Genetic biomarkers for identifying PTSD and major depression

Abstract

Provided herein is a method for detecting post-traumatic stress disorder (PTSD) in a human subject, the method comprising: obtaining a cell sample obtained from the human subject; measuring a test gene expression level of one or more PTSD-related genes from the cell sample using a first computer program executed on a computer, wherein the PTSD-related genes are selected from the group consisting of TSPAN5, HIST1H2AE, UBE3A, GPX4, EPB42, SLC4A1, NDUFAC HISTIHEH, ELOVL7, ALES, COMPT, PDZK IPI, ITGA2B, CYP4F3, EPB41L3, PRDM1, RP-II-449PI5.I, FAS, TUBB2A, and JAM3; comparing the test PTSD-related expression level to one or more control gene expression levels using a second computer program executed on a computer; determining differential expression of the one or more test PTSD-related genes; and determining a positive detection of PTSD.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 434,605, filed December 22, 2022, which is incorporated herein by reference in its entirety.

[0002] Incorporation by Reference of Sequence Listing This application contains a Sequence Listing that has been submitted through the Patent Center. The Sequence Listing, entitled 210536-010500_SL.xml, created on December 22, 2023, is 269,686 bytes in size, and is hereby incorporated by reference in its entirety.

[0003] STATEMENT REGARDING FEDERALLY FUNDED RESEARCH No federal grant was used for this application.

[0004] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, it is intended that the present specification shall take precedence and / or control over any such conflicting material. Summary of the Invention [Means for solving the problem]

[0005] Abstract Provided herein is a method for detecting post-traumatic stress disorder (PTSD) in a human subject, the method comprising: obtaining a cell sample obtained from the human subject; measuring a test gene expression level of one or more PTSD-related genes from the cell sample using a first computer program executed on a computer, wherein the PTSD-related genes are selected from the group consisting of TSPAN5, HIST1H2AE, UBE3A, GPX4, EPB42, SLC4A1, NDUFA1, HIST1HEH, ELOVL7, ALES, COMPT, PDZK1IP1, ITGA2B, CYP4F3, EPB41L3, PRDM1, RP-11-449P15.1, FAS, TUBB2A, and JAM3; comparing the test PTSD-related expression level with one or more control gene expression levels using a second computer program executed on the computer; determining differential expression of the one or more test PTSD-related genes; and determining a positive detection of PTSD based on the differential expression. In some embodiments, the test and / or control gene expression levels are of one or more, two or more, three or more PTSD-related genes, hi some embodiments, the test and / or control gene expression levels are of four or more, five or more, six or more PTSD-related genes. In some embodiments, the test gene expression levels and / or control gene expression levels are of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, or all twenty PTSD-related genes.In some embodiments, the PTSD-related gene is selected from the group consisting of TSPAN5, HIST1H2AE, UBE3A, GXP4, EPB42, SLC4A1, NDUFA1, HIST1H3H, EVOLV7, and JAM3. In some embodiments, the PTSD-related gene is selected from the group consisting of TSPAN5, HIST1H2AE, UBE3A, GXP4, and EPB42. In some embodiments, the test PTSD-related gene expression level is 2.5 x 10. -6 In some embodiments, the test PTSD-related gene expression levels are differentially expressed with a p-value lower than 9 x 10 -7 In some embodiments, the test PTSD-related gene expression level is differentially expressed with a p-value lower than 1.6 x 10 -7In some embodiments, the cell sample is obtained from a blood sample, a saliva sample, a buccal smear sample, a cerebrospinal fluid sample, saliva, skin, cerebrospinal fluid, or any combination thereof. In some embodiments, the test gene expression is messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), small nuclear RNA (snRNA), U-spliceosomal RNA (U-RNA), small nucleolar RNA (snoRNA), Piwi-interacting RNA (piRNA), repeat-associated small interfering RNA (rasiRNA), small rDNA-derived RNA (srRNA), small transfer RNA-derived RNA (tsRNA), small ribosomal RNA-derived RNA (rsRNA), long non-coding RNA-derived small RNA (lncsRNA), or messenger RNA-derived small RNA (msRNA), gapmer, mixmer, double-stranded RNA (dsRNA), single-stranded RNAi (ssRNAi), DNA-directed RNA interference (ddRNAi), or any combination thereof. In some embodiments, the expression level is evaluated at the transcriptional level. In some embodiments, the expression level is evaluated at the translational level. In some embodiments, the first computer program and the second computer program are the same. In some embodiments, the first computer program and the second computer program are different. In some embodiments, the method is performed more than once. In some embodiments, the method is performed weekly, monthly, every two months, three times per year, four times per year, twice per year, or yearly. In some embodiments, the method further comprises treating the subject for PTSD. In some embodiments, treating the subject comprises administering to the subject a medication, a psychotherapy, or a combination thereof. In some embodiments, the subject is administered a medication. In some embodiments, the medication is an SSRI, a tricyclic antidepressant, an MAOI, an antipsychotic, a beta-blocker, a benzodiazepine, a hallucinogen, or a combination thereof. In some embodiments, the medication is an SSRI. In some embodiments, the medication is a tricyclic antidepressant. In some embodiments, the medication is an MAOI.In some embodiments, the medication is an antipsychotic, in some embodiments, the medication is a beta-blocker, in some embodiments, the medication is a benzodiazepine, in some embodiments, the medication is a hallucinogen.

[0006] Also provided herein is a method for screening for PTSD in a subject, the method comprising: detecting PTSD in a subject, the method comprising any one of the detection methods described herein; and determining that the subject should be clinically diagnosed with PTSD based on a positive detection of PTSD in the subject. In some embodiments, the control population is a PTSD-negative population. In some embodiments, the method further comprises clinically diagnosing the subject with PTSD using one or more clinical PTSD diagnostic criteria. In some embodiments, the clinical PTSD diagnostic criteria are CAPS-5 or PCL-5. In some embodiments, the clinical PTSD diagnostic criteria are CAPS-5. In some embodiments, the clinical PTSD diagnostic criteria are PCL-5. In some embodiments, the detection step and the clinical diagnosis step are performed by two different entities. In some embodiments, the method further comprises assessing one or more comorbid conditions associated with PTSD. In some embodiments, the one or more comorbid conditions include generalized anxiety disorder, alcohol use disorder, anxiety-related insomnia, childhood trauma, or any combination thereof. In some embodiments, the one or more comorbid conditions are diagnosed using one or more clinical criteria, including the Generalized Anxiety Disorder 7-Item Scale (GAD-7), the Alcohol Use Disorders Identification Test (AUDIT-C), the Pittsburgh Sleep Quality Index (PSQI), Adverse Childhood Experiences (ACE), or any combination thereof. In some embodiments, the detection step, the clinical diagnosis step, and the comorbid condition diagnosis step, or any combination of two of them, are performed by two different entities.

[0007] Also provided herein is a method for selecting a subject eligible for treatment for PTSD, the method comprising: detecting PTSD in a subject, the method comprising any one of the detection methods described herein; and determining the subject as eligible for PTSD treatment based on a positive detection of PTSD in the subject. In some embodiments, the subject has been screened for PTSD according to any one of the screening methods described herein.

[0008] Also provided herein is a method for assessing the effectiveness of a subject receiving a PTSD treatment, the method comprising: detecting PTSD in the subject, comprising any one of the detection methods described herein; and determining the effectiveness of the PTSD treatment based on the detection of PTSD in the subject, wherein a negative PTSD detection indicates that the PTSD treatment is effective. In some embodiments, the PTSD treatment is a single PTSD treatment or a PTSD treatment event that is part of a series or ongoing PTSD treatment. In some embodiments, the PTSD treatment comprises cognitive behavioral therapy, cognitive processing therapy, cognitive therapy, prolonged exposure therapy, eye movement desensitization and reprocessing (EMDR) therapy, narrative exposure therapy (NET), group therapy, brief eclectic psychotherapy, selective serotonin reuptake inhibitors (SSRIs), serotonin and norepinephrine reuptake inhibitors (SNRIs), anti-anxiety medications, antidepressants, sertraline, paroxetine, fluoxetine, venlafaxine, ketamine, tricyclic antidepressants, MAOIs, antipsychotics, beta-blockers, benzodiazepines, hallucinogens, comorbidity treatments, complementary and / or alternative therapies, or any combination thereof, where complementary and / or alternative therapies comprise acupuncture, yoga, animal-assisted therapy, and meditation. In some embodiments, the method comprises detecting PTSD in the subject prior to treatment, including any one of the detection methods described herein. In some embodiments, the method includes screening for PTSD in the subject prior to treatment, including any one of the screening methods described herein. In some embodiments, the method includes selecting a subject eligible for PTSD treatment prior to treatment, including any one of the methods for selecting eligible subjects described herein.

[0009] Also provided herein is a method for determining the prevalence of PTSD in a target population, the method comprising: detecting PTSD in each subject in the target population comprising any one of the detection methods described herein; and determining the prevalence of PTSD in the target population based on a positive detection of PTSD in each subject in the target population. In some embodiments, the population is a military population. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a flow chart showing the sequencing and amplification and analysis of RNA of interest.

[0011] [Figure 2] FIG. 2 shows machine learning to determine the likely number of genes that may be associated with PTSD.

[0012] [Figure 3] Figure 3 shows the 20 highly associated genes associated with PTSD and their P values.

[0013] [Figure 4] FIG. 4 shows the demographics assessed for the generation of an objective molecular detection assay to screen for PTSD.

[0014] [Figure 5] FIG. 5 shows that the sensitivity of the molecular detection assay, or the percentage of subjects correctly identified as having the disorder, is 90%.

[0015] [Figure 6]Figure 6 shows the area under the receiver operating characteristic curve (AUC) of previous models using the same genes but with different thresholds, as achieving 85%. Figure 6 also shows the receiver operating characteristic (ROC) curves for all samples (all_in) and cross-validation with size 1 or k=1 (k1_Cross).

[0016] [Figure 7] FIG. 7 is a flow chart that is a schematic illustration of an exemplary method consistent with certain aspects of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0017] Detailed Description overview PTSD is characterized at the molecular level by disruption of the regulation of a major stress hormone system, the so-called hypothalamic-pituitary-adrenal (HPA) axis. The hypothalamic-pituitary-adrenal (HPA) axis is a key component of the neuroendocrine response to acute and chronic stress. It results in the release of corticotropin-releasing hormone (CRH) and vasopressin (AVP) from parvocellular neurons in the hypothalamus into the portal venous system, activating the synthesis and release of ACTH from the anterior pituitary gland. ACTH then stimulates the adrenal cortex to synthesize and release glucocorticoids, particularly cortisol (de Kloet et al., Nat Rev Neurosci 2005; 6: 463-75). In PTSD, the fine regulation of this HPA axis is disrupted, as evidenced by reduced levels and exaggerated responsiveness of ACTH and cortisol in these patients. Enhanced glucocorticoid receptor (GR) responsiveness appears to underlie these disturbances. These findings contrast with the findings of higher cortisol levels and reduced GR responsiveness associated with major depression (Ribeiro et al., Am J Psychiatry 1993; 150: 1618-29; Holsboer F, Neuropsychopharmacology 2000; 23: 477-501). Although initially interpreted as reflecting the long-term consequences of traumatic exposure, there is reason to suspect that HPA axis alterations in PTSD may reflect pre-traumatic risk factors. Recent studies of early childhood and adult children of parents with PTSD also support the idea that both low cortisol levels and glucocorticoid responsiveness are risk factors for PTSD. Indeed, it was recently observed that adults who experienced childhood abuse and who carry genetic variations in the FKBP5 gene (involved in regulating GR) appear to be at higher risk for PTSD symptoms as adults (Binder et al., JAMA 2008; 299: 1291-305). Two other studies have examined gene expression after traumatic exposure.In a study of individuals exposed to severe trauma encountered in the emergency room who either met criteria for PTSD (n = 8) or did not meet criteria for PTSD (n = 6) at both 1- and 4-month follow-up, there were gene expression changes associated with several interesting molecular categories associated with stress responses. Interestingly, FKBP5 expression was shown to be upregulated in this study. However, given its proximity to traumatic exposure, this finding may reflect biological changes associated with recovery from the effects of traumatic exposure, rather than with the development or persistence of chronic PTSD.

[0018] Indeed, genetic factors are known to be important in determining responses to environmental events and have been linked to PTSD, depression, schizophrenia, and other psychiatric disorders. Duncan LE, et al., Mol Psychiatry. Mar 2018;23(3):666-673; Petra Zimmermann, et al., Am J Psychiatry. 2011 October ;168(10). For example, twin studies estimate that the heritability of PTSD ranges from 24 to 72% after trauma, suggesting that an individual's genes and epigenetics influence whether they experience PTSD after a trauma or stressor. Sartor CE, et al., Arch Gen Psychiatry. Mar 2012;69(3):293-9; Stein MB, et al., Am J Psychiatry. Oct 2002;159(10):1675-81; True WR, et al. Arch Gen Psychiatry. Apr 1993;50(4):257-64; Wolf EJ, et al., Psychol Med. May 2014;44(7):1499-509. A study of Holocaust survivor families was the first to demonstrate that epigenetic changes at specific loci can be passed from parents to offspring and increase the risk of PTSD. Rachel Yehuda et al., Society of Biological Psychiatry September 1, 2016;80:372-380. Gene expression levels controlling glucocorticoid receptor regulation have been found to be dysregulated in 9 / 11 World Trade Center first responders with severe PTSD and have also been found to be altered in the brains of patients with PTSD. Casey Sarapasa et al., Disease Markers 30 (2011) 101-110; and Sophie E. Holmes et al., Proc Natl Acad Sci USA. 2017 Aug 1;114(31):8390-8395.

[0019] It has been found that more than 90% of adults in the United States have experienced at least one traumatic event in their lifetime (Breslau, N. et al., Soc Psychiatry Psychiatr Epidemiol. Aug 2016;51(8):1137-48), and approximately 8% or an estimated 13 million Americans will develop post-traumatic stress disorder (PTSD). Kessler, RC, et al., Archives of General Psychiatry, 52, 1048-1060. PTSD is a highly debilitating condition characterized by intrusive and recurrent memories of the trauma, avoidance of trauma-related stimuli, trauma-related numbing and / or negative changes in mood or cognition, and changes in reactivity and arousal.

[0020] Because PTSD can be reversible if detected early, early treatment has the potential to reduce adverse outcomes. Wlassoff V., brainblogger.com / 2015 / 01 / 24 / how-does-post-traumatic-stress-disorder-change-the-brain / , Accessed November 19, 2021; van Wingen GA, et al., Proc Natl Acad Sci USA. Sep 18 2012;109(38):15508-13; Shalev AY, Ankri Y, Israeli-Shalev Y, et al. Arch Gen Psychiatry. Feb 2012;69(2):166-76; Bisson JI, Olff M. Eur J Psychotraumatol. 2021;12(1):1824381. Several studies have found that early intervention can reduce the development of chronic PTSD by as much as 50%. Rothbaum BO, et al., Biological Psychiatry. 2012;72(11):957-963; Shalev AY, et al., Arch Gen Psychiatry. Feb 2012;69(2):166-76. Chronic PTSD has been associated with increased risk of cancer and metabolic syndrome (Cohen BE, et al., JAMA. 2009;302(5):489-492; Weiss T, et al., General Hospital Psychiatry. 2011;33(2):135-142), and increased inflammatory responses (O'Toole BI, et al., Journal of Psychosomatic Research. 2008;64(1):33-40). Psychosocial impacts may include homelessness, poverty, and incarceration (APA Clinical Practice Guideline (CPG) for PTSD).

[0021] However, early treatment is hindered by the fact that PTSD is underrecognized by healthcare providers, likely due to the lack of objective tests that can be ordered to aid in diagnosis (Joneydi R, et al., Med Care. Jun 1 2021;59(6):557-564). The lack of prompt diagnosis leads to delays in treatment, which in turn leads to the development of maladaptive coping and alcohol and substance use disorders. Studies repeatedly show very high rates of comorbid disorders. Kessler, RC, et al., Archives of General Psychiatry, 52, 1048-1060, 1995; Pietrzak RH et al., Am J Geriatr Psychiatry. May 2012;20(5):380-90; Walter KH, et al., J Trauma Stress. Dec 2018;31(6):837-844; Smith SM et al., J Psychiatr Res. Nov 2016;82:16-22. A meta-analysis found that 52% of individuals with current PTSD also had comorbid major depressive disorder. Rytwinski NK, et al., Journal of Traumatic Stress. 2013;26(3):299-309.

[0022] The typical PTSD diagnostic process can take months to years, with delays due to both patient and provider characteristics. On the patient side, very often, patients do not connect their current experiences to the traumatic event and therefore have little insight into the need for behavioral health care. In other cases, patients make the connection but want to address it themselves, often due to the stigma of appearing "weak or unresilient" if they do not. Many coping behaviors are initiated at this stage, but they are negative and subsequently interfere with personal and professional life, which ultimately prompts the patient to seek treatment from a provider.

[0023] On the provider side, the diagnosis of PTSD and other mental health conditions is typically made by skilled behavioral health providers in subspecialties. However, early diagnosis depends on the primary care provider's ability to consider the differential diagnosis and make appropriate referrals. Primary care providers generally lack the time, training, and resources to conduct lengthy clinical interviews and therefore rely on self-report surveys as screening tools to help identify patients for referral.

[0024] There are several open-source psychometric screening tools available; however, they generally all suffer from low accuracy rates and vary significantly based on the population to which they are applied (Spoont M, et al., VA Evidence-based Synthesis Program Reports, 2013). Many studies have been conducted to determine the accuracy rates of various tools, but they are difficult to directly compare because different "cutoffs" are often used. For example, one study may use a lower cutoff to maximize the detection of PTSD cases. However, this decision comes with more false positives. Using a higher cutoff can minimize false positives, but at the expense of an increased number of false negatives.

[0025] The most frequently used self-report screening instrument is the 17-item PTSD Checklist (PCL) (Scott D. McDonald, Patrick S. Calhoun Clinical Psychology Review 30 (2010) 976-987), which is widely used for clinical screening of PTSD and tracking its symptoms. Berlant, J., et al., The Journal of Clinical Psychiatry, 63, 15-20 (2002) and Blevins CA, et al., J Trauma Stress. Dec 2015;28(6):489-98. A preliminary version of the PCL-5 recommended a diagnostic cutoff of 31–33. Weathers FW, et al., ptsd.va.gov / professional / assessment / adult-sr / ptsd-checklist.asp, Accessed August 2, 2022. In a sample of 273 trauma-exposed mental health service users, at a cutoff of 33.5, the PCL-5 had a sensitivity of 95%; however, this resulted in a specificity of 52%. Roberts NP et al., Eur J Psychotraumatol. 2021;12(1):1863578.

