RNA editing as a biomarker for mood disorders

Abstract

The present invention relates to an in vitro method for predicting the risk of a patient to develop a pathology, such as a psychiatric disorder, associated with altered A-to-I editing on the PDE8A transcript from a body fluid, such as a blood sample, urine sample, or saliva sample from the patient, or a method for determining whether the patient is at risk of developing the pathology. The present invention also relates to a kit for carrying out the method.

Description

[Technical field]

[0001] The present invention relates to a method for in vitro prediction of the risk of a patient to develop a pathology, such as a psychiatric disorder, associated with altered A-to-I editing on the PDE8A transcript, or for determining whether a patient is at risk of developing said pathology, which prediction or discrimination method is performed from a body fluid, such as a blood, urine or saliva sample from said patient.The present invention also relates to a kit for carrying out said method. [Background technology]

[0002] Suicide and suicidal behavior are major global public health issues, with 1 million reported suicides in most Western countries and a predicted increase to 1.5 million by 2020. Today, biological prevention of suicide risk has not been proven effective, and clinical assessments do not adequately predict the occurrence of suicidal behavior in a given patient. Furthermore, pharmacological treatment of psychiatric disorders has shown limited effectiveness in reducing rates of attempted and completed suicide. There is an urgent public health need to develop new strategies and promote preventive measures for those at highest risk of suicide. To achieve this goal, suicide prevention strategies require identification of at-risk individuals.

[0003] Among the many factors involved in the decision to commit suicidal behavior, several recent advances in molecular genetics and genomics have revealed that many factors may contribute to susceptibility to suicidal behavior. Potential genetic predispositions that interact with environmental and epigenetic factors to modify the function of neural circuits throughout life likely mediate this susceptibility [1-3]. RNA editing of receptors and other associated proteins expressed in the synaptic cleft has been shown to be involved in the pathogenesis of various psychiatric disorders and to be associated with suicidal behavior [4-7]. Recently, Simmons and colleagues reported that mRNA expression of Adenosine Deaminase Acting on RNA (ADAR1), an RNA-editing enzyme 1, was increased in the cerebral cortex of depressed suicide victims [8]. Phosphodiesterase 8A (PDE8A), a target of ADARs, is expressed in both brain and blood tissues and is edited by ADARs in humans [9-11]. Although changes in the editing profile of PDE8A pre-mRNA can be quantitatively assessed from total RNA extracts of human tissues, including the brain, it is practically more convenient to assess them from RNA in blood samples, which can be used to monitor the patient's condition. Based on these observations, the aim is to identify body fluid biomarkers that determine disease-specific molecular signatures. The application of such body fluid tests will open up new possibilities for the assessment of the primary or secondary risk of severe psychiatric side effects.

[0004] To date, no test has been specifically approved by the U.S. Food and Drug Administration (FDA) or other drug regulatory authorities as an effective test for predicting a patient's risk of developing or exhibiting a psychiatric disorder, such as a depressive disorder or suicidal behavior.

[0005] There is great interest in reviewing any tests that may show promise for determining a patient's risk of suicidal behavior or depressive disorders.

[0006] There are currently no approved biological tests to identify such patients. Summary of the Invention

[0007] There is therefore a need to provide an in vitro test that can determine with high accuracy and high discrimination the risk that a patient will develop or experience a psychiatric disorder, such as a depressive disorder or suicidal behaviour, and which is particularly easy to use, for example from a blood sample of the patient to be tested. This is the object of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] In a first aspect, the present invention provides an in vitro method for predicting the risk that a patient is to present a condition selected from the group consisting of a psychiatric or neurological syndrome, an immunological syndrome, and a degenerative syndrome associated with altered A-to-I editing on the PDE8A transcript, or for determining whether a patient is at risk of developing said condition, comprising: from a body fluid sample of said patient containing cells expressing at least one of the editing enzymes ADAR1a, ADAR1b, and ADAR2 and PDE8A. (a) in the same intracellular RNA extract obtained from the cell-containing blood sample (i) the expression level of PDE8A mRNA, and (ii) the level of PDE8A RNA editing at at least one or a combination of editable sites in the PDE8A gene, preferably sites associated with said pathology; and / or the expression level of at least one PDE8A isoform or combination of PDE8A isoforms or non-edited transcripts (Ne), which are expressible and preferably associated with said pathology; determining a (b) determining the relative proportion of the RNA editing sites and / or the relative proportion of the PDE8A isoform level to the PDE8A mRNA expression level from the results obtained in step (a); and (c) identifying whether the patient is experiencing or at risk of developing the condition, (i) comparing the relative proportions of the RNA editing sites and / or isoforms obtained in step (b) with the relative proportions of RNA editing and / or isoforms at the sites obtained from control bodily fluid samples from patients known to exhibit or not exhibit the pathological condition or known to be or not at risk of developing the pathological condition; and / or (ii) applying an algorithm or statistical model exhibiting discrimination power using the editing sites and / or isoforms selected in step (b), or a combination thereof; and using the results or final values ​​obtained in (i) or (ii) of step (c) to determine whether said patient develops said pathological condition or is at risk of developing said pathological condition. The present invention relates to a method comprising the steps of:

[0009] The threshold determined for the control sample in step (c)-(i), or the algorithm used in step (c)-(ii), comprises the following steps: (a) selecting a patient collection consisting of patients annotated as having a proportion of risk for developing a mental disorder, or patients annotated as not having that risk, or a patient collection for confirming that the patients will develop a mental disorder; selecting one or several PDE8A RNA editing sites and / or isoforms, or a combination thereof; (b) analyzing the target PDE8A RNA editing site or target isoform to obtain an RNA editing level of said target site and / or a proportion of said target isoform for each of the patients of said collection; (c)-(i) assessing, for each RNA editing site and / or each isoform, its threshold and / or its accuracy and ability to identify the risk of developing said pathology, such as a psychiatric disorder, or to identify that a patient expresses said pathology, by univariate statistical analysis methods; and / or (c)-(ii) evaluating the accuracy and ability of each combination of RNA editing sites and / or isoforms to identify the risk of developing said pathology, in particular a psychiatric disorder, or to confirm that a patient is expressing said pathology, by multivariate statistical analysis methods; and (c)-(iii) selecting editing sites and / or isoforms, or combinations thereof, that exhibit acceptable discrimination ability; and (e) constructing an algorithm using said combination of selected editing sites and / or isoforms and using said algorithm so obtained to predict the risk of a given patient to develop said pathological condition, in particular a psychiatric disorder, or to confirm that said patient expresses said pathological condition. The compound can be obtained by a method comprising the steps of:

[0010] In a preferred embodiment, said algorithm usable in the method of the invention comprises: mROC programs that maximize the AUC (area under the curve) of the ROC, particularly for identifying linear combinations, where the equation for each combination is the following formula, where ai is the calculated coefficient, and (biomarker i) is the relative proportion of each level of the RNA editing site or isoform target: Z = a1 (biomarker 1) + a2 (biomarker 2) + ... ai (biomarker i) + ... an (biomarker n) and which can be used as a new virtual marker Z, and / or Logistic regression models applied to univariate and multivariate analyses to estimate the relative risk of patients with different RNA editing site levels or isoform values; and / or A CART (Classification and Regression Tree) approach applied to evaluate combinations of RNA editing sites and / or isoforms, and / or Random forest (RF) approaches applied to evaluate combinations of RNA editing sites and / or isoforms, specifically approaches to rank the importance of those RNA editing sites and / or isoforms and to combine the best RNA editing sites and / or isoforms to classify a "relative risk", and / or optionally Multivariate analyses applied to evaluate combinations of RNA editing sites and / or isoforms for "relative risk," e.g. Support Vector Machine (SVM) approach, Artificial Neural Network (ANN) approach, Bayesian network approach, WKNN (weighted k-nearest neighbor) approach, Partial least squares discriminant analysis (PLS-DA), Linear and quadratic discriminant analysis (LDA / QDA), and Any other mathematical method for combining biomarkers The multivariate analysis is selected from the group consisting of This is carried out by multivariate methods including

[0011] In a preferred embodiment, said body fluid sample is selected from a blood sample, a urine sample, and a saliva sample, with a blood sample being most preferred.

[0012] In a preferred embodiment, the condition is a psychiatric disorder.

[0013] In another preferred embodiment, the condition is selected from the group consisting of psychiatric disorders, bipolar disorder, depressive disorder, schizophrenia, panic disorder, social phobia, post-traumatic stress disorder (PTSD), substance dependence / abuse, abnormal eating behavior, preferably anorexia or obesity, obsessive-compulsive disorder (OCD), early stage Alzheimer's disease, and Parkinson's disease.

[0014] In a more preferred embodiment, said condition is a depressive disorder, and more particularly suicidal behavior.

[0015] In a more preferred embodiment of the method of the present invention, the method is for predicting the risk of a patient attempting suicide, wherein in step (c)-(i) a control body fluid sample is obtained from an affective disorder control group of patients.

[0016] In an even more preferred embodiment of the method of the present invention, the method is for determining whether a patient exhibits depression, wherein in step (c)-(i) a control body fluid sample is obtained from a healthy group of patients.

