Genetic biomarkers for identifying PTSD and major depression

Abstract

The present invention provides 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 with a first computer program executed on a computer, in which 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 tested PTSD-related expression level with one or more control gene expression levels with a second computer program executing on the computer; determining differential expression of the one or more tested PTSD related genes; and determining a positive detection result of the PTSD.

Description

[0001] Cross-reference

[0002] This application claims priority to U.S. Provisional Application 63 / 434,605, filed December 22, 2022, the entire contents of which are incorporated herein by reference.

[0003] The sequence list is incorporated by reference.

[0004] This application contains a sequence list, which has been submitted through the Patent Center. The sequence list is titled 210536-010500_SL.xml, created on December 22, 2023, and is 269,686 bytes in size. Its entire contents are incorporated herein by reference.

[0005] Statement on Federally Funded Research

[0006] This application did not use any federal funding.

[0007] By incorporating via reference

[0008] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference as if each individual publication, patent, or patent application were expressly and individually indicated to the extent of their inclusion herein by reference. If any publication, patent, or patent application incorporated by reference conflicts with the disclosure contained in this specification, this specification is intended to supersede and / or give precedence to any such conflicting material. Summary of the Invention

[0009] This article provides a method for detecting post-traumatic stress disorder (PTSD) in human subjects, the method comprising: obtaining a cell sample from a human subject; measuring the 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; comparing the tested PTSD-related expression levels with the expression levels of one or more control genes using a second computer program executed on a computer; determining the differential expression of the one or more tested PTSD-related genes; and determining a positive detection result for PTSD based on the differential expression. In some embodiments, the expression level of the test gene and / or the expression level of the control gene are the expression levels of one or more, two or more, or three or more PTSD-related genes. In some embodiments, the expression level of the test gene and / or the expression level of the control gene are the expression levels of four or more, five or more, or six or more PTSD-related genes. In some embodiments, the expression level of the test gene and / or the expression level of the control gene are the expression levels 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 20 PTSD-related genes. In some embodiments, the PTSD-related genes are selected from the group consisting of TSPAN5, HIST1H2AE, UBE3A, GXP4, EPB42, SLC4A1, NDUFA1, HIST1H3H, EVOLV7, and JAM3. In some embodiments, the PTSD-related gene expression level being tested is less than 2.5 × 10⁻⁶. -6 The p-value indicates differential expression. In some embodiments, the expression levels of PTSD-related genes being tested are less than 9 × 10⁻⁶. -7 The p-value indicates differential expression. In some embodiments, the expression levels of PTSD-related genes being tested are less than 1.6 × 10⁻⁶.-7 The p-value indicates differential expression. In some embodiments, the cell sample is obtained from a blood sample, saliva sample, oral smear sample, cerebrospinal fluid sample, saliva, skin, cerebrospinal fluid, or any combination thereof. In some embodiments, the expression of the test gene is messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), small nuclear RNA (snRNA), U-splice RNA (U-RNA), small nucleolar RNA (snoRNA), Piwi-interacting RNA (piRNA), repeat-associated small interfering RNA (rasiRNA), small rDNA-derived RNA (srRNA), transfer RNA-derived small RNA (tsRNA), ribosomal RNA-derived small RNA (rsRNA), large 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 assessed at the transcriptional level. In some embodiments, the expression level is assessed at the translational level. In some embodiments, the first computer program is the same as the second computer program. In some embodiments, the first computer program is different from the second computer program. In some embodiments, the method is performed more than once. In some embodiments, the method is performed weekly, monthly, every two months, three times a year, four times a year, twice a year, or once a year. In some embodiments, the method further includes a step of treating the subject's PTSD. In some embodiments, treating the subject includes administering medication, psychotherapy, or a combination thereof to the subject. In some embodiments, the subject is administered medication. In some embodiments, the medication is an SSRI, a tricyclic antidepressant, a 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 implementations, the drug treatment is a hallucinogen.

[0010] This document also provides a method for screening a subject for PTSD, the method comprising: detecting PTSD in the subject, including any of the detection methods described herein; and determining, based on a positive test result for the subject's PTSD, that the subject should be clinically diagnosed with PTSD. In some embodiments, the control group 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 comorbidities associated with PTSD. In some embodiments, the one or more comorbidities include generalized anxiety disorder, alcohol use disorder, anxiety-related insomnia, childhood trauma, or any combination thereof. In some embodiments, the one or more comorbidities are diagnosed using one or more clinical criteria, including 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. In some embodiments, the testing steps, the clinical diagnostic steps, and the comorbidity diagnostic steps, or any combination thereof, are performed by two different entities.

[0011] This document also provides a method for screening subjects who meet the criteria for PTSD treatment, the method comprising: detecting PTSD in the subject, including any of the detection methods described herein; and determining that the subject meets the criteria for PTSD treatment based on a positive test result for PTSD. In some embodiments, the subject is screened for PTSD according to any of the screening methods described herein.

[0012] This document also provides a method for evaluating the efficacy of PTSD treatment in a subject, the method comprising: detecting PTSD in the subject, including any of the detection methods described herein; and determining the efficacy of the PTSD treatment based on the subject's PTSD test results, wherein a negative PTSD test result indicates that the PTSD treatment is effective. In some embodiments, the PTSD treatment is a single PTSD treatment or a PTSD treatment event as part of a series or ongoing PTSD treatments. In some embodiments, the PTSD treatment includes 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 compromise psychotherapy, selective serotonin reuptake inhibitors (SSRIs), serotonin and norepinephrine reuptake inhibitors (SNRIs), anxiolytics, antidepressants, sertraline, paroxetine, fluoxetine, venlafaxine, ketamine, tricyclic antidepressants, MAOIs, antipsychotics, beta-blockers, benzodiazepines, hallucinogens, comorbidity treatment, complementary and / or alternative therapies (including acupuncture, yoga, animal-assisted therapy, meditation), or any combination thereof. In some embodiments, the method includes testing the subject for PTSD prior to treatment, including any of the testing methods described herein. In some embodiments, the method includes screening the subject for PTSD prior to treatment, including any of the screening methods described herein. In some implementations, the method includes screening subjects who meet the PTSD treatment criteria before treatment, including any of the methods described herein for screening eligible subjects.

[0013] This document also provides a method for determining the prevalence of PTSD in a target population, the method comprising detecting PTSD in each subject of the target population, including any of the detection methods described herein; and determining the prevalence of PTSD in the target population based on the positive PTSD test results of each subject of the target population. In some embodiments, the population is a military population. Attached Figure Description

[0014] Figure 1 This is a flowchart illustrating the sequencing, amplification, and analysis of the subject's RNA.

[0015] Figure 2 Describe the machine learning used to determine the number of genes that may be associated with PTSD.

[0016] Figure 3 Twenty genes highly associated with PTSD and their P values ​​were described.

[0017] Figure 4Demographic profiles of individuals evaluated for generating objective molecular assays to screen for PTSD.

[0018] Figure 5 The sensitivity of the molecular detection analysis, or the percentage of subjects correctly identified as having the aforementioned disorder, is 90%.

[0019] Figure 6 The area under the subject operating characteristic (AUC) curve for the previous model using the same gene at different thresholds reached 85%. Figure 6 Also plotted receiver operating characteristic (ROC) curves for the entire sample (all_in) and cross-validation of size 1 or k=1 (k1_Cross).

[0020] Figure 7 This is a flowchart, which is a schematic diagram of an exemplary method consistent with some aspects of this disclosure. Detailed Implementation

[0021] SUMMARY

[0022] PTSD is characterized at the molecular level by dysregulation of the primary stress-hormonal system, the so-called HPA (hypothalamic-pituitary-adrenal) axis. The hypothalamic-pituitary-adrenal (HPA) axis is a major component of the neuroendocrine response to acute and chronic stress, leading to the release of corticotropin-releasing hormone (CRH) and vasopressin (AVP) from small-celled neurons in the hypothalamus into the portal system to activate the anterior pituitary gland in the synthesis and release of ACTH. ACTH, in turn, 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, manifested by decreased and increased responsiveness of ACTH and cortisol levels in these patients. Increased responsiveness of glucocorticoid receptors (GR) appears to be the cause of these disturbances. These findings contrast with observations of higher cortisol levels and reduced glucocorticoid responsiveness associated with major depressive disorder (MDD) (Ribeiro et al., Am J Psychiatry 1993; 150:1618-29; Holsboer F, Neuropsychopharmacology 2000; 23:477-501). Although initially interpreted as reflecting the lasting consequences of traumatic exposure, there is reason to suspect that alterations in the HPA axis in PTSD reflect pre-traumatic risk factors. Recent studies of infants and adult children of parents with PTSD also support the view that low cortisol levels and glucocorticoid responsiveness are risk factors for PTSD. Indeed, recent observations suggest that adults who have experienced child abuse and have genetic variations in the FKBP5 gene (involved in the regulation of GR) appear to face a higher risk of PTSD symptoms in adulthood (Binder et al., JAMA 2008; 299:1291-305). Two other studies have examined gene expression following traumatic exposure. In a study of individuals exposed to severe trauma in the emergency room (meeting (n=8) or not (n=6) the diagnostic criteria for PTSD at 1-month and 4-month follow-up, respectively), changes in gene expression were associated with several interesting molecular classes related to stress response. Interestingly, this study showed upregulation of FKBP5 expression. However, given the proximity to the time of trauma exposure, these findings may reflect biological changes associated with recovery from the effects of trauma exposure, rather than those associated with the development or persistence of chronic PTSD.

[0023] Indeed, genetic factors are known to play a significant role in determining responses to environmental events and have been previously linked to PTSD, depression, schizophrenia, and other mental health disorders. (Duncan LE et al., Mol Psychiatry. 2018 Mar; 23(3):666-673; Petra Zimmermann et al., Am J Psychiatry. 2011 Oct; 168(10). For example, twin studies estimate the heritability of post-traumatic PTSD to range from 24% to 72%, suggesting that individual genes and epigenetics influence whether they experience PTSD after trauma or stressors. 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 has shown for the first time that epigenetic changes at specific gene loci can be inherited from parents to offspring, leading to an increased risk of PTSD. Rachel Yehuda et al., Society of Biological Psychiatry, Sep 1 2016; 80:372–380. The expression levels of genes controlling glucocorticoid receptor regulation were found to be dysregulated in the first responders at the 9 / 11 World Trade Center who suffered from severe PTSD, and were also found to be altered in the brains of PTSD patients. Casey Sarapasa et al., DiseaseMarkers 30 (2011) 101–110; and Sophie E. Holmes et al., Proc Natl Acad Sci USA. 2017 Aug 1; 114(31):8390-8395.

[0024] It has been found that over 90% of American adults experience at least one traumatic event in their lifetime (Breslau, N. et al., Soc Psychiatry Psychiatr Epidemiol. 2016 Aug; 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 disorder characterized by intrusive and recurrent memories of trauma, avoidance of trauma-related stimuli, numbness and / or negative alterations in trauma-related emotions or cognition, and altered reactivity and arousal.

[0025] Early intervention has the potential to reduce adverse outcomes because PTSD may be reversible if detected early. 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. September 18, 2012; 109(38):15508-13; Shalev AY, Ankri Y, Israel-Shalev Y et al., Arch Gen Psychiatry. February 2012; 69(2):166-76; Bisson JI, Olff M. Eur J Psychotraumatol. 2021; 12(1):1824381. Multiple studies have found that early intervention can reduce the incidence of chronic PTSD by up to 50%. Rothbaum BO et al., Biological Psychiatry. 2012; 72(11):957–963; ShalevAY et al., Arch Gen Psychiatry. Feb. 2012; 69(2):166–76. Chronic PTSD is associated with an increased risk of cancer, 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 enhanced inflammatory response (O'Toole BI et al., Journal of Psychosomatic Research. 2008; 64(1):33–40). Psychosocial effects include homelessness, poverty, and incarceration (APA CPG for PTSD).

[0026] However, early treatment is hampered by insufficient recognition of PTSD by healthcare providers (Joneydi R et al., Med Care. 1 June 2021; 59(6):557-564), possibly due to a lack of objective tests available to aid in diagnosis. This lack of timely diagnosis leads to delayed treatment, resulting in maladaptive coping mechanisms and disorders such as alcohol and substance use disorders. Studies have repeatedly shown extremely high rates of comorbidity. (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 patients with existing PTSD also had major depressive disorder. Rytwinski NK et al., Journal of Traumatic Stress. 2013; 26(3):299–309.

[0027] The typical PTSD diagnosis process can take months or even years, with delays occurring due to characteristics of both patients and healthcare providers. On the patient's side, they often don't connect their current experience to the traumatic event and therefore rarely realize the need for behavioral health care. In other cases, they are aware of the connection but prefer to manage it themselves; this is often driven by a sense of stigma associated with appearing "weak or lacking resilience." Many coping behaviors begin at this stage, are negative, and subsequently interfere with their personal and professional lives, ultimately prompting them to seek care from a healthcare provider.

[0028] On the healthcare provider side, PTSD and other mental health diagnoses are typically made by behavioral health service providers with subspecialty training. However, early diagnosis depends on the primary care provider's ability to consider differential diagnoses and make appropriate referrals. Primary care providers often lack the time, training, and resources to conduct lengthy clinical interviews, thus relying on self-reported surveys as a screening tool to help identify patients who should be referred.

[0029] Several open-source psychometric screening tools are available; however, they generally suffer from low accuracy, and their effectiveness varies significantly depending on the target population. (Spoont M et al., VA Evidence-based Synthesis Program Reports, 2013). Although numerous studies have determined the accuracy of various tools, direct comparisons are often difficult due to the use of different "cutoff values." For example, in one study, a lower cutoff value might be used to maximize the detection of PTSD cases. However, this decision would be accompanied by more false positives. Conversely, using a higher cutoff value minimizes false positives, but at the cost of increased false negatives.

[0030] The most commonly used self-report screening tool is the 17-item PTSD checklist (PCL) (Scott D. McDonald, Patrick S. Calhoun, Clinical Psychology Review 30 (2010) 976–987), which has been widely used for clinical screening and symptom tracking of PTSD. 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. The preliminary version of PCL-5 suggested a diagnostic cutoff value of 31 to 33. Weathers FW et al., ptsd.va.gov / professional / assessment / adult-sr / ptsd-checklist.asp, accessed August 2, 2022. In a sample of 273 traumatized mental health service users, the sensitivity of PCL-5 was 95% at a cutoff value of 33.5; however, this resulted in a specificity of only 52%. Roberts NP et al., Eur J Psychotraumatol. 2021; 12(1):1863578.

[0031] Besides the issues of accuracy, relying solely on self-reporting is problematic (Althubaiti A. J Multidiscip Healthc. 2016; 9:211-7), and current standards of care are stigmatizing (Johnson HP et al., Psychiatr Danub. 2018 Nov; 30(Suppl 7); 508-510; Melotte H et al., Eur J Psychotraumatol. 2017; 8(1); 1389-207; Wilk JE et al., Psychiatr Serv. 2016 Aug 1; 67(8); 878-82), malingering (Hall RC., Gen Hosp Psychiatry. 2006 Nov-December; 28(6); 525-35; Ali S, Jabeen S et al., Innov Clin Neurosci. 2015 Jan–Feb; 12(1–2); 12–20) and underreporting (Johnson HP, Agius M., Psychiatr Danub. 2018 Nov; 30(Suppl 7); 508–510). For example, reporting bias has been well established; patients may be dishonest due to fear of public discrimination, fear of hospitalization, embarrassment, shame, or various other factors that contribute to public, self- or structural stigma. Corrigan PW et al., Psychol Sci Public Interest. 2014 Oct; 15(2); 37–70.

[0032] This is particularly evident among former military personnel. The impact of PTSD on U.S. military personnel and veterans (11-20%) is significantly higher than on their civilian counterparts (6-8%). The U.S. military currently incorporates questionnaires into its pre-deployment, post-deployment, and periodic health assessments, as well as post-deployment health reassessments, to identify members who should be further evaluated by trained clinicians to make appropriate diagnoses (including comorbid diagnoses such as depression or traumatic brain injury) and to obtain additional information needed to plan treatment.