[0026] In addition to compatibility issues, relying solely on self-reporting is problematic (Althubaiti A. J Multidiscip Healthc. 2016;9:211-7), and current standard of care is associated with stigma (Johnson HP et al., Psychiatr Danub. Nov 2018;30(Suppl 7):508-510, Mellotte H et al., Eur J Psychotraumatol. 2017;8(1):1389207, Wilk JE, et al., Psychiatr Serv. Aug 1 2016;67(8):878-82), malingering (Hall RC, Gen Hosp Psychiatry. Nov-Dec 2006;28(6):525-35, Ali S, Jabeen S, et al., Innov Clin Neurosci. Jan-Feb 2015;12(1-2):12-20), and underreporting (Johnson HP, Agius M., Psychiatr Danub. Nov 2018;30(Suppl 7):508-510). For example, reporting bias is well documented, and patients may not tell the truth due to fear of public discrimination, fear of hospitalization, embarrassment, shame, or myriad other factors that underlie public, self, or structural stigma. Corrigan PW et al., Psychol Sci Public Interest. Oct 2014;15(2):37-70.

[0027] This may be particularly true for those who have served in the military. U.S. military personnel and veterans (11-20%) suffer from PTSD at disproportionately higher rates than their civilian counterparts (6-8%). The U.S. military currently uses survey questionnaires incorporated into pre-deployment health assessments, post-deployment health assessments, and periodic health assessments, as well as post-deployment health reassessments, to identify service members who should undergo further evaluation by trained clinicians to obtain the additional information needed to make an appropriate diagnosis, including a diagnosis of comorbid conditions such as depression or traumatic brain injury, and to plan treatment.

[0028] Examples of primary care PTSD screening used in DoD assessments: Have you had any experiences in your life in the past month that were so amazing, shocking, or upsetting? 1. Have you ever had nightmares about it or thought about it without intending to? Yes / No 2. Have you tried really hard not to think about it or done something different to avoid being in a situation that reminds you of it? Yes / No 3. Were you always on guard, on the lookout, or easily startled? Yes / No 4. Have you felt numb or detached from other people, activities, or your surroundings? Yes / No

[0029] Concerns about how answers may affect current or future assignments, promotions, compensation, benefits, and / or security clearances often influence how respondents answer these questions. Lack of identification and delayed treatment can lead to maladaptive coping strategies, increased social dysfunction, and reduced medical readiness. See, e.g., Sarapas C, et al. Dis Markers. 2011;30(2-3):101-10; Le-Niculescu H, et al., Mol Psychiatry. 2020 May;25(5):918-938; and Cer RZ, et al., Gigascience. 2014 Oct 13;3:20.

[0030] Indeed, one study found that PTSD diagnoses were consistently underreported in military medical records to reduce stigma or protect personnel's career prospects. Wilk JE, et al., Psychiatr Serv. Aug 1 2016;67(8):878-82. Another study used anonymous surveys to obtain more accurate reports from a single infantry brigade combat team (n=1712). It was found that 20.3% of combatants who screened positive for depression or PTSD reported discomfort reporting honestly on routine postdeployment screening. Warner CH, et al., Arch Gen Psychiatry. Oct 2011;68(10):1065-71. Naturally, disorders with symptoms such as avoidance of traumatic reminders may be underreported at a systematic level.

[0031] These findings also extend to studies of civilian patients. A study of a South African inpatient psychiatric ward randomly sampled 40 subjects with no previous diagnosis of PTSD and found that the prevalence of PTSD in the study group rose to 40% from the ward's original prevalence of 5.5% achieved using standard treatment methods. van Zyl M, et al., Afr J Psychiatry (Johannesbg). May 2008;11(2):119-22.

[0032] These results suggest that individuals with other comorbid disorders may not be appropriately screened or diagnosed for PTSD. Further support for this underdiagnosis trend comes from another study of the U.S. child and adolescent population. Researchers interviewed patients using a trauma-focused interview at two sites and identified PTSD in 47.7% of patients (n=44) at one site, up from an original prevalence of 2.3%, and in 44.6% (n=56) at the second site, up from an original prevalence of 5.4%. Miele D, O'Brien EJ., Journal of Traumatic Stress. 2010;23(5):591-598.

[0033] The bias introduced by the observer is rarely discussed. A study comparing rates of PTSD in hospitalized male and female veterans found only one woman diagnosed with PTSD compared with seven men (n = 62), suggesting that women may not be appropriately diagnosed with the disorder due to provider-introduced bias. Grossman LS, et al., Psychiatr Serv. Mar 1997;48(3):393-5. In addition to the aforementioned drawbacks, these self-report tools are not widely used in primary care clinics. They are most often used in high-risk primary care settings such as military or veterans' clinics. In other settings, individuals would need to self-report symptoms or request a referral to a BH for a definitive diagnosis.

[0034] Taken together, the above findings explain many of the reasons why PTSD is tragically underdiagnosed in the current standard of care. They also highlight the significant need for objective diagnostic tools that can be applied more broadly without adding time or procedural burdens to primary care providers, while at the same time enabling early identification of large numbers of individuals who would otherwise go undiagnosed and untreated.

[0035] Once patients reach a specialty care provider, the structured interview for the DSM-5 Clinician-Administered PTSD Scale (CAPS-5) is often impractically long, so a diagnosis is usually made by clinical judgment after an unstructured interview. Due to the shortage of these providers, even after referral, there are often significant delays in receiving care due to wait times or failure to follow through on referrals due to social stigma.

[0036] Given the negative impact of PTSD on overall health and functioning and the availability of effective evidence-based treatments, there is a clear need for objective tests that can be used for early identification of affected individuals. Scott D. McDonald, Patrick S. CalhounClinical Psychology Review 30 (2010) 976-987, Department of Veterans Affairs, Veterans Health Administration (2004), (VHA Directive 2004-015).

[0037] Unfortunately, no single treatment has been proven effective for all individuals with PTSD, and none even has a substantial evidence base. Therefore, current treatment approaches often involve trial and error to find the right treatment for each individual patient. The latest American Psychological Association Clinical Pathway Guidelines recommend several treatments, including trauma-focused cognitive behavioral therapy (CBT-TF), cognitive processing therapy (CPT), cognitive therapy (CT), and eye movement desensitization and reprocessing (EMDR). Bisson JI, Olff M., Eur J Psychotraumatol, 2021;12(1):1824381. However, approximately 33% of individuals across the PTSD population are resistant to first- and second-line treatments, and nonresponse rates for cognitive behavioral therapy can be as high as 50% and for selective serotonin reuptake inhibitors (SRIs) as high as 20–40%. Green B. Advances in Psychiatric Treatment, 2013;19:181-190.

[0038] There is a need for objective biomarkers of PTSD that address diagnostic and related challenges, such as stigma, symptom overlap, and observer and reporter bias. Behavioral health is the only medical field still lacking objective measures to aid in diagnosis and treatment monitoring. There is a need for prospective diagnostic and treatment monitoring categories related to the biological dysregulation underlying this disorder.

[0039] definition As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Any reference to "or" herein may be intended to include "and / or" unless indicated otherwise.

[0040] As used herein, the term "about" can mean a referenced numerical expression plus or minus 5%, 10%, 15%, or 20% of the referenced numerical expression. In some instances, "about" can mean a referenced numerical expression plus or minus 15% of the referenced numerical expression. In some instances, "about" can mean a referenced numerical expression plus or minus 20% of the referenced numerical expression.

[0041] As used herein, a plurality of items, structural elements, compositional elements, and / or materials may be presented in common lists for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Accordingly, the individual members of such lists should not be construed as de facto equivalents of any other members of the same list solely based on their presentation in a common group, unless otherwise indicated.

[0042] Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. It should be understood that such range format is used merely for convenience and brevity and, therefore, should be interpreted flexibly, not only including the numerical values ​​expressly recited as the limits of the range, but also including all individual numerical values ​​or subranges subsumed within that range, as if each numerical value and subrange were expressly recited. By way of illustration, a numerical range of "about 1 to about 5" should be interpreted not only to include the explicitly recited value of about 1 to about 5, but also to include each individual value and subrange within the stated range. Thus, individual values ​​such as 2, 3, and 4, and subranges such as 1 to 3, 2 to 4, and 3 to 5, as well as 1, 2, 3, 4, and 5, are individually included within this numerical range. This same principle also applies to ranges reciting only one numerical value as a minimum or maximum value. Moreover, such interpretation shall apply regardless of the breadth of the range or characteristic described.

[0043] As used herein, the term "adjustment disorder" can be used in relation to a stressor when the response to the stressor does not meet criteria for post-traumatic stress disorder (or other specific mental disorders), and when the symptom pattern of post-traumatic stress disorder occurs in response to a less extreme stressor (e.g., separation from a spouse, being fired). Symptoms can include avoidance, numbing, and increased arousal, which were present before exposure to the stressor and may not meet diagnostic criteria for PTSD and may require consideration of other diagnoses (e.g., brief psychotic disorder, conversion disorder, major depressive disorder), which may be made instead of or in addition to PTSD.

[0044] The terms "administer," "administering," "administration," and the like, as used herein, can refer to methods that can be used to enable delivery of a compound or composition to a desired site of biological action.

[0045] As used herein, "clinical diagnosis" and its grammatical equivalents in relation to PTSD can refer to a diagnosis of PTSD through an informal or formal clinical interview by a trained behavioral health provider.

[0046] As used herein, "complementarity" may refer to the non-covalent binding of nucleobases through hydrogen bonding, Watson-Crick base pairing, wobble base pairing, van der Waals interactions, or any combination thereof.

[0047] As used herein, "control" as used herein refers to a control subject, a control population, or control data (eg, obtained from a baseline or database).

[0048] The phrase "control gene expression level," as used herein, may refer to expression data of one or more PTSD-related genes derived from a baseline, control, control population, or dataset described herein.

[0049] As used herein, the term "detect" and its grammatical equivalents describe the presence or absence of PTSD in a subject.Positive detection of PTSD, or as used interchangeably herein, positive PTSD detection, describes that PTSD is identified or detected in a subject.Negative detection of PTSD, or as used interchangeably herein, negative PTSD detection, describes that PTSD is not identified or detected in a subject.Similarly, a subject or group (e.g., a subject or population of interest, or a control or control group) that is negative for PTSD includes, for example, a subject that is not detected with PTSD by the methods and assays described herein and / or by the clinical diagnosis described herein.

[0050] As used herein, the term "differential expression" and its grammatical equivalents describe a statistically significant difference in gene expression levels between two experimental conditions (e.g., a test group and a control group (e.g., a group clinically diagnosed with PTSD and a group negative for PTSD)). Examples of calculating differential expression are further described herein.

[0051] As used herein, the term "expression level" may refer to the degree of gene expression in terms of the expression level of pre- or post-processing mRNA (before and after intron removal) of the gene. In certain cases, the term "expression level" may refer to the translated protein that is the product of the gene. Comparing or evaluating expression levels, i.e., determining the degree of expression, may be a crucial factor in the process of detecting PTSD. Similarly, evaluating expression levels may also indicate the degree of PTSD progression in a subject. Comparing expression levels may involve arithmetically, algorithmically, or mathematically calculating differential values. In some embodiments, differential values ​​inform the analysis of more complex quantitative information (e.g., correlating the expression levels of certain PTSD-related genes, or a subset thereof, with the occurrence or likelihood of the occurrence of a desired outcome described herein (e.g., identification, monitoring, and / or progression of PTSD)). Such calculations.

[0052] The term "fragment," as used herein, may refer to a portion of a sequence, a subset that may be shorter than the full-length sequence. A fragment may be a portion of a gene. A fragment may be a portion of an oligonucleotide sequence. A fragment may be less than about 20, less than about 30, less than about 40, or less than about 50 amino acids in length. A fragment may be about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, or 70% of the full length of an amino acid or nucleotide sequence. A fragment may be less than about 20, less than about 30, less than about 40, or less than about 50 oligonucleotides in length. The term "homology" may refer to the percent identity of a sequence relative to a reference sequence. As a practical matter, any particular sequence may be at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to any sequence described herein.

[0053] The phrase "nucleic acid" or "nucleic acid sequence" as used herein may refer to an oligonucleotide, nucleotide, polynucleotide, or any fragment thereof, and may refer to DNA or RNA of genomic or synthetic origin, which may be single-stranded or double-stranded and may represent the sense or antisense strand of any DNA-like or RNA-like material. Nucleic acid molecules may be naturally occurring, recombinant, or synthetic. The term "nucleotide" may also be abbreviated as "nt." The abbreviation "NA" as used herein may refer to nucleic acid.

[0054] As used herein, the term "peptide" can describe a chain of up to 30 amino acids. As used herein, the term "protein" can describe a chain of more than 30 amino acids. Peptides and proteins can also form dimers, trimers, and higher oligomers (i.e., consisting of more than one molecule, which may be identical or non-identical). As used herein, the terms "peptide" and "protein" (wherein "protein" is used interchangeably with "polypeptide") can also refer to naturally modified peptides / proteins, where the modification is by, for example, glycosylation, acetylation, phosphorylation, etc.

[0055] The phrase "pharmaceutically acceptable excipient," as used herein, may refer to a pharmaceutically acceptable material, composition, or vehicle (e.g., a liquid or solid filler, diluent, excipient, carrier, solvent, or encapsulating material).

[0056] As used herein, "post-traumatic stress disorder" can refer to a condition characterized by the occurrence of characteristic symptoms after exposure to a traumatic stressor, such as a direct personal experience of an event involving actual or threatened death or serious injury, or other threat to physical integrity; or after witnessing an event involving the death, injury, or threat to the physical integrity of another; or after learning of an unexpected or violent death, serious harm, or threat of death or injury experienced by a family member or other close relative. A person's response to the event can include intense fear, helplessness, or horror. In children, the response can include disorganized or agitated behavior. Characteristic symptoms resulting from exposure to extreme trauma can include persistent re-experiencing of the traumatic event, persistent avoidance of stimuli associated with the trauma, and a general numbing of responsiveness, as well as persistent symptoms of increased arousal. In post-traumatic stress disorder, the stressor can be extreme in nature.

[0057] As used herein, the term "predisposition to disease" is established in the art and is used accordingly herein.

[0058] As used herein, the term "sample" can refer to a biological sample (e.g., cells, tissues (from any organ, including postmortem brain tissue), or fluids (including serum, whole blood, cerebrospinal fluid, lymph, saliva, milk, pus, urine, feces), etc., isolated or obtained from an individual or from a cell culture component of a cell culture containing cells of a subject. Tissue or fluid samples obtained from patients and / or subjects that contain cells can be used to assess the expression levels of one or more genes or proteins.

[0059] As used herein, the term "trauma" may refer to the direct personal experience of an event involving actual or threatened death or serious injury, or other threat to physical integrity; or witnessing an event involving the death, injury, or threat to the physical integrity of another; or learning of an unexpected or violent death, serious harm, or threat of death or injury experienced by a family member or other close relative.

[0060] The terms "treat," "treating," or "treatment," as used herein, can include at least partially alleviating, reducing, or ameliorating the symptoms of a disease or condition; preventing further symptoms; improving or preventing the underlying cause of the symptoms; inhibiting a disease or condition, for example, at least partially halting the onset of a disease or condition; relieving the disease or condition; causing a regression of the disease or condition; alleviating the conditions caused by the disease or condition; or at least partially halting the symptoms of a disease or condition, either preventatively or therapeutically, or both. Treatment can include treatment of conditions associated with PTSD (e.g., reducing feelings of anxiety, depression, intense fear, helplessness, or fear, disorganized or agitated behavior, re-experiencing the traumatic event, persistent avoidance of stimuli associated with the trauma, and general numbing of responsiveness, as well as persistent symptoms of increased arousal). In some aspects, the methods described herein may include administering a therapeutically effective amount of a PTSD treatment to a subject, wherein the subject may be a human or an animal subject, and the animal subject may be a mammal.

[0061] PTSD molecular detection assays and methods of use thereof Provided herein is a PTSD molecular detection assay or its use. In some embodiments, the PTSD molecular detection assay is useful in the detection, diagnosis, screening, patient selection, treatment, and / or monitoring methods described herein. In some embodiments, the PTSD molecular detection assay and its use method described herein can detect PTSD in a subject. In certain embodiments, the detection of PTSD in a subject described herein can diagnose PTSD in the subject. In some embodiments, the detection of PTSD in a subject described herein increases the likelihood that the subject will be clinically diagnosed with PTSD. In some embodiments, the detection of PTSD in a subject described herein can predict and / or establish the effectiveness of PTSD treatment in the subject.

[0062] In some embodiments, the PTSD molecular detection assay described herein measures test gene expression levels of one or more PTSD-related genes. In some embodiments, the one or more PTSD-related genes are multiple PTSD-related genes, for example, one or more genes listed in Table 1. In some embodiments, the PTSD molecular detection assay described herein measures the expression levels of PTSD-related genes in a subject to generate a test PTSD-related gene expression level. In some embodiments, the PTSD molecular detection assay described herein compares the one or more test PTSD-related gene expression levels with a control gene expression level. In some embodiments, the one or more test PTSD-related gene expression levels are compared with one or more control gene expression levels to determine the relative expression of the one or more test PTSD-related gene expression levels compared to the one or more control gene expression levels. Those skilled in the art will understand that the control genes compared correspond to the one or more test PTSD-related genes.

[0063] PTSD-related genes In some embodiments, the PTSD molecular detection assay described herein determines the relative expression of one or more PTSD-related gene expression levels (i.e., one or more test PTSD-related gene expression levels) of a subject relative to one or more control gene expression levels. In some embodiments, the one or more PTSD-related genes can be any gene associated with the occurrence of PTSD in a subject. In some embodiments, the one or more PTSD-related genes include one or more of TSPAN5, HIST1H2AE, UBE3A, GPX4, EPB42, SLC4A1, NDUFA1, HIST1H1E, ELOVL7, ALAS2, COMT, PDZK1IP1, ITGA2B, CYP4F3, EPB41L3, PRDM1, RP-11-449P15.1, FAS, TUBB2A, CHMP5, BOD1L1, OSM, JAM3, or variants thereof (e.g., splice variants), or any combination thereof. In some embodiments, the one or more PTSD-related genes include one or more of TSPAN5, HIST1H2AE, UBE3A, GPX4, EPB42, SLC4A1, NDUFA1, HIST1H1E, ELOVL7, ALAS2, COMT, PDZK1IP1, ITGA2B, CYP4F3, EPB41L3, PRDM1, RP-11-449P15.1, FAS, TUBB2A, JAM3, or variants thereof (e.g., splice variants), or any combination thereof.