[0017] In a particular embodiment of the method of the invention, the patient has already been identified as exhibiting a psychiatric disorder, in particular a depressive disorder, that may be associated with a risk of developing suicidal behavior, and is selected from among patients with or without a history of suicide attempts, and it is identified in step (c) whether the patient exhibits suicidal behavior or is at risk of developing suicidal behavior.

[0018] In another embodiment, the methods of the invention allow for monitoring patients and / or dividing patients into treatment responders and treatment non-responders.

[0019] It is known that many disorders, including psychiatric disorders, are sometimes associated with suicidality, hi some embodiments, the patient suffers from a psychiatric disorder, bipolar disorder, schizophrenia, panic disorder, social anxiety disorder, post-traumatic stress disorder (PTSD), substance addiction / abuse, eating disorders (abnormal eating behaviors such as anorexia, obesity), obsessive-compulsive disorder (OCD), early Alzheimer's disease, or Parkinson's disease.

[0020] In another embodiment, the patient may be one who has been confirmed as not exhibiting a psychiatric disorder and who is being treated for a non-psychiatric disorder, particularly a patient who is being treated for hepatitis.

[0021] Another aspect of the present invention is a method for producing a step (a) comprises determining the expression levels of the editing enzymes ADAR1a, ADAR1b and / or ADAR2 in the same cell extract; In step (c), it is determined whether the patient exhibits the condition or is at risk of developing the condition. (i) further comparing the expression levels of the editing enzymes ADAR1a, ADAR1b and / or ADAR2 obtained in step (b) with expression levels obtained for normal patients and, optionally, with expression levels obtained for patients exhibiting a pathology associated with altered A-to-I editing on the PDE8A transcript, and / or (ii) applying an algorithm or statistical model exhibiting discrimination ability using the editing sites and / or isoforms, and the expression levels of ADARs selected in steps (a) and (b), or a combination thereof; The present invention relates to the method.

[0022] In a preferred embodiment of the method of the invention, said blood sample comprises PBMCs (peripheral blood mononuclear cells).

[0023] In the method of the present invention, in step (a), it is preferable to determine the editing of PDE8A RNA for each site for at least one site selected from the editing sites listed in Table III, and it is more preferable to determine the editing for multiple sites including at least one site among O to ZZ in Table III.

[0024] In the method of the present invention, in step (a), it is preferable to determine editing of one or more sites in the PDE8A RNA for at least a site selected from the group consisting of sites A, B, C, D, E, F, G, Z, and ZZ.

[0025] In the method of the present invention, in step (a), it is more preferred to determine the editing of one or more sites in the PDE8A RNA, at least for site B.

[0026] In the method of the present invention, in step (a), it is preferable to determine the relative ratio of the level of a PDE8A RNA isoform containing one or a combination of several of the multiple editing sites to the PDE8A mRNA expression level.

[0027] In the method of the present invention, in step (a), it is preferable to determine the relative proportion of PDE8A RNA isoforms selected from the group consisting of isoforms B, BC, D, E, Ne (non-edited), BE, G, BG, BF, A, Z, C, F, BZ, and BD.

[0028] More preferably, in the method of the invention, the relative proportion of at least PDE8A RNA isoform B is determined in step (a).

[0029] In a preferred embodiment of the method of the invention, the algorithm or statistical method comprises in step (c) an mROC program that maximizes the AUC (area under the curve) of the ROC, in particular a program for identifying linear combinations, the equation for each combination being based on the following formula, where ai are the calculated coefficients and (biomarker i) is the relative proportion of each PDE8A RNA editing level of one site or one isoform target, or the expression level of ADAR1a, 1b, or ADAR2: Z = a1 (biomarker 1) + a2 (biomarker 2) + ... ai (biomarker i) + ... an (biomarker n) and which can be used as a new virtual marker Z, and / or Logistic regression models applied to univariate and multivariate analyses to estimate the relative risk of a given patient at various editing site and / or isoform and / or ADAR class values; or Any other mathematical method for combining biomarkers Includes.

[0030] In the method of the present invention, in step (c), it is preferred that the algorithm is selected from the group of mROC combinations consisting of the combinations listed in Table VI.

[0031] In the method of the present invention, in steps (a) and (b), it is preferable to determine the PDE8A mRNA expression level by qPCR and / or to determine the RNA editing level at each site by an NGS (next generation sequencing) method.

[0032] In a preferred embodiment, Seq1-Forward (SEQ ID NO:2) or Seq2-Forward (SEQ ID NO:3) as forward primer; Seq1-reverse (SEQ ID NO: 4), Seq2-reverse (SEQ ID NO: 5) or Seq3-reverse (SEQ ID NO: 6) as a reverse primer; and The following selection criteria, namely (i) an oligo between 18 and 27 nucleotides in length; (ii) Preferably has a melting point of around 60°C (57°C to 63°C) (iii) have an optimal GC content of around 50% (±5%) (iiii) the maximum allowable repeat length is 5 mononucleotides; and (vi) specifically amplifying the region of interest shown in FIG. 1; A set of forward and reverse primers that match The expression level of PDE8A mRNA and / or the RNA editing level at each site are determined by qPCR using a set of forward and reverse primers selected from the above.

[0033] In a more preferred embodiment, the method of the present invention determines the RNA editing level of PDE8A per site using the following primer set: Seq2-forward and Seq-3-reverse.

[0034] In another aspect, the invention provides a kit for determining whether a patient is at risk of developing a condition selected from the group consisting of a psychiatric or neurological syndrome, an immunological syndrome, and a degenerative syndrome associated with altered A-to-I editing on the PDE8A transcript, or for determining whether a patient is at risk of developing said condition, comprising: (1) instructions for applying the method of the invention to obtain an end value that can be analyzed to determine the risk of developing the pathological condition, said instructions including or not including an ROC curve; and (2) Seq1-Forward (SEQ ID NO:2) or Seq2-Forward (SEQ ID NO:3) as a forward primer; Seq1-reverse (SEQ ID NO: 4), Seq2-reverse (SEQ ID NO: 5) or Seq3-reverse (SEQ ID NO: 6) as a reverse primer; and The following selection criteria, namely (i) an oligo between 18 and 27 nucleotides in length; (ii) Preferably has a melting point of around 60°C (57°C to 63°C) (iii) have an optimal GC content of around 50% (±5%) (iiii) the maximum allowable repeat length is 5 mononucleotides; and (vi) specifically amplifying the region of interest shown in FIG. 1; A set of forward and reverse primers that match A set of a forward primer and a reverse primer selected from the group consisting of: The kit further comprises:

[0035] In a more preferred embodiment, the kit comprises the following primer set: Seq2-forward (SEQ ID NO:3) and Seq-3-reverse (SEQ ID NO:6).

[0036] The following examples and the description of the figures and drawings are chosen to fully explain the invention so that those skilled in the art can make and use it, and are not intended to limit the scope of the invention as contemplated by the inventors, nor are they intended to represent that only the experiments described below have been performed.

[0037] Other features and advantages of the invention will become apparent in the remainder of the specification of the examples and figures, the description of which is provided below. [Brief description of the drawings]

[0038] [Figure 1] Figure 1 shows a sequence of interest located within the 9th intron of the human PDE8A gene. The internal sequence of intron 9 (225 bp) (base positions 85,096,606 to 85,096,830, SEQ ID NO: 1) is shown. This sequence is amplified using the primer combination Seq2 forward and Seq3 reverse. Editing sites previously described by Orlowski and coworkers (A to H)

