EP3445868A1 - Methylomic and transcriptomic changes during conversion to psychosis - Google Patents

Methylomic and transcriptomic changes during conversion to psychosis

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Publication number
EP3445868A1
EP3445868A1 EP17717204.6A EP17717204A EP3445868A1 EP 3445868 A1 EP3445868 A1 EP 3445868A1 EP 17717204 A EP17717204 A EP 17717204A EP 3445868 A1 EP3445868 A1 EP 3445868A1
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Prior art keywords
psychosis
chromosome
ending
starting
subject
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EP17717204.6A
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German (de)
French (fr)
Inventor
Marie-Odile Krebs
Oussama KEBIR
Boris CHAUMETTE
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Institut National de la Sante et de la Recherche Medicale INSERM
Universite Paris Descartes
Centre Hospitalier Sainte Anne
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Institut National de la Sante et de la Recherche Medicale INSERM
Universite Paris Descartes
Centre Hospitalier Sainte Anne
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Publication of EP3445868A1 publication Critical patent/EP3445868A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention refers to an in vitro method for diagnosing the transition to psychosis in a subject at risk of psychosis and a method for predicting onset of psychosis.
  • Schizophrenia is a progressive illness, generally occurring in late adolescence, with several stages from early vulnerability, at-risk mental state (also called ultra-high risk abbreviated UHR), first-episode psychosis, to chronic disease.
  • UHR ultra-high risk abbreviated UHR
  • the molecular mechanisms triggering the progression of the illness are still largely unknown.
  • the onset of psychosis is the consequence of complex interactions between genetic vulnerability to psychosis and response to environmental and/or maturational changes. Epigenetics is hypothesized to mediate the interplay between genes and environment leading to the onset of psychosis.
  • the inventors of the present invention performed the first longitudinal prospective study of genomic DNA methylation during psychotic transition in help-seeking young individuals referred to a specialized outpatient unit for early detection of psychosis, and enrolled in a one-year follow-up.
  • the Infinium HumanMethylation450 BeadChip array was used after bisulfite conversion and longitudinal variations in methylation at 41 1 947 CpG sites were analyzed.
  • Methylation data were confirmed by pyrosequencing in the same population.
  • the 100 top CpGs associated with conversion to psychosis were subjected to exploratory analyses regarding the related gene networks and their capacity to distinguish between converters and non-converters. Cluster analysis showed that the top CpG sites correctly distinguished between converters and non- converters.
  • Methylation changes associated with conversion to psychosis preferentially occurred in gene promoters and pathways relevant for psychosis, including oxidative stress regulation, axon guidance and inflammatory pathways. Longitudinal variations in DNA methylation may reflect the biological mechanisms that precipitate some prodromal individuals into full-blown psychosis, under the influence of environmental factors and maturational processes at adolescence.
  • UHR clinical ultra-high-risk state for psychosis
  • at-risk mental state The identification of clinical ultra-high-risk state for psychosis (hereinafter UHR; also known as the "at-risk mental state”) has been a relatively recent development in the field of psychiatry, and it has provided a means to capture the pre-psychotic phase and to describe individuals with prodromal symptoms that may transition into psychosis and schizophrenia (Yung, AR and McGorry, 1996, Aust. N. Z. J. Psychiatry 30:587-99). Operational criteria for UHR have been proposed based on specific comprehensive interviews. In individuals reaching these criteria, the conversion rate of individuals at UHR to full-blown psychosis is 30 to 40% in the following 24 to 36 months.
  • the inventors further studied longitudinal variations in gene expression during the onset of psychosis.
  • a longitudinal transcriptomic analysis was performed in blood samples. 14 genes and 2 gene networks, in particular the genes CPT1 A and NRP1 , were identified by the inventors as being differentially expressed during psychotic transition.