[0033] Example of primary healthcare PTSD screening used in DoD assessment:

[0034] In your life, have you ever had any experience that made you feel extremely fearful, horrible, or unpleasant, to the point that in the past month, you:

[0035] 1. Have you had nightmares related to it or thought about it when you didn't want to? Yes / No

[0036] 2. Do you try your best not to think about it or deliberately avoid situations that remind you of it? Yes / No

[0037] 3. Are you always alert, wary, or easily startled? Yes / No

[0038] 4. Do you feel numb or alienated from others, activities, or your surroundings? Yes / No

[0039] Concerns about the impact of answers on current or future tasks, promotions, compensation and benefits, and / or safety clearances typically influence respondents’ responses to these questions. Missed identification and delayed treatment can lead to maladaptive coping strategies, increased social dysfunction, and reduced medical readiness. See, for example, Sarapas C et al., Dis Markers. 2011; 30(2-3):101-10; Le-Niculescu H et al., Mol Psychiatry. May 2020; 25(5):918-938; and CerRZ et al., Gigascience. October 13, 2014; 3:20.

[0040] In fact, one study found that PTSD diagnoses were consistently unreported in military medical records to reduce stigma or protect service members’ career prospects. Wilk JE et al., Psychiatr Serv. 2016 Aug 1; 67(8):878-82. Another study used anonymized surveys to obtain more accurate reporting from a single infantry brigade combat team (n=1712). They found that 20.3% of soldiers who tested positive for depression or PTSD indicated they were reluctant to report truthfully during routine post-deployment screenings. Warner CH et al., Arch Gen Psychiatry. 2011 Oct; 68(10):1065-71. Understandably, for a disorder whose symptoms are avoidance of traumatic reminders, it may not be accurately reported at the systemic level.

[0041] These findings have also extended to studies of civilian patients. A study of patients in a South African psychiatric ward randomly sampled 40 participants who had not previously been diagnosed with PTSD and found that the prevalence of PTSD in the study group was 40%, far higher than the original prevalence of 5.5% obtained using standard care methods in the ward. van Zyl M et al., Afr J Psychiatry (Johannesbg). May 2008; 11(2):119-22.

[0042] This result confirms that individuals with other comorbid disorders may not be properly screened for or diagnosed with PTSD. Another study of a US population of children and adolescents further supports this underdiagnosis trend. Researchers interviewed patients at two locations using trauma-focused interviews and identified PTSD in 47.7% of patients at one location, 2.3% higher than the original prevalence (n=44), and in the second location, 44.6% of patients were identified as having PTSD, 5.4% higher than the original prevalence (n=56). (Miele D, O'Brien EJ., Journal of Traumatic Stress. 2010; 23(5):591-598.)

[0043] Less discussed is observer-introduced bias. A study comparing PTSD rates in hospitalized male and female veterans found that only one woman was diagnosed with PTSD, compared to seven men (n=62), suggesting that women may be underdiagnosed due to healthcare provider bias. Grossman LS et al., Psychiatr Serv. 1997 Mar; 48(3):393-5. In addition to the aforementioned drawbacks, these self-report tools are not widely used in primary care settings. They are most commonly used in high-risk primary care settings, such as military or veterans' clinics. In other settings, individuals must voluntarily report their symptoms or request a referral to a BH for a definitive diagnosis.

[0044] Overall, these findings explain many reasons why PTSD is severely underdiagnosed under current standards of care. They also highlight the urgent need for objective diagnostic tools that can be more widely applied without increasing the time or administrative burden on primary care providers, while identifying many previously undiagnosed and untreated individuals earlier.

[0045] Once a patient arrives at a specialist care provider, diagnosis is often made through clinical judgment following unstructured interviews, as structured interviews for the DSM-5 Clinician Management of PTSD Scale (CAPS-5) are typically time-consuming and impractical. Due to the shortage of these specialist care providers, even after referral, patients often experience severe delays in care due to waiting times or social stigma preventing them from completing the referral process.

[0046] Given the negative impact of PTSD on overall health and function, and the availability of effective evidence-based treatments, there is a clear need for an objective test that can be used to identify affected individuals at an early stage. Scott D. McDonald, Patrick S. Calhoun, Clinical Psychology Review 30 (2010) 976–987; Department of Veterans Affairs, Veterans Health Administration (2004), (VHA Directive 2004-015).

[0047] Unfortunately, no single treatment (even those with a solid evidence base) has been proven effective for all patients with PTSD. Therefore, current treatment approaches are often trial-and-error, seeking the right treatment for each individual patient. The latest clinical pathway guidelines from the American Psychological Association 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 PTSD patients in the general population are resistant to first- and second-line treatments; the non-response rate for cognitive behavioral therapy can be as high as 50%, while the non-response rate for selective serotonin reuptake inhibitors is approximately 20% to 40%. (Green B., Advances in Psychiatric Treatment, 2013; 19:181–190.)

[0048] An objective biomarker for PTSD is needed to address diagnostic challenges such as stigma, symptom overlap, and observer and reporter bias. Behavioral health is the only area in medicine that still lacks objective measurements to aid in diagnosis and treatment monitoring. Advances in diagnostic and treatment monitoring categories related to the biological dysregulations that contribute to the development of disorders are needed.

[0049] DEFINITIONS

[0050] As used herein, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise. Any reference to “or” herein is intended to cover “and / or” unless otherwise stated.

[0051] As used herein, the term "about" may refer to a referenced numerical indication plus or minus 5%, 10%, 15%, or 20% of that indication. In some cases, "about" may refer to a referenced numerical indication plus or minus 15% of that indication. In other cases, "about" may refer to a referenced numerical indication plus or minus 20% of that indication.

[0052] As used herein, for convenience, multiple items, structural elements, constituent elements, and / or materials may be presented in a general list. However, these lists should be understood as each member of the list being individually identified as a separate and distinct member. Therefore, unless otherwise stated, an individual member of such a list should not be construed as being factually equivalent to any other member of the same list simply because it is presented in the same group.

[0053] Concentration, quantity, and other numerical data may be expressed or presented in this document in range form. It should be understood that this range format is used for convenience and brevity only, and therefore should be interpreted flexibly to include not only the values ​​explicitly listed as range boundaries, but also all individual values ​​or subranges covered within the range, as if each value and subrange were explicitly listed. For example, a numerical range of “about 1 to about 5” should be interpreted to include not only the explicitly listed values ​​of about 1 to about 5, but also the individual values ​​and subranges within the range shown. Thus, this numerical range includes 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 the individual values ​​1, 2, 3, 4, and 5. The same principle applies to ranges that record only one value as a minimum or maximum value. Furthermore, this interpretation applies regardless of the breadth of the range or the characteristics described.

[0054] As used herein, the term "adjustment disorder" can refer to a response to a stressor that does not meet the criteria for post-traumatic stress disorder (PTSD) (or another specific mental disorder), or to a symptom pattern of PTSD that is a response to a non-extreme stressor (e.g., separation by a spouse, job loss). Symptoms may include avoidance, numbness, and increased arousal that were present before exposure to the stressor and may not meet the diagnostic criteria for PTSD, requiring consideration of other diagnoses (e.g., transient psychotic disorder, conversion disorder, major depressive disorder) that may provide an alternative or complementary diagnosis to PTSD.

[0055] As used herein, the terms “application,” “injection,” “administration,” etc., can refer to a method that can be used to deliver a compound or composition to a desired biological site of action.

[0056] As used in this article, “clinical diagnosis” and its grammatical equivalents, when relevant to PTSD, can refer to a diagnosis of PTSD made by a trained behavioral health provider through an informal or formal clinical interview.

[0057] As used in this article, “complementary” can refer to non-covalent bonding of nucleotide bases through hydrogen bonds, Watson-Crick base pairing, wobble base pairing, van der Waals interactions, or any combination thereof.

[0058] As used in this article, “control” refers to control subjects, control populations, or control data, such as data from baseline or databases.

[0059] As used herein, the phrase “control gene expression level” may refer to expression data of one or more PTSD-related genes derived from the baseline, control, control population, or dataset described herein.

[0060] As used herein, the term “detection” and its grammatical equivalents describe the identification of the presence of PTSD in a subject. A positive PTSD test or positive PTSD test, used interchangeably herein, describes the identification or detection of PTSD in a subject. A negative PTSD test or negative PTSD test, used interchangeably herein, describes the identification or non-detection of PTSD in a subject. Similarly, a PTSD-negative subject or population (e.g., a target subject or population, or a control or control group) includes subjects for whom PTSD is not detected (as described by the methods and analyses presented herein, and / or by the clinical diagnosis presented herein).

[0061] As used in this paper, the term "differential expression" and its grammatical equivalents describe statistically significant differences in gene expression levels between two experimental conditions (e.g., test and control groups, such as a group clinically diagnosed with PTSD and a PTSD-negative group). This paper will further describe computational examples of differential expression.

[0062] As used herein, the term "expression level" can refer to the degree of gene expression in relation to the gene expression level before or after mRNA pretreatment or posttreatment (before and after intron removal). In some cases, the term "expression level" can refer to the protein translated from the gene. Comparison or assessment of expression levels (i.e., determining the degree of expression) can be a decisive factor in the detection of PTSD. Similarly, assessment of expression levels can also indicate the extent of PTSD progression in a subject. Comparisons of expression levels can include arithmetic, algorithmic, or mathematical calculations of difference values. In some implementations, difference values ​​provide more complex quantitative information for the analysis, such as the association between the expression levels of certain PTSD-related genes or subsets thereof and the occurrence or likelihood of the target outcomes described herein, such as the identification, monitoring, and / or progression of PTSD. Such calculations...

[0063] As used herein, the term "fragment" can be a portion of a sequence, a subset that may be shorter than the full-length sequence. A fragment can be a portion of a gene. A fragment can be a portion of an oligonucleotide sequence. The length of a fragment can be less than about: 20, 30, 40, or 50 amino acids. A fragment can be about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, or 70% of the total length of the amino acid or nucleotide sequence. The length of a fragment can be less than about: 20, 30, 40, or 50 oligonucleotides. The term "homology" can refer to the percentage of identity between a sequence and a reference sequence. In practical terms, any particular sequence can achieve at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with any sequence described herein.

[0064] As used herein, the phrase “nucleic acid” or “nucleic acid sequence” can refer to oligonucleotides, nucleotides, polynucleotides, or any fragment thereof, as well as DNA or RNA of genomic or synthetic origin, which can be single-stranded or double-stranded, and can represent the sense or antisense strand of any DNA- or RNA-like material. Nucleic acid molecules can be naturally occurring, recombinant, or synthetic. The term “nucleotide” can be further abbreviated as “nt”. As used herein, the abbreviation “NA” can refer to nucleic acids.

[0065] 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 further form dimers, trimers, and higher oligomers, i.e., composed of more than one molecule, whose equimolarities may be the same or different. As used herein, the terms "peptide" and "protein" (where "protein" and "polypeptide" are used interchangeably) can also refer to naturally modified peptides / proteins, wherein the modifications are influenced by, for example, glycosylation, acetylation, phosphorylation, etc.

[0066] As used herein, the phrase “pharmaceuticalally acceptable excipient” can refer to pharmaceutically acceptable materials, compositions, or carriers, such as liquid or solid fillers, diluents, excipients, carriers, solvents, or encapsulating materials.

[0067] As used herein, “post-traumatic stress disorder” can refer to a condition characterized by the development of distinctive symptoms following exposure to a traumatic stressor, such as a direct, personal experience of an event involving actual or threatening death or serious injury, or other event that threatens one’s personal integrity; witnessing an event involving the death, injury, or threat of personal integrity of another person; or learning of an accidental or violent death, serious injury, or threat of death or injury experienced by a family member or other close partner. The individual’s reaction to the event may involve intense fear, helplessness, or panic. In children, the reaction may involve disorganized or agitated behavior. Characteristic symptoms following exposure to extreme trauma may include persistent re-experiencing of the traumatic event, persistent avoidance of trauma-related stimuli and generalized numbness, as well as persistent symptoms of increased arousal. In post-traumatic stress disorder, the stressor can be of an extreme nature.

[0068] As used herein, the term "predisposition for a disease" is established in the field and is used analogously herein.

[0069] As used herein, the term "sample" can refer to a biological sample, such as cells, tissue (from any organ, including post-mortem brain tissue), or fluid (including serum, whole blood, cerebrospinal fluid, lymph, saliva, breast milk, pus, urine, and feces), isolated or obtained from an individual or from a cell culture component containing a cell culture of the subject's cells. Cell-containing tissue or fluid samples obtained from patients and / or subjects can be used to assess the expression levels of one or more genes or proteins.

[0070] As used herein, the term “trauma” can mean directly experiencing an event involving actual or threatening death or serious injury, or other events that threaten one’s personal integrity; witnessing an event involving the death, injury, or threat to the personal integrity of another person; or learning of an accidental or violent death, serious injury, or threat of death or injury experienced by a family member or other close partner.

[0071] As used herein, the terms “treatment,” “treatment course,” or “method of treatment” may include, at least in part: relieving, reducing, or improving symptoms of a disease or condition; preventing additional symptoms; improving or preventing the underlying cause of symptoms; inhibiting a disease or condition, such as at least partially halting its development; alleviating a disease or condition; causing a remission of a disease or condition; alleviating symptoms caused by a disease or condition; or preventing, treating, or both of terminating symptoms of a disease or condition. Treatment may include treating symptoms associated with PTSD, such as reducing anxiety, depression, intense fear, helplessness, or panic, disorganized or agitated behavior, re-experiencing the traumatic event, persistent avoidance of trauma-related stimuli and generalized numbness, and persistent symptoms of increased arousal. In some aspects, the methods described herein may include administering a therapeutically effective amount of PTSD treatment to a subject (which may be a human or animal subject, which may be a mammal).

[0072] PTSD molecular detection assays and methods of use thereof

[0073] This document provides a molecular diagnostic assay for PTSD or its uses. In some embodiments, the molecular diagnostic assay for PTSD can be used in the detection, diagnosis, screening, patient screening, treatment, and / or monitoring methods described herein. In some embodiments, the molecular diagnostic assay for PTSD described herein and its methods of use can detect PTSD in a subject. In some embodiments, the PTSD test for a subject described herein can diagnose PTSD in a subject. In some embodiments, the PTSD test for a subject described herein can increase the likelihood that a subject will be clinically diagnosed with PTSD. In some embodiments, the PTSD test for a subject described herein can predict and / or establish the effectiveness of PTSD treatment in a subject.

[0074] In some embodiments, the PTSD molecular detection analysis described herein measures the expression levels of one or more PTSD-related genes. In some embodiments, the one or more PTSD-related genes are multiple PTSD-related genes, such as the one or more genes listed in Table 1. In some embodiments, the PTSD molecular detection analysis described herein measures the expression levels of PTSD-related genes in a subject to generate test PTSD-related gene expression levels. In some embodiments, the PTSD molecular detection analysis described herein compares the expression levels of the one or more test PTSD-related genes with control gene expression levels. In some embodiments, the comparison of the expression levels of the one or more test PTSD-related genes with the expression levels of the one or more control genes determines the relative expression of the one or more test PTSD-related genes compared to the expression levels of the one or more control genes. Those skilled in the art will understand that the control genes being compared are those corresponding to one or more test PTSD-related genes.

[0075] PTSD-related genes

[0076] In some embodiments, the PTSD molecular detection analysis described herein determines the relative expression levels of one or more PTSD-related genes in a subject (i.e., the expression levels of the one or more tested PTSD-related genes) compared to the expression levels of one or more control genes. In some embodiments, the one or more PTSD-related genes may be any gene associated with the occurrence of PTSD in a subject. In some embodiments, the one or more PTSD-related genes comprise 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 implementations, 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.

[0077] In some implementations, 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, nineteen or more, or at most all of the following, or variants thereof (e.g., splicing variants) or any combination thereof, are measured.

[0078] Table 1 lists exemplary PTSD-related gene mRNA transcripts in DNA form with T replacing U.