[10] and by Weissmann et al. (WO 2011 / 161253) are shown in grey bold capital letters on the sequence. The new editing sites O to ZZ are shown in black underlined capital letters on the sequence. [Diagram 2] Representative electropherograms showing non-specific by-products obtained by amplification using various primer pairs. The primer pair Seq2-forward and Seq3-reverse, which did not generate detectable non-specific products, was used for amplification and ultra-deep sequencing of the target region of PDE8A. [Diagram 3] Figure 1 shows RNA editing sites identified in human leukocytes by ultra-deep sequencing.Figure 2 reports the relative proportions (mean±SEM) of RNA editing assessed for multiple sites (A-G, Z, and ZZ) of the PDE8A gene in affective disorder controls. [Figure 4]Figure 1. Expression levels of relevant genes measured by qPCR analysis in leukocytes of affective disorder control (AC) and suicide attempter (SA) populations. The mRNA expression levels of ADAR1a (A), ADAR1b (B), ADAR2 (C), and PDE8A (D) in affective disorder controls (AC) were compared with suicide attempters (SA). The mRNA expression levels of ADAR1a, ADAR1b, ADAR2, and PDE8A were quantified in 38 patients (n=18 affective disorder controls and n=20 suicide attempters). The expression of the various transcripts was normalized to the gene expression of GAPDH and β2M. Measurements were performed in three replicate experiments. The mRNA expression of ADAR1a, 1b, and PDE8A is significantly higher in suicide attempters. The p-value was calculated using the Mann-Whitney test. [Diagram 5] Figure 3. Relative percentage of RNA editing at site B of the PDE8A gene. (A) Box plot of PDE8A site B mRNA editing measured in affective disorder controls (AC) and suicide attempters (SA). (B) Measures of PDE8A mRNA editing adjusted for relative transcript levels. Data represent the mean of five independent experiments. p-values ​​were calculated using the Mann-Whitney test. [Figure 6] Figure 1 shows diagnostic performance of biomarker combinations using the mROC approach. This figure shows an example of a multivariate ROC curve comparing affective disorder controls to suicide attempters. ROC curve obtained with the mROC combination ADAR1a+ADAR2+PDE8A_site A*+PDE8A_site B*. [Figure 7] Figure 1 shows the diagnostic performance of biomarker combinations using logistic regression. This figure shows an example of a multivariate ROC curve comparing affective disorder controls to suicide attempters. ROC curve obtained with the ADAR1a+PDE8A_site B* logistic regression model. [Figure 8]Longitudinal study of Hepatitis C virus (HCV) in patients treated with IFN-α and ribavirin. PAXgene blood RNA tubes were sampled from 10 HCV-infected patients 4 weeks prior (-4) and every 2 weeks (weeks 0, 2, 4, 6, 8, 10, and 12) after the start of antiviral treatment. The treatment included weekly subcutaneous injections of PEGylated IFN-α (180 μg) and daily ribavirin tablets (1000-1200 mg). [Figure 9] FIG. 1 shows longitudinal analysis of expression levels of relevant genes (AB) and relative proportions of RNA editing at site B of the PDE8A gene (CD) in two Hepatitis C Virus (HCV)-infected patients treated with IFN-α and ribavirin. Data shown are the mean ± SD (error bars) of five independent experiments (n=5) measured for each patient at each time point. [Figure 10] Clustering analysis of 6 HCV-infected patients included in the IFN trial. K-median clusters were output using MEV software v4.9, using Pearson correlation for distance, 1000 iterations and 2 clusters as parameters. This approach resulted in two distinct clusters of patients. The first cluster, including 4 patients with a rapid and robust response to increased IFN, was observed by a steep slope during the first 4 weeks of treatment. A second cluster, including 2 patients with a blunted IFN response, could also be observed. [Figure 11] (A-B) Clinical assessment of non-repeated suicide attempters (NRSA) and repetitive suicide attempters (RSA) over a 6-month follow-up period. Hamilton (HAMD) scores, MADRS (Montgomery-Asberg Depression Rating Scale) scores, and BDI (Beck Depression Inventory) scores are presented for both groups at 0 months, the time of initial intake, and at 6 months. (C-D) Corresponding amounts of RNA editing at all sites in the PDE8A gene. Data represent the mean of five replicate experiments, and error bars represent the standard error of the mean. p-values ​​were calculated using the Wilcoxon test. [Figure 12] (A) Longitudinal analysis of ADAR1a, ADAR1b, ADAR2, and PDE8A gene expression in all HCV patients (n=10) over 16 weeks. (B) Longitudinal analysis of RNA editing at site B of the PDE8A gene in all HCV patients adjusted for relative PDE8A transcript levels (n=10). (C) Longitudinal analysis of RNA editing at other specific sites of the PDE8A gene in all HCV patients adjusted for relative PDE8A transcript levels (n=10). Means shown ± SEM (n=10).

[0039] [Figure 13] (A) Longitudinal analysis of a biomarker combination consisting of ADAR gene expression in a group of patients with (n=3) and without (n=7) a depressive episode. (B) Longitudinal analysis of a biomarker combination consisting of RNA editing in a group of patients with (n=3) and without (n=7) a depressive episode. (C) ROC curve of the biomarker combination for separating populations with and without a depressive episode.

[0040] [Figure 14] FIG. 1 shows that specific combinations of biomarkers were applied to all data points obtained over the course of IFN treatment. EXAMPLES

[0041] Example 1: Materials and Methods 1) Human participants Data are presented from two different cohorts: one including depressed patients with or without a history of suicide attempt (referred to as the Suicide Attempter Study and Follow-up Study) and one longitudinal study analyzing patients infected with Hepatitis C virus and treated with interferon alpha (referred to as the Interferon Study). Written informed consent was obtained from all subjects after a detailed discussion of the study aims and requirements. All subjects were able to understand the informed consent, which explained in detail the study goals and measures. The study was approved by the local Institutional Review Board in accordance with the licensing requirements and good clinical practice guidelines.

[0042] 2) Surveys of people who have attempted suicide and follow-up surveys of people who have attempted suicide 2.1 Survey of suicide attempters The study enrolled 38 depressed patients seen in the emergency and post-emergency psychiatry departments of the CHU in Montpellier, France (n=17) or in a psychiatric treatment center in Oviedo, Spain (n=21). The psychiatric diagnoses were established by structured clinical interview criteria. Psychiatric diagnoses were determined according to the Diagnostic and Statistical Manual of Mental Disorders IV (DSM-IV). The sample included 20 suicide attempters (SA) (52.63%) and 18 non-suicide attempters (affective disorder controls, AC) (47.37%) (see Table I(A)).

[0043] The subjects were ethnically homogenous, all of Caucasian ethnicity from Western Europe. Demographic data regarding association and gender were recorded, as well as the patients' psychiatric diagnoses, alcohol or substance consumption, and number of suicide attempts.

[0044] [Table 1]

[0045] Two groups were included for comparison: the first group, called affective controls (AC), included depressed patients with no history of suicide attempts, and the second group, called suicide attempters (SA), included depressed patients with a history of suicide attempts.

[0046] 2.2 Follow-up survey of suicide attempters The follow-up study enrolled 28 depressed suicide attempt patients seen in the emergency and post-emergency psychiatric departments of the CHU in Montpellier, France. The patients were followed up over a period of 6 months (see Table I(B)). During the follow-up, 8 of the 28 patients (28.6%) made one or several new suicide attempts ("repeated suicide attempters", RSA), whereas 20 patients (71.4%) did not make any new suicide attempts ("non-repeated suicide attempters", NRSA). Psychiatric evaluation of the patients was performed as previously described for the suicide attempter study. Three scales were used to assess the severity of depression at baseline and follow-up assessments: the Hamilton Depression Scale (HAMD), the Montgomery-Asberg Depression Rating Scale (MADRS), and the Beck Depression Inventory (BDI).

[0047] JPEG0007654918000002.jpg85145

[0048] 3) Interferon survey Ten patients from three CHU (Central University Hospital) departments in Marseille, Nancy, and Strasbourg (France) were enrolled in the study. Hepatitis C virus (HCV)-infected patients who did not meet any criteria for psychiatric disorders were enrolled before the start of antiviral treatment and followed up for up to 12 weeks after the start of antiviral treatment. An initial blood sample was obtained 4 weeks before treatment (-4). Blood was collected in PAXgene tubes at the start of treatment and every 2 weeks for up to 12 weeks (Figure 8).

[0049] 4) Blood collection, RNA extraction, and qualification evaluation A volume of 2.5 ml of whole blood samples from each patient was collected in PAXgene Blood RNA tubes and stored at -20°C for 2 months, after which they were transferred to -80°C. PAXgene™ Blood RNA tubes (PreAnalytix) contain reagents that protect RNA molecules from ribonuclease degradation and in vitro changes in gene expression. Samples were randomly distributed among the different extraction rounds. Total RNA from 2.5 ml of whole blood was extracted using the PAXgene™ Blood RNA Kit (reference number 28704, PreAnalytix). Extracted RNA was isolated using a Qiagen QIAcube system according to the manufacturer's protocol for PAXgene Blood RNA Part 1, Automated Protocol. Standard precautions were adopted during sample preparation and RNA extraction to avoid RNA degradation by ribonucleases. Total RNA concentration was determined using a Qubit fluorometer (Invitrogen) and the Quant-IT RNA BR assay (Invitrogen).

[0050] 5) Preparation of NGS libraries An NGS library was prepared using a two-step PCR method to selectively sequence the region of interest within intron 9 of the PDE8A gene. As shown in Figure 1, a new editing site was identified within the region of interest apart from previously described editing sites. The region of interest was amplified by PCR using validated PCR primers (Seq2 forward and Seq3 reverse) (Table II and Figure 1).

[0051] [Table 2]

[0052] A two-step PCR library preparation protocol was used to generate sequencing libraries containing multiple samples, which were then sequenced on a next-generation sequencing system such as the MiSeq platform (Illumina).

[0053] For PCR amplification, Q5 Hot Start High Fidelity Enzyme (New England Biolabs) was used according to the manufacturer's guidelines (reference number M0494S). PCR reactions were performed on a Peqstar 96x thermocycler using an optimized PCR protocol. After PCR, all samples were analyzed by LabChipGx (Perkin Elmer) to assess both the quantity and quality of the PCR products. Amplicon purity was determined and quantified using the fluorescence-based Qubit method. After quality control, 96 PCR reactions (microplates) were purified using magnetic beads (High Prep PCR MAGbio System, Mokascience). After purification, DNA was quantified using the Qubit system and purification yields were calculated. Samples were then individually indexed by PCR amplification using Q5 Hot Start High Fidelity PCR Enzyme (New England Biolabs) and an Illumina 96 Index Kit (Nextera XT Index Kit; Illumina). After PCR, samples were organized into libraries and purified using the Magbio PCR clean-up system. The libraries were denatured and loaded onto the sequencing cartridge following Illumina's guidelines for sequencing FASTQ on the MiSeq platform only. A commercially available human blood peripheral leukocyte total RNA pool (Clontech, ref. 636592) was incorporated into the libraries to determine the degree of variability between different sequencing flow cells over the course of the experiment. NGS libraries were sequenced at standard concentrations and library diversity was introduced into these libraries using PhiX Control V3 (Illumina). Experiments were performed five times independently.

