PCR) assays such as quantitative PCR assays are well known in the art. Methods alsoinclude assays to detect / quantify specific proteins such as immune-affinity assays (e g., ELISA, Western blot, flow cytometry, etc.).

[0086] Methods for detecting biomarkers to be assessed include protocols that examine the presence and / or expression of a desired protein. Tissue or cell samples can be conveniently assayed using, for example, an immunoassay known in the art (e.g., ELISA, Western blot, flow cytometry, etc.). In aspects, biomarkers are measured using a lateral flow assay (LFA) as described below.

[0087] In aspects, one or more biomarkers from Table 1 are measured. Levels of protein, DNA, RNA and / or metabolites can be used.Table 1 - List of Biomarkers - International Meta-AnalysisTable 2 - List of Biomarkers - Genome-wide association studies (GWAS)

[0088] In aspects, a subject is deemed “at risk” or susceptible to PTSD if they are statistically more likely to develop PTSD than not (i.e., a 51 % chance of developing signs of PTSD). In aspects, a subject is deemed susceptible if they are about 25% likely to suffer from PTSD. In aspects, a subject is deemed susceptible if they are about 10%, are about 20%, are about 30%, about 40%, about 45%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85% or more likely to suffer from PTSD.

[0089] In aspects, a subject is deemed “at risk” or susceptible to PTSD if they are statistically more likely to develop PTSD than not (i.e., a 51 % chance of suffering from PTSD) after finishing a traumatic event. In aspects, a subject is deemed susceptible if they are about 25% likely to develop PTSD. In aspects, a subject is deemed susceptible if they are about 10%, are about 20%, are about 30%, about 40%, about 45%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85% or more likely to develop PTSD.

[0090] In aspects, a subject is deemed “at risk” or susceptible to PTSD if they are statistically more likely to develop a dependency than not (i.e., a 51 % chance of suffering from PTSD) after experiencing a traumatic event. In aspects, a subject is deemed susceptible if they are about 25% likely to suffer from PTSD. In aspects, a subject is deemed susceptible if they are about 10%, are about 20%, are about 30%, about 40%, about 45%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85% or more likely to suffer from PTSD after experiencing a traumatic event.

[0091] In aspects, a subject is deemed “at risk” or susceptible to PTSD if they are statistically more likely to develop PTSD than not (i.e., a 51 % chance of suffering from PTSD after experiencing a traumatic event. In aspects, a subject is deemed susceptible if they are about 25% likely to suffer from PTSD. In aspects, a subject is deemedsusceptible if they are about 10%, are about 20%, are about 30%, about 40%, about 45%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85% or more likely to suffer from PTSD after experiencing a traumatic event.SNPs and other Factors

[0092] Causative SNPs are those SNPs that produce alterations in gene expression or in the expression, structure, and / or function of a gene product, and therefore are most predictive of a possible clinical phenotype. One such class includes SNPs falling within regions of genes encoding a polypeptide product, (i.e., cSNPs). These SNPs may result in an alteration of the amino acid sequence of the polypeptide product (i.e., non- synonymous codon changes) and give rise to the expression of a defective or other variant protein. Further, in the case of nonsense mutations, a SNP may lead to premature termination of a polypeptide product. Such variant products can result in a pathological condition (e.g., genetic disease). Examples of genes in which a SNP within a coding sequence causes a genetic disease include sickle cell anemia and cystic fibrosis.

[0093] Causative SNPs do not necessarily have to occur in coding regions; causative SNPs can occur in, for example, any genetic region that can ultimately affect the expression, structure, and / or activity of the protein encoded by a nucleic acid. Such genetic regions include, for example, those involved in transcription, such as SNPs in transcription factor binding domains, SNPs in promoter regions, in areas involved in transcript processing, such as SNPs at intron-exon boundaries that may cause defective splicing, or SNPs in mRNA processing signal sequences such as polyadenylation signal regions. Some SNPs that are not causative SNPs nevertheless are in close association with, and therefore segregate with, a disease-causing sequence In this situation, the presence of a SNP correlates with the presence of, or predisposition to, or an increased risk in developing the disease. These SNPs, although not causative, are nonetheless also useful for diagnostics, disease predisposition screening, and other uses.

[0094] Additional embodiments include the use of one or more single nucleotide polymorphisms (“SNPs”) as biomarkers to diagnose PTSD, determine one’s susceptibility to PTSD and / or monitor the progress of patients with PTSD. Accordingly, the detection of polymorphisms may be indicative of PTSD. In aspects, the detection of at least one of the polymorphisms may be indicative of an elevated predisposition to PTSD in the individual.SLC6A4

[0095] Studies have found that a high risk of developing PTSD is associated with a specific polymorphism in the promoter region of the serotonin transporter gene SLC6A4. The 5-HTTLPR polymorphism contains two predominant alleles, L (long) and S (short) representing different lengths of a polymorphic promoter region, with the S-allele associated with reduced serotonin transporter gene expression, leading to reduced serotonin reuptake. Studies have indicated that risk for PTSD development is associated with genotype (mainly S-allele carriers, also associated with depression) and high levels of trauma / stress.