[0079] Table 1: Exemplary PTSD-related genes

[0080] SEQ ID NO: Gene mRNA NCBI Accession Number SEQ ID NO: 1 TSPAN5 nucleic acid sequence XM_005262680 SEQ ID NO: 2 HIST1H2AE nucleic acid sequence AY400521 SEQ ID NO: 3 UBE3A nucleic acid sequence KR710362 SEQ ID NO: 4 GPX4 nucleic acid sequence NM_001367832 SEQ ID NO: 5 EPB42 nucleic acid sequence KR711753 SEQ ID NO: 6 SLC4A1 nucleic acid sequence KR711958 SEQ ID NO: 7 NDUFA1 nucleic acid sequence NM_004541 SEQ ID NO: 8 HIST1H1E nucleic acid sequence AF531302 SEQ ID NO: 9 ELOVL7 nucleic acid sequence AB181393 SEQ ID NO: 10 ALAS2 nucleic acid sequence KR710694 SEQ ID NO: 11 COMT nucleic acid sequence CR456422.1 SEQ ID NO: 12 PDZK1IP1 nucleic acid sequence KJ898169 SEQ ID NO: 13 ITGA2B nucleic acid sequence NM_000419 SEQ ID NO: 14 CYP4F3 nucleic acid sequence NM_000896 SEQ ID NO: 15 EPB41L3 nucleic acid sequence KJ902290 SEQ ID NO: 16 PRDM1 nucleic acid sequence NM_001198 SEQ ID NO: 17 RP-11-449P15.1 nucleic acid sequence AC073957 SEQ ID NO: 18 FAS nucleic acid sequence KC863989 SEQ ID NO: 19 TUBB2A nucleic acid sequence NM_001069 SEQ ID NO: 20 CHMP5 nucleic acid sequence KJ906176.1 SEQ ID NO: 21 BOD1L1 nucleic acid sequence NM_148894.3 SEQ ID NO: 22 JAM3 NM_032801.5

[0081] In some embodiments, one or more PTSD-related genes may be any of the genes described in Table 1, consisting of the corresponding nucleic acid sequences or variants thereof (such as splice variants) described in Table 1. 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 described in Table 1 are measured.

[0082] In some cases, gene expression levels can be determined by detecting a portion of the RNA sequence of a gene associated with PTSD to ascertain the level or amount of the entire RNA sequence. The portion detected by sequencing or hybridization techniques 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 values ​​for the RNA sequence. In some cases, contiguous regions within the sequence are detected using sequencing or hybridization techniques. In this case, the sequence length can 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, or any number in between. In some cases, multiple regions within the sequence are detected using sequencing or hybridization techniques. In this case, approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, or more regions within the RNA sequence can be detected.

[0083] In some aspects of this 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-splice RNA (U-RNA), small nucleolar RNA (snoRNA), Piwi-interacting RNA (piRNA), repeat-associated small interfering RNA (rasiRNA), small rDNA-derived RNA (srRNA), transfer RNA-derived small RNA (tsRNA), ribosomal RNA-derived small RNA (rsRNA), large 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 aspects, expression levels are determined by measuring the expression of pretreated mRNA. In some aspects, expression levels are determined by measuring the expression of nucleic acids that interfere with the expression or translation of PTSD-related genes.

[0084] Expression levels may depend on, or be at least roughly proportional to, the number of RNA molecules, peptides, or protein molecules of a given gene, thus allowing for quantitative assessment of gene expression. Depending on the means and methods used for the quantitative measurement, the determined gene expression level can be in the form of various readout parameters, such as the intensity of a radioactive signal (e.g., Northern blotting with a radioactive probe), the intensity of a fluorescent signal (e.g., DNA microarrays), or the mass-to-charge ratio (e.g., mass spectrometry).

[0085] Therefore, in some embodiments, PTSD molecular detection assays and methods of using them include measuring the expression levels of one or more PTSD-associated genes and / or one or more control genes. Gene expression level measurements, as provided herein, include methods based on multinucleotide hybridization analysis, methods based on multinucleotide sequencing, and methods based on proteomics. Exemplary methods known in the art for quantifying RNA expression in samples include Northern blotting and in situ hybridization (Parker & Barnes, Methods in Molecular Biology 106:247-283 (1999)); RNase protection assays (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)). Antibodies that recognize sequence-specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes, can be used. Representative methods for sequencing-based gene expression analysis include sequenced gene expression analysis (SAGE) and gene expression analysis via massively parallel marker sequencing (MPSS). Other methods known in the art may also be used.

[0086] Reverse transcription PCR (RT-PCR)

[0087] Typically, mRNA is isolated from the test sample (such as the biological sample described herein). The starting material may be total RNA isolated from a biological sample (such as a cell sample obtained from a subject, control, or population). Baseline samples from subjects, controls, or populations may be used as baseline controls, if necessary. mRNA may be extracted from tissue samples, such as fresh, frozen (e.g., fresh-frozen), or paraffin-embedded and fixed (e.g., formalin-fixed) samples.

[0088] General methods for mRNA extraction are well known in the art and disclosed in standard molecular biology textbooks, including Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons (1997). Methods for extracting RNA from paraffin-embedded tissues are disclosed, for example, Rupp and Locker, Lab Invest. 56:A67 (1987) and De Andres et al., BioTechniques 18:42044 (1995). Specifically, RNA isolation can be performed using purification kits, buffer sets, and proteases from commercial manufacturers such as Qiagen, according to the manufacturer's instructions. For example, total RNA from cultured cells can be isolated using Qiagen RNeasy microcolumns. Other commercially available RNA isolation kits include MasterPure. TM Whole DNA and RNA purification kits ( The total RNA in tissue samples can be isolated using the RNA Stat-60 (Tel-Test) kit (Madison, WI) and the paraffin block RNA isolation kit (Ambion, Inc.), including methods such as centrifugation via cesium chloride density gradient.

[0089] Samples containing RNA are then reverse transcribed to produce cDNA from the RNA template, which is subsequently amplified exponentially in a PCR reaction. The two most commonly used reverse transcriptases are avian medulloblastoma virus reverse transcriptase (AMV-RT) and Moloney murine leukemia virus reverse transcriptase (MMLV-RT). The reverse transcription step is typically initiated using specific primers, random hexamers, or oligo-dT primers, depending on the specific circumstances and the target of the expression profiling analysis. For example, extracted RNA can be reverse transcribed using the 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 subsequent PCR reactions.

[0090] PCR-based methods use thermostable DNA-dependent DNA polymerases, such as Taq DNA polymerase. For example, PCR typically utilizes the 5'-nuclease activity of Taq or Tth polymerase to hydrolyze hybridization probes bound to their target amplicons, but any enzyme with equivalent 5'-nuclease activity can be used. A typical amplicon of the PCR reaction product is generated using two oligonucleotide primers. A third oligonucleotide or probe can be designed to facilitate the detection of the amplicon nucleotide sequence located between the hybridization sites of the two PCR primers. Probes can be detectably labeled, for example with reporter dyes, and can be further provided as having both fluorescent dyes and quenching fluorescent dyes, such as in… Probe configuration in progress. If using... During the amplification reaction, Taq DNA polymerase cleaves the probe in a template-dependent manner. The resulting probe fragment dissociates in solution, and the signal from the released reporter dye is unaffected by the quenching effect of a second fluorophore. One reporter dye molecule is released for each new molecule synthesized, and the detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data.

[0091] RT-PCR can be performed using commercially available devices, such as high-throughput platforms (piatforras), like the ABiPRISM 7700 Sequence Detection. (Perkin-Elmer-Applied Biosystems, Foster City, CA, USA) or Lightcycler (Roche Molecular Biochemicals, Mannheim, Germany), in some embodiments, the procedure is in It runs on the 480 (Roche Diagnostics) real-time PCR system, which is a microplate-based circulation platform.

[0092] 5-Nuclease analysis data are typically initially represented as threshold cycles (Ct), where fluorescence values ​​are recorded in each cycle, representing the amount of amplified product up to that point in the amplification reaction. Threshold cycles (Ct) are generally described as the point where the fluorescence signal is first recorded as statistically significant. Alternatively, the data can be represented as crossover points (Cp). Cp values ​​are calculated by determining the second derivative of the entire qPCR amplification curve and its maximum value. The Cp value represents the cycle with the highest fluorescence increase and the start of the logarithmic phase of PCR.

[0093] Real-time PCR is compatible with quantitative competitive PCR (where normalization is achieved using internal competitors for each target sequence) and also with quantitative comparative PCR (using normalized genes present in the sample or housekeeping genes for RT-PCR). For further details, see, for example, Held et al., Genome Research 6:986-994 (1996).

[0094] The applicable procedural steps in the methods disclosed herein use fixed, paraffin-embedded tissue as the RNA source. For example, mRNA isolation, purification, primer extension, and amplification can be performed according to methods already 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 representative procedure begins with cutting a section of paraffin-embedded tumor tissue sample approximately 10 μm thick, followed by RNA extraction and removal of proteins and DNA from the RNA-containing sample. After RNA concentration analysis, RNA is reverse transcribed using gene-specific primers, followed by RT-PCR to provide cDNA amplification products.

[0095] Intron-based PCR primer and probe design

[0096] PCR primers and probes can be designed based on the exon or intron sequences present in the mRNA transcript of the target gene. Primer / probe design can be performed using publicly available software, such as DNA BLAT software developed by Kent, WJ, Genome Res. 12(4):656-64 (2002), or by BLAST software (including its variants).

[0097] When necessary or desired, repetitive sequences of a target sequence can be masked to mitigate nonspecific signals. Exemplary tools for this include the RepeatMasker program, available online at Baylor College of Medicine, which screens DNA sequences against a library of repetitive elements and returns query sequences with masked repetitive elements. The masked intron sequences can then be used to design primer and probe sequences using any commercially available or otherwise publicly disclosed primer / probe design kit, 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, Bioinformaiics Methods and Protocols: Methods in Molecular Biology, pp. 365-386 (Humana Press).

[0098] Other factors that can influence PCR primer design include primer length, melting temperature (Tm), G / C content, specificity, complementary primer sequences, and 3' end sequences. Typically, optimal PCR primer lengths are 17–30 bases, containing approximately 20–80% (e.g., approximately 50–60%) G+C bases, and exhibiting a Tm value between 50 and 80°C (e.g., approximately 50–70°C).

[0099] Further guidance on PCR primer and probe design can be found in, for example, 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 PCR, “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), all of which are explicitly incorporated herein by reference.

[0100] Mass system

[0101] In MassARRAY-based methods, such as the exemplary method developed by Sequenom, Inc. (San Diego, CA), after RNA isolation and reverse transcription, a synthetic DNA molecule (competitor) is incorporated into the resulting cDNA. This molecule matches the target cDNA region at all positions except one base and serves as an internal standard. The cDNA / competitor mixture is amplified by PCR and then treated with shrimp alkaline phosphatase (SAP) enzymes, which leads to the dephosphorylation of the remaining nucleotides. After inactivation of the alkaline phosphatase, primer extensions are performed on the PCR products from both the competitor and cDNA, generating unique mass spectrometric signals for both competitor- and cDNA-derived PCR products. After purification, these products are dispensed onto a chip array pre-loaded with the components required for matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. The presence of cDNA in the reaction is then quantified by analyzing the peak area ratios in the generated mass spectra. For further details, see, for example, Ding and Cantor, Proe. Natl. Acad. Sci. USA 100:3059-3064 (2003).

[0102] Other PCR-based methods

[0103] Other PCR-based techniques that can be used in the methods disclosed herein include, for example, Bead Technology (Ilumina, San Diego, CA; Oliphant et al., Discover of Markers for Disease (Supplement to Biotechniques), June 2002; Ferguson et al., Analytical Chemistry 72:5618 (2000)); BeadsArray for Detection of Gene (BADGE), using commercially available LuminexlGO Rapid gene expression analysis was performed using the system and multicolor-coded microspheres (Luminex Corp., Austin, TX) (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)).

[0104] microarray

[0105] The expression levels of target genes or microarrays can also be assessed using microarray technology. In this method, target polynucleotide sequences (including cDNA and oligonucleotides) are arranged on a substrate. The arranged sequences are then contacted with detectably labeled cDNA generated from RNA in the test sample under conditions suitable for specific hybridization. As with RT-PCR, the RNA source is typically total RNA isolated from tumor samples, and, if necessary, isolated from normal tissue from the same patient as an internal control or cell line. RNA can be extracted, for example, from frozen or paraffin-embedded and fixed (e.g., formalin-fixed) tissue samples.

[0106] For example, the PCR amplification insert of the cDNA clone of the gene to be tested is applied to the substrate in a dense array. Typically, at least 10,000 nucleotide sequences are applied to the substrate. For example, microarrayed genes immobilized on a microchip with 10,000 elements each are suitable for hybridization under stringent conditions.

[0107] Fluorescently labeled cDNA probes are generated via reverse transcription of RNA extracted from target tissues, incorporating fluorescent nucleotides. The labeled cDNA probes applied to the chip specifically hybridize to individual sites on the array of DNA. After washing under stringent conditions to remove non-specifically bound probes, the chip is scanned using a confocal laser microscope or another detection method (such as a CCD camera). Quantification of hybridization of each arrayed element allows assessment of the corresponding RNA abundance.

[0108] cDNA probes from two RNA sources, each labeled with a dual-color fluorescent agent, were hybridized in pairs to an array. The relative abundance of transcripts from the two sources corresponding to each specific gene was thus simultaneously determined. The miniaturized scale of the hybridization made the assessment of a large number of gene expression patterns convenient and rapid. Such methods have been shown to have the sensitivity required to detect rare transcripts (which are expressed in a few copies per cell) and to reproducibly detect expression levels that are at least about two-fold different (Schena et al., Proc. Natl. Acad. Sci. USA 93(2):106-149(1996)).

[0109] Microarray analysis can be performed using commercially available devices, following the manufacturer's instructions, such as those using Affymetrix. This is achieved using technology or Incyte's microarray technology.

[0110] Sequential gene expression analysis (SAGE)

[0111] Gene expression serial analysis (SAGE) is a method for the simultaneous quantitative analysis of a large number of gene transcripts without the need for individual hybridization probes for each transcript. First, a short sequence tag (approximately 10 to 14 bp) is generated, containing sufficient information to uniquely identify the transcript, provided that the tag is derived from a unique location within each transcript. Then, many transcripts are linked together to form a long serial molecule, which can be sequenced, simultaneously revealing the identities of multiple tags. The expression patterns 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, for example, Velculescu et al., Science 270:484-487 (1995); and Velculescu et al., Cell 88:243-51 (1997).

[0112] Gene expression was analyzed using nucleic acid sequencing.

[0113] Nucleic acid sequencing technology is a suitable method for gene expression analysis. The basic 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 called digital gene expression (DGE) to reflect the discrete numerical characteristics of the resulting data. Early methods that applied this principle were sequence gene expression analysis (SAGE) and massively parallel labeled sequencing (MPSS), see, for example, S. Brenner et al., Nature Biotechnology 18(6):630-634 (2000). In recent years, the emergence of "next-generation" sequencing technologies has made DGE simpler, higher-throughput, and more economical. As a result, more laboratories can utilize DGE to screen more gene expression in more individual patient samples compared to previous feasibility. See, for example, J. Marioni, Genome Research 18(9):1509-1517 (2008); R. Morin, Genome Research 18(4):610-621 (2008); A. Mortasavi, Nature Methods 5(7):621-628 (2008); N. Cloonan, Nature Methods 5(7):613-619 (2008).

[0114] Methods for isolating RNA for expression analysis from blood, plasma, and serum (see, for example, Enders et al., Clin Chem 48:1647-53 (2002) and references cited therein) and from urine (see, for example, R. Boom et al., J Clin Microbiol. 28:495-503 (1990) and references cited therein) have been described.

[0115] Immunohistochemistry

[0116] Immunohistochemical methods are also suitable for detecting gene expression levels and are applied to the methods disclosed herein. Antibodies that specifically bind to the gene product of the target gene (e.g., monoclonal antibodies) can be used in such methods. Antibodies can be detected by directly labeling the antibody itself, for example, by radiolabeling, fluorescent labeling, hapten labeling (e.g., biotin), or enzyme labeling (e.g., horseradish peroxidase or alkaline phosphatase). Alternatively, unlabeled primary antibodies can be used in combination with labeled secondary antibodies that specifically target the primary antibody. Immunohistochemical protocols and kits are well known and commercially available in the art.

[0117] Proteomics

[0118] A proteome is the sum of proteins present in a sample (e.g., a tissue, organism, or cell culture) at a specific time point. Proteomics, among other things, includes studying the overall changes in protein expression in a sample (also known as "expression proteomics"). Proteomics typically involves the following steps: (1) separating individual proteins in a sample by two-dimensional 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 the data using bioinformatics.