[0054] 6) Bioinformatics analysis of sequencing data Sequencing data were downloaded as fastq files from the Miseq sequencer (Illumina). An initial quality check of each raw fastq file was performed using FastQC software (version 0.11.3) to assess the sequencing quality. A preprocessing step was performed consisting of removing adapter sequences and filtering the sequences according to sequence size and quality score (all short reads (<50 nt) and reads with an average QC <30 were removed). Read trimming and filtering tools for Illumina NGS data (fastx toolkit vO.0.14 and prinseq version 0.20.4) were then used to facilitate sequence alignment and improve its quality. After the preprocessing step, an additional quality control was performed on each cleaned fastq file before further sequence processing.

[0055] Alignment of processed reads was performed using bowtie2 (version 2.2.5) in end-to-end high sensitivity mode. Alignment was performed against the latest human genome sequence annotation (GRCh38), and reads with multiple aligned regions, low alignment quality (Q below 40), or containing insertions / deletions (INDELs) were excluded from further analysis. Filtering of file alignments was performed using SAMtools software (version 1.3.1), which provides various utilities including sorting, merging, indexing for processing SAM format alignments, and creating preposition format alignments.

[0056] Next, the data from the alignments obtained simultaneously from multiple samples were overlaid using SAMtools mpileup. A homemade script was run to count the number of different ATGC nucleotides at each genomic position ("base count"). The homemade script calculates the percentage of reads with "G" for each genomic position (# of "G" reads / (# of "G" reads + # of "A" reads) x 100). Genomic positions with a percentage of "G" reads relative to the "A" criteria greater than 0.1 are automatically detected by the script and considered as "A-to-I editing sites". In the last step, the percentage of all possible isoforms of the PDE8A transcript was calculated. By definition, the relative proportion of RNA editing at a given editing "site" represents the sum of editing modifications evaluated at this unique genomic coordinate. Conversely, mRNA isoforms are unique molecules that may or may not contain multiple editing modifications on the same transcript. For example, PDE8A mRNA isoform BC contains modifications at both site B and site C within the same transcript.

[0057] 7) Reverse transcription and quantitative real-time PCR Reverse transcription was performed using a Takara kit (PrimeScript RT, Takara, ref. RR037A) and the resulting cDNA was mixed in a final volume of 20 μl with TaqMan Universal PCR Master Mix (Applied Biosystems, ref. 4369016) and the following specific gene probes: ADAR1a (Hs01020780), ADAR1b (Hs01017596), ADAR2 (Hs00210562), PDE8A (Hs00400174), GAPDH (Hs02758991), β2M (Hs00984230), HPRT1 (Hs02800695), PGK (Hs99999906) and TBP (Hs00427620) (Applied Biosystems, a Life Technologies company). Quantitative PCR was performed in 96-well plates on a StepOnePlus real-time PCR machine (Applied Biosystems) or in 384-well plates on a LightCycler 480 real-time PCR machine (Roche). For the studies performed on depressed patients and suicide attempters, quantification of target gene expression in all patients was normalized to GAPDH and β2M expression, and the change in expression of the target gene in each sample was calculated by 2-ΔΔCt. For the studies performed on chronic hepatitis C virus (HCV) patients, analysis was performed using second derivative absolute quantification, normalized by the geometric mean of four housekeeping genes (GAPDH, HPRT1, PGK, and TBP).

[0058] 8. Statistical Analysis of Data All statistics and figures were calculated using the "R / Bioconductor" statistical open source software and GraphPad Prism software (version 7.0) [12, 13]. Usually, values ​​of biomarkers (i.e., RNA editing sites and isoforms of PDE8A and mRNA expression of ADARs) are presented as mean ± standard error of mean (SEM). Analysis of differences was performed using the Mann-Whitney test, and p values ​​less than 0.05 were considered statistically significant. All data distributions are shown as median and bar graphs or box plots for the significance of each biomarker.

[0059] For both sites and isoforms, the relative proportion of RNA editing was analyzed by adjusting for the relative transcript levels of the PDE8A gene. Adjusted RNA editing values ​​were calculated as follows: Adjusted editing value*=(RNA editing value × relative transcript level) / 100

[0060] The diagnostic performance of a biomarker could be characterized by its sensitivity, which describes its ability to detect the “suicide attempter” group, and its specificity, which describes its ability to detect the “affective disorder control” group. The results of the diagnostic test can be summarized in a 2 × 2 contingency table comparing these two clearly defined groups. Fixing a cut-off value allows the classification of the two groups into categories that are classified as either positive or negative depending on the result of the test. Given a particular biomarker, we can identify a large number of (a) patients with a positive test result in the “suicide attempter” group ("true positive": TP) and (b) patients with a negative test result in the “affective disorder control” group ("true negative": TN). Similarly, (c) patients with a negative test result in the “suicide attempter” group ("false negative": FN) and (d) patients with a positive test result in the “affective disorder control” group ("false positive": FP) are also recognized. Sensitivity is defined as TP / (TP+FN), which is referred to herein as the "true positive rate". Specificity is defined as TN / (TN+FP), which is referred to herein as the "true negative rate."

[0061] Receiver operating characteristic (ROC) analysis was used to assess the accuracy of each biomarker and its discriminatory ability. The ROC curve is a graphical visualization of the correlation between the sensitivity (Se) and specificity (Sp) of a test for different values.

[0062] Furthermore, we evaluated the possible increase in sensitivity and specificity by combining all biomarkers with each other using a multivariate approach, for example the mROC program or logistic regression

[14] . mROC is a program dedicated to identifying linear combinations [15, 16], maximizing the AUC (area under the curve) of the ROC

[17] . The equation for each combination is provided as follows and can be used as a new virtual marker Z: Z = a × biomarker 1 + b × biomarker 2 + c × biomarker 3, where a, b, c are calculated coefficients and biomarker 1, 2, 3 are the levels of biomarkers.

[0063] A logistic regression model is applied using the different combinations of editing sites and / or isoforms and / or ADARs gene expression values ​​for patients to estimate relative mood disorder risk.

[0064] Patient clusters were identified from the interferon study using a K-median approach or K-means approach

[18] . For example, K-median analysis was calculated using Pearson correlation or Euclidean correlation for distances using MEV v4.9 software (http: / / www.tm4.org / mev / ). All patients present within the same cluster were considered different from patients in other clusters.

[0065] Example 2: Survey of suicide attempters The characteristics of the patients included in this clinical study are shown in Table I. This prospective study enrolled 38 patients with diagnosed psychiatric disorders and compared a group of 20 patients (SA), all of whom had attempted suicide at least once, to a group of 18 patients who had not attempted suicide (affective disorder controls, AC). Patient ages ranged from 20 to 77 years (mean ± SD: 50.16 ± 13.5). The cohort consisted of 9 men (23.68%) and 29 women (76.32%). All patients developed depressive disorders. The number of suicide attempts ranged from 1 to 20, with a mean ± SD of 3.4 ± 4.52.

[0066] Example 3: Analysis of PDE8A mRNA editing using next generation sequencing (NGS) methods We have previously described the method used to determine the editing profile of PDE8A mRNA using capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) (International Application PCT / EP2011 / 060444, filed June 24, 2011). In this invention, we developed a new experimental system to quantify the editing level of PDE8A RNA based on next-generation sequencing (NGS), which provides sufficient per-base depth to allow reliable quantification for all sites tested. Since this method is PCR-based, we first selected several primer pairs (Table II) to amplify the PDE8A mRNA region of interest. Different primer combinations that met the following selection criteria were tested to specifically amplify the region. That is, 1) an oligo between 18-27 nucleotides in length, 2) preferably with a melting temperature near 60°C (57-63°C), 3) with an optimal GC content near 50%, 4) with an allowable maximum repeat length of 5 mononucleotides, and 5) specifically amplify the region of interest shown in Figure 1. As some primer pairs generated non-specific amplification products that interfered with accurate quantification of RNA editing, we provide optimal primer pairs including Seq2-forward and Seq-3-reverse (see Table II).

[0067] Example 4: Identification of a novel editing site within intron 9 of the human PDE8A gene by ultra-deep sequencing Orlowski and coworkers previously identified eight editing sites (A to H) within intron 9 of the human PDE8A gene (see Table III)

[10] . We subsequently identified six additional editing sites (I to N) within this same region (International Application PCT / EP2011 / 060444, filed June 24, 2011). Several studies have indicated that most ADAR activity occurs in clusters [19, 20]. Not surprisingly, we confirmed the previously identified editing sites within intron 9 of the human PDE8A gene and identified a new A-to-I editing site using NGS-based sequencing on the SH-SY5Y human neuroblastoma cell line. These new editing sites were named O, P, Q, R, S, T, U, V, W, X, Y, Z, and ZZ (see Table III, Figure 1). The presence of most of these editing sites (AH, JM, O, P, RU, and XZZ) was also confirmed in blood samples from several patients in this study using depressed and suicide attempters.