[0096] Other studies have demonstrated a correlation between methylation of SLC6A4 and PTSD symptoms following traumatic events. Individuals with more traumatic events were found to be at increased risk for PTSD, but only when they had lower methylation levels at the SLC6A4 locus. At higher methylation levels, individuals with more traumatic events were protected from developing PTSD (24, 25).SLC6A3

[0097] A polymorphism in the gene encoding the dopamine transporter (SLC6A3 also known as DAT or DAT1 ) has a 40-base pair repeat that is polymorphic in the population. Genetic variants in dopamine transporters have been associated with PTSD.DRD2

[0098] The dopamine receptor D2 (DRD2) contains rs1800497, a single nucleotide polymorphism (SNP) with T (A1 ) and C (A2) alleles. This allele is associated with PTSD in Caucasian war veterans (33) and excessive alcohol intake (34). The dopamine D3 receptor (DRD3) gene (involved in relevant processes such as executive functioning and emotional reactivity) has also been implicated in PTSD.Glucocorticoid Receptor Gene

[0099] Several polymorphisms of the glucocorticoid receptor gene have been observed in individuals with PTSD. Studies have shown that PTSD is often characterized by a dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis, such that the system is altered and hyper-responsive to cortisol feedback. These effects may be observed in PTSD patients, of which a large number display consistently lower cortisol responses in dexamethasone suppression tests indicating enhanced negative feedback in the HPA axis. The glucocorticoid receptor (GR) mediates negative feedback of the HPA axis, thus the HPA axis effects in individuals with PTSD are thought to occur, in part, via enhanced sensitivity of GR-mediated feedback mechanism, which acts to suppress stress-related cortisol release.FKBP5

[0100] Four SNPs in FKBP5 (rs9296158, rs3800373, 1360780, rs9470080, that are in high linkage disequilibrium with each other) have been shown to interact with child abuse severity to predict adult PTSD. More recently, one of these polymorphisms in FKBP5 (rs1360780), that increases the risk of developing stress-related psychiatric disorders in adulthood, was found to be dependent on changes in DNA methylation occurring as a consequence of childhood trauma-dependent stress. Other studies indicate that genetic variation in FKBP5 influences the risk of anxiety and / or depressive disorders in early life and interact with childhood abuse to increase the risk of PTSD.PACAP

[0101] The neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), is involved in regulating corticotrophin-releasing hormone (CRH) a key mediator of the stress axis.PAC1 receptor

[0102] Recently, a genetic variant in the PAC1 receptor (ADCYAP1 R1 ; rs2267735) that disrupts a putative estrogen response element, has been found to be associated with PTSD. In addition, genetic variation at the ADCYAP1 R1 locus interacts with childhood maltreatment to influence the risk of developing PTSD later in life, specifically in women. Finally, PTSD patients display changes in peripheral blood DNA methylation and mRNA expression of the ADCYAP1 R1 transcript.BDNF

[0103] Brain-derived neurotrophic factor (BDNF), a key regulator of neuronal plasticity, is known to be associated with depression as well as the extinction of fear, a key process disrupted in PTSD. A SNP in the gene encoding human BDNF gives rise to a functional variant at codon 66 where the amino acid valine (Vai) is substituted by methionine (Met), also referred to as the Val66Met polymorphism, and has been suggested to be relevant in PTSD.GAB A A receptor

[0104] GABAergic systems are known to play a role in the pathophysiology of anxiety and depression, both of which are common in individuals suffering from PTSD. Three polymorphisms in the GABAA receptor subunit alpha 2 (GABRA2) were found to have significant interactions with childhood trauma to predict PTSD.RGS2

[0105] Regulator of G-protein Signaling 2 (RGS2) has been implicated in learning and memory processes. An association with RGS2 (rs4606) and PTSD after traumatic hurricane experience was found under conditions of high stress and low social support.CCK

[0106] Cholecystokinin (CCK) is a neuropeptide that has been implicated fear acquisition and extinction in rodents (81 ). A study in war veterans has demonstrated that a single nucleotide polymorphism in the promoter region of the CCK gene was associated with an increased prevalence of PTSD as well as with severity of PTSD symptoms.CNR1

[0107] Other studies have reported associations between PTSD and cannabinoid receptor (CNR1 ) gene variants. A polymorphism in the CNR1 gene (rs1049353) interacts with childhood physical abuse to predict posttraumatic threat symptoms, specifically to increase severity of threat or fear. A different study examined the association between genetic variation in the nicotinic receptor gene family and the PTSD. A novel association between rs12898919 in the cholinergic receptor nicotinic alpha-5 (CHRNA5) gene and PTSD was observed in patients suggesting that CHRNA5 gene is associated with increased risk for PTSD.

[0108] In these embodiments, the detection of a polymorphism may be indicative of PTSD and / or elevated predisposition to PTSD in the individual. One or more of the biomarkers described in Table 1 (mRNA or protein) can be used in addition to one or more polymorphisms in the diagnosis or prognosis of PTSD.

[0109] The presence of one or more of the polymorphisms can be compared to a control, such as a standard set or reference group of polymorphisms that have been associated with the risk of developing PTSD, a clinical outcome of PTSD, or responsiveness to a treatment of PTSD, as determined according to a statistical procedure for risk prediction.

[0110] In one embodiment of this method, the presence of the polymorphisms can be detected by obtaining a genomic DNA sample from the individual and determining the presence or absence of the polymorphism at the specific locus. In some embodiments,the presence or absence of the polymorphism is determined by at least one method selected from multiplexed locus-specific PCR amplification, multiplexed single-based extension (SBE) from locus-specific amplicons, and multiplexed resolution of SBE products using matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry.Up Converting Phosphors (UCPs)

[0111] Conventional laboratory tests are not suited for quantifying most biomarkers.For example, colorimetric reporters (e.g., for covid or pregnancy) are used with in-home tests are qualitative; they can provide a positive or negative result. The signal is binary (i.e. , not quantitative) and must also be read within one hour due to signal bleaching. The signal is unreliable after a certain amount of time (usually an hour). A quantitative test is required to determine variations in levels of a protein or biomarker.

[0112] It is possible to use an ELISA assay to measure levels of biomarkers. Common fluorescent reporter ELISA tests in laboratory settings are quantitative. However, they also quench and must be read within a short period of time using expensive laboratory equipment. Moreover, the ELISA reports used today react to produce a background signal with body fluid component. Because they auto-fluoresce, they reduce sensitivity and complicate sample processing.