[0064] In some embodiments, one or more, two or more, three or more, four or more, five or more of TSPAN5, HIST1H2AE, UBE3A, GPX4, EPB42, SLC4A1, NDUFA1, HIST1H1E, ELOVL7, ALAS2, COMT, PDZK1IP1, ITGA2B, CYP4F3, EPB41L3, PRDM1, RP-11-449P15.1, FAS, TUBB2A, JAM3, or variants thereof (e.g., splice variants) are selected from the group consisting of: The expression levels of as many as 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or up to all, or any combination thereof, are measured.

[0065] Table 1 lists exemplary PTSD-related gene mRNA transcripts in DNA form, where T is substituted for U. [Table 1-1] [Table 1-2]

[0066] In some embodiments, the one or more PTSD-related genes can be any one of the genes listed in Table 1, consisting of the corresponding nucleic acid sequences listed in Table 1, or variants (e.g., splice variants) thereof. In some embodiments, the expression levels of one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, twenty or more, twenty one or more, or up to all of the PTSD-related genes listed in Table 1 are measured.

[0067] In certain cases, gene expression level can be detected by detecting a portion of the RNA sequence of the gene associated with PTSD to determine the level or amount of the entire RNA sequence.The portion detected by sequencing or hybridization technique can be approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any number between these.In certain cases, continuous regions in the sequence are detected by sequencing or hybridization technique. In such cases, the sequence may be approximately 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 nucleotides in length, or any number therebetween. In certain cases, multiple regions within the sequence are detected by sequencing or hybridization techniques. In such cases, about 2, 3, 4, 5, 6, 7, 8, 9, 10, or more regions within the RNA sequence may be detected.

[0068] In certain embodiments of the present disclosure, gene expression levels can be determined from any form of RNA, such as messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), small nuclear RNA (snRNA), U-spliceosomal RNA (U-RNA), small nucleolar RNA (snoRNA), Piwi-interacting RNA (piRNA), repeat-associated small interfering RNA (rasiRNA), small rDNA-derived RNA (srRNA), small transfer RNA-derived RNA (tsRNA), small ribosomal RNA-derived RNA (rsRNA), large non-coding RNA-derived small RNA (lncsRNA), or messenger RNA-derived small RNA (msRNA), gapmers, mixmers, double-stranded RNA (dsRNA), single-stranded RNAi (ssRNAi), DNA-directed RNA interference (ddRNAi), or any combination thereof. In certain embodiments, expression levels are determined by measuring expression of pre-processed mRNA. In certain embodiments, the expression level is determined by measuring the expression of a nucleic acid that interferes with the expression or translation of a PTSD-related gene.

[0069] For a given gene, expression level can depend on the number of RNA molecules or peptide molecules or protein molecules, and can be at least roughly proportional thereto, thereby enabling quantitative evaluation of gene expression.Depending on the means and method used for quantitative measurement, the gene expression level that is determined can be in the form of various reading parameters, such as the intensity of radioactive signal (for example, Northern blot using radioactive probe), the intensity of fluorescent signal (for example, DNA microarray), or mass-to-charge ratio (for example, mass spectrometry).

[0070] Therefore, in some embodiments, the PTSD molecular detection assay and its use method include measuring one or more test PTSD-related gene expression levels and / or one or more control gene expression levels. The gene expression level measurement provided herein includes polynucleotide hybridization analysis-based methods, polynucleotide sequencing-based methods, and proteomics-based methods. Exemplary methods known in the art for quantifying RNA expression in a sample include Northern blotting and in situ hybridization (Parker & Barnes, Methods in Molecular Biology 106:247-283 (1999)), RNAse protection assay (Hod, Biotechniques 13:852-854 (1992)), and PCR-based methods, such as reverse transcription PCR (RT-PCR) (Weis et al., Trends in Genetics 8:263-264 (1992)). Can utilize the antibody that can recognize sequence-specific double strand, including DNA double strand, RNA double strand, and DNA-RNA hybrid double strand or DNA-protein double strand.The representative method of sequencing-based gene expression analysis includes serial analysis of gene expression (SAGE) and massively parallel signature sequencing (MPSS) gene expression analysis, and can also use other methods known in the art.

[0071] Reverse transcription PCR (RT-PCR) Typically, mRNA is isolated from a test sample (e.g., a biological sample as described herein). The starting material can be total RNA isolated from a biological sample (e.g., a cell sample obtained from a subject, control, or population). If necessary, a baseline sample obtained from a subject, control, or population can be used as a baseline control. mRNA can be extracted from tissue samples, for example, from fresh samples, frozen (e.g., snap-frozen) samples, or paraffin-embedded and fixed (e.g., formalin-fixed) samples.

[0072] General methods for mRNA extraction are well known in the art and are disclosed in standard molecular biology textbooks, including Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons (1997). Methods for RNA extraction from paraffin-embedded tissues are disclosed, for example, in Rupp and Locker, Lab Invest. 56:A67 (1987) and De Andres et al., BioTechniques 18:42044 (1995). In particular, RNA isolation can be carried out using a purification kit, buffer set, and protease from a commercial manufacturer (e.g., Qiagen) according to the manufacturer's instructions. For example, total RNA from cells in culture can be isolated using Qiagen RNeasy minicolumns. Other commercially available RNA isolation kits include the MasterPure™ Complete DNA and RNA Purification Kit (EPICENTRE®, Madison, WI) and Paraffin Block RNA Isolation Kit (Ambion, Inc.), and total RNA from tissue samples can be isolated using RNA Stat-60 (Tel-Test), including by, for example, cesium chloride density gradient centrifugation.

[0073] The RNA-containing sample is then subjected to reverse transcription to produce cDNA from the RNA template, followed by exponential amplification in a PCR reaction. The two most commonly used reverse transcriptases are avian myeloblastosis virus reverse transcriptase (AMV-RT) and Moloney murine leukemia virus reverse transcriptase (MMLV-RT). The reverse transcription step is typically primed using specific primers, random hexamers, or oligo-dT primers, depending on the environment and goal of expression profiling. For example, extracted RNA can be reverse transcribed using a GeneAmp RNA PCR kit (Perkin Elmer, CA, USA) according to the manufacturer's instructions. The derived cDNA can then be used as a template in a subsequent PCR reaction.

[0074] PCR-based methods use thermostable DNA-dependent DNA polymerases, such as Taq DNA polymerase. For example, TaqMan® PCR typically utilizes the 5' nuclease activity of Taq polymerase or Tth polymerase to hydrolyze hybridization probes bound to its target amplicon, although any enzyme with equivalent 5' nuclease activity can be used. Two oligonucleotide primers are used to generate an amplicon typical of a PCR reaction product. A third oligonucleotide or probe can be designed to facilitate detection of the nucleotide sequence of the amplicon located between the hybridization sites of the two PCR primers. The probe can be detectably labeled, for example, with a reporter dye, and may further comprise both a fluorescent dye and a quencher fluorescent dye, as in the Taqman® probe configuration. When a Taqman® probe is used, the Taq DNA polymerase enzyme cleaves the probe in a template-dependent manner during the amplification reaction. The resulting probe fragments dissociate in solution, and the signal from the released reporter dye is not quenched by the second fluorophore. One reporter dye molecule is released for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data.

[0075] TaqMan® RT-PCR can be performed using commercially available equipment, such as high-throughput platforms such as the ABi PRISM 7700 Sequence Detection System® (Perkin-Elmer-Applied Biosystems, Foster City, CA, USA) or the Lightcycler (Roche Molecular Biochemicals, Mannheim, Germany); in some embodiments, the procedure is performed on a LightCycler® 480 (Roche Diagnostics) real-time PCR system, which is a microwell plate-based cycler platform.

[0076] 5-nuclease assay data is generally first expressed as the threshold cycle (Ct), where the fluorescence value recorded during each cycle represents the amount of product amplified up to that point in the amplification reaction. The threshold cycle (Ct) is generally described as the point at which the fluorescence signal is first recorded as statistically significant. Alternatively, the data may be expressed as the crossing point (Cp). The Cp value is calculated by determining the second derivative and their maximum value of the entire qPCR amplification curve. The Cp value represents the cycle at which the increase in fluorescence is highest and the exponential phase of PCR begins.

[0077] Real-time PCR is compatible with both quantitative competitive PCR, which uses the internal competitor of each target sequence for normalization, and the quantitative competitive PCR, which uses the normalization gene contained in sample or uses housekeeping gene for RT-PCR.For further details, see, for example, Held et al., Genome Research 6:986-994 (1996).

[0078] The steps of the protocol suitable for use in the method of the present disclosure use fixed paraffin-embedded tissue as an RNA source. For example, mRNA isolation, purification, primer extension, and amplification can be performed according to methods available in the art. (See, for example, Godfrey et al. J. Molec. Diagnostics 2: 84-91 (2000); Speeht et al., Am. J. Pathol. 158: 419-29 (2001)). Briefly, a typical process begins with cutting out a paraffin-embedded tumor tissue sample piece approximately 10 μm thick, and then RNA is extracted, and protein and DNA are depleted from the RNA-containing sample. After analyzing the RNA concentration, the RNA is reverse-transcribed using gene-specific primers, followed by RT-PCR to obtain cDNA amplification products.

[0079] Intron-based PCR primer and probe design PCR primers and probes can be designed based on the sequences of exons or introns present in the mRNA transcript of the gene of interest. Primer / probe design can be performed using publicly available software, such as the DNA BLAT software developed by Kent, WJ., Genome Res. 12(4):656-64 (2002), or BLAST software, including its variants.

[0080] If necessary or desired, the repeat sequence of target sequence can be masked to reduce non-specific signals.An exemplary tool for achieving this includes the Repeat Masker program available online through Baylor College of Medicine, which screens DNA sequence against a library of repeat elements and returns the query sequence with the repeat elements masked.The masked intron sequence can then be used to design primer and probe sequences using any commercially available or otherwise publicly available primer / probe design package, such as Primer Express (Applied Biosystems), MGB assay-by-design (Applied Biosystems), Primer3 (Steve Rozen and Helen J. Skaletsky (2000) Primer3 on the WWW for general users and for biologist programmers).See S. Rrawetz, S. Misener, Bioinformatics Methods and Protocols: Methods in Molecular Biology, pp.365-386 (Humana Press).

[0081] Other factors that can affect PCR primer design include primer length, melting temperature (Tm), G / C content, specificity, complementary primer sequence, and 3' end sequence. In general, optimal PCR primers are generally 17-30 bases long, contain about 20-80%, for example, about 50-60%, G+C bases, and exhibit a Tm of 50-80°C, for example, about 50-70°C.

[0082] For further guidelines regarding the design of PCR primers and probes, see, e.g., Dieffenbach, CW. et al., "General Concepts for PCR Primer Design" in: PCR Primer, A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1995, pp. 133-155; Tunis and Geifand, "Optimization of PCRs" in: PCR Protocols, A Guide to Methods and Applications, CRC Press, London, 1994, pp. 5-11; and Plasterer, TN Primerselect: Primer and probe design. Methods Mol. Biol 70:520-527 (1997), the entire disclosures of which are expressly incorporated herein by reference.

[0083] Mass ARRAY® System In MassARRAY-based methods, such as an exemplary method developed by Sequenom, Inc. (San Diego, CA), after RNA isolation and reverse transcription, the resulting cDNA is spiked with a synthetic DNA molecule (competitor) that matches the target cDNA region at all positions except for a single base and serves as an internal standard. The cDNA / competitor mixture is PCR-amplified and subjected to post-PCR shrimp alkaline phosphatase (SAP) enzyme treatment, which results in dephosphorylation of the remaining nucleotides. After inactivation of the alkaline phosphatase, the PCR products from the competitor and the cDNA are subjected to primer extension, resulting in different mass signals for the competitor-derived PCR product and the cDNA-derived PCR product. After purification, these products are dispensed onto a chip array pre-loaded with the components required for analysis by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). The cDNA present in the reaction is then quantified by analyzing the ratio of peak areas in the resulting mass spectrum. For further details, see, e.g., Ding and Cantor, Proc. Natl. Acad. Sci. USA 100:3059-3064 (2003).

[0084] Other PCR-based methods Additional PCR-based techniques that may find use in the methods disclosed herein include, for example, Bead Array® technology (Ilumina, San Diego, CA; Oliphant et al., Discover)'s Markers for Disease (Supplement to Biotechniques), June 2002; Ferguson et al., Analytical Chemistry 72:5618 (2000)); BeadsArray for Detection of Gene Expression® (BADGE), which uses the commercially available LuminexlGO LabMAP® system and multiple color-coded microspheres (Luminex Corp., Austin, TX) in a rapid assay for gene expression (Yang et al., Genome Res. 31:1888-1898 (2001)); and high coverage expression profiling (HiCEP) analysis (Fukumura et al., Nucl. Acids. Res. 31(16) e94 (2003)).

[0085] Microarray The expression level of target gene or microarray can also be evaluated using microarray technology.In this method, the target polynucleotide sequence (including cDNA and oligonucleotide) is arrayed on a substrate.The arrayed sequence is then contacted with detectably labeled cDNA produced from the RNA of test sample under conditions suitable for specific hybridization.As in the above-mentioned RT-PCR method, the source of RNA is typically the total RNA isolated from tumor sample and, if necessary, from the normal tissue of the same patient as internal control or cell line.RNA can be extracted from, for example, frozen or archived, paraffin-embedded and fixed (for example, formalin-fixed) tissue sample.

[0086] For example, PCR-amplified inserts of the cDNA clones of the genes to be assayed are applied to the substrate in a high-density array.Usually, at least 10,000 nucleotide sequences are applied to the substrate.For example, the genes arranged in the microarray, each of which has 10,000 elements, are immobilized on a microchip, and are suitable for hybridization under stringent conditions.

[0087] Fluorescently labeled cDNA probes can be produced by reverse transcription of the RNA extracted from the tissue of interest through the incorporation of fluorescent nucleotides.The labeled cDNA probes applied to chips are specifically hybridized with the DNA of each spot on the array.After washing under stringent conditions to remove non-specifically bound probes, the chips are scanned by confocal laser microscope or other detection methods, such as CCD camera.The hybridization quantification of each arrayed element allows the evaluation of corresponding RNA abundance.

[0088] Using dual-color fluorescence, the separately labeled cDNA probes produced from two RNA sources are hybridized to the array in pairs.The relative abundance of the transcripts from the two sources corresponding to each designated gene is thus determined simultaneously.By miniaturizing the scale of hybridization, the expression pattern of a large number of genes can be conveniently and rapidly evaluated.This method has been shown to have the required sensitivity to detect rare transcripts that are expressed at a few copies per cell, and to reproducibly detect at least approximately two-fold differences in expression level (Schena et al., Proc. Natl. Acad. ScL USA 93(2): 106-149 (1996)).

[0089] Microarray analysis can be performed using commercially available equipment according to the manufacturer's protocols, for example, by using AlTymctrix GenChip® technology or Incyte's microarray technology.

[0090] Serial Analysis of Gene Expression (SAGE) Serial analysis of gene expression (SAGE) is a method that allows for the simultaneous and quantitative analysis of multiple gene transcripts without the need to provide individual hybridization probes for each transcript. First, short sequence tags (approximately 10 bp to 14 bp) containing sufficient information to uniquely identify the transcripts are generated, provided that the tags are derived from unique locations within each transcript. Multiple transcripts are then linked together to form long, continuous molecules that can be sequenced to simultaneously reveal the identities of multiple tags. The expression pattern of any transcript population can be quantitatively assessed by determining the abundance of individual tags and identifying the genes corresponding to each tag. For further details, see, e.g., Velculeseu et al., Science 270:484-487 (1995) and Velculeseu et al., Cell 88:243-51 (1997).

[0091] Gene expression analysis by nucleic acid sequencing Nucleic acid sequencing technology is a preferred method for analyzing gene expression. The underlying principle of these methods is that the number of times a cDNA sequence is detected in a sample is directly related to the relative expression of the RNA corresponding to that sequence. These methods are sometimes referred to by the term digital gene expression (DGE), reflecting the distinct numerical characteristics of the resulting data. Early methods that applied this principle were serial analysis of gene expression (SAGE) and massively parallel signature sequencing (MPSS). See, for example, S. Brenner, et al., Nature Biotechnology 18(6):630-634 (2000). More recently, the emergence of "next-generation" sequencing technologies has made DGE simpler, more high-throughput, and less expensive. As a result, many laboratories can use DGE to screen more gene expression in more individual patient samples than was previously possible. See, for example, J. Marioni, Genome Research 18(9): 1509-1517 (2008), R. Morin, Genome Research 18(4):610-621 (2008), A. Mortazavi, Nature Methods 5(7):621-628 (2008), N. Cloonan, Nature Methods 5(7):613-6! 9 (2008).

[0092] Methods for isolating RNA for expression analysis from blood, plasma, and serum (see, e.g., Enders, et al., Clin Chem 48:1647-53 (2002) and references cited therein) and urine (see, e.g., R, Boom, et al., J Clin Microbiol. 28, 495-503 (1990) and references cited therein) have been described.

[0093] Immunohistochemistry Immunohistochemistry is also suitable for detecting gene expression levels and is applied to the methods disclosed herein.Antibodies (e.g., monoclonal antibodies) that specifically bind to the gene product of the gene of interest can be used in such methods.Antibodies can be detected by directly labeling the antibody itself, for example, with a radioactive label, a fluorescent label, a hapten label (e.g., biotin), or an enzyme (e.g., horseradish peroxidase or alkaline phosphatase).Alternatively, unlabeled primary antibodies can be used together with labeled secondary antibodies specific to the primary antibodies.Immunohistochemistry protocols and kits are well known in the art and are commercially available.

[0094] Proteomics Proteome is the totality of proteins present in a sample (for example, tissue, organism, or cell culture) at a certain time.Proteomics includes, among other things, the study of the overall changes in protein expression in a sample (also referred to as "expression proteomics").Proteomics typically includes the following steps: (1) separating the individual proteins in a sample by 2-D gel electrophoresis (2-D PAGE); (2) identifying the individual proteins recovered from the gel, for example, by mass spectrometry or N-terminal sequencing; and (3) analyzing data using bioinformatics.

[0095] Control gene expression levels In some embodiments, the one or more control gene expression levels are determined, for example, by using the PTSD molecular detection assays and / or methods of use described herein. In some embodiments, the one or more control gene expression levels are pre-determined, for example, through the use of the PTSD molecular detection assays and / or methods of use described herein, or from an existing database.