[0068] [Table 3]

[0069] For practical reasons, optimization of standard conditions for deep sequencing of loci of interest within the PDE8A transcript was performed on human samples from the SH-SY5Y neuroblastoma cell line. This identified new editing sites not previously reported within intron 9 of the human PDE8A gene (underlined and bold, numbers 15-27). The new editing sites are named O, P, Q, R, S, T, U, V, W, X, Y, Z, and ZZ. The genomic coordinates are compared against PDE8A on chromosome 15 of the human genome (GRCh38:84,980,440). The SH-SY5Y cell line column indicates that an editing site was identified in that particular human neuroblastoma cell line. The blood column indicates that white blood cells were collected in a PAXgene blood RNA tube. X indicates that the editing site was evaluated in at least one patient with a cutoff value of >0.1%.

[0070] Example 5: RNA editing sites identified in human leukocytes by ultra-deep sequencing Only RNA editing sites (mean ± SEM) assessed at a cutoff value above 0.1% from the affective disorder control group are reported in Table IV and Figure 3 (see Table IV and Figure 3 ).

[0071] [Table 4]

[0072] By definition, the relative proportion of RNA editing at a given editing "site" represents the sum of editing modifications assessed at this unique genomic coordinate. Conversely, mRNA isoforms are unique molecules that may or may not contain multiple editing modifications on the same transcript. For example, PDE8A mRNA isoform BC contains modifications at both site B and site C within the same transcript.

[0073] Example 6: ADAR1a, 1b, and 2 mRNA expression is increased in the blood of suicide attempters We analyzed the mRNA expression of ADAR1a, ADAR1b, and ADAR2 genes by the so-called delta-delta CT method using TaqMan® real-time PCR assay (Figure 4A, Figure 4B, and Figure 4C, respectively). The expression levels of ADAR1a, ADAR1b, and ADAR2 varied from 2.6 to 25 (mean ± SD: 10.2 ± 6), from 2.04 to 14.09 (mean ± SD: 5.9 ± 3.3), and from 1.25 to 50.5 (mean ± SD: 12.4 ± 13.1), respectively, in AC. In SA (n=20), the mRNA expression levels of ADAR1a, 1b, and ADAR2 comprised between 4.2 and 49.5 (mean ± SD: 17.8 ± 11.7), between 3 and 33.8 (mean ± SD: 10.6 ± 7), and between 3.8 and 62 (mean ± SD: 17.8 ± 16.1), respectively. Expression of ADAR1a and ADAR1b genes was significantly higher in SA compared to AC, with mean values ​​of 17.8 and 10.6 versus 10.2 and 5.9, respectively (p=0.0435 and 0.0047).

[0074] Example 7: PDE8A mRNA expression is high in suicide attempters Similarly, changes in PDE8A expression levels were observed in the SA group compared to the AC group (Figure 4D). Indeed, PDE8A expression varied from 4.7 to 19.9 (mean ± SD: 9.2 ± 3.3) in AC, while its levels ranged between 7.5 and 32.8 (mean ± SD: 15.7 ± 7.1) in SA. Similar to ADARs, PDE8A expression was significantly higher in the SA group than in the affective disorder group (p = 0.0013) (Figure 4D). This suggests that PDE8A may provide an additional biomarker for suicidal behavior.

[0075] Example 8: mRNA editing at site B of PDE8A is increased and nonedited (Ne) PDE8A transcripts are decreased in suicide attempters A) mRNA editing at site B of PDE8A We analyzed the PDE8A mRNA editing profile between AC and SA patients using an NGS assay. As shown in Figure 5A, we found that the relative percentage of PDE8A mRNA edited at site B was higher in SA compared to AC. In AC (n=18), PDE8A edited transcripts ranged from 11.2 to 21.8%, with a mean value of 17.4% (SD ± 2.8%). In SA (n=20), the levels of edited variants ranged from 12.3 to 26.7%, with a mean value of 20.2% (SD ± 3.7%). There was a significant difference between the values ​​in AC and SA (p=0.01). Furthermore, we adjusted the relative percentage of RNA editing at site B of the PDE8A gene for the relative expression level of PDE8A mRNA (site B*). The results are presented in Figure 5B. Adjusting for RNA editing at site B further accentuated the difference between AC and SA groups, as indicated by the p-value (p<0.0001).

[0076] B) Non-edited PDE8A transcripts We analyzed the relative proportion of nonedited PDE8A transcripts in affective disorder controls (AC) and suicide attempters (SA). The relative proportion of nonedited PDE8A transcripts was higher in AC compared to SA. In AC, the relative proportion of PDE8A nonedited transcripts ranged from 77.1 to 87.9%, with a mean value of 81.7% (SD ± 2.9%). In SA, the level of nonedited transcripts ranged from 71.5 to 87.2%, with a mean value of 78.9% (SD ± 3.8%). There was a significant difference between the values ​​in AC and SA (p = 0.0094).

[0077] Example 9: PDE8A mRNA editing regulated by ADAR1a and ADAR2 mRNA expression as blood biomarkers of suicidality We evaluated the performance of this diagnostic using the expression of ADARs and PDE8A (see Table V(A)), adjusted PDE8A mRNA editing at different sites (sites A*-G*, Z*, and ZZ*, Table V(B)), and different isoforms (Table V(C)) as biomarkers of suicidality using receiver operating characteristic (ROC) curves. Table V shows the results for the individual biomarkers. We found the highest area under the curve (AUC) with a value of 0.878 for PDE8A site B* mRNA editing (Table VB). The same results were obtained with PDE8A mRNA isoform B (Table V(C)).

[0078] [Table 5]

[0079] JPEG0007654918000007.jpg74138

[0080] JPEG0007654918000008.jpg94156

[0081] Table V(A) presents the diagnostic performance of the relative expression of ADAR1a, ADAR1b, ADAR2, and PDE8A. Table V(B) presents the diagnostic performance of the individual editing sites of PDE8A. Table V(C) presents the diagnostic performance of the individual editing isoforms or non-edited transcripts (Ne) of PDE8A. Only the most relevant editing isoforms and editing sites, defined as having a p-value less than 0.1, are presented. p-values ​​were calculated using the Mann-Whitney test. The symbol "*" indicates that the editing site or editing isoform of PDE8A is adjusted for the relative expression of its mRNA.

[0082] We further investigated whether our ability to predict future suicide could be enhanced by a combined approach including PDE8A mRNA editing together with other biomarkers such as other PDE8A mRNA editing sites or isoforms and mRNA expression of ADARs.

[0083] For example, we found that the mROC curve improved from an AUC of 0.878 using only PDE8A site B* editing to an AUC of 0.956 by adding PDE8A site A* mRNA editing together with ADAR1a and ADAR2 mRNA expression (see Table VI, Table VII, line 6, and FIG. 6). Other combinations listed in Table VII provided favorable diagnostic performance with AUCs between 0.878 (ADAR1b mRNA expression and PDE8A site B*, line 30) and 0.964 (ADAR1a and ADAR2 mRNA and PDE8A isoform B, isoform BC, and isoform D mRNA editing, line 1).

[0084] [Table 6]

[0085] FIG. 13 is a table of the composite diagnostic performance of the multivariate ROC curve comparing affective disorder controls obtained with the combination ADAR1a+ADAR2+PDE8A_site A*+PDE8A_site B* to suicide attempters.

[0086] Statistical analysis using a logistic regression model or mROC approach yielded similar results (Table VIII, FIG. 7). Both methods showed identical accuracy for ADAR1a mRNA expression + PDE8A site B* mRNA editing (Table VII, line 29 and Table VIII, line 3, and / or FIG. 7).

[0087] [Table 7]

[0088] JPEG0007654918000011.jpg128149

[0089] All combinations of biomarkers were performed by mROC approach. Combinations could be, for example, combinations of ADARs mRNA expression and PDE8A RNA site editing (adjusted* or not adjusted), combinations of ADARs mRNA expression and PDE8A RNA isoform editing (adjusted* or not adjusted), combinations of PDE8A RNA site editing (adjusted* or not adjusted), combinations of PDE8A RNA isoform editing (adjusted* or not adjusted). For example, the equation for the combination of biomarkers ADAR1a+ADAR2+PDE8A_siteA*+PDE8A_siteB* calculated by mROC program could be (a)Z=0.02327×[ADAR1a]-0.026474×[ADAR2]-16.6903×PDE8A_siteA*+0.99311×[PDE8A_siteB*]. These equations were used to calculate new virtual markers (Z) to perform patient diagnosis. The symbol "*" indicates that the editing site or editing isoform of PDE8A is adjusted for the relative expression of its mRNA.