[0119] Control gene expression levels

[0120] In some embodiments, the expression levels of the one or more control genes are determined, for example, by using PTSD molecular detection analysis and / or the methods described herein. In some embodiments, the expression levels of the one or more control genes are determined in advance, for example, by using PTSD molecular detection analysis and / or the methods described herein, or from a pre-existing database.

[0121] In some implementations, the control gene expression level is derived from the baseline control, control, or control population.

[0122] The control or control population can be any suitable comparative control or control population. In some embodiments, the control or control population does not have PTSD. In some embodiments, the control or control population is negative for PTSD, for example, by a clinical diagnostic method 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 the exposure to trauma. In such embodiments where the control or control population does not develop PTSD, such a control or control population may be referred to herein as a negative control or negative control population.

[0123] In some embodiments, the control or control population has PTSD. In some embodiments, the control or control population has PTSD but is not receiving PTSD treatment. In some embodiments, the control or control population has PTSD and is receiving PTSD treatment. In such embodiments where the control or control population has PTSD, such control or control population may be referred to herein as a positive control or positive control population.

[0124] In some implementations, the control or control population is obtained from database entries, which may be negative or positive controls, depending on the selected database entries.

[0125] In some cases, controls are selected from i) controls who have been exposed to trauma but have not developed PTSD; ii) a group of PTSD-negative subjects representative of the study population; and iii) database entries. Alternatively, expression level ranges obtained from a group of PTSD-negative subjects representative of the study population can be used as normal values. Such normal values ​​can be used to standardize data collected from subjects when performing the methods described herein. Those skilled in the art will understand how to select appropriate controls based on the methods performed, the data to be captured, the information to be analyzed, etc.

[0126] In some cases, to improve the reliability of data related to expression levels, samples from control subjects may be evaluated more than once, or multiple samples from said control subjects may be obtained. The data may be further aggregated to calculate the mean or median for each control subject, and variance may be calculated as needed. In some cases, expression levels are compared with the expression levels of the corresponding gene in samples from more than one control subject (e.g., at least 2, 10, 20, or more control subjects). In some cases, expression levels from control subject samples may be aggregated, and a mean or median may be calculated, with variance calculated as needed. In these embodiments, the mean or median expression level may be referred to herein as the mean expression level or median expression level. These values ​​may, for example, be stored in a database as standardized values ​​for each gene and retrieved from the database when needed, thus eliminating the need to experimentally evaluate expression levels in control samples each time expression levels in patient samples are evaluated. Therefore, controls may also be database entries. Furthermore, by using the variance of expression levels in control samples, the statistical significance of the deviation from the control mean in the sample being evaluated can be determined. Finally, age- or sex-specific controls may be used where appropriate.

[0127] In control samples from a database of subjects without PTSD, RNA or protein levels are expressed in specific proportions, contributing synergistically to normal bodily functions. In the absence of endogenous rescue mechanisms to compensate for interference, disruptions to gene expression levels (such as upregulation and / or downregulation of one or more genes) can lead to medical conditions. Several steps in the gene expression process can be modulated to regulate gene expression levels, such as the transcription initiation step involving the number of transcription factors, the presence of enhancer / repressor sequences, the translation step involving the half-life of the mRNA to be translated, and post-translational modifications of proteins that affect protein half-life, secretion, and folding. Gene regulation confers cellular control over structure and function and is fundamental to events such as cellular or multicellular differentiation, morphogenesis, or adaptation.

[0128] In some implementations, control subjects or control populations that test negative for PTSD can be identified using one or more clinical diagnostic classification schemes, such as those described herein. Clinical diagnostic classification schemes for PTSD include: the DSM-5 Clinician-Managed PTSD Scale (CAPS-5), the DSM-5 PTSD Checklist (PCL-5), or both. Similarly, control subjects or control populations that test negative for PTSD may also test negative for one or more PTSD comorbidities, identified using 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.

[0129] DSM-5 Clinician Management of PTSD Scale (CAPS-5)

[0130] The DSM-5 Clinician Management of PTSD Scale (CAPS-5) is a structured interview considered the gold standard for assessing trauma severity and diagnosing PTSD. This interview, conducted by trained clinicians, covers all the criteria for PTSD in the DSM-5. It assesses the frequency and intensity of various PTSD symptoms and their impact on daily functioning.

[0131] To score the structured interviews, assessors combine information related to the frequency and intensity of items 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 severity scores of individual items for the symptoms corresponding to a given DSM-5 cluster: Criterion B (items 1 to 5); Criterion C (items 6 to 7); Criterion D (items 8 to 14); and Criterion E (items 15 to 20). The dissociative symptom cluster score is also calculated by summing items 19 and 20.

[0132] To be diagnosed with PTSD, a patient must have: at least one symptom of criterion B; at least one symptom of criterion C; at least two symptom of criterion D; at least two symptom of criterion E; meet criterion F (interference lasting for one month); and meet criterion G (interference causing clinically significant distress or functional impairment).

[0133] DSM-5 PTSD List

[0134] The DSM-5 or PCL-5 PTSD Checklist is a self-report questionnaire specifically designed to assess PTSD symptoms based on the standards outlined in the DSM-5. It is widely used in clinical and research settings. It takes approximately 5 to 10 minutes to complete.

[0135] The PCL-5 consists of 20 items that measure the severity and frequency of PTSD symptoms, covering four symptom clusters: intrusiveness, avoidance, negative changes in cognition and emotion, and hypervigilance. Individuals who have experienced a traumatic event are asked to rate the degree to which each symptom has bothered them in the past month on a scale of 0 (none at all) to 4 (extremely severe).

[0136] PCL-5 should be interpreted by a clinician. PCL-5 can be scored in different ways:

[0137] The overall symptom severity score (range -0 to 80) can be obtained by summing the scores of each of the 20 items.

[0138] The severity score of a DSM-5 symptom cluster can be obtained by summing the scores of items within a given cluster, such as cluster B (items 1 to 5), cluster C (items 6 to 7), cluster D (items 8 to 14), and cluster E (items 15 to 20).

[0139] A provisional PTSD diagnosis can be made by considering each item rated 2 = “moderate” or higher as an accepted symptom, and then following the DSM-5 diagnostic criteria, which require at least: 1 B item (questions 1 to 5), 1 C item (questions 6 to 7), 2 D items (questions 8 to 14), and 2 E items (questions 15 to 20).

[0140] By summing the scores of the items, an overall severity score for PTSD symptoms can be obtained. The PCL-5 provides a quantitative measurement of PTSD symptomatology and can assist in the assessment, monitoring, and diagnosis of PTSD. It is not intended to provide a formal diagnosis on its own, but can be used as a screening tool or as part of a comprehensive evaluation conducted by a healthcare professional.

[0141] Generalized Anxiety Disorder 7-Item Scale

[0142] The Generalized Anxiety Disorder 7-Item Scale (GAD-7) is a self-report screening tool for generalized anxiety disorder. A GAD-7 score is calculated by assigning scores of 0, 1, 2, and 3 to response categories such as “never,” “a few days,” “more than half the days,” and “almost every day,” respectively, and then summing the scores of all seven items.

[0143] Scores of 5, 10, and 15 are used as cutoff points for mild, moderate, and severe anxiety, respectively. When used as a screening tool, further evaluation is recommended for scores of 10 or higher. Using a threshold score of 10, the GAD-7 has 89% sensitivity and 82% specificity for GAD. It performs well in moderate 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 (PTSD) (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. 2016 Aug 1;67(8):878-82.doi:10.1176 / appi.ps.201500292.

[0144] Alcohol Use Disorder Identification Test

[0145] The Alcohol Use Disorder Identification Test (AUDIT-C) is an alcohol screening test that helps identify dangerous drinkers or patients with active alcohol use disorders, including alcohol abuse or dependence. AUDIT-C uses a scale of 0 to 12 (0 reflects no drinking). In men, a score of 4 or higher is considered positive; in women, a score of 3 or higher is considered positive. Generally, a higher AUDIT-C score indicates a greater likelihood that drinking affects the patient's health and safety.

[0146] Pittsburgh Sleep Quality Index

[0147] The Pittsburgh Sleep Quality Index (PSQI) is a self-report questionnaire that assesses sleep quality and disturbances over a one-month time interval. The PSQI consists of 19 items that measure several different aspects of sleep, providing seven sub-scores and a composite score. The sub-scores are composed of subjective sleep quality, sleep latency (time to fall asleep), sleep duration, habitual sleep efficiency (percentage of time spent in bed), sleep disturbances, use of sleeping pills, and daytime dysfunction.

[0148] Each item is weighted on a scale of 0 to 3. The overall PSQI score is then calculated by summing the scores of the seven sub-items, providing an overall score from 0 to 21, where a lower score indicates healthier sleep quality.

[0149] Adverse childhood experiences (ACE)

[0150] The Adverse Childhood Experiences Questionnaire (ACE-Q) is a 10-item measure to quantify adverse or traumatic experiences a client had before age 18. The ACE-Q examines a client's childhood exposure to psychological, physical, and sexual abuse, as well as family dysfunction, including domestic violence, substance use, and incarceration. The ACE-Q has been used in adverse childhood experiences (ACE) research (Felitti et al., 1998), which found that ACE-Q scores are associated with mental health challenges in later life, as well as health-risk behaviors (including substance abuse) and serious health problems. These include depression, suicide attempts, alcoholism, substance abuse, smoking, 50 or more sexual partners, lack of exercise, severe obesity, increased risk of sexually transmitted infections, fractures, heart disease, lung disease, liver disease, and multiple cancers (Felitti et al., 1998). Positive responses are summed to provide an overall ACE-Q score (out of 10). A score of 4 or higher is considered clinically significant. A small percentage (5% to 10%) of the general population with a score of 4 or higher had the most significant long-term health consequences (Hughes, K. et al. (2017). Lancet Public Health, 2(8), e356–e366).

[0151] Differential expression

[0152] As described herein, in some embodiments, PTSD molecular detection analysis and / or its usage methods include determining differential expression of PTSD-related genes for one or more tests compared to one or more control genes, and determining a PTSD detection result based on said differential expression. In some embodiments, PTSD molecular detection analysis and its usage methods measure the expression levels of PTSD-related genes for one or more tests in samples obtained from subjects, compare the expression levels of said PTSD-related genes for one or more tests with the expression levels of said control genes to determine differential expression of said PTSD-related genes for one or more tests; and determine a positive PTSD detection result based on said differential expression.

[0153] In the context of this disclosure, any suitable method may be used to determine differentially expressed genes. In some embodiments, differential expression can be calculated by standardizing expression data between two experimental conditions (e.g., a group clinically diagnosed with PTSD and a PTSD-negative group) and identifying statistically significant genes associated with the condition (e.g., PTSD). In some embodiments, differential expression can be calculated by gene expression ranking, which involves ranking standardized expression data between two experimental conditions (e.g., a group clinically diagnosed with PTSD and a PTSD-negative group) and identifying statistically significant genes associated with the condition (e.g., PTSD).

[0154] Examples of standardization methods applicable to this disclosure include, but are not limited to, global standardization, Lowess standardization, truncated mean (TMM), quantile standardization, scaling standardization, variance stabilization (VSN), and invariance method (IN). In some embodiments, gene expression can be standardized relative to a set of control genes (including a database or a pre-determined discrete genome).

[0155] In some implementations, gene expression levels can be standardized using normal quantile transformation (NQT). In some implementations, gene expression levels can be standardized using the following NQT: random numbers are drawn from a normal distribution based on the number of observations (e.g., the number of mRNA species) and sorted according to Equation 1. Then, Equations 2 and 3 are used to specify the quantiles for each level. Unless otherwise stated, all equations included herein are R language commands.

[0156] Equation 1:

[0157] rn = sort(rnorm(no_of_mRNAs))

[0158] Equation 2:

[0159] qt = rank(x) / length(x)

[0160] Equation 3:

[0161] nqt = rn[rank(qt)]

[0162] In some implementations, differentially expressed genes associated with PTSD can be identified after standardizing gene expression levels. In the context of this disclosure, any suitable method can be used to identify differentially expressed genes. Examples include regression models, such as linear or logistic regression models. For instance, using regression models (e.g., linear or logistic), quantile-transformed data can be analyzed to identify any mRNA species showing increased or decreased expression. Using NQT data from different time points, the corresponding p-values, intercepts, and slopes can be calculated using equations 4 through 8, and any mRNA with p < 0.05 is considered statistically significant.

[0163] Equation 4:

[0164] lmod=lm(y~x)where x=time,y=NQT da t a

[0165] Equation 5:

[0166] s = summary(lmod)

[0167] Equation 6:

[0168] p=as.character(pf(s$fstatistic[1], s$fstatistic[2],))(s$fstatistic[3], lower.tail=FALSE)

[0169] Equation 7:

[0170] intercept = s$coefficients[1]

[0171] Equation 8:

[0172] slope = s$coefficients[2]

[0173] The discussion of the above analysis 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), which is incorporated herein by reference in its entirety.

[0174] In some implementations, 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, up to all tested PTSD-related genes are tested at a concentration of less than 0.05, 0.01, or 5 × 10⁻⁶. -3 1×10 -3 5×10 -4 1×10 -4 1×10 -5 9.9×10 -6 9.8×10 -6 9.7×10 -6 9.6×10 -6 9.5×10 -6 9.4×10 -6 9.3×10 -6 9.2×10 -6 9.1×10 -6 9×10 -6 8.9×10 -6 8.8×10 -6 8.7×10 -6 8.6×10 -6 8.5×10 -6 8.4×10 -6 8.3×10 -6 8.2×10 -6 8.1×10-6 、8×10 -6 、7.9×10 -6 、7.8×10 -6 、7.7×10 -6 、7.6×10 -6 、7.5×10 -6 、7.4×10 -6 、7.3×10 -6 、7.2×10 -6 、7.1×10 -6 、7×10 -6 、6.9×10 -6 、6.8×10 -6 、6.7×10 -6 、6.6×10 -6 、6.5×10 -6 、6.4×10 -6 、6.3×10 -6 、6.2×10 -6 、6.1×10 -6 、6×10 -6 、5.9×10 -6 、5.8×10 -6 、5.7×10 -6 、5.6×10 -6 、5.5×10 -6 、5.4×10 -6 、5.3×10 -6 、5.2×10 -6 、5.1×10 -6 、5×10 -6 、4.9×10 -6 、4.8×10 -6 、4.7×10 -6 、4.6×10 -6 、4.5×10 -6 、4.4×10 -6 、4.3×10 -6 、4.2×10 -6 、4.1×10 -6 、4×10 -6 、3.9×10 -6 、3.8×10 -6 、3.7×10 -6 、3.6×10 -6 、3.5×10 -6 、3.4×10 -6 、3.3×10 -6 、3.2×10 -6 、3.1×10 -6、3×10 -6 、2.9×10 -6 、2.8×10 -6 、2.7×10 -6 、2.6×10 -6 、2.5×10 -6 、2.4×10 -6 、2.3×10 -6 、2.2×10 -6 、2.1×10 -6 、2×10 -6 、1.9×10 -6 、1.8×10 -6 、1.7×10 -6 、1.6×10 -6 、1.5×10 -6 、1.4×10 -6 、1.3×10 -6 、1.2×10 -6 、1.1×10 -6 、1×10 -6 、9.9×10 -7 、9.8×10 -7 、9.7×10 -7 、9.6×10 -7 、9.5×10 -7 、9.4×10 -7 、9.3×10 -7 、9.2×10 -7 、9.1×10 -7 、9×10 -7 、8.9×10 -7 、8.8×10 -7 、8.7×10 -7 、8.6×10 -7 、8.5×10 -7 、8.4×10 -7 、8.3×10 -7 、8.2×10 -7 、8.1×10 -7 、8×10 -7 、7.9×10 -7 、7.8×10 -7 、7.7×10 -7 、7.6×10 -7 、7.5×10 -7 、7.4×10 -7 、7.3×10 -7 、7.2×10 -7 、7.1×10 -7 、7×10-7 、6.9×10 -7 、6.8×10 -7 、6.7×10 -7 、6.6×10 -7 、6.5×10 -7 、6.4×10 -7 、6.3×10 -7 、6.2×10 -7 、6.1×10 -7 、6×10 -7 、5.9×10 -7 、5.8×10 -7 、5.7×10 -7 、5.6×10 -7 、5.5×10 -7 、5.4×10 -7 、5.3×10 -7 、5.2×10 -7 、5.1×10 -7 、5×10 -7 、4.9×10 -7 、4.8×10 -7 、4.7×10 -7 、4.6×10 -7 、4.5×10 -7 、4.4×10 -7 、4.3×10 -7 、4.2×10 -7 、4.1×10 -7 、4×10 -7 、3.9×10 -7 、3.8×10 -7 、3.7×10 -7 、3.6×10 -7 、3.5×10 -7 、3.4×10 -7 、3.3×10 -7 、3.2×10 -7 、3.1×10 -7 、3×10 -7 、2.9×10 -7 、2.8×10 -7 、2.7×10 -7 、2.6×10 -7 、2.5×10 -7 、2.4×10 -7 、2.3×10 -7 、2.2×10 -7 、2.1×10 -7 、2×10 -71.9×10 -7 1.8×10 -7 1.7×10 -7 1.6×10 -7 1.5×10 -7 1.4×10 -7 1.3×10 -7 1.2×10 -7 1.1×10 -7 Or 1×10 -7 The p-value difference is expressed.