[0113] Embodiments include a lateral flow assay system that includes multiplexed panels of quantifiable protein biomarkers which incorporate a unique reporter system (UCP-reporters) with output signals that can be collectively analyzed. The test can be performed with finger-prick blood samples, saliva, urine, etc. which can be collected at a clinical or home setting.

[0114] The reporter system includes uniform nanocrystals created from rare-earth elements, known as up-converting phosphors (UCPs). Unlike fluorescent and colorimetric reporters, UPCs do not quench and emit a stable lifetime signal which can be read and accurately analyzed even after three years. In comparison, the fluorescentreporter counterparts must be analyzed within about one hour. The UCP reporters do not auto-fluoresce, and thus produce zero background signal for biological samples. These characteristics improve sensitivity from 10 to 10,000-fold over existing methods because there is no interference when using whole blood. By comparison, fractionated serum must be used in the current ELISA tests.

[0115] Compared with gold nanoparticles, a lateral flow test using upconverting phosphor nanoparticles (UCNPs) is more sensitive (approximately tenfold) and robust, due to the unique feature of using the lower energy 980 nm infrared light (excitation light) to generate higher energy visual light (emission light). This light process is called “up-conversion,” which does not occur in nature. Thus, UCNPs as a reporter label do not generate background fluorescence (autofluorescence) compared with conventional fluorescent labels, such as fluorescently labeled nanoparticles and quantum dots. Moreover, UCNPs do not fade, allowing the lateral flow strips based on UCNPs to be stored in the long term. More importantly, the lateral flow test based on UCNPs has no interference from red blood cell hemolysis that is a problem sometimes encountered in lateral flow test based on colloidal gold-labeled using blood.

[0116] Thus, the test can be run directly from finger-pricked, heel-pricked, dried blood samples (DBS), saliva, urine, etc. The test can be completed at home setting or at a clinic. The completed strips can be shipped to a reference lab for accurate analysis up to weeks after the test was run. Alternatively, they can be analyzed with an at-home optical reader that uploads the scanned strip, data uploaded into the cloud. The UCPs have been shown to accurately measure biomarkers on dried strips, five years after the test was run. The test strips can multiplex measurements for accurate assessment of complex disease states.

[0117] There are several benefits of the UCP-reporter lateral flow assay described herein, including:• UCP-LFAs provide stable, optical signals with 10 - 1000 times higher sensitivity than in existing lateral flow technology.• UCP-LFAs have zero background signal, neither from bodily fluids nor LFA cartridge substrate materials.• Signal generation is confined to UCP particles only at the capture strip.• There is no quenching, fading or bleaching of the signal which is stable even if stored for years.• There is unlimited signal integration time with simultaneous excitation and emission.• Optical reader-assisted algorithms can prevent operator error in diagnosis of disease states.• There is a low limit of detection (i.e. , 1 pg for protein analytes and 1 fg for DNA).

[0118] The LFA tests can be used to detect a range and / or quantify levels of hormones, proteins, DNA, RNA and metabolites. Other omic data can also be analyzed (e.g., genomics and epigenomics). Data from the reader can be transferred to the cloud and analyzed. Predictive machine learning engines can use the input data and Al tools to identify causal biomarkers and calculate ratios and correlations between biomarker levels.Method of Testing Biomarkers

[0119] Relevant biomarkers can be identified and levels determined using antibodybased methods, such as, an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA) or a lateral flow immunoassay (LFA). In one embodiment, the method uses a lateral flow assay that can detect protein biomarkers at low levels. The levels of protein can also be determined by analyzing the amount of an indicator of fluorescence.

[0120] LFA-based tests are well known in the art. A typical LFA can include the following components: sample pad, conjugate release pad, membrane with immobilized antibodies and adsorbent pad. The components of the strip are usually fixed to an inert backing material. A biological sample is applied to a portion of the strip (i.e., sample pad) of a lateral flow assay, resulting in the accumulation of a labelled binding reagent,in an analyte concentration-dependent manner (proportional or inversely proportional) at a detection zone on the test strip. The test strip can then be analyzed to detect / quantify the amount of target analytes (i.e., biomarkers) by assay reading components mounted on the PCBA. A microprocessor, ASIC or the like can analyze and interpret the readings and display the assay results to a user and / or health care provider.

[0121] In one embodiment, the method includes a multi-line lateral flow test strip that uses a sandwich-antibody capture technique to quantify one or more biomarkers. The method can use an up-converting phosphor (UCP) detection system that provides high sensitivity, zero biological background florescent which results in ten-fold dynamic range increase over other antibody detection systems. Further, the UCP is quantitative and lasts for over twenty years.

[0122] The amount of an analyte present in a sample can be determined in absolute terms (e.g., in terms of a numerical value per unit volume) or in relative terms (e.g., by reference to a predetermined threshold). In particular, the “relative amount of one or more biomarkers” is not intended to mean that the concentrations of different biomarkers in a sample are compared with one another, but rather that the concentration of one or more such analytes may be compared to a predetermined threshold.

[0123] The measurement of the assay and / or interpretation of the assay result can include one or more data processing steps, in which assay data are subjected to one or more computations or other type of processing. Such processing can be performed by a digital electronic device such as a microprocessor or the like, which will typically form part of an extrinsic, or an integral, assay result reader. For example, the data processing can include the calculation of a ratio.

[0124] One or more of the biomarkers can be used in a method of predicting the susceptibility that an PTSD. In this manner, one biomarker or a combination ofbiomarkers can be used in the method. In this manner, at least one biomarker or a combination of biomarkers can be used in the method.

[0125] In a first step, the expression levels of one or more proteins are measured in plasma samples from subjects who suffered from PTSD. In an embodiment, the expression levels can be used to generate a footprint or signature for subsequent diagnosis of patients. In an embodiment, the expression levels can be used to generate a footprint or signature for subsequent diagnosis of patients.