[0090] [Table 8]

[0091] Logistic regression was used to evaluate the accuracy of biomarker combinations and their discriminatory power. Combinations could be, for example, the combination of ADARs expression and PDE8A RNA site editing (adjusted* or not adjusted), the combination of ADARs mRNA expression and PDE8A RNA isoform editing (adjusted* or not adjusted), the combination of PDE8A RNA site editing (adjusted* or not adjusted), and the combination of PDE8A RNA isoform editing (adjusted* or not adjusted). For example, the equation for the biomarker combination ADAR1a+PDE8A_siteB* calculated by logistic regression could be P=Logit-1(0.0564×[ADAR1a]+1.9012×[PDE8A_siteB*-4.68849]. These equations were used to calculate the probability P to perform the patient diagnosis. P(logit) is the probability that the patient belongs to the category of "suicide attempters". The symbol "*" indicates that the PDE8A editing site or editing isoform is adjusted for the relative expression of its mRNA.

[0092] Example 10: Increased PDE8A mRNA editing in Hepatitis C Virus (HCV)-infected patients during IFN-α therapy Major depression during IFN-α therapy for chronic hepatitis C virus (HCV) infection is common, with an incidence of up to 45%

[21] . This model offers a unique opportunity to evaluate individuals for depression and / or drug-induced psychiatric side effects within a short period (weeks). Longitudinal studies have the great advantage that each patient acts as his or her own reference control, ruling out genetic variability and environmental influences. Furthermore, ADAR1a is inducible by stimulation with interferon

[22] . In this study, we investigate whether PDE8A mRNA editing changes during IFN-α treatment of HCV patients. Six HCV patients were evaluated prospectively, 4 weeks before treatment, and every 2 weeks over the course of a 12-week treatment period with IFN-α and ribavirin (Figure 8). Overall, we observed an increase in PDE8A mRNA editing at site B in all patients after IFN-α treatment. For example, by further analyzing the relative proportions of RNA editing at the individual sites of PDE8A and gene expression of ADARs in two representative patients, we could observe clear inter-individual differences in the response to IFN-α treatment. In patient 1, we could not observe induction of ADARs gene expression in response to IFN-α, but a gradual increase in PDE8A RNA editing (Figures 9A, 9C). Conversely, in patient 2, we could observe a rapid and strong increase in the expression of ADARs (Figure 9B), as well as a rapid and strong increase in PDE8A RNA editing (Figure 9D). Overall, using the k-median approach, clustering of all six patients confirmed the different IFN-α-mediated responses, suggesting two distinct patient groups (Figure 10). The first patient group (cluster 1) has a significant and rapid IFN-α-mediated response to both ADAR1a transcript levels and PDE8A editing at site B, as seen by the steeper slope of the curves (Figure 10). The second group of patients (cluster 2) has a milder response compared to the other patients, as observed for patient 1 (Figures 9A-9C). All patients were clinically evaluated using the Montgomery-Asberg Depression Scale (MADRS), Mini-Instrumental Interview (MINI), Manic Depression Remission Scale (MAThyS), and Young Manic Rating Scale (YMRS).Psychiatric evaluation clearly demonstrated changes in mood, including irritability and / or depression as measured by the tests described above.

[0093] Example 11: Reduction in PDE8A mRNA editing over time in non-repeated suicide attempters Suicide attempts constitute one of the strongest risk factors for future suicide attempts [23, 24]. The rate of re-attempted suicide during the year after suicide ranges from 12 to 15% [25, 26]. In a follow-up study including 28 patients (Table 1(B)), we analyzed the clinical assessments of these patients at the first visit and after 6 months. We divided the whole group into two different subgroups: the first group (n = 20) who did not attempt suicide again (NRSA) and the second group (n = 8) consisting of patients who attempted suicide at least once during the period (RSA). The mean number of suicide attempts in this group during the 6-month follow-up period was 1.875. The patients were monitored using three independent clinical assessment scores (Hamilton, MADRS, and BDI), and the NRSA group showed a significant improvement in the BDI score during the follow-up (Figure 11A). On the other hand, the RSA group showed no improvement in the clinical assessments and even a significant deterioration in the MADRS score (Figure 11B). To compare their clinical assessment against the biomarker (RNA editing of the PDE8A gene), we analyzed RNA editing in leukocytes of these patients at follow-up evaluation (Figures 11C and 11D). We investigated whether PDE8A mRNA editing was modulated between non-repeated suicide attempters (NRSA) and repetitive suicide attempters (RSA) over a 6-month period. Interestingly, all PDE8A mRNA editing sites showed significant changes in the NRSA group, while only site B changed in the RSA group, suggesting an improvement in the mental status of these patients.

[0094] Example 12: Changes in PDE8A mRNA editing in Hepatitis C Virus (HCV)-infected patients undergoing IFN-α therapy Major depression during IFN-α therapy for chronic hepatitis C virus (HCV) infection is common, with an incidence of up to 45%

[21] . This model offers a unique opportunity to evaluate individuals for depression and / or pharmacogenic psychiatric side effects within a short period (weeks). In this study, we exploit the well-documented and well-characterized mood changes observed in patients infected with hepatitis C virus undergoing antiviral treatment with IFN and ribavirin. A small cohort of 10 individuals with hepatitis C virus, without a history of psychiatric disorders, was recruited in different hospitals in France. The patients underwent a harmonized psychiatric evaluation at recruitment and repeated at regular intervals during the treatment period. In this particular setting, each patient acts as his own control, allowing the evolution in him to be monitored over time. All patients were clinically evaluated using the Montgomery-Asberg Depression Scale (MADRS), MINI (Mini-Interface for Mental Illnesses), MAThyS (Manic Depressive Remission Scale), and YMRS (Young Manic Rating Scale). Psychiatric evaluation clearly indicated changes in mood, such as irritability and / or depression, as measured by the above-mentioned tests. To further investigate in more detail what happens to each patient treated with IFN, we analyzed the MADRS scores during the 12-week treatment period. We considered each patient with a high MADRS score along with emotional reactivity to have an altered mood (become depressed). Three out of ten patients were classified as having a depressive episode.

[0095] Longitudinal studies have the great advantage that each patient acts as his or her own reference control, ruling out genetic variability and environmental influences. Moreover, ADAR1a is inducible by stimulation with interferon

[22] . Although ADAR2 gene expression did not change during the treatment period, a clear upregulation of ADAR1a and ADAR1b transcript levels was observed as early as 2 weeks after the start of treatment (Figure 12A, Table IX). The similar expression profile observed between ADAR1a, ADAR1b, and PDE8A suggests that these genes are specifically altered by antiviral treatment. The RNA editing of the PDE8A gene adjusted for the relative PDE8A transcript levels was analyzed by applying an ultra-deep sequencing approach and analyzed in silico (Figure 12B). Since PDE8A gene expression increased significantly during the treatment period, the RNA editing of the PDE8A gene adjusted for the relative PDE8A transcript levels was analyzed. Remarkably, the increase in RNA editing of PDE8A transcripts was highly uniform in the entire study population, with total editing at site B of PDE8A transcripts doubling within 2 weeks. The overall increase in RNA editing at these sites reached a plateau level between 2 and 4 weeks of treatment initiation (Figure 12C). Consistent with previous data obtained in SH-SY5Y, IFN injection in patients infected with Hepatitis C virus induced a rapid and robust increase in RNA editing activity in their leukocytes. By combining gene expression data, a striking difference was observed between groups with and without depressive episodes (Figure 13A). Interestingly, a similar separation was observed by specifically analyzing RNA editing of PDE8A transcripts in combination with gene expression data (Figure 13B). Combining RNA editing and specific gene expression biomarkers showed high specificity and sensitivity in identifying IFN-treated patients at risk of experiencing a psychiatric event during treatment (Table X, Figure 13D). Finally, we tested a combination of biomarkers to identify patients at risk by applying a specific algorithm to all patients at all time points (Figure 14).The combination of biomarkers was very robust over the entire pilot period in detecting the three patients who would develop depression, with 3 of 18 calls being false negative (16%). On the other hand, only 3 of 42 results were false positive (7%) in the group of patients without clinical signs of depression. Taken together, the identified blood biomarkers allow for accurate and robust detection of patients at risk of developing depression in the setting of drug treatment such as IFN.

[0096] [Table 9]

[0097] The p-values, fold induction, and areas under the ROC curve (AUC) obtained by Wilcoxon test are summarized in Table IX. ADAR2 expression did not change during the study period while ADAR1a, ADAR1b, and PDE8A were significantly increased.