[0096] In some embodiments, the control gene expression level is derived from a baseline control, a control, or a control population.

[0097] The control or control population can be any suitable comparator or control population. In some embodiments, the control or control population does not have PTSD. In some embodiments, the control or control population tests negative for PTSD, for example, by a clinical diagnostic method (e.g., as described herein). In some embodiments, the control or control population has been exposed to trauma, but has not developed PTSD, for example, within 6 months of exposure to the trauma. In such embodiments, where the control or control population does not suffer from PTSD, such a control or control population can be referred to herein as a negative control or negative control population.

[0098] In some embodiments, the control or control population suffers from PTSD. In some embodiments, the control or control population suffers from PTSD but is not undergoing PTSD treatment. In some embodiments, the control or control population suffers from PTSD and is undergoing PTSD treatment. In such embodiments, the control or control population suffers from PTSD, such control or control population may be referred to herein as a positive control or positive control population.

[0099] In some embodiments, the control or control population is obtained from a database entry, which may be a negative control or a positive control based on the database entry selected.

[0100] In certain cases, the control is selected from: i) a control who has been exposed to trauma but has not developed PTSD; ii) a group of subjects who are negative for PTSD and representative of the study population; and iii) a database entry. Alternatively, the range of expression levels obtained from a group of subjects who are negative for PTSD and representative of the study population may be used as a normal value. Such normal values ​​can be used to normalize the data collected from the subjects when performing the methods described herein. Those skilled in the art will understand how to select an appropriate control depending on the method being performed, the data to be captured, the information being analyzed, etc.

[0101] In certain cases, to increase the reliability of data related to expression levels, a control subject sample can be evaluated more than once, or multiple samples of the control subject can be obtained. Data can be further pooled to calculate the mean or median and, if necessary, the variance for each control subject. In certain cases, the expression level is compared with the expression level of the corresponding gene in samples from more than one control subject (e.g., at least 2, 10, 20, or more control subjects). In certain cases, the expression levels of the control subject samples can be pooled, and the mean or median and, if necessary, the variance can be calculated. In such embodiments, the mean or median expression level can be referred to herein as the mean expression level or median expression level. These values ​​can be entered into a database, for example, as the normalized value of each gene, and can be retrieved from the database if necessary, thereby eliminating the need to experimentally evaluate the expression level in a control sample every time the expression level in a patient sample is evaluated. Therefore, the control can also be a database entry. Furthermore, the variance of the expression levels of the control samples can be used to determine the statistical significance of the deviation from the mean of the control in the sample to be evaluated. Finally, age or sex specific controls may be used where deemed appropriate.

[0102] In control samples obtained from a database of subjects without PTSD, the levels of RNA or protein are expressed in a certain ratio, contributing to a coordinated effect on normal bodily functions. Disturbances in the gene expression levels of one or more genes, such as up-regulation and / or down-regulation, can cause medical conditions if there is no endogenous recovery mechanism that can compensate for the disturbance. Some steps in the gene expression process can be modulated to regulate the expression level of a gene, such as the step of initiating the transcription process, which involves the amount of transcription factors or the presence of enhancer / inhibitory sequences; the step of translation, which involves the half-life of the mRNA being translated; the half-life of proteins, protein secretion, and post-translational modification of proteins, which affect protein folding. Gene regulation provides cells with control over structure and function, and is the basis for events such as the differentiation of cells or multicellular organisms, their morphogenesis, or their adaptability.

[0103] In some embodiments, the control subject or control population who tests negative for PTSD can be determined by one or more clinical diagnostic classification schemes (such as those described herein). Clinical diagnostic classification schemes for PTSD include the Clinician Administered PTSD Scale for DSM-5 (CAPS-5), the PTSD Checklist for DSM-5 (PCL-5), or both. Similarly, the control subject or control population who tests negative for PTSD can also test negative for one or more PTSD comorbidities as determined by one or more clinical diagnostic classification schemes for PTSD comorbidities. Such clinical diagnostic classification schemes for PTSD comorbidities include the Generalized Anxiety Disorder 7-Item Scale (GAD-7), the Alcohol Use Disorder Identification Test (AUDIT-C), the Pittsburgh Sleep Quality Index (PSQI), Adverse Childhood Experiences (ACE), or any combination thereof.

[0104] Clinician-Administered PTSD Scale for DSM-5 (CAPS-5) The Clinician-Administered PTSD Scale for DSM-5 (CAPS-5) is a structured interview that is considered the gold standard for assessing the severity of trauma and diagnosing PTSD. This interview is administered by a trained clinician and covers all DSM-5 criteria for PTSD. It assesses the frequency and intensity of various PTSD symptoms and their impact on daily functioning.

[0105] To score the structured interview, raters combine information about item frequency and intensity into a single severity rating. The CAPS-5 total symptom severity score is calculated by summing the severity scores of the 20 DSM-5 PTSD symptoms. Similarly, the CAPS-5 symptom cluster severity score is calculated by summing the individual item severity scores for symptoms corresponding to a given DSM-5 cluster: Criterion B (items 1–5), Criterion C (items 6–7), Criterion D (items 8–14), and Criterion E (items 15–20). A symptom cluster score may also be calculated for dissociation by summing items 19 and 20.

[0106] To make a diagnosis of PTSD, a patient must have at least one criterion B symptom, at least one criterion C symptom, at least two criterion D symptoms, at least two criterion E symptoms, meet criterion F (the disturbance has lasted for one month), and meet criterion G (the disturbance causes either clinically significant distress or functional impairment).

[0107] DSM-5 PTSD Checklist The PTSD Checklist for DSM-5, or PCL-5, is a self-report questionnaire specifically designed to assess PTSD symptoms based on the criteria outlined in DSM-5. It is widely used in both clinical and research settings. It takes approximately 5-10 minutes to complete.

[0108] The PCL-5 consists of 20 items that measure the severity and frequency of PTSD symptoms across four symptom clusters: intrusion, avoidance, negative changes in cognition and mood, and hyperarousal. Individuals who have experienced a traumatic event are asked to rate the extent to which each symptom has bothered them over the past month using a scale ranging from 0 (not at all) to 4 (very much).

[0109] Interpretation of the PCL-5 should be performed by a clinician. The PCL-5 can be scored in different ways. A total symptom severity score (range 0–80) can be obtained by summing the scores for each of the 20 items. DSM-5 symptom cluster severity scores can be obtained by summing the scores of items within a given cluster: Cluster B (items 1–5), Cluster C (items 6–7), Cluster D (items 8–14), and Cluster E (items 15–20). A provisional PTSD diagnosis can be made by treating each item rated 2 = "moderate" or higher as a supported symptom, and then following the DSM-5 diagnostic rules requiring at least one B item (questions 1-5), one C item (questions 6-7), two D items (questions 8-14), and two E items (questions 15-20).

[0110] The item scores can be summed to obtain an overall severity score of PTSD symptoms. The PCL-5 provides a quantitative measure of PTSD symptomatology and may aid in the assessment, monitoring, and diagnosis of PTSD. It is not intended to provide a formal diagnosis by itself, but can be used as a screening tool or as part of a comprehensive assessment administered by a medical professional.

[0111] Generalized Anxiety Disorder 7-Item Scale The Generalized Anxiety Disorder 7-Item Scale (GAD-7) is a self-report screening tool for generalized anxiety disorder. The GAD-7 score is calculated by assigning scores of 0, 1, 2, and 3 to response categories of "not at all," "a few days," "more than half the days," and "almost every day," respectively, and adding together the scores for the seven questions.

[0112] Scores of 5, 10, and 15 are interpreted as cutoff points for mild, moderate, and severe anxiety, respectively. When used as a screening tool, a score of 10 or higher suggests further evaluation. Using a threshold score of 10, the GAD-7 has a sensitivity of 89% and a specificity of 82% for GAD. It is moderately good at screening for three other common anxiety disorders: panic disorder (sensitivity 74%, specificity 81%), social anxiety disorder (sensitivity 72%, specificity 80%), and post-traumatic stress disorder (sensitivity 66%, specificity 81%). Wilk JE, Herrell RK, Carr AL, West JC, Wise J, Hoge CW. Diagnosis of PTSD by Army Behavioral Health Clinicians: Are Diagnoses Recorded in Electronic Health Records? Psychiatr Serv. Aug 1 2016;67(8):878-82. doi:10.1176 / appi.ps.201500292.

[0113] Alcohol Use Disorder Specific Test The Alcohol Use Disorder Identification Test (AUDIT-C) is an alcohol screen that can help identify patients who are risky drinkers or have an active alcohol use disorder (including alcohol abuse or alcohol dependence). The AUDIT-C is scored on a scale of 0 to 12 (a score of 0 reflects no alcohol use). For men, a score of 4 or higher is considered positive, and for women, a score of 3 or higher is considered positive. Generally, the higher the AUDIT-C score, the more likely the patient's drinking is affecting their health and safety.

[0114] Pittsburgh Sleep Quality Index The Pittsburgh Sleep Quality Index (PSQI) is a self-assessment questionnaire that assesses sleep quality and disturbance over a one-month interval. Consisting of 19 items, the PSQI measures several different aspects of sleep and provides seven component scores and one composite score. The component scores include subjective sleep quality, sleep latency (i.e., the time it takes to fall asleep), sleep duration, habitual sleep efficiency (i.e., the percentage of time asleep relative to the time spent in bed), sleep disturbance, use of sleeping medication, and daytime dysfunction.

[0115] Each item is weighted on an interval scale of 0 to 3. The overall PSQI score is then calculated by summing the seven component scores to provide an overall score ranging from 0 to 21, with lower scores indicating healthier sleep quality.

[0116] Adverse Childhood Experiences (ACEs) The Adverse Childhood Experiences Questionnaire (ACE-Q) is a 10-item scale designed to quantify clients' instances of adverse or traumatic experiences before age 18. The ACE-Q assesses clients' childhood exposure to psychological, physical, and sexual abuse, as well as family dysfunction, including domestic violence, substance use, and incarceration. The ACE-Q was used in the Adverse Childhood Experiences (ACE) study (Felitti et al., 1998), which found that ACE-Q scores correlated with future mental health problems, health risk behaviors (including substance abuse), and serious health problems. These included depression, suicide attempts, alcoholism, drug abuse, smoking, 50 or more sexual partners, physical inactivity, severe obesity, increased risk of sexually transmitted diseases, fractures, heart disease, lung disease, liver disease, and several types of cancer (Felitti et al., 1998). Each positive response is summed to give an overall ACE-Q score (out of 10). A score of 4 or higher is considered clinically significant. A small proportion (5%-10%) of the overall population had a score of 4 or higher, with the most pronounced adverse overall long-term health outcomes (Hughes, K., et. al. (2017). Lancet Public Health, 2(8), e356-e366).

[0117] Differential expression As described herein, in some embodiments, the PTSD molecular detection assay and / or method of use thereof comprises determining the differential expression of one or more test PTSD-related genes compared to one or more control genes, and determining the detection of PTSD based on the differential expression. In some embodiments, the PTSD molecular detection assay and method of use thereof comprises measuring the expression levels of one or more test PTSD-related genes in a sample obtained from a subject, comparing the expression levels of the one or more test PTSD-related genes with the expression levels of one or more control genes to determine the differential expression of the one or more test PTSD-related genes, and determining a positive detection of PTSD based on the differential expression.

[0118] Any suitable method for determining differential gene expression can be utilized in the context of the present disclosure.In some embodiments, differential expression can be calculated by normalizing expression data between two experimental conditions (for example, a group clinically diagnosed with PTSD and a PTSD-negative group) and identifying the statistically significant genes that correlate with the condition (for example, PTSD).In some embodiments, differential expression can be calculated by ranking gene expression, including ranking the normalized expression data between two experimental conditions (for example, a group clinically diagnosed with PTSD and a PTSD-negative group), and identifying the statistically significant genes that correlate with the condition (for example, PTSD).

[0119] Examples of normalization methods suitable for the present disclosure include, but are not limited to, global normalization, Lowess normalization, trimmed mean method (TMM), quantile normalization, scaling normalization, variance stabilization (VSN), and invariant method (IN). In some embodiments, gene expression normalization may be performed against a set of reference genes, including a database or a predetermined distinct set of genes.

[0120] In some embodiments, gene expression levels may be normalized using Normal Quantile Transformation (NQT). In some embodiments, gene expression levels may be normalized using NQT as follows: random numbers are drawn from a normal distribution based on the number of observations (e.g., number of mRNA species) and classified based on Equation 1. Quantiles are then assigned according to their respective ranks using Equations 2-3. All equations contained herein are R language commands unless otherwise specified.

[0121] Equation 1: rn=sort(rnorm(number of mRNA))

[0122] Equation 2: qt=rank(x) / length(x)

[0123] Equation 3: nqt=rn[rank(qt)]

[0124] In some embodiments, normalizing gene expression levels can identify differentially expressed genes that correlate with PTSD. Any suitable method for identifying differentially expressed genes can be used in the context of the present disclosure. Examples include regression models, such as linear regression models or logistic regression models. For example, a regression model (e.g., linear or logistic) can be used to analyze quantile-transformed data to identify any mRNA species that show either increased or decreased expression. Using the NQT data for different time points, the corresponding P values, intercept, and slope values ​​can be calculated using equations 4-8, and any mRNA with P<0.05 is considered statistically significant:

[0125] Equation 4: lmod=lm(y~x), where x=time, y=NQT data

[0126] Equation 5: s=summary(lmod)

[0127] Equation 6: p=as.character(pf(s$fstatistic[1],s$fstatistic[2],))(s$fstatistic[3],lower.tail=FALSE)

[0128] Equation 7: intercept=s$coefficients[1]

[0129] Equation 8: slope=s$coefficients[2]

[0130] A discussion of the above analyses can be found in (Cer RZ, Herrera-Galeano JE, Anderson JJ, Bishop-Lilly KA, Mokashi VP. miRNA Temporal Analyzer (mirnaTA): a bioinformatics tool for identifying differentially expressed microRNAs in temporal studies using normal quantile transformation. Gigascience. 2014 Oct 13;3:20. doi: 10.1186 / 2047-217X-3-20. PMID: 25379175; PMCID: PMC4212236.), the entire contents of which are incorporated herein by reference.

[0131] In some embodiments, one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, twenty, and up to all of the tested PTSD-related genes have a nucleotide sequence of less than 0.05, less than 0.01, less than 5×10 -3 Lower than 1×10 -3 Lower than 5×10 -4 Lower than 1×10 -4 Lower than 1×10 -5 Lower than 9.9 × 10 -6 lower than 9.8 × 10 -6 lower than 9.7 × 10 -6 lower than 9.6 × 10 -6 Lower than 9.5 x 10 -6 lower than 9.4 × 10 -6 lower than 9.3 × 10 -6 lower than 9.2 × 10 -6 lower than 9.1 × 10 -6 Lower than 9×10 -6 Lower than 8.9 × 10 -6 lower than 8.8 × 10 -6 lower than 8.7 × 10 -6 lower than 8.6 × 10 -6 Lower than 8.5 × 10 -6 lower than 8.4 × 10 -6 lower than 8.3 × 10 -6 lower than 8.2 × 10 -6 lower than 8.1 × 10 -6 Lower than 8×10 -6 Lower than 7.9 × 10 -6 lower than 7.8 × 10 -6 lower than 7.7 × 10 -6 lower than 7.6 × 10-6 Lower than 7.5 x 10 -6 lower than 7.4 × 10 -6 lower than 7.3 × 10 -6 lower than 7.2 × 10 -6 lower than 7.1 × 10 -6 Lower than 7×10 -6 lower than 6.9 × 10 -6 lower than 6.8 × 10 -6 lower than 6.7 × 10 -6 lower than 6.6 × 10 -6 Lower than 6.5 × 10 -6 lower than 6.4 × 10 -6 lower than 6.3 × 10 -6 lower than 6.2 × 10 -6 lower than 6.1 × 10 -6 Lower than 6×10 -6 lower than 5.9 × 10 -6 lower than 5.8 × 10 -6 lower than 5.7 × 10 -6 lower than 5.6 × 10 -6 Lower than 5.5 x 10 -6 lower than 5.4 × 10 -6 lower than 5.3 × 10 -6 lower than 5.2 × 10 -6 lower than 5.1 × 10 -6 Lower than 5×10 -6 lower than 4.9 × 10 -6 lower than 4.8 × 10 -6 lower than 4.7 × 10 -6 lower than 4.6 × 10 -6 Lower than 4.5 × 10 -6 lower than 4.4 × 10 -6 lower than 4.3 × 10 -6 lower than 4.2 × 10 -6 lower than 4.1 × 10 -6 Lower than 4×10 -6 lower than 3.9 × 10 -6 lower than 3.8 × 10 -6 lower than 3.7 × 10 -6 lower than 3.6 × 10 -6Lower than 3.5 × 10 -6 lower than 3.4 × 10 -6 lower than 3.3 × 10 -6 lower than 3.2 × 10 -6 lower than 3.1 × 10 -6 Lower than 3×10 -6 lower than 2.9 × 10 -6 lower than 2.8 × 10 -6 lower than 2.7 × 10 -6 lower than 2.6 × 10 -6 Lower than 2.5 × 10 -6 lower than 2.4 × 10 -6 lower than 2.3 × 10 -6 lower than 2.2 × 10 -6 lower than 2.1 × 10 -6 Lower than 2×10 -6 lower than 1.9 × 10 -6 lower than 1.8 × 10 -6 lower than 1.7 × 10 -6 lower than 1.6 × 10 -6 Lower than 1.5 x 10 -6 lower than 1.4 × 10 -6 lower than 1.3 × 10 -6 lower than 1.2 × 10 -6 lower than 1.1 × 10 -6 Lower than 1×10 -6 Lower than 9.9 × 10 -7 lower than 9.8 × 10 -7 lower than 9.7 × 10 -7 lower than 9.6 × 10 -7 Lower than 9.5 x 10 -7 lower than 9.4 × 10 -7 lower than 9.3 × 10 -7 lower than 9.2 × 10 -7 lower than 9.1 × 10 -7 Lower than 9×10 -7 Lower than 8.9 × 10 -7 lower than 8.8 × 10 -7 lower than 8.7 × 10 -7 lower than 8.6 × 10 -7Lower than 8.5 × 10 -7 lower than 8.4 × 10 -7 lower than 8.3 × 10 -7 lower than 8.2 × 10 -7 lower than 8.1 × 10 -7 Lower than 8×10 -7 Lower than 7.9 × 10 -7 lower than 7.8 × 10 -7 lower than 7.7 × 10 -7 lower than 7.6 × 10 -7 Lower than 7.5 x 10 -7 lower than 7.4 × 10 -7 lower than 7.3 × 10 -7 lower than 7.2 × 10 -7 lower than 7.1 × 10 -7 Lower than 7×10 -7 lower than 6.9 × 10 -7 lower than 6.8 × 10 -7 lower than 6.7 × 10 -7 lower than 6.6 × 10 -7 Lower than 6.5 × 10 -7 lower than 6.4 × 10 -7 lower than 6.3 × 10 -7 lower than 6.2 × 10 -7 lower than 6.1 × 10 -7 Lower than 6×10 -7 lower than 5.9 × 10 -7 lower than 5.8 × 10 -7 lower than 5.7 × 10 -7 lower than 5.6 × 10 -7 Lower than 5.5 x 10 -7 lower than 5.4 × 10 -7 lower than 5.3 × 10 -7 lower than 5.2 × 10 -7 lower than 5.1 × 10 -7 Lower than 5×10 -7 lower than 4.9 × 10 -7 lower than 4.8 × 10 -7 lower than 4.7 × 10 -7 lower than 4.6 × 10 -7Lower than 4.5 × 10 -7 lower than 4.4 × 10 -7 lower than 4.3 × 10 -7 lower than 4.2 × 10 -7 lower than 4.1 × 10 -7 Lower than 4×10 -7 lower than 3.9 × 10 -7 lower than 3.8 × 10 -7 lower than 3.7 × 10 -7 lower than 3.6 × 10 -7 Lower than 3.5 × 10 -7 lower than 3.4 × 10 -7 lower than 3.3 × 10 -7 lower than 3.2 × 10 -7 lower than 3.1 × 10 -7 Lower than 3×10 -7 lower than 2.9 × 10 -7 lower than 2.8 × 10 -7 lower than 2.7 × 10 -7 lower than 2.6 × 10 -7 Lower than 2.5 × 10 -7 lower than 2.4 × 10 -7 lower than 2.3 × 10 -7 lower than 2.2 × 10 -7 lower than 2.1 × 10 -7 Lower than 2×10 -7 lower than 1.9 × 10 -7 lower than 1.8 × 10 -7 lower than 1.7 × 10 -7 lower than 1.6 × 10 -7 Lower than 1.5 x 10 -7 lower than 1.4 × 10 -7 lower than 1.3 × 10 -7 lower than 1.2 × 10 -7 lower than 1.1 × 10 -7 Lower than or 1 × 10 -7 are differentially expressed with p-values ​​lower than