[0126] Next, expression levels of the same proteins are measured in plasma, blood or tissue samples from healthy subjects (i.e., a patient who is not prone to PTSD). This is used as a control. Thereafter, samples from healthy patients can be compared to identifying proteins that have altered levels of expression in the plasma samples from the subjects with an ailment. A biomarker fingerprint or signature can be created from the proteins with altered levels of expression. This can be used for diagnosing or determining the prognosis of an ailment in the test subject by comparing of levels of proteins from plasma of the test subject. Conventional statistical analysis can be used to determine, for example, confidence levels.

[0127] In embodiments, SNPs that occur naturally in the human genome are provided as isolated nucleic acid molecules. These SNPs are associated with PTSD and related pathologies. In particular the SNPs are associated with either an increased or decreased risk of developing PTSD. As such, they can have a variety of uses in the diagnosis and / or treatment of PTSD and related pathologies.

[0128] In yet another embodiment, a reagent for detecting a SNP in the context of its naturally-occurring flanking nucleotide sequences (which can be, e.g., either DNA or mRNA) is provided. In particular, such a reagent may be in the form of, for example, a hybridization probe or an amplification primer that is useful in the specific detection of a SNP of interest. In an alternative embodiment, a protein detection reagent is used to detect a variant protein that is encoded by a nucleic acid molecule containing a SNPdisclosed herein. A preferred embodiment of a protein detection reagent is an antibody or an antigen-reactive antibody fragment.

[0129] Various embodiments of the invention also provide kits comprising SNP detection reagents, and methods for detecting the SNPs disclosed herein by employing detection reagents. In a specific embodiment, the present invention provides for a method of identifying an individual having an increased or decreased risk of developing liver fibrosis by detecting the presence or absence of one or more SNP alleles disclosed herein. In another embodiment, a method for diagnosis of liver fibrosis and related pathologies by detecting the presence or absence of one or more SNP alleles disclosed herein is provided.

[0130] The nucleic acid molecules of the invention can be inserted in an expression vector, such as to produce a variant protein in a host cell. Thus, the present invention also provides for a vector comprising a SNP-containing nucleic acid molecule, genetically-engineered host cells containing the vector, and methods for expressing a recombinant variant protein using such host cells. In another specific embodiment, the host cells, SNP-containing nucleic acid molecules, and / or variant proteins can be used as targets in a method for screening and identifying therapeutic agents or pharmaceutical compounds useful in the treatment of liver fibrosis and related pathologies.Diagnostic Kit for Diagnosing PTSD or Determining Susceptibility to PTSD

[0131] The following working example is based on configurations described above. Embodiments of the invention can be compiled into a diagnostic kit for diagnosing an ailment or determining that a subject is vulnerable to PTSD.

[0132] For example, immunoassay kits can be used as described herein. Such kits can include, (a) antibodies having binding specificity for the polypeptides identified as biomarkers; and (b) anti-antibody immunoglobulins. This immunoassay kit can be utilized for the practice of the various methods provided herein. The antibodies and theanti-antibody immunoglobulins can be provided in an amount of about 0.001 mg to 100 grams, and more preferably about 0.01 mg to 1 gram. The anti-antibody immunoglobulin may be a polyclonal immunoglobulin, protein A or protein G or functional fragments thereof, which may be labeled prior to use by methods known in the art. In embodiments, the immunoassay kit includes antibodies that specifically bind to butyrylcholinesterase. The kit can also include reagents that can be used to identify variations in expression levels of one or more proteins in a sample from a test subject.

[0133] An indicator compound can be used to determine the amount of each biomarker. The indicator compound can be any compound, chemical, or biological component which may interact with a target or byproduct of a target. For example, an indicator compound may comprise an antibody, a reactive chemical compound, a labeled molecule, or any combination thereof. Antibodies used in an embodiment of the present disclosure may be monoclonal or polyclonal and derived from any species (e.g., human, rat, mouse, rabbit, pig). Further, indicator molecules may be aptamers, proteins, peptides, small organic molecules, natural compounds, non-peptide polymers, MHC multimers (including MHC-dextramers, MHC-tetramers, MHC-pentamers and other MHC-multimers), or any other molecules that specifically and efficiently bind to other molecules are also marker molecules.

[0134] Labeled molecules, for use as indicator compounds, may be any molecule that absorbs, excites, or modifies radiation, such as the absorption of light (e.g., dyes and chromophores) and the emission of light after excitation (fluorescence from fluorochromes). Additionally, labeled molecules may have an enzymatic activity, by which it catalyzes a reaction between chemicals in the near environment of the labeling molecules, producing a signal which include production of light (chemi-luminescence) or precipitation of chromophores, dyes, or a precipitate that can be detected by an additional layer of detection molecules.

[0135] The indictor signal in at least one embodiment of the present disclosure may comprise any detectable signal, including but not limited to, color change, fluorescence,and chemical / structural change of the target (and / or indicator compound) so as to be amenable to reacting with a secondary detection marker. Further, the detection of a signal may involve a secondary reactive molecule.

[0136] The target of an exemplary indicator compound of the present disclosure may be any diagnostic marker or diagnostic condition for a disease state (or pre-disease state) of an individual. For example, the disease state may be diabetes, pre-diabetes or metabolic syndrome.

[0137] A kit can include one biomarker or a combination of biomarkers relevant to PTSD. The skilled artisan will appreciate that the number of biomarkers may be varied without departing from the nature of the disclosure, and thus other combinations of biomarkers are also encompassed by the disclosure. The skilled artisan will know which one biomarker or a combination of biomarkers to use. It is also contemplated that additional medical information can be used in determining the susceptibility and / or vulnerability of a patient to PTSD.