[0098] [Table 10]

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(2008) Altered editing in cyclic nucleotide phosphodiesterase 8A1 gene transcripts of systemic lupus erythematosus T lymphocytes. Immunology 125: 408-419. 11. Morse DP, Aruscavage PJ, Bass BL (2002) RNA hairpins in noncoding regions of human brain and Caenorhabditis elegans mRNA are edited by adenosine deaminases that act on RNA. Proc Natl Acad Sci U S A 99: 7906-7911. 12. Youngchao Ge SD, Terence P. Speed (2003) Resampling-based multiple testing for microarray data analysis. Test 12: 1-77. 13. Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, et al. (2004) Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 5: R80. 14. Kramar A FD, Fortune A, Reiser B (2001) mROC: A computer program for combining tumour predicting disease states. Comput Methods Programs Biomed 66: 199-207. 15. Su JQ LJ (1993) Linear combinations of multiple diagnostic markers. Journal of the American Statistical Association. 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Asnis GM, De La Garza R, 2nd (2006) Interferon-induced depression in chronic hepatitis C: a review of its prevalence, risk factors, biology, and treatment approaches. J Clin Gastroenterol 40: 322-335. 21. Asnis GM, De La Garza R, 2nd (2006) Interferon-induced depression in chronic hepatitis C: a review of its prevalence, risk factors, biology, and treatment approaches. J Clin Gastroenterol 40: 322-335. 22. George CX, Samuel CE (1999) Human RNA-specific adenosine deaminase ADAR1 transcripts possess alternative exon 1 structures that initiate from different promoters, one constitutively active and the other interferon inducible. Proc Natl Acad Sci U S A 96: 4621-4626. 23. Borges G, Angst J, Nock MK, Ruscio AM, Walters EE, et al. (2006) A risk index for 12-month suicide attempts in the National Comorbidity Survey Replication (NCS-R). Psychol Med 36: 1747-1757. 24. Brown GK, Beck AT, Steer RA, Grisham JR (2000) Risk factors for suicide in psychiatric outpatients: a 20-year prospective study. J Consult Clin Psychol 68: 371-377. 25. Cedereke M, Ojehagen A (2005) Prediction of repeated parasuicide after l-12 months. Eur Psychiatry 20: 101-109. 26. Heyerdahl F, Bjornaas MA, Dahl R, Hovda KE, Nore AK, et al. (2009) Repetition of acute poisoning in Oslo: 1-year prospective study. Br J Psychiatry 194: 73-79. The present invention includes the following aspects. <1> 1. An in vitro method for predicting the risk that a patient will present with a condition selected from the group consisting of a psychiatric or neurological syndrome, an immunological syndrome, and a degenerative syndrome associated with altered A-to-I editing on the PDE8A transcript, or for determining whether a patient is at risk for developing said condition, comprising: from a body fluid sample of said patient containing cells expressing at least one of the editing enzymes ADAR1a, ADAR1b, and ADAR2 and PDE8A. (a) in the same intracellular RNA extract obtained from the cell-containing blood sample (i) the expression level of PDE8A mRNA, and (ii) the level of PDE8A RNA editing at at least one or a combination of editable sites in the PDE8A gene, preferably sites associated with said pathology; and / or the expression level of at least one PDE8A isoform or a combination of PDE8A isoforms which are expressible and which are preferably associated with said pathology; determining a (b) determining the relative proportion of the RNA editing sites and / or the relative proportion of the PDE8A isoform level to the PDE8A mRNA expression level from the results obtained in step (a); and (c) comparing the relative proportions of the RNA editing sites and / or isoforms obtained in step (b) with (i) the relative proportions of RNA editing and / or isoforms at the sites obtained from control body fluid samples from patients known to exhibit or not exhibit the pathological condition, or known to be or not at risk for developing the pathological condition, and / or (ii) applying an algorithm or statistical model exhibiting discrimination power using the editing sites and / or isoforms selected in step (b), or a combination thereof; identifying whether the patient is experiencing or at risk of developing the condition by using the results or end values ​​obtained in (i) or (ii) of step (c) to determine whether said patient develops said pathological condition or is at risk of developing said pathological condition. The method comprising: <2> The condition is a mental illness. <1> The method according to <3> The pathology is selected from the group consisting of depressive disorder, psychotic disorder, bipolar disorder, schizophrenia, panic disorder, social anxiety disorder, post-traumatic stress disorder (PTSD), substance dependence / abuse, abnormal eating behavior, obsessive-compulsive disorder (OCD), early Alzheimer's disease, and Parkinson's disease. <1> or <2> The method according to <4> The patient is selected from among patients who have already been identified as having a psychiatric disorder and who have or have not had a history of suicide attempts, and in step (c), it is identified whether the patient has exhibited suicidal behavior or is at risk of developing suicidal behavior. <1> The method according to <5> The patient is a patient who has already been treated for a non-psychiatric disorder. <1> The method according to <6> A method for predicting a patient's risk of attempting suicide, comprising obtaining a control body fluid sample from a control group of patients suffering from an affective disorder in step (c)(i). <1> The method according to <7> A method for determining whether a patient exhibits depression, comprising obtaining a control body fluid sample from a healthy group of patients in step (c)(i), <1> The method according to <8> The method allows monitoring patients and / or dividing patients into treatment responders and treatment non-responders. <1> The method according to <9> step (a) comprises determining the expression levels of the editing enzymes ADAR1a, ADAR1b and / or ADAR2 in the same cell extract; In step (c), it is determined whether the patient exhibits the condition or is at risk of developing the condition. (i) further comparing the expression levels of the editing enzymes ADAR1a, ADAR1b and / or ADAR2 obtained in step (b) with expression levels obtained for normal patients and, optionally, with expression levels obtained for patients exhibiting a pathology associated with altered A-to-I editing on the PDE8A transcript, and / or (ii) applying an algorithm or statistical model exhibiting discrimination ability using the editing sites and / or isoforms, and the expression levels of ADARs selected in steps (a) and (b), or a combination thereof; The above is determined by <1> ~ <8> 2. The method according to claim 1 . <10> The body fluid sample is selected from a blood sample, a urine sample, and a saliva sample, preferably a sample containing PBMC (peripheral blood mononuclear cells), more preferably a blood sample. <1> ~ <9> 2. The method according to claim 1 . <11> In step (a), the determination of PDE8A RNA editing per site is performed for at least one site selected from the editing sites listed in Table III, preferably for a plurality of sites including at least one site among sites O to ZZ in Table III, more preferably for sites selected from the group consisting of sites A, B, C, D, E, F, G, Z, and ZZ. <1> ~ <10> 2. The method according to claim 1 . <12> In step (a), a relative ratio of the level of a PDE8A isoform containing the one or more editing sites to the level of PDE8A mRNA expression is determined. <1> ~ <11> 2. The method according to claim 1 . <13> In step (a), the relative proportion of the PDE8A RNA isoforms selected from the group consisting of isoforms B, BC, D, E, Ne (non-edited), BE, G, BG, BF, A, Z, C, F, BZ, and BD is determined. <12> The method according to <14> In step (c), the algorithm is selected from the group of mROC combinations consisting of the combinations listed in Table VI. <1> ~ <13> 2. The method according to claim 1 . <15> In the steps (a) and (b), the PDE8A mRNA expression level is determined by qPCR, and / or the RNA editing level at each site is determined by NGS (next generation sequencing). <1> ~ <14> 2. The method according to claim 1 . <16> Seq1-Forward (SEQ ID NO:2) or Seq2-Forward (SEQ ID NO:3) as forward primer; Seq1-reverse (SEQ ID NO: 4), Seq2-reverse (SEQ ID NO: 5) or Seq3-reverse (SEQ ID NO: 6) as a reverse primer; and The following selection criteria, namely (i) an oligo between 18 and 27 nucleotides in length; (ii) Preferably has a melting point of around 60°C (57°C to 63°C) (iii) have an optimal GC content of around 50% (±5%) (iiii) the maximum allowable repeat length is 5 mononucleotides; and (vi) specifically amplifying the region of interest shown in FIG. 1; A set of forward and reverse primers that match The RNA editing level at each site of PDE8A mRNA is determined by a set of primers selected from the group consisting of: <1> ~ <15> 2. The method according to claim 1 . <17> 1. A kit for determining whether a patient is at risk of developing a condition selected from the group consisting of a psychiatric or neurological syndrome, an immunological syndrome, and a degenerative syndrome associated with an altered A-to-I editing on the PDE8A transcript, or for determining whether a patient is at risk of developing said condition, comprising: (1) to obtain a final value by which the risk of causing the pathological condition can be determined, <1> ~ <16> and instructions, with or without a ROC curve, for applying the method according to any one of claims 1 to 5. (2) Seq1-Forward (SEQ ID NO:2) or Seq2-Forward (SEQ ID NO:3) as a forward primer; Seq1-reverse (SEQ ID NO: 4), Seq2-reverse (SEQ ID NO: 5) or Seq3-reverse (SEQ ID NO: 6) as a reverse primer; and The following selection criteria, namely (i) an oligo between 18 and 27 nucleotides in length; (ii) Preferably has a melting point of around 60°C (57°C to 63°C) (iii) have an optimal GC content of around 50% (±5%) (iiii) the maximum allowable repeat length is 5 mononucleotides; and (vi) specifically amplifying the region of interest shown in FIG. 1; A set of forward and reverse primers that match A set of primers selected from the group consisting of: The kit comprising: <18> The primer set is a set consisting of Seq2-forward (SEQ ID NO: 3) and Seq3-reverse (SEQ ID NO: 6). <16> or the method according to <17> The kit according to claim 1,