  • the present invention thus refers to an in vitro method for diagnosing the transition to psychosis in a subject at risk of psychosis, comprising
  • step c based on the comparison of step b), determining differentially methylated regions (DMR) in the sample of step a), and
  • DMR differentially methylated region
  • said differentially methylated region is located in a gene, wherein said gene is selected from the group consisting of GSTT1 , GSTP1 , GSTM5, LOC441228, ZNF514, RNF34, COQ2, DOK7, HIST1 H2AM, UNGP3, KIAA1274, LRP2BP, CDH13, TRAF3IP2, PTPRN2, COL9A2, TRIM39, NRP1 , GGA3, CHL1 , SIM1 , TFAP2E, SCRN1 , RIPPLY3, UHRF1 BP1 L, KIA1026, t-RNA-Leu, MIR3683, ATRN, EFNA3, CBS, ZNF282, BAH , NFIX, PPM1 E, CEP152,IL17RE, LZTS2, FBRSL1 , EREG, CRELD1 , KCNIP4, AHSP, ASAM, INPP5A, LRWD1 , COX10, MRC2,
  • the invention further refers to a method for predicting onset of psychosis in a subject at risk of psychosis comprising
  • step c based on the comparison of step b), determining differentially methylated regions (DMR) in the sample of step a), and
  • the invention further refers to the use of at least one differentially methylated region (DMR) located in a chromosomal region in a chromosomal as defined herein above in the 'in vitro method for diagnosing the transition to psychosis'.
  • DMR differentially methylated region
  • the invention further refers to a DNA methylation inhibitor and/or a gene expression modulator, said gene being selected from the group consisting of GSTT1 , GSTP1 , GSTM5, LOC441228, ZNF514, RNF34, COQ2, DOK7, HIST1 H2AM, UNGP3 (also called STON2), KIAA1274, LRP2BP, CDH13, TRAF3IP2, PTPRN2, COL9A2, TRIM39, NRP1 , GGA3, CHL1 , SIM1 , TFAP2E, SCRN1 , RIPPLY3 (also called MRPL20P1 ), UHRF1 BP1 L, KIA1026, T-RNA-Leu, MIR3683 (also called LOC100121257), ATRN, EFNA3, CBS, ZNF282, BAH , NFIX, PPM1 E, CEP152,IL17RE, LZTS2, FBRSL1 , EREG, CRELD1 , KCNIP4,
  • the invention refers to a method of treatment of psychosis or transition in psychosis in a subject in need thereof which method comprises administering said subject with a therapeutically effective amount of a DNA methylation inhibitor and/or a gene expression modulator, said gene being as defined herein above.
  • the invention refers to a method of treatment of psychosis or transition in psychosis in a subject in need thereof which method comprises
  • the present invention further refers to an in vitro method for diagnosing the transition to psychosis in a subject at risk of psychosis, said method comprising a) determining the level of expression of at least one gene selected from the group consisting of CPT1 A, NRP1 , CREB3L3, EPB49, EREG, GYPA, HBB, HBD, HSPB7, MUC16, OSBP2, PEG3, TBX1 , TMOD1 , TRIM58, AKT1 , CHL1 , GSTM5 and IL17RE in a biological sample from said subject,
  • step b) comparing said level of expression of the at least one gene determined in step a) to a reference value
  • step c) deducing from the comparison of step b) if the subject is in transition to psychosis.
  • the present invention further refers to a method for predicting onset of psychosis in a subject at risk of psychosis comprising
  • step b) deducing from the comparison of step b) if the subject is at risk of onset of psychosis.
  • the methods based on CpG methylations and the methods based on the level of expression of at least one gene may be combined.
  • the present invention further refers to an in vitro method for diagnosing the transition to psychosis in a subject at risk of psychosis, said method comprising
  • step aiii based on the comparison of step aii), determining differentially methylated regions (DMR) in the sample of step ai), and
  • step bi) determining the level of expression of at least one gene selected from the group consisting of CPT1 A, NRP1 , CREB3L3, EPB49, EREG, GYPA, HBB, HBD, HSPB7, MUC16, OSBP2, PEG3, TBX1 , TMOD1 , TRIM58, AKT1 , CHL1 , GSTM5 and IL17RE in a biological sample from said subject, ii) comparing said level of expression of step bi) with the at least one gene determined in step bii) to a reference value; and
  • DMR differentially methylated region
  • steps a) including ai), aii) and aiii) and b) including bi) and bii) may be switched, i.e. the steps as indicated under b) may be performed first, followed by the steps as indicated under a).
  • the present invention further refers to a method for predicting onset of psychosis in a subject at risk of psychosis comprising
  • step aiii based on the comparison of step aii), determining differentially methylated regions (DMR) in the sample of step ai), and
  • DMR differentially methylated region
  • steps a) including ai), aii) and aiii) and b) including bi) and bii) may be switched, i.e. the steps as indicated under b) may be performed first, followed by the steps as indicated under a).
  • the sample used in steps a) and b) in particular step ai) and bi) is a sample from the same subject and the same origin. For example, one blood sample is obtained from said subject and then divided into two, one part of said sample being used for the steps a) of the combined methods of the invention and the other part of said sample being used for the steps b) of the combined methods of the invention.