[0138] In a specific embodiment, a kit includes a single biomarker or a combination of biomarkers disclosed herein. The kit can further optionally include instructions for use. The kit can further optionally include tubes, applicators, vials or other storage container with the above-mentioned biomarker and / or vials containing one or more of the biomarkers. In an embodiment, each biomarker is in its own tube, applicator, vial or storage container.

[0139] The kits, regardless of type, will generally include one or more containers into which the one biomarker or a combination of biomarkers are placed and, preferably, suitably aliquoted. The components of the kits may be packaged either in aqueous media or in lyophilized form.Operating Environment:

[0140] The system is typically comprised of a central server that is connected by a data network to a user's (e.g., healthcare provider’s) computer. The central server can be comprised of one or more computers connected to one or more mass storage devices. The precise architecture of the central server does not limit the claimed invention. Further, the user's (i.e. healthcare practitioner’s) computer can be a laptop or desktop type of personal computer. It can also be a cell phone, smart phone or other handheld device, including a tablet. The precise form factor of the user's computer does not limit the claimed invention. Examples of well-known computing systems, environments, and / or configurations that may be suitable for use with the invention include, but are not limited to, personal computers, server computers, hand held, laptop or mobile computer or communications devices such as cell phones and PDA's, multiprocessor systems, microprocessor-based systems, set top boxes, programmable consumer electronics, network PCs, minicomputers, mainframe computers, distributed computing environments that include any of the above systems or devices, and the like. The precise form factor of the user's computer does not limit the claimed invention. In one embodiment, the user's computer is omitted, and instead a separate computing functionality provided that works with the central server. In this case, a user would log into the server from another computer and access the system through a user environment.

[0141] The user environment can be housed in the central server or operatively connected to it. Further, the user can receive from and transmit data to the central server by means of the Internet, whereby the user accesses an account using an Internet web-browser and browser displays an interactive web page operatively connected to the central server. The central server transmits and receives data in response to data and commands transmitted from the browser in response to the customer's actuation of the browser user interface. Some steps of the invention may be performed on the user's computer and interim results transmitted to a server. These interim results may be processed at the server and final results passed back to the user.

[0142] The methods described herein can be executed on a computer system, generally comprised of a central processing unit (CPU) that is operatively connected to a memory device, data input and output circuitry (I / O) and computer data network communication circuitry. Computer code executed by the CPU can take data received by the data communication circuitry and store it in the memory device. In addition, the CPU can take data from the I / O circuitry and store it in the memory device. Further, the CPU can take data from a memory device and output it through the I / O circuitry or the data communication circuitry. The data stored in memory may be further recalled from the memory device, further processed or modified by the CPU in the manner described herein and restored in the same memory device or a different memory device operatively connected to the CPU including by means of the data network circuitry. The memory device can be any kind of data storage circuit or magnetic storage or optical device, including a hard disk, optical disk or solid state memory. The I / O devices can include a display screen, loudspeakers, microphone and a movable mouse that indicate to the computer the relative location of a cursor position on the display and one or more buttons that can be actuated to indicate a command.

[0143] The computer can display on the display screen operatively connected to the I / O circuitry the appearance of a user interface. Various shapes, text and other graphical forms are displayed on the screen as a result of the computer generating data that causes the pixels comprising the display screen customer’s actuation of the browser user interface. Some steps of the invention can be performed on the user's computer and interim results transmitted to a server. These interim results can be processed at the server and final results passed back to the user.

[0144] The invention may also be entirely executed on one or more servers. A server may be a computer comprised of a central processing unit with a mass storage device and a network connection. In addition, a server can include multiple of such computers connected together with a data network or other data transfer connection, or, multiple computers on a network with network accessed storage, in a manner that provides such functionality as a group. Practitioners of ordinary skill will recognize that functions thatare accomplished on one server may be partitioned and accomplished on multiple servers that are operatively connected by a computer network by means of appropriate inter process communication. In addition, the access of the website can be by means of an Internet browser accessing a secure or public page or by means of a client program running on a local computer that is connected over a computer network to the server. A data message and data upload or download can be delivered over the Internet using typical protocols, including TCP / IP, HTTP, TCP, UDP, SMTP, RPC, FTP or other kinds of data communication protocols that permit processes running on two remote computers to exchange information by means of digital network communication. As a result a data message can be a data packet transmitted from or received by a computer containing a destination network address, a destination process or application identifier, and data values that can be parsed at the destination computer located at the destination network address by the destination application in order that the relevant data values are extracted and used by the destination application. The precise architecture of the central server does not limit the claimed invention. In addition, the data network may operate with several levels, such that the user's computer is connected through a fire wall to one server, which routes communications to another server that executes the disclosed methods.

[0145] Computer program logic implementing all or part of the functionality previously described herein may be embodied in various forms, including, but in no way limited to, a source code form, a computer executable form, and various intermediate forms (e.g., forms generated by an assembler, compiler, linker, or locator.) Source code may include a series of computer program instructions implemented in any of various programming languages (e.g., an object code, an assembly language, or a high-level language such as C, C-HF, C#, Action Script, PHP, EcmaScript, JavaScript, JAVA, or 5 HTML) for use with various operating systems or operating environments. The source code may define and use various data structures and communication messages. The source code may be in a computer executable form (e.g., via an interpreter), or the source code may be converted (e.g., via a translator, assembler, or compiler) into a computer executable form.