Claims

1. 1. An in vitro method for predicting the risk of a patient to present a condition that is a psychiatric disorder associated with altered A-to-I editing on the PDE8A transcript, comprising: from a blood sample of said patient containing cells expressing at least one of the editing enzymes ADAR1a, ADAR1b, and ADAR2 and PDE8A. (Aa) In the same intracellular RNA extract obtained from the cell-containing blood sample (i) the expression level of PDE8A mRNA, and (ii) the level of PDE8A RNA editing at at least one or a combination of editable sites in the PDE8A gene that are associated with the pathology; or the expression level of at least one PDE8A isoform or a combination of PDE8A isoforms that is expressible and associated with said pathology; determining (Ab) determining an RNA editing value, which is the relative proportion of the RNA editing site or the relative proportion of the PDE8A isoform level to the PDE8A mRNA expression level, from the results obtained in step (Aa) and calculating an adjusted RNA editing value as follows: Adjusted RNA editing value = (RNA editing value x relative transcript level) / 100; and (Ac) comparing the adjusted RNA editing value of the RNA editing site or the isoform obtained in step (Ab) with (i) the adjusted RNA editing value of the RNA editing site or the isoform obtained from a control blood sample of a patient known to exhibit or not exhibit the pathology; and (ii) applying an algorithm or statistical model exhibiting discrimination power using the editing sites or isoforms selected in step (Ab), or a combination thereof; identifying whether the patient is developing the pathological condition by using the results or final values ​​obtained in (i) and (ii) of step (Ac) to determine whether said patient develops said pathological condition; The threshold determined for the control sample in step (Ac)-(i) or the algorithm used in step (Ac)-(ii) comprises the following steps: (Ba) selecting a patient collection of patients annotated as having a proportion of risk for developing a mental illness, or annotated as not having that risk, or a patient collection for confirming that the patients will develop a mental illness; Selecting one or several PDE8A RNA editing sites or isoforms, or a combination thereof; (Bb) analyzing the target PDE8A RNA editing site or target isoform to obtain an RNA editing level of the target site or a proportion of the target isoform for each of the patients of the collection; (Bc)-(i) evaluating the threshold or its accuracy and ability for each RNA editing site or each isoform to identify that the patient is experiencing the pathology by univariate statistical analysis methods, or (Bc)-(ii) evaluating the accuracy and its ability for each combination of RNA editing sites or isoforms to identify that the patient is experiencing the pathology by multivariate statistical analysis methods, and (Bc)-(iii) selecting editing sites or isoforms, or combinations thereof, that show acceptable discrimination ability, and (Bd) constructing an algorithm using the selected combination of editing sites or isoforms and using the algorithm so obtained to identify the patient as expressing the pathology; and The algorithm that can be used in the method is mROC program for identifying linear combinations that maximize the AUC (area under the curve) of the ROC, the equation for each combination being: ｉ is the calculated coefficient and (biomarker i) is the adjusted RNA editing value for the individual level of the RNA editing site or isoform target: Z = a １ (Biomarker 1) + a ２ (Biomarker 2) + a ｉ (biomarker i)+ . . . a ｎ (biomarker n) and which can be used as a new virtual marker Z; or Logistic regression models applied to univariate and multivariate analyses to estimate the relative risk of patients with different RNA editing site levels or isoform values; or A CART (Classification and Regression Trees) approach applied to evaluate combinations of RNA editing sites or isoforms; or A random forest (RF) approach is applied to evaluate combinations of RNA editing sites or isoforms, ranking the importance of those RNA editing sites or isoforms and combining the best RNA editing sites or isoforms to classify a "relative risk"; or A multivariate analysis applied to evaluate combinations of RNA editing sites or isoforms for "relative risk", Support Vector Machine (SVM) approach, Artificial Neural Network (ANN) approach, Bayesian network approach, WKNN (weighted k-nearest neighbor) approach, Partial least squares discriminant analysis (PLS-DA), Linear and quadratic discriminant analysis (LDA / QDA), and Any other mathematical method of combining biomarkers; The method is carried out by a multivariate method comprising the multivariate analysis selected from the group consisting of Law.

2. 2. The method of claim 1, wherein the psychiatric disorder is selected from the group consisting of depressive disorder, psychotic disorder, bipolar disorder, schizophrenia, panic disorder, social anxiety disorder, post-traumatic stress disorder (PTSD), substance dependence / abuse, abnormal eating behavior, obsessive-compulsive disorder (OCD), early Alzheimer's disease, and Parkinson's disease.

3. 2. The method of claim 1, wherein the patient has already been identified as exhibiting a psychiatric disorder and is selected from among patients with or without a history of suicide attempts, and in step (Ac) it is identified whether the patient is exhibiting suicidal behavior.

4. The method of claim 1 , wherein the patient is already receiving treatment for a non-psychiatric disorder.

5. 2. The method for predicting the risk of a patient attempting suicide according to claim 1, wherein the control blood sample in step (Ac)(i) is a control blood sample obtained from a patient of an affective disorder control group.

6. 2. The method according to claim 1, for determining whether a patient exhibits depression, wherein the control blood sample in step (Ac)(i) is a control blood sample obtained from a patient of a healthy group.

7. 10. The method of claim 1, which allows for monitoring patients or dividing patients into treatment responders and treatment non-responders.

8. Step (Aa) comprises determining the expression level of the editing enzymes ADAR1a, ADAR1b or ADAR2 in the same cell extract; In step (Ac), whether the patient develops the pathological condition is determined. (i) further comparing the expression levels of the editing enzymes ADAR1a, ADAR1b or ADAR2 obtained in step (Ab) with the expression levels obtained for normal patients and with the expression levels obtained for patients exhibiting a pathology associated with altered A-to-I editing on the PDE8A transcript; and (ii) applying an algorithm or statistical model showing discrimination ability using the editing sites or isoforms, and the expression levels of ADARs selected in steps (Aa) and (Ab), or a combination thereof; The method according to any one of claims 1 to 7, wherein the determination is made by:

9. The method according to any one of claims 1 to 8, wherein in step (Aa), the determination of PDE8A RNA editing per site is carried out for at least one site selected from the editing sites listed in Table III.

10. 10. The method of claim 9, wherein in step (Aa), the determination of PDE8A RNA editing per site is performed for a plurality of sites including at least one site of sites O through ZZ of Table III.

11. 10. The method of claim 9, wherein in step (Aa), the determination of PDE8A RNA editing for each site is performed for a plurality of sites including at least one selected from the group consisting of sites A, B, C, D, E, F, G, Z, and ZZ.

12. The method according to any one of claims 1 to 11, wherein in step (Aa) the relative proportion of the level of a PDE8A isoform containing one editing site or multiple editing sites to the PDE8A mRNA expression level is determined.

13. 13. The method of claim 12, wherein in step (Aa) the relative proportion of the PDE8A RNA isoforms selected from the group consisting of isoforms B, BC, D, E, Ne (non-edited), BE, G, BG, BF, A, Z, C, F, BZ, and BD is determined.

14. The method according to any one of claims 1 to 13, wherein in steps (Aa) and (Ab), the PDE8A mRNA expression level is determined by qPCR, and the RNA editing level per site is determined by NGS (next generation sequencing).

15. Seq1-Forward (SEQ ID NO:2) or Seq2-Forward (SEQ ID NO:3) as the forward primer; Seq1-reverse (SEQ ID NO:4), Seq2-reverse (SEQ ID NO:5) or Seq3-reverse (SEQ ID NO:6) as a reverse primer; and The following selection criteria, namely (i) an oligo between 18 and 27 nucleotides in length (ii) having a melting point of around 60°C (57°C to 63°C); (iii) have an optimal GC content of around 50% (±5%) (iv) the maximum allowable repeat length is 5 mononucleotides; and (v) specifically amplifying the region of interest shown in FIG. A set of forward and reverse primers that match The method according to any one of claims 1 to 14, wherein the RNA editing level at each site of PDE8A mRNA is determined by a set of primers selected from the group consisting of:

16. A kit for determining whether a patient is at risk of developing a condition that is a psychiatric disorder associated with altered A-to-I editing on the PDE8A transcript, comprising the steps of: (1) instructions for applying the method according to any one of claims 1 to 15 in order to obtain a final value, the analysis of which allows determining the risk of causing said pathological condition; (2) Seq1-forward (SEQ ID NO:2) or Seq2-forward (SEQ ID NO:3) as a forward primer; Seq1-reverse (SEQ ID NO:4), Seq2-reverse (SEQ ID NO:5) or Seq3-reverse (SEQ ID NO:6) as a reverse primer; and The following selection criteria, namely (i) an oligo between 18 and 27 nucleotides in length (ii) having a melting point of around 60°C (57°C to 63°C); (iii) have an optimal GC content of around 50% (±5%) (iv) the maximum allowable repeat length is 5 mononucleotides; and (v) specifically amplifying the region of interest shown in FIG. A set of forward and reverse primers that match A set of primers selected from the group consisting of: The kit comprising:

17. The primer set is Seq2-forward (SEQ ID NO: 3) and Seq3-reverse (SEQ ID NO: 6).

18. The kit according to claim 16, wherein the set of primers consists of Seq2-forward (SEQ ID NO: 3) and Seq3-reverse (SEQ ID NO: 6).

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