[0132] In some embodiments, one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, twenty, up to all of the tested PTSD-related genes, are selected from a total of 2.5×10 -6 Lower than 9×10 -7 lower than, or 1.6 × 10 -7 are differentially expressed with p-values ​​lower than

[0133] In some embodiments, the differential expression of one or more test PTSD-related genes can be determined to establish a significant relationship, if any, between one or more test PTSD-related genes and the detection of PTSD. Those skilled in the art will recognize that there are numerous statistical methods that can be used to determine whether there is a significant relationship between a desired outcome (e.g., the detection of PTSD) and the expression levels of the marker genes described herein.

[0134] Similarly, those skilled in the art will recognize that numerous co-expression analysis methods, currently known or to be developed in the future, will fall within the scope and spirit of the present disclosure. These methods may incorporate, for example, correlation coefficients, co-expression network analysis, clique analysis, etc., and may be based on expression data obtained from RT-PCR, microarrays, sequencing, and other similar technologies. For example, gene expression clusters can be identified using pairwise correlation analysis based on Pearson's or Spearman's correlation coefficients. (See, for example, Pearson K. and Lee A,, Bioraetrika 2, 357 (1902); C. Spearman, Amer. J. Psychol 15:72-101 (1904); J. Myers, A. Well, Research Design and Statistical Analysis, p. 508 (2nd Ed., 2003)).

[0135] Furthermore, values ​​obtained from the PTSD molecular detection assays and / or methods described herein can be measured and / or compared using algorithms, models, and / or classifiers. Similarly, values ​​obtained from the PTSD molecular detection assays and / or methods described herein can be used to train algorithms, models, and / or classifiers for use in the PTSD molecular detection assays and / or methods described herein. Thus, in some embodiments, the PTSD molecular detection assays and methods described herein include algorithms, models, and / or classifiers. Without intending to be limiting, the classifiers described herein can be logistic regression classifiers, support vector machine classifiers, or other classifiers. The classifiers can be any one of a number of methods for classification or regression known in the art. The model can be an artificial neural network, a Bayesian graphical model, a Gaussian process, a logistic regression, a support vector machine, a decision tree, a hidden Markov model, or a k-nearest neighbor. K-fold cross-validation can also be used.

[0136] The ensemble of models can be used by boosting or other ensemble methods.The model can be any one of many methods for classification or regression known in the art.The model can be artificial neural network, Bayesian graphical model, Gaussian process, logistic regression, support vector machine, decision tree, hidden Markov model or k-nearest neighbor method.K-fold cross-validation can also be used.

[0137] In some embodiments, the accuracy rate, sensitivity, positive predictive value (PPV), negative predictive value (NPV), area under the curve (AUC), or any combination thereof, of the assays and methods described herein (e.g., assays or methods for detecting PTSD) may be greater than about 50%. In some embodiments, the accuracy rate of the assays and methods described herein is greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, greater than about 97%, greater than about 98%, or greater than about 99%. In some embodiments, the sensitivity of the assays and methods described herein is greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, greater than about 97%, greater than about 98%, or greater than about 99%. In some embodiments, the PPV of the assays and methods described herein is greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, greater than about 97%, greater than about 98%, or greater than about 99%. In some embodiments, the NPV of the assays and methods described herein is greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, greater than about 97%, greater than about 98%, or greater than about 99%. In some embodiments, the AUC of the assays and methods described herein is greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, greater than about 97%, greater than about 98%, or greater than about 99%. Methods for calculating accuracy, sensitivity, positive predictive value (PPV), negative predictive value (NPV), and area under the curve (AUC) are standard to those skilled in the art.

[0138] In some embodiments, determining the differential expression of one or more test PTSD-related genes can be used to predict the likelihood that PTSD will be identified and / or detected in a subject, to identify and / or detect PTSD in a subject, to predict the likelihood that a subject will be clinically diagnosed with PTSD, to diagnose a subject with PTSD, to predict the likelihood that a subject will respond to a PTSD treatment, to establish the efficacy of a PTSD treatment in a subject, or combinations thereof.

[0139] Therefore, the PTSD molecular detection assay and related information provided by implementing the method of the present disclosure facilitates diagnosis and treatment decisions in the diagnosis of PTSD, the selection of patients for PTSD treatment, and the monitoring of the effectiveness of PTSD treatment.For example, such molecular detection assays allow primary care providers to identify patients with a high probability of having PTSD, thereby facilitating the primary care providers to refer patients to specialists who can clinically diagnose PTSD in the patient.Similarly, such molecular detection assays will allow PTSD treatment specialists to identify patients with a high probability of responding to PTSD treatment.Furthermore, such molecular detection assays will allow PTSD treatment specialists to monitor patients undergoing PTSD treatment by evaluating the effectiveness of PTSD treatment.

[0140] Thus, in some embodiments, a method for detecting PTSD in a subject is provided herein. In some embodiments, the method for detecting PTSD in a subject includes obtaining a biological sample (e.g., a cell sample) from the subject, analyzing the sample using a PTSD molecular detection assay described herein to determine differential expression of one or more test PTSD-related gene expression levels, and determining the detection of PTSD based on the differential expression of the one or more test PTSD-related genes compared to one or more control gene expression levels.

[0141] In some embodiments, a method of detecting PTSD in a subject includes obtaining a biological sample (e.g., a cell sample) from the subject, measuring expression levels of one or more test PTSD-related genes in the sample, comparing the expression levels of the one or more test PTSD-related genes to one or more control gene expression levels, determining differential expression of the one or more test PTSD-related genes, and determining a positive detection of PTSD based on the differential expression.

[0142] In some embodiments, a method of detecting PTSD in a subject includes obtaining a biological sample (e.g., a cell sample) from the subject; measuring expression levels of one or more test PTSD-related genes in the sample using a first computer program executed on a computer; comparing the expression levels of the one or more test PTSD-related genes to one or more control gene expression levels using a second computer program executed on a computer; determining differential expression of the one or more test PTSD-related genes; and determining a positive detection of PTSD based on the differential expression.

[0143] In some embodiments, the one or more control gene expression levels are derived from a control or control population. In some embodiments, the control or control population is a negative control or negative control population. In some embodiments, the one or more control gene expression levels are derived from a database. In some embodiments, the first computer program and the second computer program are the same. In some embodiments, the first computer program and the second computer program are different.

[0144] In certain embodiments, detecting PTSD in a subject by the methods and assays described herein can diagnose or indicate the diagnosis of PTSD in the subject.In some embodiments, diagnosing PTSD by the methods, systems, assays, devices, and kits described herein can be a laboratory diagnosis of PTSD, which is separate from and / or auxiliary to the clinical diagnosis of PTSD.In some embodiments, diagnosing PTSD by the methods, systems, assays, devices, and kits described herein can replace the clinical diagnosis of PTSD.

[0145] In some embodiments, the detection methods described herein further comprise determining that the subject should be clinically diagnosed with PTSD. In some embodiments, the detection methods described herein further comprise clinically diagnosing the subject with PTSD. Clinically diagnosing the subject with PTSD comprises diagnosing PTSD through an informal or formal clinical interview by a trained behavioral health provider. Formal clinical interviews include published clinical diagnostic criteria for PTSD, including the Clinician-Administered PTSD Scale for DSM-5 (CAPS-5) and the PTSD Checklist for DSM-5 (PCL-5), both of which are described in more detail below.

[0146] In yet further embodiments, provided herein are methods for screening for PTSD in a subject. In some embodiments, the method for screening for PTSD in a subject comprises detecting PTSD in the subject as described herein, and determining, based on a positive detection of PTSD in the subject, that the subject should be evaluated for a clinical diagnosis of PTSD.

[0147] In some embodiments, a method of screening for PTSD includes obtaining a biological sample (e.g., a cell sample) from a subject; measuring expression levels of one or more test PTSD-related genes; comparing the expression levels of the one or more test PTSD-related genes to one or more control gene expression levels; determining differential expression of the one or more test PTSD-related genes; determining a positive detection of PTSD based on the differential expression; and determining that the subject should be clinically evaluated for PTSD based on a positive detection of PTSD in the subject.

[0148] In some embodiments, a method of screening for PTSD includes obtaining a biological sample (e.g., a cell sample) from a subject; measuring expression levels of one or more test PTSD-related genes using a first computer program executed on a computer; comparing the expression levels of the one or more test PTSD-related genes to one or more control gene expression levels using a second computer program executed on a computer; determining differential expression of the one or more test PTSD-related genes; determining a positive detection of PTSD based on the differential expression; and determining that the subject should be clinically evaluated for PTSD based on a positive detection of PTSD in the subject.

[0149] In some embodiments, the one or more control gene expression levels in the method for screening for PTSD are derived from a control or control population. In some embodiments, the control or control population is a negative control or negative control population. In some embodiments, the method for screening for PTSD further comprises clinically diagnosing the subject with PTSD using one or more informal or formal interviews. In some embodiments, the formal interview comprises the use of PTSD clinical diagnostic criteria. In some embodiments, the one or more PTSD clinical diagnostic criteria include CAPS-5 or PCL-5. In some embodiments, the PTSD clinical diagnostic criteria is CAPS-5. In some embodiments, the PTSD clinical diagnostic criteria is PCL-5. In some embodiments, the detection of PTSD as described herein and the clinical diagnosis of PTSD in the subject are performed by the same entity. In some embodiments, the detection of PTSD as described herein and the clinical diagnosis of PTSD in the subject are performed by two entities (e.g., a primary care provider and a specialist). In some embodiments, the first computer program and the second computer program are the same. In some embodiments, the first computer program and the second computer program are different.

[0150] In some embodiments, other clinical criteria can be used to assess comorbidity with PTSD (e.g., the Generalized Anxiety Disorder 7-Item Scale (GAD-7), the Alcohol Use Disorder Identification Test (AUDIT-C), the Pittsburgh Sleep Quality Index (PSQI), Adverse Childhood Experiences (ACE), or any combination thereof). Accordingly, in some embodiments, the methods described herein further include analyzing the subject for one or more comorbid conditions not consistent with PTSD, including anxiety disorders (e.g., generalized anxiety disorder), substance use disorders (e.g., alcohol use disorder), sleep disorders (e.g., anxiety-related insomnia), prior trauma (e.g., childhood trauma), and the like. In certain embodiments, methods for providing a differential PTSD diagnosis are provided herein. In some embodiments, detecting PTSD, clinically diagnosing PTSD in a subject, and assessing comorbid conditions and / or providing a differential diagnosis, or any combination thereof, are performed by the same entity or two or more entities.

[0151] In some embodiments, the method of screening for PTSD further comprises selecting a subject eligible for treatment for PTSD.

[0152] In yet further embodiments, provided herein are methods for selecting a subject eligible for treatment for PTSD. In some embodiments, the method for selecting a subject eligible for treatment for PTSD comprises detecting PTSD in a subject as described herein, and determining that the subject is eligible for PTSD treatment based on a positive detection of PTSD in the subject.

[0153] In some embodiments, a method of selecting a subject eligible for treatment for PTSD includes obtaining a biological sample (e.g., a cell sample) from the subject; measuring expression levels of one or more test PTSD-related genes; comparing the expression levels of the one or more test PTSD-related genes to one or more control gene expression levels; determining differential expression of the one or more test PTSD-related genes; determining a positive detection of PTSD based on the differential expression; and determining that the subject is eligible for PTSD treatment based on the positive detection of PTSD in the subject.

[0154] In some embodiments, a method for selecting a subject eligible for treatment for PTSD includes obtaining a biological sample (e.g., a cell sample) from the subject; measuring expression levels of one or more test PTSD-related genes using a first computer program executed on a computer; comparing the expression levels of the one or more test PTSD-related genes to one or more control gene expression levels using a second computer program executed on a computer; determining differential expression of the one or more test PTSD-related genes; determining a positive detection of PTSD based on the differential expression; and determining that the subject is eligible for PTSD treatment based on the positive detection of PTSD in the subject.

[0155] In some embodiments, one or more control gene expression levels are derived from a control or control population. In some embodiments, the control or control population is a negative control or a negative control population. In some embodiments, the first computer program and the second computer program are the same. In some embodiments, the first computer program and the second computer program are different.

[0156] In some embodiments, the method for selecting a subject eligible for PTSD treatment described herein further comprises determining that the subject should be clinically evaluated for PTSD. In some embodiments, the method for selecting a subject eligible for PTSD treatment described herein further comprises clinically diagnosing the subject for PTSD. In some embodiments, the step of determining that the subject should be clinically evaluated for PTSD and / or the step of clinically diagnosing the subject for PTSD can occur before or after the subject is determined to be eligible for PTSD treatment. For example, in some embodiments, the method for selecting a subject eligible for PTSD treatment comprises detecting PTSD in the subject as described herein (wherein, if PTSD is detected in the subject, the method comprises determining that the subject should be clinically evaluated for PTSD), and clinically diagnosing the subject for PTSD (wherein, if PTSD is clinically diagnosed, the method comprises selecting the subject for PTSD treatment).

[0157] In some embodiments, the methods described herein further comprise treating the subject with a PTSD treatment. Examples of PTSD treatments encompassed by the present disclosure include psychotherapy (e.g., cognitive behavioral therapy, cognitive processing therapy, cognitive therapy, prolonged exposure therapy, eye movement desensitization and reprocessing (EMDR) therapy, narrative exposure therapy (NET), group therapy, or a combination thereof), psychodynamic therapy (e.g., brief eclectic psychotherapy), medications or active agents (e.g., selective serotonin reuptake inhibitors (SSRIs), serotonin and norepinephrine reuptake inhibitors (SNRIs), anti-anxiety medications, antidepressants, or combinations thereof, including sertraline, paroxetine, fluoxetine, venlafaxine, ketamine, tricyclic antidepressants, MAOIs, antipsychotics, beta-blockers, benzodiazepines, hallucinogens), comorbidity treatments (e.g., substance abuse treatments), complementary and / or alternative therapies (e.g., acupuncture, yoga, animal-assisted therapy, meditation, or a combination thereof), and any combination thereof. Further described herein are methods of treatment, eg, using one or more medicaments formulated as pharmaceutical compositions.

[0158] In some embodiments, provided herein are methods for assessing the effectiveness of a treatment for PTSD in a subject receiving such treatment. In some embodiments, the methods for assessing the effectiveness of a treatment for PTSD in a subject include detecting PTSD in the subject as described herein, and determining the effectiveness of the PTSD treatment based on the detection of PTSD.

[0159] In some embodiments, a method for assessing the effectiveness of a treatment in a subject receiving a PTSD treatment includes obtaining a biological sample (e.g., a cell sample) from the subject receiving a PTSD treatment, measuring expression levels of one or more test PTSD-related genes, comparing the expression levels of the one or more test PTSD-related genes to one or more control gene expression levels, determining differential expression of the one or more test PTSD-related genes, determining a positive detection of PTSD based on the differential expression, and determining the effectiveness of the PTSD treatment based on the detection of PTSD.

[0160] In some embodiments, a method for assessing the effectiveness of a treatment in a subject receiving a PTSD treatment includes obtaining a biological sample (e.g., a cell sample) from the subject receiving the PTSD treatment; measuring expression levels of one or more test PTSD-related genes using a first computer program executed on a computer; comparing the expression levels of the one or more test PTSD-related genes to one or more control gene expression levels using a second computer program executed on a computer; determining differential expression of the one or more test PTSD-related genes; determining a positive detection of PTSD based on the differential expression; and determining the effectiveness of the PTSD treatment based on the detection of PTSD.

[0161] In some embodiments, the PTSD treatment is effective based on a negative detection of PTSD. In some embodiments, the PTSD treatment is not effective (i.e., at that time) based on a positive detection of PTSD. In some embodiments, the one or more control gene expression levels are one or more control gene expression levels from a control or control population. In some embodiments, the control or control population is a negative control or negative control population. In some embodiments, the one or more control gene expression levels are from a baseline. In some embodiments, the baseline is a subject prior to PTSD treatment. In some embodiments, the first computer program and the second computer program are the same. In some embodiments, the first computer program and the second computer program are different.

[0162] In some embodiments, the detection of PTSD in subject can be compared before and after receiving PTSD treatment.Therefore, the detection of PTSD in subject can be compared with the baseline detection of PTSD.In some embodiments, the effectiveness of PTSD treatment can be determined by comparing the detection in subject before and after treatment.