[0146] The invention may be described in the general context of computer-executable instructions, such as program modules, being executed by a computer. Generally, program modules include routines, programs, objects, components, data structures, etc., that perform particular tasks or implement particular abstract data types. The computer program and data may be fixed in any form (e.g., source code form, computer executable form, or an intermediate form) either permanently or transitorily in a tangible storage medium, such as a semiconductor memory device (e.g., a RAM, ROM, PROM, EEPROM, or Flash-Programmable RAM), a magnetic memory device (e.g., a diskette or fixed hard disk), an optical memory device (e.g., a CD-ROM or DVD), a PC card (e.g., PCMCIA card), or other memory device. The computer program and data may be fixed in any form in a signal that is transmittable to a computer using any of various communication technologies, including, but in no way limited to, analog technologies, digital technologies, optical technologies, wireless technologies, networking technologies, and internetworking technologies. The computer program and data may be distributed in any form as a removable storage medium with accompanying printed or electronic documentation (e.g., shrink wrapped software or a magnetic tape), preloaded with a computer system (e.g., on system ROM or fixed disk), or distributed from a server or electronic bulletin board over the communication system (e.g., the Internet or World Wide Web.) It is appreciated that any of the software components of the present invention may, if desired, be implemented in ROM (read-only memory) form. The software components may, generally, be implemented in hardware, if desired, using conventional techniques.

[0147] The invention may also be practiced in distributed computing environments where tasks are performed by remote processing devices that are linked through a communications network. In a distributed computing environment, program modules may be located in both local and remote computer storage media including memory storage devices. Practitioners of ordinary skill will recognize that the invention may be executed on one or more computer processors that are linked using a data network, including, for example, the Internet. In another embodiment, different steps of theprocess can be executed by one or more computers and storage devices geographically separated but connected by a data network in a manner so that they operate together to execute the process steps. In one embodiment, a user's computer can run an application that causes the user's computer to transmit a stream of one or more data packets across a data network to a second computer, referred to here as a server. The server, in turn, may be connected to one or more mass data storage devices where the database is stored. The server can execute a program that receives the transmitted packet and interpret the transmitted data packets in order to extract database query information. The server can then execute the remaining steps of the invention by means of accessing the mass storage devices to derive the desired result of the query. Alternatively, the server can transmit the query information to another computer that is connected to the mass storage devices, and that computer can execute the invention to derive the desired result. The result can then be transmitted back to the user's computer by means of another stream of one or more data packets appropriately addressed to the user's computer. In one embodiment, the relational database may be housed in one or more operatively connected servers operatively connected to computer memory, for example, disk drives. In yet another embodiment, the initialization of the relational database may be prepared on the set of servers and the interaction with the user's computer occur at a different place in the overall process.

[0148] It should be noted that the flow diagrams are used herein to demonstrate various aspects of the invention and should not be construed to limit the invention to any particular logic flow or logic implementation. The described logic may be partitioned into different logic blocks (e.g., programs, modules, functions, or subroutines) without changing the overall results or otherwise departing from the true scope of the invention. Oftentimes, logic elements may be added, modified, omitted, performed in a different order, or implemented using different logic constructs (e.g., logic gates, looping primitives, conditional logic, and other logic constructs) without changing the overall results or otherwise departing from the true scope of the invention.EXAMPLES

[0149] The following non-limiting examples are provided for illustrative purposes only in order to facilitate a more complete understanding of representative embodiments now contemplated. These examples are intended to be a mere subset of all possible contexts in which the components of the formulation may be combined. Thus, these examples should not be construed to limit any of the embodiments described in the present specification, including those pertaining to the type and amounts of components of the formulation and / or methods and uses thereof.Example 1Prediction of PTSD following a Traumatic Event

[0150] Post-traumatic stress disorder (PTSD) is a mental illness that can manifest after exposure to stressful or life-threatening events. Common symptoms of PTSD include agitation, nervousness and anxiety, problems with concentration or thinking, problems with memory, headaches, depression and crying spells, suicidal thoughts or attempts, and mood swings. Although estimates suggest that 90% of individuals will be exposed to a significant traumatic event in their lifetime, only about 5 - 10% of the general population suffers from PTSD. These studies suggest that individual vulnerability and resilience are key factors to consider in the pathology of PTSD.

[0151] Because of the limitations of accurately detecting PD, it is difficult to detect in its early stages when therapies can be most effective. It is also difficult to study disease progression and develop drugs / therapies. Further, it is difficult for drug developers to properly recruit patients for their clinical trials. Accordingly, there is a need for accurate and affordable noninvasive methods of diagnosing PD.

[0152] The lifetime prevalence of PTSD is approximately 8% in the general population but can be as high as 31 % in veterans or individuals with higher frequency of severe psychological stress or trauma exposure. PTSD symptoms can be debilitating and last for years.

[0153] Suicide is among the ten leading causes of death for all ages and it is also among the top three causes of death for people aged 15 - 34. Suicidal tendencies are difficult to diagnose, as well as the progression of those tendencies. Usually, suicidal tendencies can only be preventatively diagnosed through a mental status examination, but more commonly after an attempt at suicide was made. Suicide is a challenging mental health concern since all major mental disorders carry an increased risk of suicide. Psychiatric disorders, such as BP, MDD, SCZ or PTSD, are present in about 90% of people who commit suicide and contribute to most of the population at risk for suicide. The incidence of these mental disorders in suicide victims at the time of their death ranges from 87.3% to 98%.

[0154] Suicide prevention has become another major issue for military organizations. In the U.S Army alone, the incidence of suicide has been increasing steadily during the present economic crisis and the Iraq and Afghanistan wars. Although much effort has been expended in an effort to understand the biological factors that can increase the risk of suicide and to identify the biological changes that occur in those with a propensity towards self-harm, at present, there are no objective biological markers to prevent suicide. A recent study of PTSD found a substantially increased risk of suicide and suicide attempts in PTSD patients. About 62 percent of individuals diagnosed with PTSD have suicidal ideation with traumatic events generally increasing a person’s suicide risk. The likelihood for suicide attempts of individuals with PTSD is approximately fifteen times higher than in individuals without it. Thus, there exists a need for determining suicide in risk of patients who suffer from PTSD.