[0175] In some implementations, 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, up to all tested PTSD-related genes are included in a sample size of less than 2.5 × 10⁻⁶. -6 Less than 9×10 -7 or less than 1.6 × 10 -7 The p-value difference is expressed.

[0176] In some implementations, identifying differential expression of PTSD-related genes across one or more tests can confirm the existence of a significant relationship between the PTSD-related genes and PTSD detection. Those skilled in the art will recognize that numerous statistical methods can be used to determine whether a significant relationship exists between a target outcome (e.g., PTSD detection) and the expression levels of marker genes as described herein.

[0177] Similarly, those skilled in the art will recognize that many existing or subsequently developed methods for co-expression analysis fall within the scope and spirit of this disclosure. These methods may include, for example, correlation coefficients, co-expression network analysis, cluster analysis, etc., and are based on expression data from RT-PCR, microarrays, sequencing, and other similar technologies. For example, pairwise correlation analysis based on Pearson or Spearman correlation coefficients can be used to identify gene expression clusters. (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 edition, 2003)).

[0178] Furthermore, numerical values ​​from the PTSD molecular detection analysis and / or methods described herein can be measured and / or compared using algorithms, models, and / or classifiers. Similarly, numerical values ​​from the PTSD molecular detection analysis and / or methods described herein can be used to train algorithms, models, and / or classifiers for the PTSD molecular detection analysis and / or methods described herein. Therefore, in some embodiments, the PTSD molecular detection analysis and methods described herein include algorithms, models, and / or classifiers. Without being intended to limit, the classifiers described herein can be logistic regression classifiers, support vector machine classifiers, or other classifiers. The classifier can be any of the many methods known in the art for classification or regression. The model can be an artificial neural network, a Bayesian graphical model, a Gaussian process, logistic regression, support vector machine, decision tree, hidden Markov model, or k-nearest neighbor. K-fold cross-validation may also be used.

[0179] Model ensembles can be used through enhancement or another ensemble method. The model can be any of a variety of classification or regression methods known in the art. The model can be an artificial neural network, Bayesian graphical model, Gaussian process, logistic regression, support vector machine, decision tree, hidden Markov model, or k-nearest neighbor. K-fold cross-validation can also be used.

[0180] In some embodiments, the accuracy, sensitivity, positive predictive value (PPV), negative predictive value (NPV), area under the curve (AUC), or any combination thereof of the analyses and methods described herein (such as analyses or methods for detecting PTSD) may be greater than about 50%. In some embodiments, the accuracy of the analyses 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 analyses 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 analyses 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 analyses 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 implementations, the AUC of the analyses 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 practice for those skilled in the art.

[0181] In some implementations, identifying differential expression of PTSD-related genes for one or more tests can be used to: predict the likelihood of identifying and / or detecting PTSD in a subject; identify and / or detect PTSD in a subject; predict the likelihood that a subject will be clinically diagnosed with PTSD; diagnose a subject with PTSD; predict the likelihood that a subject will respond to PTSD treatment; determine the effectiveness of PTSD treatment in a subject; or any combination thereof.

[0182] Therefore, the PTSD molecular diagnostic analyses and related information provided by the implementation of the methods disclosed herein contribute to PTSD diagnosis, patient screening for PTSD treatment, and diagnostic and treatment decisions for monitoring the effectiveness of PTSD treatment. For example, such molecular diagnostic analyses will enable primary care providers to identify patients with a high probability of having PTSD, thereby prompting primary care providers to refer these patients to specialists capable of clinically diagnosing PTSD in these patients. Similarly, such molecular diagnostic analyses will enable PTSD treatment specialists to identify patients with a high probability of responding to PTSD treatment. Furthermore, such molecular diagnostic analyses will enable PTSD treatment specialists to monitor patients receiving PTSD treatment by evaluating the effectiveness of PTSD treatment.

[0183] Therefore, in some embodiments, this document provides a method for detecting PTSD in a subject. In some embodiments, a method for detecting PTSD in a subject includes: obtaining a biological sample (e.g., a cell sample) from the subject; analyzing the sample using the PTSD molecular detection analysis described herein to determine differential expression levels of PTSD-related genes for one or more tests; and determining a PTSD detection result based on the differential expression levels of the PTSD-related genes for the one or more tests and the one or more control genes.

[0184] In some implementations, a method for detecting PTSD in a subject includes: obtaining a biological sample (e.g., a cell sample) from the subject; measuring the expression levels of PTSD-related genes for one or more tests in the sample; comparing the expression levels of the PTSD-related genes for the one or more tests with the expression levels of one or more control genes; determining differential expression of the PTSD-related genes for the one or more tests; and determining a positive PTSD test result based on the differential expression.

[0185] In some implementations, a method for detecting PTSD in a subject includes: obtaining a biological sample (e.g., a cell sample) from the subject; measuring the expression levels of PTSD-related genes in the sample for one or more tests using a first computer program executed on a computer; comparing the expression levels of the PTSD-related genes for the one or more tests with the expression levels of one or more control genes using a second computer program executed on a computer; determining differential expression of the PTSD-related genes for the one or more tests; and determining a positive PTSD test result based on the differential expression.

[0186] In some embodiments, the expression levels of one or more control genes are derived from controls or control populations. In some embodiments, the controls or control populations are negative controls or control populations. In some embodiments, the expression levels of one or more control genes 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.

[0187] In some embodiments, PTSD testing of subjects using the methods and analyses described herein can diagnose or indicate that a subject has PTSD. In some embodiments, PTSD diagnosis using the methods, systems, analyses, devices, and kits described herein can be a laboratory diagnosis of PTSD, distinct from and / or supplemental to a clinical diagnosis of PTSD. In some embodiments, PTSD diagnosis using the methods, systems, analyses, devices, and kits described herein can replace a clinical diagnosis of PTSD.

[0188] In some implementations, the detection methods described herein further include determining that a subject should be clinically diagnosed with PTSD. In some implementations, the detection methods described herein also include performing a clinical diagnosis of PTSD on the subject. Performing a clinical diagnosis of PTSD on the subject involves a PTSD diagnosis conducted by a trained behavioral health provider through informal or formal clinical interviews. Formal clinical interviews include published clinical diagnostic criteria for PTSD, including the DSM-5 Clinician Management of PTSD Scale (CAPS-5) and the DSM-5 PTSD Checklist (PCL-5), both of which are described in further detail below.

[0189] In other embodiments, this document provides methods for screening subjects for PTSD. In some embodiments, the methods for screening subjects for PTSD include: testing the subject for PTSD as described herein, and determining, based on a positive PTSD test result, whether the subject should undergo evaluation for a clinical diagnosis of PTSD.

[0190] In some implementations, a method for screening for PTSD includes: obtaining a biological sample (e.g., a cell sample) from a subject; measuring the expression levels of PTSD-related genes for one or more tests; comparing the expression levels of the PTSD-related genes for one or more tests with the expression levels of one or more control genes; identifying differential expression of the PTSD-related genes for one or more tests; determining a positive PTSD test result based on the differential expression; and determining that the subject should undergo clinical evaluation for PTSD based on the subject's positive PTSD test result.

[0191] In some implementations, a method for screening for PTSD includes: obtaining a biological sample (e.g., a cell sample) from a subject; measuring the expression levels of one or more PTSD-related genes using a first computer program executed on a computer; comparing the expression levels of the one or more PTSD-related genes with the expression levels of one or more control genes using a second computer program executed on a computer; identifying differential expression of the PTSD-related genes in the one or more tests; determining a positive PTSD test result based on the differential expression; and determining that the subject should undergo clinical evaluation for PTSD based on the subject's positive PTSD test result.

[0192] In some embodiments, the expression levels of one or more control genes in the PTSD screening method are derived from a control or control population. In some embodiments, the control or control population is a negative control or negative control group. In some embodiments, the PTSD screening method further includes performing a clinical diagnosis of PTSD on the subject using one or more informal or formal interviews. In some embodiments, the formal interview includes the use of clinical diagnostic criteria for PTSD. In some embodiments, the one or more clinical diagnostic criteria for PTSD include CAPS-5 or PCL-5. In some embodiments, the clinical diagnostic criteria for PTSD is CAPS-5. In some embodiments, the clinical diagnostic criteria for PTSD is PCL-5. In some embodiments, the PTSD testing and the clinical diagnosis of PTSD on the subject, as described herein, are performed by the same entity. In some embodiments, the PTSD testing and the clinical diagnosis of PTSD on the subject, as described herein, are performed by two entities, such as a primary care provider and a specialist. In some embodiments, the first computer program is the same as the second computer program. In some embodiments, the first computer program is different from the second computer program.

[0193] In some embodiments, other clinical criteria may be used to assess comorbidities of 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). Therefore, in some embodiments, the methods described herein also include performing an analysis of one or more comorbidities consistent with PTSD in the subject, said comorbidities including: anxiety disorders (e.g., generalized anxiety disorder), substance use disorders (e.g., alcohol use disorder), sleep disorders (e.g., anxiety-related insomnia), past trauma (e.g., childhood trauma), etc. In some embodiments, methods for providing a differential diagnosis of PTSD are provided herein. In some embodiments, the detection of PTSD, the clinical diagnosis of PTSD in the subject, comorbidity assessment, and / or the provision of a differential diagnosis, or any combination thereof, are performed by the same entity or two or more entities.

[0194] In some implementations, the method for screening PTSD also includes screening subjects who meet the criteria for PTSD treatment.

[0195] In some further embodiments, this document provides methods for screening subjects who meet the criteria for PTSD treatment. In some embodiments, the methods for screening subjects who meet the criteria for PTSD treatment include: testing the subject for PTSD as described herein, and determining the subject's eligibility for PTSD treatment based on a positive PTSD test result.

[0196] In some implementations, the method for screening subjects eligible for PTSD treatment includes: obtaining biological samples (e.g., cell samples) from the subject; measuring the expression levels of PTSD-related genes for one or more tests; comparing the expression levels of the PTSD-related genes for one or more tests with the expression levels of one or more control genes; identifying differential expression of the PTSD-related genes for one or more tests; determining a positive PTSD test result based on the differential expression; and determining that the subject is eligible for PTSD treatment based on the subject's positive PTSD test result.

[0197] In some implementations, a method for screening subjects eligible for PTSD treatment includes: obtaining biological samples (e.g., cell samples) from the subject; measuring the expression levels of PTSD-related genes for one or more tests using a first computer program executed on a computer; comparing the expression levels of the PTSD-related genes for the one or more tests with the expression levels of one or more control genes using a second computer program executed on a computer; determining differential expression of the PTSD-related genes for the one or more tests; determining a positive PTSD test result based on the differential expression; and determining that the subject is eligible for PTSD treatment based on the subject's positive PTSD test result.

[0198] In some embodiments, the expression levels of one or more control genes are derived from controls or control populations. In some embodiments, the controls or control populations are negative controls or control populations. In some embodiments, the first computer program is the same as the second computer program. In some embodiments, the first computer program is different from the second computer program.

[0199] In some embodiments, the method for screening subjects eligible for PTSD treatment described herein further includes determining that the subject should undergo a PTSD clinical assessment. In some embodiments, the method for screening subjects eligible for PTSD treatment described herein further includes performing a PTSD clinical diagnosis on the subject. In some embodiments, determining that the subject should undergo a PTSD clinical assessment and / or performing a PTSD clinical diagnosis on the subject may be performed before or after being determined to be eligible for PTSD treatment. For example, in some embodiments, a method for screening subjects eligible for PTSD treatment includes: detecting PTSD in the subject as described herein; wherein, after detecting PTSD in the subject, the method includes determining that the subject should undergo a PTSD clinical assessment; performing a PTSD clinical diagnosis on the subject; wherein, after performing a PTSD clinical diagnosis, the method includes screening the subject for PTSD treatment.

[0200] In some embodiments, the methods described herein also include treating the subject with PTSD treatment methods. Examples of PTSD treatments covered by this 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 combinations thereof), psychodynamic therapy (e.g., brief compromise psychotherapy), medicines or active agents (e.g., selective serotonin reuptake inhibitors (SSRIs), serotonin and norepinephrine reuptake inhibitors (SNRIs), anxiolytics, antidepressants, or combinations thereof, including sertraline, paroxetine, fluoxetine, venlafaxine, ketamine, tricyclic antidepressants, MAOIs, antipsychotics, beta-blockers, benzodiazepines, hallucinogens), comorbidity treatment (e.g., substance abuse treatment), complementary and / or alternative treatments (e.g., acupuncture, yoga, animal-assisted therapy, meditation, or combinations thereof), and any combination thereof. Treatment methods (e.g., medicines formulated as pharmaceutical compositions in one or more forms) will be further described herein.

[0201] In some embodiments, this document provides methods for assessing the effectiveness of PTSD treatment in subjects. In some embodiments, the methods for assessing the effectiveness of PTSD treatment in subjects include: detecting PTSD in the subject as described herein, and determining the effectiveness of the PTSD treatment based on the PTSD detection results.

[0202] In some implementations, methods for assessing the treatment efficacy in subjects receiving PTSD treatment include: obtaining biological samples (e.g., cell samples) from the subjects receiving PTSD treatment; measuring the expression levels of PTSD-related genes in one or more tests; comparing the expression levels of the PTSD-related genes in the one or more tests with the expression levels of one or more control genes; identifying differential expression of the PTSD-related genes in the one or more tests; determining a positive PTSD test result based on the differential expression; and determining the efficacy of PTSD treatment based on the PTSD test result.

[0203] In some implementations, a method for assessing the treatment efficacy in subjects receiving PTSD treatment includes: obtaining biological samples (e.g., cell samples) from the subjects receiving PTSD treatment; measuring the expression levels of one or more PTSD-related genes using a first computer program executed on a computer; comparing the expression levels of the one or more PTSD-related genes using a second computer program executed on a computer with the expression levels of one or more control genes; determining differential expression of the PTSD-related genes using the one or more tests; determining a positive PTSD test result based on the differential expression; and determining the efficacy of PTSD treatment based on the PTSD test result.

[0204] In some implementations, PTSD treatment is effective based on a negative PTSD test result. In some implementations, PTSD treatment is ineffective (i.e., at that time point) based on a positive PTSD test result. In some implementations, the one or more control gene expression levels are one or more control gene expression levels derived from a control or control population. In some implementations, the control or control population is a negative control or negative control group. In some implementations, the one or more control gene expression levels are derived from baseline. In some implementations, the baseline is the subject's state before PTSD treatment. In some implementations, the first computer program is the same as the second computer program. In some implementations, the first computer program is different from the second computer program.

[0205] In some implementations, PTSD test results can be compared before and after PTSD treatment in subjects. Therefore, the subject's PTSD test results can be compared to baseline PTSD test results. In these implementations, the effectiveness of PTSD treatment can be determined relative to the subject's test results before and after treatment.

[0206] In some implementations, PTSD treatment may be an initial PTSD treatment or a PTSD treatment event as part of a series or ongoing PTSD treatment. In some implementations, methods for assessing the effectiveness of PTSD treatment in subjects may be repeated once, more than once, twice, after each or certain PTSD treatment events, or as required by the treatment provider monitoring the effectiveness of PTSD treatment. In some implementations, the methods described herein may be used to provide data to individuals, healthcare providers, or both, which can be used to assess the effectiveness of PTSD treatment over time or at specific time points.