  • determining includes qualitative and/or quantitative detection (i.e. detecting and/or measuring the level of expression of at least one gene as defined herein above or determining CpG methylations as defined herein above) with or without reference to a control.
  • Psychosis is a clinical term and herein refers to a family of serious mental illnesses, referred to as psychotic disorders.
  • Psychosis in general, is characterized by radical changes in personality, impaired functioning, and a distorted or nonexistent sense of objective reality. Accordingly, subjects suffering from psychosis have impaired reality testing; that is, they are unable to distinguish personal subjective experience from the reality of the external world.
  • Primary symptoms of psychosis include hallucinations, delusions, disorganized speech (except for substance/medication-induced psychotic disorder and psychotic disorder due to another medical condition), abnormal psychomotor behavior, as well as dimensional assessments of depression and mania.
  • Symptoms of psychosis may be relatively constant or come and go, and is therefore also referred to as "psychotic symptoms", “psychotic episode” or “psychotic experience”.
  • Psychosis refers to psychotic disorders, as mentioned above, and as further defined herein below, but psychosis can also occur for many other reasons, including substance abuse, brain injury, seizure disorders, or conditions of extreme sleep deprivation or isolation.
  • Psychotic disorders include, but are not limited to, schizotypal (Personality) disorders, delusional disorder, brief psychotic disorder, schizophreniform disorder, schizophrenia, schizoaffective disorder, substance/medication-induced psychotic disorder, psychotic disorder due to another medical condition, catatonia, depression and bipolar disorders. Accordingly, in one embodiment, psychosis herein refers to psychotic disorders such as schizotypal (Personality) disorders, delusional disorder, brief psychotic disorder, schizophreniform disorder, schizophrenia, schizoaffective disorder, substance/medication- induced psychotic disorder, psychotic disorder due to another medical condition, catatonia, depression and bipolar disorders.
  • psychosis is not schizophrenia.
  • psychosis is not chronic schizophrenia
  • Transition to psychosis herein refers to a "pre-psychotic” or “prodromal stage” of psychosis, which has been defined as the period marked by changes from a person's premorbid mental state and level of functioning up to the appearance of psychotic features (Yung, AR and McGorry, 1996, Aust. N. Z. J. Psychiatry 30:587-99 and Yung AR, McGorry PD. 1996, Schizophr. Bull. 22(2):353-70).
  • Those changes may be, for example, changes in drive, perception, beliefs, attention, concentration, mood, affect, and behavior (Yung, AR and McGorry, 1996, Aust. N. Z. J. Psychiatry 30:587-99 and Yung AR, McGorry PD. 1996, Schizophr. Bull. 22(2):353-70). It will be understood by the skilled in the art, that a precondition for early intervention is the accurate detection of prodromal states, i.e., knowing who may be at high risk of developing a psychotic disease.
  • CAARMS Comprehensive Assessment of At-Risk Mental States
  • UHR Ultra-high-risk state for psychosis
  • CAARMS Comprehensive Assessment of At-Risk Mental States
  • CAARMS includes the following subscales: disorders of thought content (e.g. delusional mood, overvalued ideas and delusions), perceptual abnormalities (e.g. distortions, illusions and hallucinations), conceptual disorganization (e.g. subjectively experienced difficulties with forming thoughts and objective assessment of formal thought disorder), motor changes (e.g.
  • Scores for each subscale range from 0 to 6. Accordingly, a subject obtains a CAARMS score depending on his assessment results in each of the above mentioned subscales. Typically, the assessment is done by a trained psychiatrist.
  • subject refers to an animal, preferably a non- human or human mammal.
  • non-human mammals include rodents and primates.
  • the subject is a human.
  • the "subject” denotes herein an individual that is under medical care, surveillance or treatment.
  • the present invention refers to the diagnosis of the transition to psychosis in a subject at risk of psychosis, or, in other words, diagnosing in a subject at risk of psychosis a high-risk state for psychosis or a Ultra-high-risk state for psychosis (UHR) using the methods of the invention.
  • the subject does not have
  • Manifest symptoms of psychosis herein preferably refers to fulfilling typically the DSM-IV criteria for psychosis.
  • the subject in context of the present invention, is at risk of psychosis.
  • a subject "at risk of psychosis” herein refers to a subject having alterations in global functioning.