[0155] In this example, a health care provider suspects that a patient is suffering from PTSD. The patient indicates that he served several combat missions and suffers from anxiety. A physician conducts a mental status examination and determines that the patient also suffers from: (a) irritability and anger outburst, (b) excessive fear and worry, (c) headaches and fatigue, (d) depression and (e) insomnia. Further, family members also express their concern for the patient’s wellbeing.

[0156] Blood and saliva samples are analyzed using the methods described herein. An analysis of biomarkers indicates that the patient is susceptible to PTSD.Specifically, the patient has an 85% chance of PTSD based on levels of biomarkers and additional biomedical information. The physician also identifies multiple traumatic events that the patient experienced during combat missions.

[0157] The physician advises counseling and medication. Professional therapy is directed at helping the patient understand his thoughts and cope with his challenges. Medications (i.e. , selective serotonin reuptake inhibitors) are administered to help reduce tension or irritability or to improve sleep. In this example, the patient’s anxiety (and related symptoms) are relieved within about three months of treatment. At this stage, the patient continues therapy but chooses not to continue with medication.

[0158] Certain embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the present invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described embodiments in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0159] Groupings of alternative embodiments, elements, or steps of the present invention are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other group members disclosed herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group asmodified thus fulfilling the written description of all Markush groups used in the appended claims.

[0160] Unless otherwise indicated, all numbers expressing a characteristic, item, quantity, parameter, property, term, and so forth used in the present specification and claims are to be understood as being modified in all instances by the term “about.” As used herein, the term “about” means that the characteristic, item, quantity, parameter, property, or term so qualified encompasses a range of plus or minus ten percent above and below the value of the stated characteristic, item, quantity, parameter, property, or term. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical indication should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and values setting forth the broad scope of the invention are approximations, the numerical ranges and values set forth in the specific examples are reported as precisely as possible. Any numerical range or value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Recitation of numerical ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate numerical value falling within the range. Unless otherwise indicated herein, each individual value of a numerical range is incorporated into the present specification as if it were individually recited herein.

[0161] Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term “consisting of” excludes any element, step, or ingredient not specified in the claims. The transition term “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s).Embodiments of the present invention so claimed are inherently or expressly described and enabled herein.

[0162] Groupings of alternative embodiments, elements, or steps of the present invention are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other group members disclosed herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0163] All patents, patent publications, and other publications referenced and identified in the present specification are individually and expressly incorporated herein by reference in their entirety for the purpose of describing and disclosing, for example, the compositions and methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0164] In closing, it is to be understood that although aspects of the present specification are highlighted by referring to specific embodiments, one skilled in the art will readily appreciate that these disclosed embodiments are only illustrative of the principles of the subject matter disclosed herein. Therefore, it should be understood that the disclosed subject matter is in no way limited to a particular methodology, protocol, and / or reagent, etc., described herein. As such, various modifications or changes to or alternative configurations of the disclosed subject matter can be made in accordancewith the teachings herein without departing from the spirit of the present specification. Lastly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Accordingly, the present invention is not limited to that precisely as shown and described.

Claims

CLAIMSWhat is claimed is:1 . A method of detecting PTSD or determining a prognosis of a subject with PTSD, the method comprising steps of: a) measuring the expression level of at least one mRNA in a test sample from the subject, b) receiving the expression level of the at least one mRNA in the test sample by a computer and c) comparing the expression level of the at least one mRNA in the test sample to a level in a base sample for the same at least one mRNA, and d) receiving a result comparing the expression levels of the at least one mRNA in the test sample measured in a) and the base sample measured in c), e) detecting or determining the prognosis of PTSD based on altered expression of the at least one mRNA in the test sample as compared to the base sample, and f) treating the subject based on the detection or prognosis of PTSD.

2. The method of claim 1 , wherein the test sample is a blood sample or a brain tissue sample.

3. The method of claim 1 , wherein the PTSD is preclinical PTSD or asymptomatic PTSD.

4. The method of claim 1 , wherein the at least one mRNA is selected from ZDHHC14, PARK2, KAZN, TMEM51-AS1 , ZNF813, LINC02335, MIR5007, TUC338, LINC02571 , HLA-B, SH3RF3, PODXL, EX0C6, RAB27B, CCBL2, CDC42BPB, INPP4A, CTNND1 , TSFM, NCK1 , KHK, LM0D1 and PLCG1.

5. The method of claim 1 , wherein the at least one mRNA is comprised of 2, 3, 4, 5,6. 7, 8, 9, 10 or 11 mRNAs.

6. The method of claim 1 , further comprising steps of measuring the expression level of at least one protein in the test sample from the subject and comparing the expression level of the at least one protein in the test sample to a level in a base sample for the same at least one protein.

7. The method of claim 6, wherein the at least one protein is selected from ZDHHC14, PARK2, KAZN, TMEM51-AS1 , ZNF813, LINC02335, MIR5007, TUC338, LINC02571 , HLA-B, SH3RF3, PODXL, EXOC6, RAB27B, CCBL2, CDC42BPB, INPP4A, CTNND1 , TSFM, NCK1 , KHK, LMOD1 and PLCG1.

8. The method of claim 1 , further comprising a step of evaluating additional biomedical information of the subject.

9. The method of claim 1 , further comprising a step of compiling a clinical history, conducting a mental status examination, conducting a clinically structured interview, reviewing patient symptoms and / or reviewing patient reports.

10. The method of claim 1 , further comprising a step of comparing one or more single nucleotide polymorphisms (“SNPs”).11 . The method of claim 10, wherein the one or more single nucleotide polymorphisms (“SNPs”) are coding regions of SLC6A4, SLC6A3, DRD2, Glucocorticoid Receptor Gene, FKBP5, PACAP, PAC1 receptor, BDNF, GABAA receptor, RGS2, CCK or CNRI .