[0163] In some embodiments, the PTSD treatment may be a first PTSD treatment, or may be a PTSD treatment instance that is part of a series or ongoing PTSD treatment. In some embodiments, the method for assessing the treatment effectiveness of a subject receiving PTSD treatment may be repeated once, more than once, twice, or as many times as necessary for the treatment provider to monitor the effectiveness of the PTSD treatment after each PTSD treatment instance or several PTSD treatment instances. In some embodiments, the methods described herein may be used to provide data to an individual, a healthcare provider, or both, and the data may be used to evaluate the effectiveness of the PTSD treatment over time or at a specific time point.

[0164] In some embodiments, provided herein are methods for determining the prevalence of PTSD in a population of interest. In some embodiments, the method for determining the prevalence of PTSD in a population of interest comprises detecting PTSD in each subject of the population of interest and determining the prevalence of PTSD in the population of interest.

[0165] In some embodiments, a method for determining the prevalence of PTSD in a population of interest includes obtaining a biological sample (e.g., a cell sample) from each subject in the population of interest, measuring expression levels of one or more test PTSD-related genes in each sample, comparing the expression levels of the one or more test PTSD-related genes in each sample to one or more control gene expression levels, determining differential expression of the one or more test PTSD-related genes, determining detection of PTSD in each subject in the population of interest based on the differential expression, and determining the prevalence of PTSD in the population of interest based on the detection of PTSD in each subject in the population of interest.

[0166] In some embodiments, a method for determining the prevalence of PTSD in a population of interest includes obtaining a biological sample (e.g., a cell sample) from each subject in the population of interest; measuring expression levels of one or more test PTSD-related genes in each sample using a first computer program implemented on a computer; comparing the expression levels of the one or more test PTSD-related genes in each sample to one or more control gene expression levels using a second computer program implemented on a computer; determining differential expression of the one or more test PTSD-related genes; determining detection of PTSD in each subject in the population of interest based on the differential expression; and determining the prevalence of PTSD in the population of interest based on the detection of PTSD in each subject in the population of interest.

[0167] In some embodiments, determining the prevalence of PTSD in the population of interest comprises assessing the number of subjects in which PTSD is detected. In some embodiments, the first computer program and the second computer program are the same. In some embodiments, the first computer program and the second computer program are different.

[0168] In some embodiments, the one or more control gene expression levels are derived from a control or control population. In some embodiments, the control or control population is a negative control or negative control population. In some embodiments, the population of interest is a military population or a female population.

[0169] subject In some embodiments, the subject may include a mammal suitable for testing for PTSD as described herein. Examples of such mammals include humans and non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.). The mammal may be of any age or developmental stage, e.g., the mammal may be neonatal, juvenile, adolescent, adult, or fetal. The mammal may be male or female. In some cases, the human may be about 1 day to about 7 days old, about 1 week to about 5 weeks old, about 1 month to about 12 months old, about 1 year to about 6 years old, about 5 years to about 15 years old, about 14 years to about 30 years old, about 25 years to about 50 years old, about 40 years to about 75 years old, about 70 years to about 100 years old, about 85 years to about 110 years old, or about 100 years to about 130 years old.

[0170] The subject may be a patient, for example, a patient who is undergoing treatment for trauma that can cause the symptoms of PTSD.In some cases, the subject may be at risk of developing a condition or disease such as PTSD.The subject may be in remission from PTSD.The subject may be healthy.

[0171] In some aspects, the method may further include diagnosing the subject as having the disease. In some aspects, the diagnosing step may include utilizing in vitro diagnosis. In some aspects, the in vitro diagnosis may be a companion diagnostic. In other cases, the diagnosing step may include in vivo diagnosis.

[0172] Diagnostic tests may include imaging procedures, blood count analysis, histopathology analysis, biomarker analysis, biopsy, magnetic resonance imaging procedure, physical examination, urinalysis, ultrasound procedure, genetic testing, liver function tests, positron emission tomography procedure, x-ray, serology, angiography procedure, electrocardiogram procedure, endoscopy, diagnostic polymerase chain reaction test (PCR), pap smear, hematocrit test, skin allergy test, urinalysis, colonoscopy, enzyme-linked immunosorbent assay (ELISA), microscopic analysis, bone marrow test, rapid diagnostic test, pregnancy test, organ function test, toxicology test, infectious disease test, body fluid test, or any combination thereof.

[0173] In some embodiments, the diagnosis may include a physical examination, radiological imaging, blood tests, antibody tests, or any combination thereof. In some embodiments, the diagnosis may include radiological imaging, which may include computed tomography (CT) imaging, X-ray imaging, magnetic resonance imaging (MRI), ultrasound imaging, or any combination thereof.

[0174] Administration and Treatment In some aspects, disclosed herein are methods of administering a treatment (e.g., a pharmaceutical composition) described herein to a subject, where the subject may be in need thereof. In some cases, a method of treating or preventing a disease may include administering a medicament described herein. In some embodiments, provided herein are pharmaceutical compositions comprising a medicament (e.g., a PTSD treatment) and a pharmaceutically acceptable carrier, excipient, and / or diluent for treating a subject in need thereof.

[0175] In some embodiments, the subject may be a subject in need thereof. In some embodiments, the subject may have a disease, for example, post-traumatic stress disorder. Treatment may include reducing fear, reducing helplessness, reducing disorganized behavior, reducing anxiety, reducing depression, reducing psychosis, reducing panic attacks, reducing substance abuse, reducing suicidal thoughts, reducing insomnia, and increasing appetite.

[0176] Delivery of medications for the treatment of PTSD may include direct application to the affected tissue or area of ​​the body. Delivery may include parenchymal injection, intrathecal injection, intraventricular injection, or intracisternal injection. The compositions provided herein can be administered by any method. The method of administration may be by inhalation, intraarterial injection, intraventricular injection, intracisternal injection, intramuscular injection, intraorbital injection, intraparenchymal injection, intraperitoneal injection, intraspinal injection, intrathecal injection, intravenous injection, intraventricular injection, stereotactic injection, subcutaneous injection, or any combination thereof. Delivery may include parenteral administration (including intravenous, subcutaneous, intrathecal, intraperitoneal, intramuscular, intravascular, or infusion), oral administration, inhalation administration, intraduodenal administration, or rectal administration. Delivery may include topical administration (e.g., lotion, cream, ointment) to a surface, such as the external surface of the skin. In some cases, the subject may administer the composition without supervision. In some cases, the subject may administer the composition under the supervision of a medical professional (e.g., a doctor, nurse, physician's assistant, staff member, hospice worker, etc.). In some cases, the medical professional may administer the composition.

[0177] The step of selecting a treatment may include the additional step of applying and monitoring the treatment, and the treatment may be selected based on the results. For example, a subject diagnosed as being at risk of developing PTSD and receiving preventive treatment may be monitored, and if necessary, their treatment may be subsequently adjusted or changed depending on whether and to what extent PTSD symptoms occur. A treatment may also be applied to a patient with acute PTSD, and the effect of the treatment may be routinely monitored and recorded. The recorded data provides a basis for evaluating whether the applied treatment is beneficial to the patient. Based on the evaluation, a person skilled in the art (likely a clinician in this case) may adjust the currently applied treatment, for example, by increasing / decreasing the dosage regimen or dosage of the treatment, or may decide to completely switch to another treatment.

[0178] In some embodiments, the pharmaceutical composition can be administered to a subject in a suitable unit dose. The pharmaceutical composition may be in unit dose form. In some cases, a unit dose may refer to a pharmaceutical product in a form sold for use, in which a specific mixture of active ingredients and inactive ingredients, diluents, or excipients is in a specific composition and allocated to a specific dose to be delivered. In some cases, a unit dose may also include non-reusable packaging, although the FDA distinguishes between unit dose "packaging" or "dispensing." More than one unit dose may refer to different pharmaceutical products packaged together, or to a single pharmaceutical product containing multiple drugs and / or multiple doses. In some cases, the term unit dose may also refer to particles containing a pharmaceutical composition, and any mixtures involved. In some cases, the type of unit dose may vary depending on the route of administration of the drug delivery and the substance being delivered. In some embodiments, administration may include intravenous, intraperitoneal, intraarterial, intratumoral, subcutaneous, intramuscular, intranasal, topical, oral, or intradermal administration. In some cases, administration may include inhalation administration. In some embodiments, the dosing regimen may be determined by the attending physician and clinical factors. In some embodiments, the dosage for a subject may depend on numerous factors, including the subject's size, body surface area, age, sex, general health, the compound to be administered, the time and route of administration, other drugs administered concomitantly, or any combination thereof. In some embodiments, the dose range may include 0.001 to 1000 μg. In some embodiments, the dose may be below or above such ranges. In some embodiments, the typical dosing regimen for the pharmaceutical composition may be within the range of 1 μg to 10 mg. In some embodiments, the typical dosing regimen for the pharmaceutical composition may be 10 μg to 10 mg per day, week, or month. 2 Units ~ 10 10In some embodiments, if the regimen involves continuous infusion, it can also be in the range of 1 μg to 10,000 mg per kilogram of body weight per minute of the pharmaceutical composition, or the engineered polynucleotide, or the DNA encoding the engineered polynucleotide, or the vector comprising or encoding the engineered polynucleotide, respectively. In certain cases, the range is 1 mg per kilogram of body weight to 1000 mg per kilogram of body weight. In some embodiments, progress can be monitored by periodic assessment.

[0179] In some embodiments of the present disclosure, when the pharmaceutical composition is a liquid, it may be administered in a liquid dosage form such as about 1 ml to about 5 ml, about 5 ml to 10 ml, about 15 ml to about 20 ml, about 25 ml to about 30 ml, about 30 ml to about 50 ml, about 50 ml to about 100 ml, about 100 ml to 150 ml, about 150 ml to about 200 ml, about 200 ml to about 250 ml, about 250 ml to about 300 ml, about 300 ml to about 350 ml, about 350 ml to about 400 ml, about 400 ml to about 450 ml, about 450 ml to 500 ml, about 500 ml to 750 ml, or about 750 ml to 1000 ml.

[0180] In some embodiments, the compositions described herein can be administered to the subject in need thereof for one or more days.In some embodiments, administration can be carried out for approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days or about 31 days. In some embodiments, administration may occur for approximately 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or about 24 months. In some embodiments, administration can occur for approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 years or more. In some cases, administration can occur for life. In some embodiments, the pharmaceutical compositions described herein can be administered for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or more. In some cases, the compositions described herein can be administered on consecutive days or non-consecutive days. In some cases, the compositions described herein can be administered to a subject more than once per day. In some cases, the compositions described herein can be administered to a subject 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times per day.

[0181] In some embodiments, methods of using the compositions disclosed herein are disclosed herein. In some embodiments, the daily oral dosage regimen can be about 0.1 milligrams per kilogram of total body weight (mg / kg) to about 80 mg / kg, about 0.2 mg / kg to about 30 mg / kg, or about 0.5 mg / kg to about 15 mg / kg. In some embodiments, the daily parenteral dosage regimen can include about 0.1 mg to about 10,000 mg / kg, about 0.2 mg / kg to about 5,000 mg / kg, or about 0.5 mg / kg to about 1,000 mg / kg per kilogram of total body weight. In some embodiments, the daily topical dosage regimen can be about 0.1 mg to about 500 mg. In some embodiments, the daily inhalation dosage regimen can be about 0.01 mg / kg to about 1,000 mg / kg per day. In some embodiments, the optimal amount and interval of individual doses of the composition can be determined by the nature and extent of the condition being treated, the form of administration, the route of administration, and the site of administration, as well as the specific subject being treated; such optimal conditions can preferably be determined by the methods described herein. In some embodiments, the number of doses of the composition to be provided per day for a given number of days can be ascertained by those skilled in the art using conventional treatment-determining testing procedures. In some embodiments, the dosing regimen can be determined by the attending physician and other clinical factors. In some embodiments, the dosage for any one subject can depend on numerous factors. In some embodiments, factors affecting dosage can include the subject's size, body surface area, age, the specific compound to be administered, sex, time and route of administration, general health, other drugs administered simultaneously, or any combination thereof. In some embodiments, progress can be monitored by periodic assessment.

[0182] The pharmaceutical formulation may be administered in a daily oral dosage regimen that can be about 0.1 milligrams per kilogram (mg / kg) of total body weight to about 80 mg / kg, about 0.2 mg / kg to about 30 mg / kg, or about 0.5 mg / kg to about 15 mg / kg. In some embodiments, the daily parenteral dosage regimen can include about 0.1 mg to about 10,000 mg / kg of total body weight, about 0.2 mg / kg to about 5,000 mg / kg, or about 0.5 mg / kg to about 1,000 mg / kg. In some embodiments, the daily topical dosage regimen can be about 0.1 mg to about 500 mg. In some embodiments, the daily inhalation dosage regimen can be about 0.01 mg / kg to about 1,000 mg / kg per day. In some embodiments, the optimal amount and interval of individual doses of the composition can be determined by the nature and extent of the condition being treated, the form of administration, the route of administration, and the site of administration, as well as the specific subject being treated; such optimal conditions can preferably be determined by the methods described herein. In some embodiments, the number of doses of the composition to be provided per day for a given number of days can be ascertained by those skilled in the art using conventional treatment-determining testing procedures. In some embodiments, the dosing regimen can be determined by the attending physician and other clinical factors. In some embodiments, the dosage for any one subject can depend on numerous factors. In some embodiments, factors affecting dosage can include the subject's size, body surface area, age, the specific compound to be administered, sex, time and route of administration, general health, other drugs administered simultaneously, or any combination thereof. In some embodiments, progress can be monitored by periodic assessment.

[0183] system Systems including the PTSD molecular detection assays described herein or uses thereof are provided herein. The systems provided herein further include materials for use in the disclosed molecular detection assays, methods, compositions, kits, devices, or any combination thereof. For example, the systems provided herein include agents, which may include gene-specific or gene-selective probes and / or primers for quantifying the expression of the disclosed genes to predict prognosis or response to treatment. Such systems may optionally include reagents for RNA extraction from samples (e.g., fixed, paraffin-embedded tissue samples) and / or reagents for RNA amplification. Additionally, the systems may optionally include reagents with identifying descriptions, labels, or instructions related to their use in the disclosed methods. In some embodiments, the systems include a collection container for obtaining a biological sample. Such a collection container may be any sample collection device capable of collecting a suitable amount of human blood, tissue, or saliva, including specific cell types (e.g., peripheral blood mononuclear cells (PBMCs), epithelial cells, or other human cell types) containing nucleic acids from which a whole genome ribonucleic acid sample can be isolated for testing.Examples of specimen collection devices include PAXgene® Blood RNA Tubes (Becton-Dickinson Biosciences Catalog Number: 762165), OMNIgene™ Saliva DNA and RNA Device OMR-610 (DNA Genotek Catalog Number: OMR-610), Oragene Discover OCR-100 Saliva Collection Device (DNA Genotek Catalog Number: OCR-100), DNA / RNA Shield SafeCollect Saliva Collection Kit (Zymo Research Catalog Number: R1211), DNA / RNA Shield Blood Collection Tubes (Zymo Research Catalog Number: R1150), SpeciMAX Saliva Collection Kit (ThermoFisher Catalog Number: A50696), Tempus™ Blood RNA Tubes (ThermoFisher Catalog Number: 4342792), Buccal Swab Collection and Stabilization Kit (CanVax Catalog Number: SWC001), TAP® Micro Select (YourBio Health - FDA 510k approved), or a combination thereof.

[0184] The system may include containers (including microliter plates suitable for use in automated implementation of the method), each of which contains one or more of the various materials or reagents (typically in concentrated form) utilized in the method, including, for example, chromatography columns, pre-fabricated microarrays, buffers, appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP, and dTTP; or rATP, rCTP, rGTP, and UTP), reverse transcriptase, DNA polymerase, RNA polymerase, and one or more probes and primers of the present disclosure (e.g., poly(T) or random primers of appropriate length linked to a promoter that reacts with RNA polymerase). Mathematical algorithms used to infer or quantify prognostic or predictive information are also suitable potential components of the systems provided herein.

[0185] The values ​​obtained from the above-mentioned assays, for example, expression data, can be calculated and stored manually. Aspects of the present disclosure also provide a system for fully or partially implementing the methods of the present disclosure. The present disclosure therefore provides a computer program product, including a computer-readable storage medium on which a computer program is stored. When the program is read by a computer, it can perform related calculations based on the values ​​obtained from the analysis of one or more biological samples from an individual (e.g., gene expression levels, normalization, standardization, threshold determination, and conversion of the values ​​obtained from the assay into scores and / or textual or graphical representations of PTSD progression and related information). The computer program product has a computer program for performing calculations stored therein.

[0186] The system may include, for example, i) a computer and ii) a non-transitory storage medium containing computer-executable instructions that, when executed by the computer, cause the computer to perform any one or more steps of the methods described herein. The system may include, for example, i) a computer and ii) a non-transitory storage medium containing computer-executable instructions that, when executed by the computer, cause the computer to perform the steps of: a) receiving at least one test gene expression sequence (e.g., RNA expression sequence) from a tissue sample, and b) determining the gene expression level of the at least one sequence. In some embodiments, the non-transitory storage medium includes computer-executable instructions that, when executed by the computer, cause the computer to perform the steps of: c) obtaining a control gene expression level (e.g., from a control) of the at least one sequence or an average control gene expression level (e.g., from a control population) of the at least one sequence, and d) detecting the relative expression of the at least one test gene expression sequence relative to the control gene expression level.

[0187] The system also generally includes an output device capable of displaying the results obtained by the computer during or as a result of (e.g., at) the expression step. The non-transitory storage medium may be located on the computer's hard drive or on a portable device, such as a disk, CD, DVD, thumb drive, flash drive, laptop, portable computer (e.g., PC or other type), or other such device. Alternatively, the non-transitory storage medium may be located at a location (e.g., a remote location or database accessible via the Internet), or may be stored in the cloud or on or in another computer or computer system accessible by the system's computer. The non-transitory storage medium may also include instructions for causing the computer to receive at least one genomic DNA sequence as input from a nucleotide sequencing device or database. The database may be downloaded from a remote location (e.g., via the Internet) and / or may be located (stored) on the computer, or on another computer or computer system accessible by the system's computer, or even on the portable device described above. In other aspects, the data is downloaded from a genetic sequencing device, and the system may also include such a device. If present, the device is operably electronically linked to the computer in such a manner that data collected or measured by the expression level testing device can be output and transmitted by, and received as input by, the computer.