  • “Alterations in global functioning” herein refers to a social and occupational functioning assessment scale score of typically less than 70, preferably, obtained during the during the year preceding the time point of the application of the methods of the invention.
  • the subject at risk of psychosis does not have a CAARMS score above the psychosis threshold as defined by CAARMS.
  • the CAARMS score, threshold and methods to determine the CAARMS score of an individual at risk of psychosis are known to the skilled in the art and further described in "Diagnostic and Statistical Manual of Mental disorders » DSM-5, American Psychiatic Association, 2013 or DSM-4, American Psychiatic Association, 1994.
  • the subject is not under antipsychotic treatment for more than 12 weeks, wherein antipsychotic treatment preferably refers to more than 10Omg of a Chlorpromazine equivalent.
  • the subject has no psychoactive substance dependence or abuse during the year preceding the time point of the application of the methods of the invention and/or during more than 5 years.
  • the subject does not suffer from serious or non- stabilized somatic and neurological disorders.
  • the subject does not suffer from a head injury.
  • the subject has an intelligent quotient of more than 70.
  • the subject has any age; preferably, the age of the subject is 16 to 30.
  • Symptoms that might indicate a risk of psychosis are generally known to the skilled in the art and are, for example, further described in "Diagnostic and Statistical Manual of Mental disorders » DSM-5, American Psychiatic Association, 2013 or in the articles of, for example, Yung, AR and McGorry (see Yung AR, McGorry PD. 1996a. Aust. N. Z. J. Psychiatry 30:587-99, Yung AR, McGorry PD. 1996b. Schizophr. Bull. 22(2):353-70, Yung AR et al. 1996 Schizophr. Bull. 22(2):283-303, and Yung AR, et al. 2005. Aust. N. Z. J. Psychiatry 39(1 1 -12):964-71 ).
  • a "subject that is at risk of psychosis” shows at least one symptom selected from the group constituted of subtle changes in drive, subtle changes in affect, subtle changes in thought, subtle changes in speech, subtle changes in perception, subtle changes in motor activity, first vegetative symptoms, social difficulties, academic problems, vocational problems, mood symptoms (anxiety, depression); social withdrawal, impaired social cognition (affect recognition; understanding others' emotions, actions, intentions), attenuated psychotic symptoms such as unusual thoughts, suspiciousness, paranoia, playful, thinking, and primary symptoms of psychosis such as hallucinations, delusions, disorganized speech, abnormal psychomotor behavior, as well as dimensional assessments of depression and mania.
  • a subject that is at risk of psychosis shows at least one symptom selected from the group constituted of disorders of thought content (e.g. delusional mood, overvalued ideas and delusions), perceptual abnormalities (e.g. distortions, illusions and hallucinations), conceptual disorganization (e.g. subjectively experienced difficulties with forming thoughts and objective assessment of formal thought disorder), motor changes (e.g. subjectively experienced difficulties with movement and objective signs of catatonia), concentration and attention (assessing both the subjective experience and objective rating), emotion and affect (assessing subjective sense of change in emotions and objective rating of blunting of affect), subjectively impaired energy (a basic symptom) and impaired tolerance to normal stress (a basic symptom).
  • disorders of thought content e.g. delusional mood, overvalued ideas and delusions
  • perceptual abnormalities e.g. distortions, illusions and hallucinations
  • conceptual disorganization e.g. subjectively experienced difficulties with forming thoughts and objective assessment of formal thought disorder
  • motor changes e.g. subjectively experienced difficulties
  • At least one symptom herein refers to at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more symptoms as defined above.
  • the subject that is at risk of psychosis might suffer from, for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more symptoms selected from the list of symptoms mentioned above, preferably, the subject that is at risk of psychosis suffers from 3, 4, 5 or more symptoms selected from the group of symptoms indicated above.
  • the subject at risk of psychosis is at ultra-high-risk for psychosis, wherein ultra-high-risk for psychosis is defined as herein above, preferably,
  • the subject at risk of psychosis has beforehand been identified by a physician, in particular by a psychiatrist, as being at risk of psychosis, in particular, using standard criteria for best-estimate diagnosis, more particularly using comprehensive assessment for at risk mental state.
  • ultra-high-risk for psychosis is determined in a subject using the comprehensive assessment for at risk mental state (CAARMS).
  • CAARMS Comprehensive assessment for at risk mental state
  • CAARMS manual provides detailed definitions, questions, and anchor points for eliciting and rating 27 symptoms across seven dimensions of psychopathology, including positive symptoms, negative symptoms, deterioration of role functioning, sleep disturbance, and impaired tolerance to normal stress (see Yung AR, et al. 2005. Aust. N. Z. J. Psychiatry 39(1 1 -12):964-71 ).