12. The method of claim 10, wherein the one or more single nucleotide polymorphisms (“SNPs”) affect expression, structure and / or activity of SLC6A4, SLC6A3, DRD2, Glucocorticoid Receptor Gene, FKBP5, PACAP, PAC1 receptor, BDNF, GABAA receptor, RGS2, CCK or CNRI .

13. A method of detecting PTSD or determining a prognosis of a test subject with PTSD, the method comprising steps of: a) measuring the expression level of at least one protein in a test sample from the subject, b) receiving the expression level of the at least one protein in the test sample by a computer and c) comparing the expression level of the at least one protein in the test sample to a level in a base sample for the same at least one protein, and d) receiving a result comparing the expression levels of the at least one protein in the test sample measured in a) and the base sample measured in c), e) detecting or determining the prognosis of PTSD based on altered expression of the at least one protein in the test sample as compared to the base sample, and f) treating the subject based on the detection or prognosis of PTSD.14 The method of claim 11 , wherein the test sample is a blood sample or a brain tissue.

15. The method of claim 11 , wherein the PTSD is preclinical PTSD or asymptomatic Alzheimer’s disease.

16. The method of claim 11 , wherein the at least one protein is selected from ZDHHC14, PARK2, KAZN, TMEM51-AS1 , ZNF813, LINC02335, MIR5007, TUC338, LINC02571 , HLA-B, SH3RF3, PODXL, EX0C6, RAB27B, CCBL2, CDC42BPB, INPP4A, CTNND1 , TSFM, NCK1 , KHK, LM0D1 and PLCG1.

17. The method of claim 11 , wherein the at least one protein is comprised of 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 proteins.

18. The method of claim 11 , wherein a lateral flow immunoassay is used in the step of measuring the expression level of at least one protein in a test sample from the subject.

19. The method of claim 16, wherein up converting phosphors (UCPs) are used in the lateral flow immunoassay.

20. The method of claim 11 , further comprising steps of measuring the expression level of at least one mRNA in the test sample from the subject and comparing the expression level of the at least one protein in the test sample to a level in a base sample for the same at least one mRNA.21 . The method of claim 11 , wherein the at least one mRNA is selected from ZDHHC14, PARK2, KAZN, TMEM51-AS1, ZNF813, LINC02335, MIR5007, TUC338, LINC02571 , HLA-B, SH3RF3, PODXL, EXOC6, RAB27B, CCBL2, CDC42BPB, INPP4A, CTNND1 , TSFM, NCK1 , KHK, LMOD1 and PLCG1.

22. The method of claim 11 , further comprising a step of evaluating additional biomedical information of the subject.

23. The method of claim 11 , further comprising a step of compiling a clinical history, conducting a mental status examination, conducting a clinically structured interview, reviewing patient symptoms and / or reviewing patient reports.

24. The method of claim 11 , further comprising a step of comparing one or more single nucleotide polymorphisms (“SNPs”).

25. The method of claim 24, wherein the one or more single nucleotide polymorphisms (“SNPs”) are coding regions of SLC6A4, SLC6A3, DRD2, Glucocorticoid Receptor Gene, FKBP5, PACAP, PAC1 receptor, BDNF, GABAA receptor, RGS2, CCK or CNRI .

26. The method of claim 24, wherein the one or more single nucleotide polymorphisms (“SNPs”) affect expression, structure and / or activity of SLC6A4, SLC6A3, DRD2, Glucocorticoid Receptor Gene, FKBP5, PACAP, PAC1 receptor, BDNF, GABAA receptor, RGS2, CCK or CNRI .

27. A method of diagnosing PTSD or determining a prognosis of a subject with PTSD, comprising the steps of: a) measuring the expression level of at least one mRNA or protein in a test sample from plasma of the subject, b) receiving the results of the measurement a) by a computer and c) comparing the expression level of the at least one mRNA or protein in the test sample to a level in a base sample for the same at least one mRNA or protein, and c) receiving a result comparing the expression levels in the test sample measured in a) and the base sample measured in c), d) diagnosing or determining the prognosis of PTSD based on altered expression of least one mRNA or protein in the test sample as compared to the base sample, and e) treating the subject for PTSD based on the diagnoses or prognosis.

28. The method of claim 27, wherein the at least one mRNA or protein is selected from ZDHHC14, PARK2, KAZN, TMEM51-AS1 , ZNF813, LINC02335, MIR5007, TUC338, LINC02571 , HLA-B, SH3RF3, PODXL, EX0C6, RAB27B, CCBL2, CDC42BPB, INPP4A, CTNND1 , TSFM, NCK1 , KHK, LM0D1 and PLCG1.

29. The method of claim 27, further comprising a step of evaluating additional biomedical information of the subject.

30. The method of claim 27, further comprising a step of compiling a clinical history, conducting a mental status examination, conducting a clinically structured interview, reviewing patient symptoms and / or reviewing patient reports.31 . The method of claim 27, further comprising a step of comparing one or more single nucleotide polymorphisms (“SNPs”).

32. The method of claim 31 , wherein the one or more single nucleotide polymorphisms (“SNPs”) are coding regions of SLC6A4, SLC6A3, DRD2, Glucocorticoid Receptor Gene, FKBP5, PACAP, PAC1 receptor, BDNF, GABAA receptor, RGS2, CCK or CNRI .

33. The method of claim 31 , wherein the one or more single nucleotide polymorphisms (“SNPs”) affect expression, structure and / or activity of SLC6A4, SLC6A3, DRD2, Glucocorticoid Receptor Gene, FKBP5, PACAP, PAC1 receptor, BDNF, GABAA receptor, RGS2, CCK or CNRI .

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