[0188] The output device may be of any suitable type, including, but not limited to, a printer, a display (e.g., a monitor that displays the results as a list, as a graph, or in some other suitable format), or a modem that transmits the information (e.g., to another output device, another computer, or a storage device, e.g., DVD, CD, etc.).

[0189] Such a system may include a computer having a non-transitory storage medium. The computer may be operably linked to (or may be connected to, functionally connected to, or in electrical communication with) an output device. In some cases, the computer may also be operably linked to a nucleic acid sequencing device, and data from the nucleic acid sequencing device (e.g., a genomic nucleotide sequence, generally a DNA sequence) may be output and transmitted to the computer for analysis by the methods of the present disclosure, and received as input by the computer. In other cases, the computer may be operably linked to a database, and information and / or data may be output from the database and transmitted to the computer for input by the computer. The non-transitory storage medium may include computer-executable instructions (e.g., code, computer program, etc.) that are executed by the computer and cause the computer to perform the steps of the methods described herein.

[0190] device In some embodiments, a PTSD detection device or use thereof is provided herein. In some embodiments, the PTSD detection device includes a PTSD molecular detection assay described herein. In some embodiments, the PTSD detection device may include any one or more of the components or materials of the systems, compositions, devices, and / or methods described herein (e.g., the molecular detection assays provided herein). In some embodiments, the PTSD detection device includes one or more assays for quantifying gene expression levels as described herein. In some embodiments, the PTSD detection device includes a collection container for collecting a biological sample from a subject. In some embodiments, the device includes a collection container for obtaining a biological sample, wherein the container includes one or more fixation or preservation reagents. The collection container may be any sample collection device capable of collecting a suitable amount of human blood, tissue, or saliva, including specific cell types (e.g., peripheral blood mononuclear cells (PBMCs), epithelial cells, or other human cell types) that contain nucleic acids from which a whole genome ribonucleic acid sample can be isolated for testing.

[0191] Examples of specimen collection containers include PAXgene® Blood RNA Tubes (Becton-Dickinson Biosciences Catalog Number: 762165), OMNIgene™ Saliva DNA and RNA Device OMR-610 (DNA Genotek Catalog Number: OMR-610), Oragene Discover OCR-100 Saliva Collection Device (DNA Genotek Catalog Number: OCR-100), DNA / RNA Shield SafeCollect Saliva Collection Kit (Zymo Research Catalog Number: R1211), DNA / RNA Shield Blood Collection Tubes (Zymo Research Catalog Number: R1150), SpeciMAX Saliva Collection Kit (ThermoFisher Catalog Number: A50696), Tempus™ Blood RNA Tubes (ThermoFisher Catalog Number: 4342792), Buccal Swab Collection and Stabilization Kit (CanVax Catalog Number: SWC001), TAP® Micro Select (YourBio Health - FDA 510k approved), or a combination thereof.

[0192] In some embodiments, a result report is generated after quantifying gene expression. In some embodiments, the method of the present disclosure may generate a report or summary of the information obtained from the method described herein. For example, the report may include information about the expression level of one or more genes, detection of PTSD or the patient's risk of relapse, the patient's possible prognosis or risk classification, clinical and pathological factors, and / or other information. The method and report of the present disclosure may further include storing the report in a database. The method may create a record for the subject in the database and enter the data into the record. The report may be a written report, an audio report, or an electronic report. The report may be displayed and / or stored on a computing device (e.g., a handheld device, a desktop computer, a smart device, a website, etc.). It is contemplated that the report will be provided to the physician and / or the patient. Receiving the report may further include establishing a network connection to a server computer containing the data and report and requesting the data and report from the server computer.

[0193] In some embodiments, after quantifying gene expression, a report of results is generated indicating whether the subject screens positive or negative for PTSD. A positive result for detecting PTSD warrants referral to a specialized medical provider for further evaluation and diagnosis. kit

[0194] The present disclosure further provides a kit for testing for PTSD in a subject. The kit provided herein may include any one or more of the components or materials of the systems, compositions, devices, and / or methods (e.g., molecular detection assays) described herein, as well as instructions for use. In some embodiments, the kit includes one or more collection containers. In some embodiments, the kit includes a collection container for obtaining a biological sample, wherein the container contains one or more fixation or preservation reagents. The collection container may be any sample collection device capable of collecting a suitable amount of human blood, tissue, or saliva, including specific cell types (e.g., peripheral blood mononuclear cells (PBMCs), epithelial cells, or other human cell types) containing nucleic acids from which a whole genome ribonucleic acid sample can be isolated for testing.

[0195] Examples of specimen collection containers include PAXgene® Blood RNA Tubes (Becton-Dickinson Biosciences Catalog Number: 762165), OMNIgene™ Saliva DNA and RNA Device OMR-610 (DNA Genotek Catalog Number: OMR-610), Oragene Discover OCR-100 Saliva Collection Device (DNA Genotek Catalog Number: OCR-100), DNA / RNA Shield SafeCollect Saliva Collection Kit (Zymo Research Catalog Number: R1211), DNA / RNA Shield Blood Collection Tubes (Zymo Research Catalog Number: R1150), SpeciMAX Saliva Collection Kit (ThermoFisher Catalog Number: A50696), Tempus™ Blood RNA Tubes (ThermoFisher Catalog Number: 4342792), Buccal Swab Collection and Stabilization Kit (CanVax Catalog Number: SWC001), TAP® Micro Select (YourBio Health - FDA 510k approved), or a combination thereof.

[0196] In some embodiments, the kits provided herein include a sample collection container, instructions, and / or materials for mailing the container to a processing facility for processing by the methods, devices, and / or assays described herein. The processing facility may be any suitable laboratory or laboratories capable of performing the methods described herein or utilizing the molecular detection assays or devices described herein.

[0197] In some embodiments, the kits provided herein include a sample collection container and instructions for processing the sample at a point-of-care facility. The point-of-care facility may be a patient care facility, such as a doctor's office, clinic, or hospital, or it may be any location where a subject receives medical care, such as at home. In some embodiments, the kits provided herein include all assay components necessary to perform the methods described herein at home. Suitable point-of-care testing systems may be used in the kits described herein, and may include, for example, membrane-based test strips.

[0198] In some embodiments, the kits provided herein may include a kit housing containing assay components operable to detect differential expression levels of RNA or protein from a tissue sample obtained from a subject, instructions describing how to conduct the assay using the assay components, and a control pattern of RNA or protein expression levels for comparison with the differential expression levels of RNA or protein.

[0199] Various kit packages are contemplated, and are sufficient to contain assay components, instructions, and control expression levels of subjects without PTSD. Such kits can include a collection of elements required to carry out such methods and tests for assaying the expression levels of genes associated with PTSD or proteins corresponding to these genes to identify PTSD, and any useful instructions. In some embodiments, the kit package can include a container for storing and / or transporting tissue samples.

[0200] Additionally, in some embodiments, the kit may include genomic arrays, PCR materials, etc. Furthermore, the assay components included in the kit can be used to perform any assay capable of differential RNA expression of genes associated with PTSD. Non-limiting examples can include real-time PCR, Northern blotting, nucleic acid microarrays, Western blotting, immunoassays, quantitative PCR, etc. (including combinations of techniques), as well as tools for data analysis and / or data interpretation (e.g., computational tools). The kit may also include a sample collection device capable of collecting a suitable amount of human blood, tissue, or saliva containing a specific cell type (e.g., peripheral blood mononuclear cells (PBMCs), epithelial cells, or other human cell types) containing nucleic acids from which a whole genome ribonucleic acid sample can be isolated for testing. Examples of specimen collection devices include PAXgene® Blood RNA Tubes (Becton-Dickinson Biosciences Catalog Number: 762165), OMNIgene™ Saliva DNA and RNA Device OMR-610 (DNA Genotek Catalog Number: OMR-610), Oragene Discover OCR-100 Saliva Collection Device (DNA Genotek Catalog Number: OCR-100), DNA / RNA Shield SafeCollect Saliva Collection Kit (Zymo Research Catalog Number: R1211), DNA / RNA Shield Blood Collection Tubes (Zymo Research Catalog Number: R1150), SpeciMAX Saliva Collection Kit (ThermoFisher Catalog Number: A50696), Tempus™ Blood RNA Tubes (ThermoFisher Catalog Number: 4342792), Buccal Swab Collection and Stabilization Kit (CanVax Catalog Number: SWC001), TAP® Micro Select (YourBio Health - FDA 510k approved). [Example]

[0201] Example 1: Prediction of altered RNA expression levels to identify PTSD Blood samples were collected from patients with PTSD and compared with a control group. Approximately 2.5 ml of blood was collected from each subject. 1003 unique genes were measured at each time to determine abnormal expression levels.

[0202] First, RNA from the subject's blood was subjected to reverse transcription PCR to generate a series of amplicons, i.e., sequences with primer sequences at their 3' and 5' ends. The amplicons were then partially digested and ligated to adapters for quantification. A flowchart is shown in Figure 1. The sequences were run on an Illumina sequencing instrument. After sequencing, raw data were collected to generate FASTQ files. The FASTQ files were compared with a bioinformatics system to generate normalized differential gene expression data.

[0203] Artificial intelligence and machine learning were used for strategic gene selection as shown in Figure 2.

[0204] A minimum of 5 million reads were run per sample, and a percentage of aligned reads representing at least 75% were accurately mapped back to the human genome.

[0205] The 20 best-fit genes after expression data corresponding to subjects with PTSD are shown in Figure 3. All genes determined to be statistically significant were within the 2.5 x 10 -6 or had a lower P value.

[0206] Example 2: Development of an objective molecular detection assay to screen for PTSD This example describes the development of an objective molecular detection assay for screening for PTSD. Blood samples were collected from 87 subjects with PTSD and 141 control subjects (a total of 228 subjects), the demographics of which can be seen in Figure 4. Approximately 2-3 ml of blood was collected from each subject. The RNA expression levels of 1003 gene targets and their sequencing were performed using the ADAPT Panel™. Expression data were analyzed using normal quantile transformation (NQT) as described herein (see the Differential Expression section, see Equations 1-7). Specifically, gene expression levels were normalized, and the following genes were found to be statistically significantly correlated with PTSD (Table 2). [Table 2]

[0207] Analysis of the molecular detection assay indicates that the sensitivity of the molecular detection assay, or the percentage of subjects correctly identified as having the disorder, is 90% (Figure 5). Test sensitivity is the most important attribute when using a test for screening purposes, and researchers in the field generally consider a sensitivity of greater than 80% to be adequate. Currently used surveys, such as those described herein, have sensitivities ranging from 77 to 91% in research settings where subjects participate voluntarily; however, in practice, this can be biased in either direction depending on the motivation or fear of the responder. The analysis also indicates the specificity of the molecular detection assay, or the percentage of subjects correctly identified as not having the disorder, as 73% (Figure 5). The specificity of the molecular detection assay was found to be better than that of most screening tests currently in use.

[0208] Previous molecular detection assays using the same genes but at different thresholds have shown promising results, achieving an area under the receiver operating characteristic curve (AUC) of 85% (Figure 6).

[0209] The results demonstrate that a consistent, reliable, objective molecular detection assay has been generated that indicates whether a subject is predicted to have PTSD without being hindered by self-report reporter bias.

[0210] Example 3: Validation and Calibration of Molecular Detection Assays Validating the performance of molecular detection assays in larger, independent datasets is necessary to provide further evidence of their utility. This will require larger biorepositories with diverse populations to further train and test molecular detection assays to enhance their reliability and generalizability.

[0211] Example 3.1. Validation against industry-standard psychological assessments This example describes an effort to validate the molecular detection assay by comparing it with clinically accepted psychological assessments related to PTSD. The primary assessment for this example is the Clinician-Administered PTSD Scale for DSM-5 (CAPS-5). The CAPS-5 is considered the "gold standard" for diagnosing PTSD and is a structured interview. Another primary assessment is the Post-Traumatic Stress Disorder Checklist for DSM-5 (PCL-5). Other psychological assessments include the Patient Health Questionnaire-9 (PHQ-9), the Alcohol Use Identification Test (AUDIT-C), the Generalized Anxiety Disorder-7 (GAD-7), and the Pittsburgh Sleep Quality Index (PSQI).

[0212] Example 3.1.1. Study. 500 subjects diagnosed with PTSD and 500 control subjects are recruited. Subjects diagnosed with PTSD are individuals with a CAPS-5 score that meets the diagnostic criteria for PTSD, and control subjects are individuals without a CAPS-5 score that meets the diagnostic criteria for PTSD. Each subject undergoes PTSD screening using the RNA screening assay, which is analyzed by the molecular detection assay and by psychometric analysis using an accepted psychological assessment. For RNA assessment, approximately 2.5 mL of blood is drawn, and RNA expression levels of genes, including those listed in Table 2, are assessed. For psychometric analysis, subjects are analyzed using standard protocols for the corresponding psychological assessment. A schematic diagram of the above workflow can be seen in Figure 7.

[0213] Example 3.1.2. Data Reprocessing. The ADAPT panel is run on an NGS platform to obtain a FASQfile output. Expression data is analyzed by using NQT as described herein. Specifically, the following steps are included in data preprocessing: ·Quality control, Data cleaning, ·Normalization, Feature selection, · Genomic and clinical data integration, Batch effect removal, Dimensionality reduction, Data scaling, Cross-validation, and · Validation and reproducibility.

[0214] As a first step in preprocessing, quality control (QC) checks are performed on the collected genomic and clinical data to ensure the reliability and integrity of the data by identifying and addressing issues (e.g., missing values, outliers, batch effects, or any other anomalies).

[0215] Any data errors, missing values, or outliers identified during the QC process will be addressed through data cleaning procedures. Imputation methods will be used to fill in missing values ​​or, where applicable, to exclude data points with severe issues from further analysis.

[0216] After data cleaning, data normalization is performed to remove technical variability while preserving biological significance. In this study, normalized quantile transformation (NQT) is selected as the normalization method. NQT is a rank-based procedure that scales data within each quantile separately and is known for its reduced bias and robustness compared to other normalization methods for microRNA sequencing data.

[0217] Feature selection is essential to reduce the dimensionality of predictive models and select the most relevant genomic features (e.g., genes or microRNA species). We can use a variety of methods, including statistical tests, correlation analyses, and machine learning algorithms, to identify the most informative features associated with PTSD.

[0218] This study includes both genomic and clinical data. Therefore, the two data types are integrated for comprehensive analysis. This step involves mapping clinical variables to corresponding genomic samples to ensure data consistency.

[0219] To account for systematic variability between batches, batch effect removal techniques are applied, ensuring that observed effects are not confounded by batch-specific variation.

[0220] When dealing with a large number of features, dimensionality reduction techniques (e.g., principal component analysis (PCA) or t-SNE)) can be applied to reduce the complexity of the data while preserving important patterns and structure.

[0221] For some machine learning algorithms, data may need to be scaled to ensure that all features have similar ranges. Common scaling methods include standardization (mean centering and scaling by standard deviation) or min-max scaling (scaling to a predetermined range).

[0222] To accurately assess the performance of a predictive model, cross-validation techniques (e.g., k-fold cross-validation) can be used to divide the data into subsets for iteratively training and testing the model.

[0223] After preprocessing, model performance should be validated using an independent dataset to ensure the generalizability and reproducibility of results. We use NQT as described in the normal quantile transformation (Cer RZ, Herrera-Galeano JE, Anderson JJ, Bishop-Lilly KA, Mokashi VP. miRNA Temporal Analyzer (mirnaTA): a bioinformatics tool for identifying differentially expressed microRNAs in temporal studies using normal quantile transformation. Gigascience. 2014 Oct 13;3:20. doi: 10.1186 / 2047-217X-3-20. PMID: 25379175; PMCID: PMC4212236). Custom pipelines will be written in R. Additionally, all analyses will be written using R statistical software (v4.1.2, R Core Team 2021).

[0224] Example 3.1.3. Evaluation. After an initial molecular detection assay is generated, the model components of the molecular detection assay are cross-validated as described below and evaluated and / or adjusted, if necessary, based on one or more of the following tests:

[0225] Cross-validation: Cross-validation is performed to evaluate the robustness and generalization of a molecular detection assay to new data. Cross-validation is a widely used technique for evaluating the performance of predictive molecular detection assays while mitigating potential overfitting issues. Here, k-fold cross-validation is utilized to validate the performance of the algorithm. The dataset is randomly divided into k (fold) subsets, and k-1 subsets are used to train the molecular detection assay, while the remaining subsets are used for testing. This process is repeated k times, each time using a different fold as the test set. Performance metrics (e.g., accuracy, AUC, precision, recall) are averaged across all folds to obtain an estimate of the overall performance of the molecular detection assay.

[0226] Holdout Validation Using an Independent Test Set: In addition to cross-validation, an independent test set is set aside from the collected data. This test set is completely separate from the data used for training and cross-validation. After training a molecular detection assay using the cross-validation process, it is evaluated on an independent test set to assess its generalization performance to new, unseen data. The performance metrics obtained from this evaluation provide a more realistic indication of the algorithm's ability to predict PTSD in real-world situations.

[0227] Stratified Sampling: To ensure that both the training and test sets represent the overall data distribution, stratified sampling is utilized. Stratified sampling maintains the same class distribution (e.g., controls and PTSD cases) in both the training and test sets to ensure that the molecular detection assay is not biased toward the majority class.

[0228] Performance Metrics: Various performance metrics (e.g., accuracy, sensitivity (recall), specificity, precision, and area under the receiver operating characteristic curve (AUC)) are used to assess the performance of the algorithm. These metrics provide a comprehensive assessment of how well a molecular detection assay can classify individuals with and without PTSD.

[0229] Overfitting detection and model selection: Cross-validation makes it possible to detect overfitting, a situation where a model performs well on training data but poorly on unseen data. If significant overfitting is detected, the model's hyperparameters can be adjusted or feature selection methods can be refined to improve generalization.

[0230] Cross-validation iterations (if necessary): In situations where the dataset is relatively small, cross-validation iterations can be performed to obtain more robust performance estimates. This technique involves randomly shuffling the data and running cross-validation multiple times, then calculating the average performance metric.

[0231] Example 3.1.4. Model Performance Metrics. Model performance metrics for the model components of the molecular detection assay are shown in Table 3 below. [Table 3]

[0232] Example 3.1.5. Statistical Analysis Plan. The following describes how the data derived by carrying out Example 3.1.1 will be analyzed.

[0233] Primary Endpoints. The performance metrics described in Table 3 above are calculated based on the model's predictions and actual labels (PTSD cases and controls) in both cross-validation and independent test sets.