[0207] In some implementations, this document provides a method for determining the prevalence of PTSD in a target population. In some implementations, the method for determining the prevalence of PTSD in a target population includes detecting PTSD in each subject within the target population and determining the prevalence of PTSD in the target population.

[0208] In some implementations, methods for determining the prevalence of PTSD in a target population include: obtaining biological samples (e.g., cell samples) from subjects in the target population; measuring the expression levels of PTSD-related genes for one or more tests in each sample; comparing the expression levels of PTSD-related genes for one or more tests in each sample with the expression levels of one or more control genes; determining the differential expression of PTSD-related genes for the one or more tests; determining the PTSD detection result based on the differential expression in each subject in the target population; and determining the prevalence of PTSD in the target population based on the PTSD detection result in each subject in the target population.

[0209] In some implementations, a method for determining the prevalence of PTSD in a target population includes: obtaining biological samples (e.g., cell samples) from each subject in the target population; measuring the expression levels of PTSD-related genes in each sample for one or more tests using a first computer program executed on a computer; comparing the expression levels of PTSD-related genes in each sample for one or more tests with the expression levels of one or more control genes using a second computer program executed on a computer; determining differential expression of PTSD-related genes for said one or more tests; determining the detection result of PTSD based on the differential expression of each subject in the target population; and determining the prevalence of PTSD in the target population based on the PTSD detection result of each subject in the target population.

[0210] In some implementations, determining the prevalence of PTSD in the target population includes assessing the number of subjects diagnosed with PTSD. In some implementations, the first computer program is the same as the second computer program. In some implementations, the first computer program is different from the second computer program.

[0211] In some embodiments, the expression levels of one or more control genes are derived from controls or control populations. In some embodiments, the controls or control populations are negative controls or control groups. In some embodiments, the target population is a military population or a female population.

[0212] Subjects

[0213] In some respects, subjects may include mammals willing to undergo the PTSD test as described herein. Examples of such mammals may include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.). Mammals may be at any age or developmental stage; for example, mammals may be newborns, infants, adolescents, adults, or in the womb. Mammals may be male or female. In some cases, human ages may be approximately: 1 day to 7 days old, 1 week to 5 weeks old, 1 month to 12 months old, 1 year to 6 years old, 5 years to 15 years old, 14 years to 30 years old, 25 years to 50 years old, 40 years to 75 years old, 70 years to 100 years old, 85 years to 110 years old, or approximately 100 years to 130 years old.

[0214] Participants may be patients, such as those undergoing trauma treatment that may cause PTSD symptoms. In some cases, participants may be at risk of developing a condition or illness (such as PTSD). Participants may be in remission of PTSD. Participants may be healthy.

[0215] In some aspects, the method may further include diagnosing the subject with the said disease. In some aspects, the diagnosis may include the use of in vitro diagnostics. In some aspects, the in vitro diagnostic may be a companion diagnostic. In other cases, the diagnosis may include in vivo diagnostics.

[0216] Diagnostic tests may include imaging procedures, blood cell count analysis, histopathological analysis, biomarker analysis, biopsy, magnetic resonance imaging procedures, physical examination, urine tests, ultrasound examination procedures, genetic testing, liver function tests, positron emission tomography (PET) procedures, X-ray, serology, angiography, electrocardiography, endoscopy, diagnostic polymerase chain reaction (PCR) tests, cervical smear tests, hematocrit tests, skin allergy tests, urine tests, colonoscopy, enzyme-linked immunosorbent assay (ELISA), microscopy, bone marrow examination, rapid diagnostic tests, pregnancy tests, organ function tests, toxicology tests, infectious disease tests, body fluid tests, or any combination thereof.

[0217] In some respects, a diagnosis may include a physical examination, radiological imaging, blood tests, antibody tests, or any combination thereof. In other respects, a diagnosis may include radiological imaging, which may include: computed tomography (CT) images, X-ray images, magnetic resonance imaging (MRI) images, ultrasound images, or any combination thereof.

[0218] ADMINISTRATION AND TREATMENT

[0219] In some respects, this document discloses methods for administering treatments (e.g., pharmaceutical compositions) as described herein to subjects who may require them. In some cases, methods for treating or preventing disease may include administering a drug as described herein. In some embodiments, this document provides a pharmaceutical composition comprising a drug (e.g., PTSD treatment) and a pharmaceutically acceptable carrier, excipient, and / or diluent for treating a subject in need.

[0220] In some respects, participants may be those in need. In other respects, participants may have pre-existing conditions such as post-traumatic stress disorder. Treatment may include reducing feelings of fear, helplessness, disorganized behavior, anxiety, depression, psychosis, panic attacks, substance abuse, suicidal ideation, insomnia, and appetite.

[0221] Drug delivery for the treatment of PTSD may include direct application to the affected tissues or areas of the body. Delivery may include intraparenchymal injection, intrathecal injection, intraventricular injection, or intracisional injection. The compositions provided herein may be administered by any method. Administration methods may include inhalation, intra-arterial injection, intracranial injection, intracisional injection, intramuscular injection, intraorbital injection, intraparenchymal injection, intraperitoneal injection, spinal 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, duodenal administration, or rectal administration. Delivery may include topical application (e.g., lotion, cream, ointment) to an external surface (e.g., skin). In some cases, the composition may be administered to the subject without supervision. In some cases, the composition may be administered to the subject under the supervision of a medical professional (e.g., physician, nurse, physician assistant, service provider, hospice worker, etc.).

[0222] The process of selecting a therapy may include additional steps of applying and monitoring the treatment, and selecting the therapy based on its results. For example, subjects diagnosed as being at risk of PTSD and receiving preventative treatment may be monitored, and, if necessary, their therapy may be adjusted or changed subsequently based on the presence and severity of PTSD symptoms. Furthermore, therapy may be administered to patients with acute PTSD, and the treatment effect may be routinely monitored and recorded. The recorded data provides a basis for assessing whether the administered therapy is beneficial to the patient. Based on this assessment, those skilled in the art (in this case, a clinician) will be able to adjust the currently administered therapy, for example, by increasing / decreasing the dosing regimen or dosage, or decide to switch to another therapy entirely.

[0223] In some respects, a pharmaceutical composition may be administered to a subject in an appropriate unit dose. A pharmaceutical composition may be in unit dose form. In some cases, a unit dose means a pharmaceutical product in its marketed form, having active and inactive ingredients, diluents, or excipients mixed in a specific configuration and dispensed into a specific dose to be delivered. In some cases, a unit dose may also sometimes encompass non-reusable packaging, although the FDA distinguishes between unit dose “packaging” and “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 doses. In some cases, the term unit dose may also sometimes 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 and the substance being delivered. In some respects, 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 respects, the dosing regimen may be determined by the attending physician and clinical factors. In some respects, the dosage for a subject may depend on a number of factors, including the subject's size, body surface area, age, sex, general health condition, the compound to be administered, the time and route of administration, other concurrent medications, or any combination thereof. In some respects, the dosage range may include 0.001 to 1000 μg. In some respects, the dosage may be lower or higher than this range. In some respects, the usual administration regimen of the pharmaceutical composition may be in the range of 1 μg to 10 mg. In some respects, the usual administration regimen of the pharmaceutical composition may be daily, weekly, or monthly. 2 Units up to 10 10Within a range of units. In some aspects, if the regimen involves continuous infusion, it may also be a pharmaceutical composition or engineered polynucleotide or DNA encoding said engineered polynucleotide or a vector containing or encoding said engineered polynucleotide in the range of 1 μg to 10,000 mg per kilogram of body weight per minute. In some cases, the range is from 1 mg to 1000 mg per kilogram of body weight. In some aspects, progress can be monitored by periodic assessment.

[0224] In some aspects of this disclosure, when the pharmaceutical composition is a liquid, it can be administered in liquid dosage form, for example, 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.

[0225] In some respects, the compositions described herein may be administered to a subject in need for one or more days. In some respects, administration may occur for about: 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, or about 31 days. In some respects, administration may occur for about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or about 24 months. In some respects, administration may 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 approximately 50 or more years. In some cases, administration may occur for a lifetime. In some respects, the pharmaceutical compositions described herein may be administered for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more days. In some cases, the compositions described herein may be administered on consecutive or non-consecutive days. In some cases, the compositions described herein may be administered to the subject more than once a day. In some cases, the compositions described herein may be administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more times a day.

[0226] In some aspects, the methods of using the compositions disclosed herein are also disclosed herein. In some aspects, the daily oral dosing regimen may be from about 0.1 mg / kg to about 80 mg / kg, from about 0.2 mg / kg to about 30 mg / kg, or from about 0.5 mg / kg to about 15 mg / kg of total body weight. In some aspects, the daily parenteral dosing regimen may comprise from about 0.1 mg / kg to about 10,000 mg / kg, from about 0.2 mg / kg to about 5,000 mg / kg, or from about 0.5 mg / kg to about 1,000 mg / kg of total body weight. In some aspects, the daily topical dosing regimen may be from about 0.1 mg to about 500 mg. In some aspects, the daily inhalation dosing regimen may be from about 0.01 mg / kg to about 1,000 mg / kg daily. In some aspects, the optimal amount and interval of individual doses of the composition may be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the specific subject being treated, and such optimal amount and interval may preferably be determined by the methods described herein. In some respects, the number of doses of the composition administered daily within a defined number of days can be determined by a test performed using standard treatment procedures by a person skilled in the art. In some respects, the dosing regimen can be determined by the attending physician and other clinical factors. In some respects, the dose for any subject can depend on numerous factors. In some respects, factors influencing the dose may include the subject's size, body surface area, age, the specific compound to be administered, sex, time and route of administration, general health condition, other concurrent medications, or any combination thereof. In some respects, progress can be monitored through periodic assessments.

[0227] The pharmaceutical formulation can be administered orally daily in doses ranging from about 0.1 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 of total body weight. In some aspects, daily parenteral administration may comprise about 0.1 mg / kg 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 of total body weight. In some aspects, daily topical administration may comprise about 0.1 mg to about 500 mg. In some aspects, daily inhalation administration may comprise about 0.01 mg / kg to about 1,000 mg / kg daily. In some aspects, the optimal amount and interval of individual doses of the composition may be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the specific subject being treated, and such optimal amount and interval may preferably be determined by the methods described herein. In some respects, the number of doses of the composition administered daily within a defined number of days can be determined by a test performed using standard treatment procedures by a person skilled in the art. In some respects, the dosing regimen can be determined by the attending physician and other clinical factors. In some respects, dosing in any subject can depend on numerous factors. In some respects, factors influencing dosage may include the subject's size, body surface area, age, the specific compound to be administered, sex, time and route of administration, general health condition, other concurrent medications, or any combination thereof. In some respects, progress can be monitored through periodic assessments.

[0228] SYSTEM

[0229] This document provides a system or its use for PTSD molecular detection analysis as described herein. The system provided herein also includes materials, methods, compositions, kits, devices, or any combination thereof for molecular detection analysis as described herein. For example, the system provided herein includes reagents that may include gene-specific or gene-selective probes and / or primers for quantifying the expression of the disclosed gene to predict prognostic outcomes or response to treatment. Such systems may optionally contain reagents for extracting RNA from a sample (e.g., a paraffin-embedded fixed tissue sample) and / or reagents for RNA amplification. Furthermore, the system may optionally include reagents with identifiable descriptions or labels or instructions relating to their use in the methods of this disclosure. In some embodiments, the system includes a collection container for acquiring biological samples. Such collection containers may be any sample collection device capable of collecting adequate amounts of human blood, tissue, or saliva, including specific cell types such as peripheral blood mononuclear cells (PBMCs), epithelial cells, or other nucleic acid-containing human cell types, from which a total genomic RNA sample can be isolated for testing. Examples of sample collection devices include… Blood RNA tubes (Becton-Dickinson Biosciences, catalog number: 762165), OMNIgene TM • 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 ShieldSafeCollect Saliva Collection Kit (Zymo Research catalog number: R1211), DNA / RNA Shield Blood Collection Tube (Zymo Research catalog number: R1150), SpeciMAX Saliva Collection Kit (ThermoFisher catalog number: A50696), Tempus TM Blood RNA tubes (ThermoFisher, catalog number: 4342792), oral swab collection and stabilization kit (CanVax, catalog number: SWC001) Micro Select (YourBio Health – FDA 510k approved) or a combination thereof.

[0230] The system may comprise containers (including microplates suitable for automated implementation of the method), each containing one or more of a variety of materials or reagents (typically in concentrated form) for the method, including, for example, chromatography columns, pre-fabricated microarrays, buffers, suitable nucleoside 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 this disclosure (e.g., poly(T) or random primers of appropriate length linked to a promoter that reacts with RNA polymerase). Mathematical algorithms for estimating or quantifying prognostic or predictive information are also suitable potential components of the system provided herein.

[0231] The values ​​analyzed above (such as expression data) can be manually calculated and stored. This disclosure also provides systems for performing the methods of this disclosure in whole or in part. Therefore, this disclosure provides a computer program product comprising a computer-readable storage medium containing a computer program. When read by a computer, the program can perform relevant calculations based on values ​​obtained from the analysis of one or more biological samples from an individual (e.g., gene expression levels, standardization, normalization, thresholding, and conversion of the analyzed values ​​into scores and / or textual or graphical descriptions of PTSD progression and related information). The computer program product contains a computer program for performing the calculations.

[0232] The system may include, for example, i) a computer; and ii) a non-transient storage medium containing computer-executable instructions that are executed by the computer and 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-transient storage medium containing computer-executable instructions that are executed by the computer and cause the computer to perform the following steps: a) receiving at least one test gene expression sequence (e.g., an RNA expression sequence) from a tissue sample and b) determining the gene expression level of at least one sequence. In some embodiments, the non-transient storage medium contains computer-executable instructions that are executed by the computer and cause the computer to perform the following steps: c) obtaining a control gene expression level (e.g., from a control) of at least one sequence or an average control gene expression level (e.g., from a control population) of at least one sequence; and d) detecting the relative expression of at least one test gene expression sequence relative to the control gene expression level.

[0233] The system typically also includes an output device that can present the results obtained by the computer during or through the expression step (e.g., in the expression step). The non-transient storage medium may be located on the computer's hard drive, or on a portable device (such as a disk, CD, DVD, USB flash drive, flash drive, laptop, portable computer (e.g., PC or other type)) or other such device. Alternatively, the non-transient storage medium may be located at a remote location or in a database accessible via the Internet, or in a location stored in the cloud, or on or within another computer or computer system accessible through the system's computer. The non-transient storage medium may also include instructions for causing the computer to receive (as input) at least one genomic DNA sequence from a nucleotide sequencing device or from a database. The database may be downloaded from a remote site (e.g., via the Internet) and / or may be located (stored) on a computer, or on another computer or computer system accessible through the system's computer, or even on a portable device as described above. In other respects, data is downloaded from a gene sequencing device, and the system may also include such a device. If present, the device is operatively electronically connected to a computer so that data collected or measured by the expression level testing device can be output and transmitted to and received by the computer (as input).

[0234] The output device can be any suitable type, including but not limited to a printer, a display (such as a monitor that displays the results as a list, chart, or some other suitable format) or a modem that transmits information (such as to another output device, another computer, or a storage device (such as a DVD, CD, etc.)).

[0235] Such systems allow the use of a computer with a non-transient storage medium. The computer is operatively connected to (or functionally connected to, or electrically connected to) an output device. In some cases, the computer is also operatively connected to a nucleic acid sequencing device, and data from the nucleic acid sequencing device (e.g., genomic nucleotide sequences, typically DNA sequences) can be output to and transmitted to the computer and received thereas (as input) for analysis using the methods of this disclosure. In other cases, the computer is operatively connected to a database, and information and / or data can be output from and transmitted to the database and received thereas (as input). The non-transient storage medium may contain computer-executable instructions (e.g., code, computer programs, etc.) that are executed by the computer and cause the computer to perform the steps of the methods described herein.