[0234] Hypothesis testing. Each gene is tested individually in the panel, with the null hypothesis being that gene expression in controls is equivalent to that in cases. No contribution with a large effect is expected, but testing for it is necessary.

[0235] Receiver Operating Characteristic (ROC) Curve Analysis. ROC curve analysis is used to visualize the trade-off between sensitivity and specificity at different classification thresholds. The area under the ROC curve (AUC) is calculated to compare the discriminatory power of the algorithm with that of existing screening methods. A higher AUC indicates better discriminatory ability.

[0236] Confidence intervals. To estimate the uncertainty around a performance metric, a confidence interval can be calculated. The confidence interval provides a range of values ​​within which the true performance of the algorithm is likely to lie.

[0237] Power Analysis. If a study involves comparing the performance of an algorithm to existing methods and a specific effect size is aimed for, a power analysis can be performed to determine the sample size needed to achieve adequate statistical power.

[0238] Model calibration. Model calibration refers to the agreement between the predicted probabilities generated by an algorithm and the observed positive case frequencies. Calibration plots can be generated to assess the calibration of the model and help determine whether the predicted probabilities are accurate.

[0239] Subgroup Analysis: If there are specific subgroups of interest (e.g., different age groups, genders, trauma types), subgroup analysis can be conducted to evaluate the performance of the algorithm in these subgroups. This analysis can provide insight into potential variations in the performance of the algorithm based on different participant characteristics.

[0240] Comparison of Sensitivity and Specificity. When existing screening methods have different sensitivity and specificity tradeoffs, receiver operating characteristic comparison (ROCC) curves can be used to visualize and compare the performance of the two approaches.

[0241] Statistical Significance. Appropriate statistical tests are used to determine the statistical significance of any observed differences in performance metrics between the algorithm and existing screening methods.

[0242] Example 3.1.6. Potential Confounders and Bias. The following describes how to deal with potential confounders and bias.

[0243] Demographic factors. Participant demographics, such as age, gender, ethnicity, socioeconomic status, and education level, can affect gene expression patterns and may also be associated with PTSD risk. To mitigate the influence of these factors, subgroup analysis or inclusion of demographic variables as covariates in the prediction model can be performed.

[0244] Trauma type. The type of traumatic event experienced by participants can vary significantly and differentially affect gene expression profiles. Considering trauma type as a potential confounder can help isolate the specific effects of gene expression on PTSD risk.

[0245] Medication Use. Certain medications, such as antidepressants or anti-anxiety medications, can affect gene expression levels and potentially influence PTSD outcomes. Participants' medication history should be collected and controlled for in analyses.

[0246] Comorbidities. PTSD often co-occurs with other mental health disorders, such as depression and anxiety. These comorbidities may influence gene expression patterns, potentially confounding the association with PTSD. Comorbid conditions should be carefully assessed and considered in this analysis.

[0247] Sample selection bias. If participant recruitment is not representative of the larger population of individuals with PTSD, results may not be generalizable. To minimize sample selection bias, a diverse and inclusive recruitment strategy should be utilized.

[0248] Batch effects. When gene expression data are collected in multiple batches, batch effects may introduce systematic variation that is not related to PTSD status. Appropriate statistical methods, such as batch effect correction techniques, can be applied to address this issue.

[0249] Measurement Error. Technical errors in RNA gene expression measurements can occur during laboratory processing, resulting in measurement noise. Quality control procedures and normalization methods should be applied to minimize measurement error.

[0250] Data imputation bias. When data are missing, the imputation method can introduce bias into the results. It is essential to select an appropriate imputation method and assess the sensitivity of the results to different imputation approaches.

[0251] Overfitting. Overfitting occurs when a predictive model performs well on training data but poorly on new, unseen data. Regularization techniques and proper validation methods (e.g., cross-validation) can help prevent overfitting and ensure the model's ability to generalize.

[0252] Publication bias. Publication bias can occur when studies with positive or significant results are more likely to be published, which can lead to an overestimation of the performance of a predictive model. To mitigate publication bias, researchers should consider preregistering their studies and analyzing all relevant outcomes regardless of their statistical significance.

[0253] Example 3.1.7. Results. Assume that the present molecular detection assay can achieve sensitivity greater than 90% across a range of trauma types, while maintaining specificity greater than 75%, with NPV greater than 95% and PPV greater than 50%. These characteristics are ideal for a screening tool, as they mean that individuals with PTSD can be correctly identified and promptly referred for treatment. With current tools, to achieve this level of sensitivity (>90%), specificity, or the ability to correctly rule out PTSD in those without, drops to below 50%. Current tools rely on accurate reporting, which is subject to reporter bias and, furthermore, may be impossible (due to suppression of painful memories) or may be self-modified due to conscious and / or unconscious fear, concern, and / or stigma. Some trauma types are more associated with social stigma, which is likely why accuracy metrics of current self-report tools vary significantly across trauma types. This molecular detection assay is the first and only tool currently available that provides interpretable precision metrics based on objective measurements.

[0254] Without being bound by theory, it is assumed that this molecular detection assay can achieve the same sensitivity and / or specificity as one or more psychological assessments, and if necessary, can achieve enhanced sensitivity and / or specificity as one or more psychological assessments.Therefore, this molecular detection assay can be used to detect the onset of PTSD, select suitable subjects for PTSD treatment, and / or monitor the progress of subjects undergoing PTSD treatment.Furthermore, it is assumed that this molecular detection assay will provide gene targets for the development of new precision therapeutics related to PTSD treatment.

[0255] Example 3.2. Use of Molecular Detection Assays to Establish Subject Selection for PTSD Treatment This example describes efforts to establish and / or calibrate a molecular detection assay to determine whether a subject is a suitable candidate for PTSD treatment.

[0256] The psychological assessment group described in Example 3.1 is treated according to industry standards (e.g., stellate ganglion block, Trauma Informed Talk Therapy, ketamine infusion alone, or ketamine infusion in combination with the aforementioned treatments). Data is collected by comparing the results of an initial screening (e.g., CAPS-5 interview) conducted by a trained provider before treatment with a second screening (e.g., PCL-5 or informal interview) conducted 30±3 days after treatment.

[0257] Without being bound by theory, it is hypothesized that the collected data can predict a subject's responsiveness to a treatment modality and thus train the present molecular detection assay for subject selection for PTSD treatment.

[0258] Example 3.3. Use of molecular detection assays to establish PTSD treatment responsiveness. This example describes efforts to generate a molecular detection assay and / or calibrate the molecular detection assay to compare it with clinically accepted PTSD-related psychological assessments to determine whether a subject will be responsive to a PTSD treatment.

[0259] The groups described in Example 3.1 will be treated according to industry standards. If necessary, subjects to be treated will be selected based on the molecular detection assays described in Example 3.2.

[0260] Periodically (e.g., every 2-3 months, quarterly, twice a year, and / or thereafter as needed), RNA expression levels are collected and analyzed as described in Example 3.1. Subjects with PTSD that have improved RNA expression patterns compared to baseline levels are determined to be responsive to PTSD treatment. Without being bound by theory, it is hypothesized that the collected data can train the present molecular detection assay to monitor a subject's responsiveness to treatment modalities.

[0261] Example 4: PTSD detection device or kit Example 4.1. PTSD detection device. This example describes a PTSD detection device. The PTSD detection device includes a molecular genomic test that uses quantitative gene expression to detect clinically meaningful changes in the expression of RNA isolated from human blood collected from individuals aged 18 years and older using an FDA-approved collection device (e.g., an RNA PAXgene tube or any other suitable sample collection container, such as those described in the Devices section herein). The molecular genomic test may include the ADAPT panel, which uses quantitative next-generation sequencing (NGS), or any other molecular genomic test described herein, and can measure the expression levels of gene transcripts, including the genes listed in Table 2. The device is calibrated to measure changes in transcription patterns relative to a predetermined threshold level using the molecular detection assay described herein. After quantifying the transcription pattern, a result report is generated indicating whether the subject screens positive or negative for PTSD. A positive result for PTSD detection warrants referral to a specialized medical provider for further evaluation and diagnosis.

[0262] Example 4.2. PTSD Detection Kit. This example describes a PTSD detection device. A PTSD detection kit includes any component or combination of components described in Example 4.1 and instructions for use to detect PTSD.

[0263] Example 5: Use in diagnosis, patient selection, and / or treatment Example 5.1. Diagnosis. This example describes the use of the present molecular detection assays, devices, and / or kits in a method for diagnosing PTSD. The molecular detection assay described in Example 2 or 3, the device described in Example 4.1, or the kit described in Example 4.1 is used as a screening tool for PTSD. A positive result for the detection of PTSD warrants referral to a specialized medical provider for further evaluation and diagnosis.

[0264] Example 5.2. Patient Selection. This example describes the use of the present molecular detection assays, devices, and / or kits in a method for selecting subjects for PTSD treatment. The molecular detection assay described in Example 2 or 3, the device described in Example 4.1, or the kit described in Example 4.1 is used as a screening tool to determine whether a subject will be responsive to a PTSD treatment, and optionally to a specific PTSD treatment.

[0265] Example 5.3. Treatment. This example describes the use of the present molecular detection assays, devices, and / or kits in methods for treating PTSD. After diagnosing PTSD in a subject, including by using the method described in Example 5.1, and optionally after selecting the subject for treatment, including by using the method described in Example 5.2, the subject is treated for PTSD using a therapeutically effective treatment or a clinically tolerated and / or approved treatment. The molecular detection assay described in Example 2 or 3, the device described in Example 4.1, or the kit described in Example 4.1 is used to monitor the subject's responsiveness to treatment and / or to monitor the progress of the subject undergoing treatment.

[0266] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein can be used to implement the methods presented in this disclosure. It is intended that the following claims define the scope of the disclosure, and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. 1. A method for detecting post-traumatic stress disorder (PTSD) in a human subject, comprising: a) obtaining a cell sample obtained from a human subject; b) measuring test gene expression levels of one or more PTSD-related genes from the cell sample using a first computer program executed on a computer, wherein the PTSD-related genes are selected from the group consisting of TSPAN5, HIST1H2AE, UBE3A, GPX4, EPB42, SLC4A1, NDUFA1, HIST1HEH, ELOVL7, ALES, COMPT, PDZK1IP1, ITGA2B, CYP4F3, EPB41L3, PRDM1, RP-11-449P15.1, FAS, TUBB2A, and JAM3; c) comparing said test PTSD-related expression level with one or more control gene expression levels using a second computer program executed on a computer; d) determining the differential expression of said one or more test PTSD-related genes; e) determining a positive detection of PTSD based on said differential expression; A method comprising:

2. 2. The method of claim 1, wherein the test gene expression levels and / or the control gene expression levels are of one or more, two or more, three or more PTSD-related genes.

3. 2. The method of claim 1, wherein the test gene expression levels and / or the control gene expression levels are of four or more, five or more, six or more PTSD-related genes.

4. 2. The method of claim 1, wherein the test gene expression levels and / or the control gene expression levels are of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, or all twenty PTSD-related genes.

5. 5. The method of any one of claims 1 to 4, wherein the PTSD-related gene is selected from the group consisting of TSPAN5, HIST1H2AE, UBE3A, GXP4, EPB42, SLC4A1, NDUFA1, HIST1H3H, EVOLV7, and JAM3.

6. 5. The method of any one of claims 1 to 4, wherein the PTSD-related gene is selected from the group consisting of TSPAN5, HIST1H2AE, UBE3A, GXP4, and EPB42.

7. The test PTSD-related gene expression level is 2.5 x 10 －６ 7. The method of claim 1, wherein the nucleotide sequence is differentially expressed with a p-value lower than

8. The test PTSD-related gene expression level is 9×10 －７ 7. The method of claim 1, wherein the nucleotide sequence is differentially expressed with a p-value lower than

9. The test PTSD-related gene expression level is 1.6 x 10 －７ 7. The method of claim 1, wherein the nucleotide sequence is differentially expressed with a p-value lower than

10. 10. The method of any one of 1 to 9, wherein the cell sample is obtained from a blood sample, a saliva sample, a buccal smear sample, a cerebrospinal fluid sample, saliva, skin, cerebrospinal fluid, or any combination thereof.

11. 11. The method of any one of claims 1 to 10, wherein the test gene expression is messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), small nuclear RNA (snRNA), U-spliceosomal RNA (U-RNA), small nucleolar RNA (snoRNA), Piwi-interacting RNA (piRNA), repeat-associated small interfering RNA (rasiRNA), small rDNA-derived RNA (srRNA), small transfer RNA-derived RNA (tsRNA), small ribosomal RNA-derived RNA (rsRNA), small long non-coding RNA-derived RNA (lncsRNA), or messenger RNA-derived small RNA (msRNA), gapmer, mixmer, double-stranded RNA (dsRNA), single-stranded RNAi (ssRNAi), DNA-directed RNA interference (ddRNAi), or any combination thereof.

12. 12. The method of claim 1, wherein the expression level is assessed at the transcriptional level.

13. 13. The method of claim 1, wherein the expression level is assessed at the translational level.

14. 14. The method of claim 1, wherein the first computer program and the second computer program are the same.

15. 14. The method of claim 1, wherein the first computer program and the second computer program are different.

16. 16. The method of any one of claims 1 to 15, wherein the method is performed more than once.

17. 17. The method of claim 16, wherein the method is performed weekly, monthly, bimonthly, three times per year, four times per year, two times per year, or yearly.

18. 18. The method of any one of claims 1 to 17, further comprising treating the subject for PTSD.

19. 20. The method of claim 18, wherein treating the subject comprises administering to the subject a drug therapy, a psychological therapy, or a combination thereof.

20. 17. The method of claim 16, wherein the subject is administered a drug therapy.

21. 18. The method of claim 17, wherein the medication is an SSRI, a tricyclic antidepressant, an MAOI, an antipsychotic, a beta-blocker, a benzodiazepine, a hallucinogen, or a combination thereof.

22. 21. The method of claim 20, wherein the medication is an SSRI.

23. 21. The method of claim 20, wherein the medication is a tricyclic antidepressant.

24. 21. The method of claim 20, wherein the medication is an MAOI.

25. 21. The method of claim 20, wherein the medication is an antipsychotic.

26. 21. The method of claim 20, wherein the medication is a beta-blocker.

27. 21. The method of claim 20, wherein the medication is a benzodiazepine.

28. 21. The method of claim 20, wherein the medication is a hallucinogen.

29. 1. A method of screening for PTSD in a subject, comprising: a. detecting PTSD in a subject, comprising the method of any one of claims 1 to 28; b. determining that the subject should be clinically diagnosed with PTSD based on a positive detection of PTSD in the subject; A method comprising:

30. 30. The method of claim 29, wherein the control population is a PTSD-negative population.

31. the method comprising clinically diagnosing the subject with PTSD using one or more clinical PTSD diagnostic criteria.

31. The method of claim 29 or 30, further comprising:

32. 32. The method of claim 31, wherein the clinical PTSD diagnostic criteria is CAPS-5 or PCL-5.

33. 33. The method of claim 32, wherein the clinical PTSD diagnostic criterion is CAPS-5.

34. 33. The method of claim 32, wherein the clinical PTSD diagnostic criterion is PCL-5.

35. The method of any one of claims 31 to 34, wherein the detection step and the clinical diagnosis step are performed by two different entities.

36. assessing one or more comorbidities associated with PTSD 36. The method of any one of claims 29 to 35, further comprising:

37. 37. The method of claim 36, wherein the one or more co-morbid conditions comprise generalized anxiety disorder, alcohol use disorder, anxiety-related insomnia, childhood trauma, or any combination thereof.

38. 38. The method of any one of claims 36-37, wherein the one or more comorbid conditions are diagnosed using one or more clinical criteria comprising the Generalized Anxiety Disorder 7-item scale (GAD-7), the Alcohol Use Disorders Identification Test (AUDIT-C), the Pittsburgh Sleep Quality Index (PSQI), Adverse Childhood Experiences (ACE), or any combination thereof.

39. 39. The method of any one of claims 36 to 38, wherein the detection step, the clinical diagnosis step, and the comorbidity diagnosis step, or any combination of two of them, is performed by two different entities.

40. 1. A method for selecting a subject eligible for treatment of PTSD, comprising: a. detecting PTSD in a subject, comprising the method of any one of claims 1 to 28; b. determining that the subject is eligible for PTSD treatment based on a positive detection of PTSD in the subject; A method comprising:

41. 41. The method of claim 40, wherein the subject is screened for PTSD according to the method of any one of claims 29 to 39.

42. 1. A method for assessing treatment efficacy in a subject receiving treatment for PTSD, comprising: a. detecting PTSD in a subject, comprising the method of any one of claims 1 to 28; b. determining the effectiveness of the PTSD treatment based on detecting PTSD in the subject, wherein a negative PTSD detection indicates that the PTSD treatment is effective; A method comprising:

43. 43. The method of claim 42, wherein the PTSD treatment is a single PTSD treatment or a PTSD treatment event that is part of a series or ongoing PTSD treatment.

44. 44. The method of any one of claims 42-43, wherein the PTSD treatment comprises cognitive behavioral therapy, cognitive processing therapy, cognitive therapy, prolonged exposure therapy, eye movement desensitization and reprocessing (EMDR) therapy, narrative exposure therapy (NET), group therapy, brief eclectic psychotherapy, selective serotonin reuptake inhibitors (SSRIs), serotonin and norepinephrine reuptake inhibitors (SNRIs), anti-anxiety medications, antidepressants, sertraline, paroxetine, fluoxetine, venlafaxine, ketamine, tricyclic antidepressants, MAOIs, antipsychotics, beta-blockers, benzodiazepines, hallucinogens, co-morbidity treatments, complementary and / or alternative therapies, or any combination thereof, wherein the complementary and / or alternative therapies comprise acupuncture, yoga, animal-assisted therapy, meditation.

45. 45. The method of any one of claims 42 to 44, wherein the method comprises detecting PTSD in the subject prior to treatment comprising the method of any one of claims 1 to 28.

46. 46. ​​The method of any one of claims 42 to 45, wherein the method comprises screening for PTSD in the subject prior to treatment comprising the method of any one of claims 29 to 39.

47. 47. The method of any one of claims 42 to 46, wherein the method comprises a step of selecting the subject as eligible for the PTSD treatment prior to treatment comprising the method of any one of claims 40 to 41.

48. 1. A method for determining the prevalence of PTSD in a population of interest, comprising: a. detecting PTSD in each subject in a population of interest, comprising the method of any one of claims 1 to 28; b. determining the prevalence of PTSD in the target population based on the positive detection of PTSD in each subject in the target population; A method comprising:

49. 39. The method of claim 38, wherein the group is a military group.

Images