[0236] DEVICE

[0237] In some embodiments, this document provides a PTSD detection device or its use. In some embodiments, the PTSD detection device includes a PTSD molecular detection assay as described herein. In some embodiments, the PTSD detection device may include any one or more components or materials from the systems, compositions, devices, and / or methods described herein, such as the molecular detection assays provided herein. In some embodiments, the PTSD detection device includes one or more analyses that quantify gene expression levels as described herein. In some embodiments, the PTSD detection device includes a collection container that collects a biological sample from a subject. In some embodiments, the device includes a collection container to obtain a biological sample, wherein the container contains one or more fixatives or preservatives. The collection container may be any collection container capable of collecting an appropriate amount of human blood, tissue, or saliva, including specific cell types such as peripheral blood mononuclear cells (PBMCs), epithelial cells, or other nucleic acid-containing human cell types from which a total genomic RNA sample can be isolated for testing.

[0238] Examples of sample collection containers include Blood RNA tubes (Becton-Dickinson Biosciences, catalog number: 762165), OMNIgene TM• 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 Tube (Zymo Research catalog number: R1150), SpeciMAX Saliva Collection Kit (ThermoFisher catalog number: A50696), Tempus TM Blood RNA tubes (ThermoFisher, catalog number: 4342792), oral swab collection and stabilization kit (CanVax, catalog number: SWC001) Micro Select (YourBio Health – FDA 510k approved) or a combination thereof.

[0239] In some embodiments, a results report is generated after gene expression quantification. In some embodiments, the methods of this disclosure can generate information reports or summaries obtained from the methods described herein. For example, the report may include information and / or other information regarding the expression levels of one or more genes, detection of PTSD or patient recurrence risk, possible prognosis or risk classification of the patient, clinical and pathological factors. The methods and reports of this disclosure may further include storing the report in a database. The methods may create records for subjects in the database and populate the records with data. The report may be a paper report, an auditory report, or an electronic record. 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.). The report is expected to be provided to physicians and / or patients. Receiving the report may further include establishing a network connection with a server computer (which includes data and reports) and requesting the data and reports from the server computer.

[0240] In some implementations, after gene expression quantification, a results report is generated to indicate whether the subject's PTSD screening is positive or negative. A positive PTSD test result requires referral to a specialist care provider for further evaluation and diagnosis.

[0241] KITS

[0242] This disclosure also provides kits for testing PTSD in subjects. The kits provided herein may contain any one or more components or materials from the systems, compositions, devices, and / or methods described herein, such as molecular assays and instructions for use. In some embodiments, the kit includes one or more collection containers. In some embodiments, the kit includes a collection container to obtain a biological sample, wherein the container contains one or more fixatives or preservatives. The collection container may be any collection container capable of collecting an appropriate amount of human blood, tissue, or saliva, including specific cell types such as peripheral blood mononuclear cells (PBMCs), epithelial cells, or other nucleic acid-containing human cell types from which a total genomic RNA sample can be isolated for testing.

[0243] Examples of sample collection containers include Blood RNA tubes (Becton-Dickinson Biosciences, catalog number: 762165), OMNIgene TM • 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 Tube (Zymo Research catalog number: R1150), SpeciMAX Saliva Collection Kit (ThermoFisher catalog number: A50696), Tempus TM Blood RNA tubes (ThermoFisher, catalog number: 4342792), oral swab collection and stabilization kit (CanVax, catalog number: SWC001) Micro Select (YourBio Health – FDA 510k approved) or a combination thereof.

[0244] In some embodiments, the kits provided herein comprise a sample collection container, instructions for the container, and / or consumables, which are mailed to a processing facility for processing by the methods, apparatus, and / or analyses described herein. The processing facility may be any suitable one or more laboratories capable of performing the methods described herein, or utilizing the molecular assays or apparatus described herein.

[0245] In some embodiments, the kits provided herein include a sample collection container and instructions for processing the sample in a point-of-care facility. Point-of-care facilities may be patient care facilities, such as a doctor's office, clinic, or hospital, or any location where the subject is receiving care, such as at home. In some embodiments, the kits provided herein contain all the analytical components required to perform the methods described herein at home. Suitable point-of-care testing systems may be used with the kits described herein and may include, for example, membrane-based test strips.

[0246] In some implementations, the kits provided herein may include a kit shell containing an analytical component operable to detect differentially expressed levels of RNA or protein from a tissue sample of a subject, instructions describing how to use the analytical component for analysis, and a control pattern of RNA or protein expression levels for comparison with differentially expressed levels of RNA or protein.

[0247] Considering the variety of kit casings, it is sufficient to contain analytical components, instructions, and control expression levels from subjects without PTSD. Such kits may contain assemblies of elements required to perform such methods and tests, and any helpful instructions for analyzing the expression levels of genes associated with PTSD or the proteins corresponding to those genes to identify PTSD. In some aspects, the kit casing may contain containers for containing and / or transporting tissue samples.

[0248] In addition, in some aspects, the kit may contain genomic arrays, PCR materials, etc. Furthermore, the analytical components included in the kit can be used to analyze differential RNA expression of any gene associated with PTSD. Non-limiting examples may include real-time PCR, Northern blotting, nucleic acid microarrays, Western blotting, immunoassay, quantitative PCR, etc., including combinations of technologies; and tools for data analysis and / or data interpretation (e.g., computational tools).

[0249] The kit may also include a sample collection device capable of collecting an appropriate amount of human blood, tissue, or saliva, including specific cell types such as peripheral blood mononuclear cells (PBMCs), epithelial cells, or other human cell types, from which a total genomic RNA sample can be isolated for testing. Examples of sample collection devices include... Blood RNA tubes (Becton-Dickinson Biosciences, catalog number: 762165), OMNIgene TM• 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 Tube (Zymo Research catalog number: R1150), SpeciMAX Saliva Collection Kit (ThermoFisher catalog number: A50696), Tempus TM Blood RNA tubes (ThermoFisher, catalog number: 4342792), oral swab collection and stabilization kit (CanVax, catalog number: SWC001) Micro Select (YourBio Health – FDA 510k approved).

[0250] Example

[0251] Example 1: Predicting altered RNA expression levels to identify PTSD

[0252] Blood samples were collected from patients with PTSD and compared with a control group. In this analysis, approximately 2.5 ml of blood was collected from each subject. 1003 unique genes were measured in each run to determine aberrant expression levels.

[0253] First, reverse transcription PCR is performed on RNA from the subject's blood to generate a series of amplicons, or sequences with primer sequences at their 3' and 5' ends. Next, the amplicons are partially digested and ligated to adapters for quantification. The flowchart is shown below. Figure 1 The sequences were run on an Illumina sequencing device. After the sequencing run, raw data were collected to generate FASTQ files. The FASTQ files were compared with a bioinformatics system to generate standardized differential gene expression data.

[0254] Artificial intelligence and machine learning are being used in strategic gene selection, such as Figure 2 As shown.

[0255] Each sample underwent at least five million reads, and the aligned read percentages showed at least 75% accurate mapping back to the human genome.

[0256] After analyzing the expression data, the twenty most predictive genes corresponding to subjects with PTSD were shown. Figure 3 All genes identified as statistically significant had a 2.5 × 10⁻⁶ characteristic value. -6Or a lower P value, such as Figure 3 As shown.

[0257] Example 2: Generation of objective molecular detection analysis for PTSD screening

[0258] This embodiment describes the generation of objective molecular assays for PTSD screening. Blood samples were collected from 87 subjects with PTSD and 141 control subjects (a total of 228 subjects), whose demographics are detailed below. Figure 4 Approximately 2 to 3 ml of blood was collected from each subject. An ADAPT Panel was used. TM RNA expression levels and sequencing were performed on 1003 gene targets. Expression data were analyzed using normal quantile transformation (NQT) as described herein (see Differential Expression section, referencing equations 1–7). Specifically, gene expression levels were normalized, and the following genes were statistically significantly associated with PTSD (Table 2).

[0259] Table 2: Exemplary Integrated IDs for Analyzed Genes

[0260] Integrated ID Gene Symbol ENSG00000004939 SLC4A1 ENSG00000005961 ITGA2B ENSG00000026103 FAS ENSG00000057657 PRDM1 ENSG00000082397 EPB41L3 ENSG00000093010 COMT ENSG00000114062 UBE3A ENSG00000125356 NDUFA1 ENSG00000137267 TUBB2A ENSG00000158578 ALAS2 ENSG00000162366 PDZK1IP1 ENSG00000164181 ELOVL7 ENSG00000166086 JAM3 ENSG00000166947 EPB42 ENSG00000167468 GPX4 ENSG00000168785 TSPAN5 ENSG00000186529 CYP4F3 ENSG00000257607 RP11-449P15.1 ENSG00000277075 HIST1H2AE ENSG00000278828 HIST1H3H

[0261] Molecular detection analysis confirms its sensitivity, or the percentage of subjects correctly identified with the disorder, is 90%. Figure 5 Test sensitivity is the most important attribute when a test is used for screening purposes; generally, researchers in the field consider a sensitivity of >80% to be sufficient. Current surveys, such as those described herein, have sensitivity ranging from 77% to 91% in study scenarios with voluntary participation; however, in practice, this can be biased in either direction due to respondents' motivation or fear. The analysis also confirmed the specificity of the molecular assay, or the percentage of subjects correctly identified without the disorder, at 73%. Figure 5 The molecular detection analysis was found to be more specific than most screening tests currently in use.

[0262] Promising results were shown in previous molecular assays using the same gene but with different thresholds, achieving an area under the receiver operating characteristic (AUC) of 85%. Figure 6 ).

[0263] The results confirm the generation of consistent, reliable, and objective molecular assays that indicate whether a subject is expected to have PTSD, free from self-reporting bias.

[0264] Example 3: Molecular detection analysis verification and calibration

[0265] The performance of molecular detection analyses needs to be validated on larger, independent datasets to provide additional evidence of their usefulness. This requires a larger biobank with diverse populations to further train and test molecular detection analyses, thereby enhancing their reliability and versatility.

[0266] Example 3.1: Validation relative to industry standard psychological assessment.

[0267] This embodiment describes the efforts to be made to validate this molecular assay against clinically accepted PTSD-related psychological assessments. The primary assessment relevant to this embodiment is the DSM-5 Clinician Management of PTSD Scale (CAPS-5). CAPS-5 is considered the “gold standard” for diagnosing PTSD and is a structured interview. Another primary assessment is the DSM-5 PTSD Checklist (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).

[0268] Example 3.1.1. Study. Five hundred participants diagnosed with PTSD and five hundred control participants were recruited. Participants diagnosed with PTSD were individuals whose CAPS-5 scores met the diagnostic criteria for PTSD, while control participants were individuals whose CAPS-5 scores did not meet the diagnostic criteria for PTSD. Each participant underwent PTSD screening using this RNA screening analysis conducted by this molecular assay and a psychometric analysis using an accredited psychological assessment. For RNA assessment, approximately 2.5 mL of blood was collected, and the RNA expression levels of genes (including those listed in Table 2) were assessed. For psychometric analysis, participants were analyzed using the standard protocol for the corresponding psychological assessment. A schematic diagram of the above workflow can be found in [link to diagram]. Figure 7 .

[0269] Example 3.1.2. Data reprocessing. The ADAPT Panel runs on the NGS platform, outputting FASQ files. The expressed data will be analyzed using NQT as described in this document. Specifically, data preprocessing involves the following steps:

[0270] Quality control

[0271] Data cleaning

[0272] ·standardization

[0273] Feature selection

[0274] • Integration of genomic and clinical data

[0275] Batch effect removal

[0276] Dimensional reduction

[0277] Data scaling

[0278] Cross-validation, and

[0279] • Verification and reproducibility

[0280] As a first step in preprocessing, the collected genomic and clinical data will undergo quality control (QC) checks. This will ensure the reliability and integrity of the data by identifying and addressing issues such as missing values, outliers, batch effects, or any other anomalies.

[0281] Any data errors, missing values, or outliers identified during the QC process will be addressed through data cleaning procedures. Missing values ​​will be filled using imputation, or data points with serious issues will be excluded from further analysis (if applicable).

[0282] After data cleaning, data standardization will be performed to remove technical variability while preserving biological significance. In this study, Normal Quantile Transformation (NQT) was chosen as the standardization method. NQT is a sorting-based procedure that scales data within each quantile individually, and it is known for its reduced bias and robustness compared to other standardization methods used for microRNA sequencing data.

[0283] Feature selection is crucial for dimensionality reduction and for choosing the most relevant genomic features (e.g., gene or microRNA species) for predictive models. Various methods, such as statistical tests, correlation analysis, and machine learning algorithms, can be used to identify the most informative features associated with PTSD.

[0284] The study will include both genomic and clinical data. Therefore, these two data types will be integrated for comprehensive analysis. This step involves mapping clinical variables to corresponding genomic samples and ensuring data consistency.

[0285] Batch effect removal techniques will be applied to account for systematic variation between batches. This will ensure that observed effects are not confounded by batch-specific variations.

[0286] When dealing with a large number of features, dimensionality reduction techniques (such as principal component analysis (PCA) or t-distributed random nearest neighbor embedding (t-SNE)) can be applied to reduce the complexity of the data while preserving important patterns and structures.

[0287] For some machine learning algorithms, data may need to be scaled to ensure that all features have a similar range. Common scaling methods include standardization (scaling by the mean and standard deviation) or min-max scaling (scaling to a predetermined range).

[0288] To accurately evaluate the performance of a predictive model, cross-validation techniques (such as k-fold cross-validation) can be used to divide the data into subsets to iteratively train and test the model.

[0289] After preprocessing, the model's performance should be validated using independent datasets to ensure the generalizability and reproducibility of the results. NQT, as described in 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.), was used. Custom workflows were written in R. Furthermore, all analyses were performed using R statistical software (v4.1.2; R Core Team 2021).

[0290] Example 3.1.3. Evaluation. After generating the initial molecular detection analysis, the model components of the molecular detection analysis will be cross-validated as described below, and evaluated and / or adjusted based on one or more of the following tests as needed.

[0291] Cross-validation: Cross-validation is performed to assess the robustness and generalizability of the molecular detection analysis to new data. Cross-validation is a widely used technique for evaluating the performance of predictive molecular detection analyses while mitigating potential overfitting. Here, the algorithm's performance is validated using k-fold cross-validation. The dataset is randomly divided into k subsets (folds), where k-1 subsets are used to train the molecular detection analysis, and the remaining subsets are used for testing. This process is repeated k times, each time using a different fold as the test set. The performance metrics of all folds (e.g., accuracy, AUC, precision, recall) are averaged to obtain an overall performance estimate of the molecular detection analysis.

[0292] Validation with an independent test set: In addition to cross-validation, a separate test set will be reserved from the collected data. This test set will be completely separate from the data used for training and cross-validation. Once the molecular detection analysis is trained using the cross-validation process, it will be evaluated on the independent test set to assess its general performance on new, unseen data. The performance metrics obtained from this evaluation will provide a more realistic indication of the algorithm's ability to predict PTSD in real-world scenarios.

[0293] Stratified sampling: To ensure that both the training and test sets represent the overall data distribution, stratified sampling will be used. Stratified sampling maintains the same class distribution (e.g., controls and PTSD cases) in both the training and test sets, ensuring that molecular analysis is not biased towards the majority class.

[0294] Performance metrics: The algorithm's performance will be evaluated using various performance metrics, such as accuracy, sensitivity (recall), specificity, precision, and area under the receiver operating characteristic (AUC). These metrics provide a comprehensive assessment of how well the molecular detection analysis classifies individuals with and without PTSD.

[0295] Overfitting detection and model selection: Cross-validation can be used to detect overfitting, i.e., a model that performs well on training data but poorly on unseen data. If significant overfitting is detected, the model hyperparameters can be adjusted or the feature selection method can be improved to enhance generalizability.

[0296] Repeated cross-validation (as needed): When the dataset is relatively small, repeated cross-validation can be performed to obtain a more robust performance estimate. This technique involves randomly shuffling the data and running cross-validation multiple times to calculate the average performance metric.

[0297] Example 3.1.4. Model performance indicators. The model performance metrics of the molecular detection and analysis model components are shown in Table 3 below.

[0298] Table 3: Performance Metrics of Exemplary Models

[0299]

[0300] Example 3.1.5. Statistical analysis plan. The following describes how the data obtained by performing Example 3.1.1 will be analyzed.

[0301] Key endpoints. The performance metrics described in Table 3 above will be calculated based on the model’s predictions and the true labels (PTSD cases and controls) in both cross-validation and independent test sets.

[0302] Hypothesis testing. Each gene will be tested individually in a panel, with the null hypothesis being that the gene expression in the control group is equal to that in the cases. A large effect contribution is not expected, but testing is necessary.

[0303] Receiver Operating Characteristic (ROC) curve analysis. ROC curve analysis will be used to visualize the trade-off between sensitivity and specificity at different classification thresholds. The area under the ROC curve (AUC) will be calculated to compare the discriminative power of the algorithm with that of existing screening methods. A higher AUC indicates better discriminative power.

[0304] Confidence intervals. To estimate the uncertainty of performance metrics, confidence intervals can be calculated. A confidence interval provides a range of values ​​within which the algorithm's true performance is likely to fall.

[0305] Power analysis. If the study involves comparing the performance of an algorithm with existing methods and is interested in a specific effect size, a power analysis can be performed to determine the sample size required to achieve sufficient statistical power.

[0306] Model calibration. Model calibration refers to the consistency between the predicted probabilities generated by the algorithm and the observed frequency of positive cases. Calibration plots can be created to evaluate the model's calibration and help determine whether the predicted probabilities are accurate.

[0307] Subgroup analysis. If specific target subgroups exist (e.g., different age groups, genders, trauma types), subgroup analysis can be performed to evaluate the algorithm's performance within these subgroups. This analysis can provide insights into the potential variations in algorithm performance based on different participant characteristics.

[0308] Comparison of sensitivity and specificity. If existing screening methods have different trade-offs in sensitivity and specificity, receiver operating characteristic (ROCC) curves can be used to visualize and compare the performance of the two methods.

[0309] Statistical significance. Appropriate statistical tests will be used to determine any observed statistical significance differences in performance metrics between the algorithm and existing screening methods.

[0310] Example 3.1.6. Potential confounding and bias. The following describes how potential confounding and bias will be handled.

[0311] Demographic factors. Participants' demographic characteristics (such as age, sex, race, socioeconomic status, and education level) may influence 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 predictive model can be performed.

[0312] Trauma type. The types of traumatic events experienced by participants can vary significantly and may affect gene expression profiles in different ways. Interpreting trauma type as a potential confounding factor helps to isolate the specific effects of gene expression on PTSD risk.

[0313] Medication use. Certain medications (such as antidepressants or anxiolytics) may affect gene expression levels and potentially influence PTSD outcomes. Participants' medication use history should be collected and controlled for in the analysis.

[0314] Comorbidities. PTSD often occurs concurrently with other mental health disorders, such as depression or anxiety. These comorbidities may affect gene expression patterns and may confound associations with PTSD. In this analysis, comorbidities should be carefully assessed and considered.

[0315] Sample selection bias. If the recruitment of participants does not represent the broader population of individuals with PTSD, the results may not be universally applicable. To minimize sample selection bias, diverse and inclusive recruitment strategies should be employed.

[0316] Batch effects. If gene expression data are collected in multiple batches, batch effects may introduce systematic variation independent of PTSD status. Appropriate statistical methods (such as batch effect correction techniques) can be applied to address this issue.

[0317] Measurement error. Technical errors may occur during laboratory processing in RNA gene expression measurements, leading to measurement noise. Quality control procedures and standardized methods should be applied to minimize measurement error.

[0318] Data imputation bias. In cases of missing data, imputation methods may introduce bias into the results. It is essential to select an appropriate imputation method and evaluate the sensitivity of the results to different imputation methods.

[0319] Overfitting occurs when a predictive model performs well on training data but poorly on new, unseen data. Regularization techniques and appropriate validation methods (like cross-validation) can help prevent overfitting and ensure the model's generalizability.

[0320] Publication bias. If studies with positive or significant results are more likely to be published, publication bias may occur, leading to an overestimation of the predictive model's performance. To mitigate publication bias, researchers should consider pre-registering studies and analyzing all relevant results, regardless of their statistical significance.

[0321] Example 3.1.7. Results.It is hypothesized that this molecular assay can achieve a sensitivity greater than 90% across a range of trauma types, while maintaining a specificity greater than 75%, a negative predictive value (NPV) greater than 95%, and a positive predictive value (PPV) greater than 50%. These characteristics are ideal as a screening tool, as they mean that individuals with PTSD can be correctly identified and referred for treatment immediately. With current tools, to achieve this level of sensitivity (>90%), the specificity, or the ability to correctly exclude the disease in those who do not have PTSD, drops below 50%. Current tools rely on accurate reporting, which is subject to reporter bias and may even be unattainable (due to the repression of painful memories) or self-modified by conscious and / or unconscious fear, worry, and / or shame. This may explain the significant variation in the accuracy metrics of current self-reporting tools across different trauma types, as some trauma types are more associated with social stigma. This molecular assay is currently the first and only available tool based on objective measurements to achieve the aforementioned accuracy metrics.

[0322] Unbound by theoretical constraints, this molecular detection analysis is hypothesized to achieve sensitivity and / or specificity comparable to one or more psychological assessments, and, if needed, enhanced sensitivity and / or specificity relative to one or more psychological assessments. Therefore, this molecular detection analysis can be used to detect the incidence of PTSD, screen subjects suitable for PTSD treatment, and / or monitor the progression of PTSD in subjects undergoing treatment. Furthermore, it is hypothesized that this molecular detection analysis will provide genetic targets for the development of novel precision therapies related to PTSD treatment.

[0323] Example 3.2: Using molecular detection analysis to establish subject screening for PTSD treatment.

[0324] This embodiment describes the efforts to be made to establish and / or calibrate molecular assays to determine whether a subject is a suitable candidate for PTSD treatment.

[0325] The psychological assessment group described in Example 3.1 was treated according to industry standards (e.g., stellate ganglion block, trauma informed communication therapy, ketamine infusion alone or in combination with the aforementioned therapies). Data will be collected by comparing the results of a first screening conducted by a trained provider before treatment (e.g., a CAPS-5 interview) with a second screening conducted 30+ / - 3 days after treatment (e.g., a PCL-5 or informal interview).

[0326] Unbound by theory, it is hypothesized that the collected data can be used to train molecular detection and analysis to predict subjects’ responsiveness to treatment modalities, and thus screen subjects for PTSD treatment.

[0327] Example 3.3: Using molecular detection analysis to establish PTSD treatment responsiveness.

[0328] This embodiment describes the efforts to be made to generate molecular assays and / or calibrate these molecular assays to determine whether a subject is responding to PTSD treatment relative to clinically accepted PTSD-related psychological assessments.

[0329] The group described in Example 3.1 will be treated according to industry standards. Subjects to be treated will be screened based on molecular diagnostic analyses described in Example 3.2, if necessary.

[0330] RNA expression levels were collected and analyzed periodically (e.g., every 2 to 3 months, quarterly, semi-annually, and / or as needed), as described in Example 3.1. Subjects with PTSD who showed an improved RNA expression pattern relative to baseline levels were identified as responsive to PTSD treatment. Without being bound by theory, it is hypothesized that the collected data could be used to train molecular detection analyses to monitor subject responsiveness to treatment modalities.

[0331] Example 4: PTSD detection device or kit

[0332] Example 4.1: PTSD detection device.

[0333] This embodiment describes a PTSD detection device. The PTSD detection device includes a molecular genomics assay that uses quantitative gene expression detection to identify clinically relevant changes in RNA expression isolated from human blood collected from individuals aged 18 years and older using an FDA-approved collection device (such as an RNA PAXgene tube) or any other suitable sample collection container (as described in the Devices section of this document). The molecular genomics assay may include an ADAPT Panel (which uses quantitative next-generation sequencing (NGS)) or any other molecular genomics assay described herein and measures the expression levels of gene transcripts (including those listed in Table 2). The device is calibrated to measure changes in a transcriptional pattern relative to predetermined threshold levels using the molecular assays described herein. After quantifying the transcriptional pattern, a results report is generated to indicate whether the subject's PTSD screening result is positive or negative. A positive PTSD test result requires referral to a specialist care provider for further evaluation and diagnosis.

[0334] Example 4.2: PTSD detection kit.

[0335] This embodiment describes a PTSD detection device. The PTSD detection kit includes any of the components or combinations of components described in Example 4.1, along with instructions for detecting PTSD.

[0336] Example 5: Uses in diagnosis, patient screening, and / or treatment

[0337] Example 5.1: Diagnosis.

[0338] This embodiment describes the use of the molecular assay, device, and / or kit in PTSD diagnostic methods. The molecular assay, as described in Example 2 or 3, the device, or the kit as described in Example 4.1, is used as a screening tool for PTSD. A positive PTSD test result requires referral to a specialist care provider for further evaluation and diagnosis.

[0339] Example 5.2: Patient screening.

[0340] This embodiment describes the use of the molecular assay, device, and / or kit in a method for screening subjects for PTSD treatment. The molecular assay, as described in Example 2 or 3, the device, or the kit, as described in Example 4.1, is used as a screening tool to determine whether a subject is responding to PTSD treatment (or specific PTSD treatment as needed).

[0341] Example 5.3: Treatment.

[0342] This embodiment describes the use of the molecular detection analysis, device, and / or kit in the treatment of PTSD. After a subject is diagnosed with PTSD (including by using the method described in Example 5.1), and after screening (including by using the method described in Example 5.2) the subject is treated with a therapeutically effective treatment or a clinically recognized and / or approved treatment. The molecular detection analysis as described in Example 2 or 3, the device as described in Example 4.1, or the kit as described in Example 4.1 is used to monitor the subject's responsiveness to treatment and / or to monitor the progression of the disease in a treated subject.

[0343] While preferred aspects of this disclosure are shown and described herein, it will be apparent to those skilled in the art that such aspects are provided by way of example only. Many variations, alterations, and substitutions will now occur to those skilled in the art without departing from this disclosure. It should be understood that various alternatives to the aspects of this disclosure described herein can be used to practice the methods presented herein. The following claims are intended to define the scope of this disclosure and should cover the methods and structures within the scope of these claims and their equivalents.

Claims

1. A method for detecting post-traumatic stress disorder (PTSD) in human subjects, the method comprising: a) Obtaining cell samples from human subjects; b) Measure the 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 were 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) Using a second computer program executed on a computer, the PTSD-related expression level of the test is compared with the expression levels of one or more control genes; d) Determine the differential expression of PTSD-related genes for one or more of the tests; and e) Determine the positive detection result of PTSD based on the differential expression.

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

3. The method according to claim 1, wherein, The expression levels of the test genes and / or the control genes are the expression levels of 4 or more, 5 or more, or 6 or more PTSD-related genes.

4. The method according to claim 1, wherein, The expression levels of the test genes and / or the control genes are the expression levels of 2 or more, 3 or more, 4 or more, 5 or more, 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 all 20 PTSD-related genes.

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

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

7. The method according to any one of claims 1 to 6, wherein, The expression levels of PTSD-related genes tested were less than 2.5 × 10⁻⁶. -6 The p-value difference is expressed.

8. The method according to any one of claims 1 to 6, wherein, The expression levels of PTSD-related genes tested were less than 9 × 10⁻⁶. -7 The p-value difference is expressed.

9. The method according to any one of claims 1 to 6, wherein, The expression levels of PTSD-related genes tested were less than 1.6 × 10⁻⁶. -7 The p-value difference is expressed.

10. The method according to any one of claims 1 to 9, wherein, The cell samples are obtained from blood samples, saliva samples, oral smear samples, cerebrospinal fluid samples, saliva, skin, cerebrospinal fluid, or any combination thereof.

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

12. The method according to any one of claims 1 to 11, wherein, The expression level is assessed at the transcriptional level.

13. The method according to any one of claims 1 to 12, wherein, The level of expression is assessed at the translational level.

14. The method according to any one of claims 1 to 13, wherein, The first computer program is the same as the second computer program.

15. The method according to any one of claims 1 to 13, wherein, The first computer program is different from the second computer program.

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

17. The method according to claim 16, wherein, The method can be performed once a week, once a month, once every two months, three times a year, four times a year, twice a year, or once a year.

18. The method according to any one of claims 1 to 17, further comprising the step of treating the subject's PTSD.

19. The method according to claim 18, wherein, Treatment of the subject includes administering drug therapy, psychotherapy, or a combination thereof to the subject.

20. The method of claim 16, wherein, The subjects were treated with medication.

21. The method according to claim 17, wherein, The drug treatment is SSRI, tricyclic antidepressants, MAOI, antipsychotics, beta-blockers, benzodiazepines, hallucinogens, or combinations thereof.

22. The method according to claim 20, wherein, The drug treatment is the SSRI.

23. The method according to claim 20, wherein, The drug treatment is the tricyclic antidepressant.

24. The method of claim 20, wherein, The drug treatment is the MAOI.

25. The method according to claim 20, wherein, The drug treatment is the aforementioned antipsychotic drug.

26. The method of claim 20, wherein, The drug treatment is a β-blocker.

27. The method of claim 20, wherein, The drug treatment is the benzodiazepine drug.

28. The method according to claim 20, wherein, The drug treatment is the hallucinogen.

29. A method for screening a subject for PTSD, the method comprising: a. Detecting PTSD in a subject, comprising the method according to any one of claims 1 to 28; and b. Based on the subject's positive PTSD test result, determine that the subject should be clinically diagnosed with PTSD.

30. The method according to claim 29, wherein, The control group consisted of people who were PTSD-negative.

31. The method according to claim 29 or 30, wherein, The method also includes clinically diagnosing the subject with PTSD using one or more clinical PTSD diagnostic criteria.

32. The method according to claim 31, wherein, The clinical diagnostic criteria for PTSD are CAPS-5 or PCL-5.

33. The method according to claim 32, wherein, The clinical diagnostic criteria for PTSD are CAPS-5.

34. The method according to claim 32, wherein, The clinical diagnostic criteria for PTSD are PCL-5.

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

36. The method according to any one of claims 29 to 35, wherein, The method also includes assessing one or more comorbidities associated with PTSD.

37. The method of claim 36, wherein, The one or more comorbidities include generalized anxiety disorder, alcohol use disorder, anxiety-related insomnia, childhood trauma, or any combination thereof.

38. The method according to any one of claims 36 to 37, wherein, The one or more comorbidities are diagnosed using one or more clinical criteria, which 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.

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

40. A method for screening subjects who meet the criteria for PTSD treatment, the method comprising: a. Detecting PTSD in a subject, comprising the method according to any one of claims 1 to 28; and b. Based on the subject's positive PTSD test result, determine that the subject meets the criteria for PTSD treatment.

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

42. A method for evaluating the efficacy of treatment in subjects receiving PTSD therapy, the method comprising: a. Detecting PTSD in a subject, comprising the method according to any one of claims 1 to 28; and b. Based on the PTSD test results of the subjects, determine the efficacy of the PTSD treatment, wherein, A negative PTSD test result indicates that the PTSD treatment is effective.

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

44. The method according to any one of claims 42 to 43, wherein, The treatment of PTSD includes 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 compromise psychotherapy, selective serotonin reuptake inhibitors (SSRIs), serotonin and norepinephrine reuptake inhibitors (SNRIs), anxiolytics, antidepressants, sertraline, paroxetine, fluoxetine, venlafaxine, ketamine, tricyclic antidepressants, MAOIs, antipsychotics, beta-blockers, benzodiazepines, hallucinogens, comorbidity treatment, complementary and / or alternative therapies (including acupuncture, yoga, animal-assisted therapy, meditation) or any combination thereof.

45. The method according to any one of claims 42 to 44, wherein, The method includes detecting the subject's PTSD before treatment, and includes the method according to any one of claims 1 to 28.

46. ​​The method according to any one of claims 42 to 45, wherein, The method includes screening the subject for PTSD prior to treatment, and includes the method according to any one of claims 29 to 39.

47. The method according to any one of claims 42 to 46, wherein, The method includes screening subjects who meet the PTSD treatment criteria before treatment, and includes the method according to any one of claims 40 to 41.

48. A method for determining the prevalence of PTSD in a target population, the method comprising: a. Detecting PTSD in subjects within a target population, comprising the method according to any one of claims 1 to 28; and b. Based on the PTSD positive test results of each subject in the target population, determine the prevalence of PTSD in the target population.

49. The method according to claim 38, wherein, The group in question is a military personnel.

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