  • the subject at risk of psychosis has been beforehand diagnosed by a trained psychiatrist using the comprehensive assessment for at risk mental state (CAARMS).
  • methylome refers to the DNA methylations in an organism's genome or in a particular cell.
  • Methodslomic changes refer accordingly to changes of the DNA methylation in an organism's genome or in a particular cell.
  • DNA methylation is a process by which methyl groups are added to DNA. Methylation modifies the function of the DNA. When located in a gene promoter, DNA methylation typically acts to repress gene transcription. DNA methylation is essential for normal development and is associated with a number of key processes including genomic imprinting, X-chromosome inactivation, repression of repetitive elements, aging and carcinogenesis. Two of DNA's four nucleotides, cytosine and adenine, can be methylated. Adenine methylation is restricted to prokaryotes.
  • Cytosine methylation involves the addition of a methyl group at the 5-carbon position of cytosine thus forming 5-methylcytosine.
  • cytosine methylation is the most common epigenetic modification of DNA, which occurs typically at symmetric CG sites on the DNA double helix and is referred to as CpG dinucleotides.
  • the CpG sites or CG sites refer to DNA nucleotide sequence wherein a cytosine nucleotide is followed by a guanine nucleotide in the linear sequence of bases along its 5' ⁇ 3' direction.
  • CpG is shorthand for 5'-C-phosphate-G-3', that is, cytosine and guanine separated by only one phosphate; phosphate links any two nucleosides together in DNA.
  • the CpG notation is used to distinguish this single-stranded linear sequence from the CG base-pairing of cytosine and guanine for double-stranded sequences.
  • CpG methylation herein refers to cytosine methylation of cytosine within CpG dinucleotides. CpG methylation occurs typically in humans in adult somatic cells.
  • CpG methylation can be determined in a biological sample with techniques that are well known from the one skilled in the art. These methods include, without being limited, chemical modification of the DNA with bisulfite (represented by bisulfite genomic sequencing), restriction enzyme digestion of the DNA (represented by methylation-sensitive restriction endonucleases) and affinity-based isolation of methylated DNA (represented by methylated DNA immunoprecipitation).
  • CpG methylation can be determined in a biological sample containing DNA using chemical modification of said DNA with bisulfite also referred to as "bisulfite conversion" of said DNA and further using standard methods, known to the skilled in the art, such as methylation specific restriction enzymes, Sanger sequencing or pyrosequencing, microarrays, and/or PCR Techniques such as bisulfite specific PCR (BSP) or COBRA (determination of methylation at specific restriction enzyme sites within PCR amplicon).
  • BSP bisulfite specific PCR
  • COBRA determination of methylation at specific restriction enzyme sites within PCR amplicon
  • the presence of CpG methylation can be determined using bisulfite conversion and microarrays, in particular by high-throughput micro arrays.
  • bisulfite conversion and pyosequencing are used to determine CpG methylations.
  • Determining CpG methylation herein refers to determining the presence of a CpG methylation and at the same time determining the region or the position of said methylated CpG site.
  • Regular herein refers to a stretch of DNA, preferably to a region of a chromosome, particularly to a gene, more preferably to region of a gene.
  • chromosome refers to a packaged and organized structure containing most of the DNA of a living organism. In case of eukaryotes the chromosomes are located in the nucleus.
  • gene means a DNA sequence that codes for, or corresponds to, a particular sequence of amino acids which comprises all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. Some genes, which are not structural genes, may be transcribed from DNA to RNA, but are not translated into an amino acid sequence. Other genes may function as regulators of structural genes or as regulators of DNA transcription. In particular, the term gene may be intended for the genomic sequence encoding a protein, i.e. a sequence comprising regulator, promoter, intron and exon sequences.
  • the gene is the promoter, intron or exon sequence of said gene.
  • DMR differentially methylated region
  • a "region” or “region of DNA” as defined herein above, such as a chromosomal region or a gene, contains more than one CpG site, preferably at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 CpG sites, more preferably at least 1 , 2, 3, 4,
  • a region as herein defined contains 1 , 2, 3, 4, 5,
  • the DMR contains at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 CpG sites, more preferably at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, CpG sites, in particular, a region as herein defined contains 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 CpG sites.
  • DMR differentially methylated region
  • the differentially methylated region preferably contains at least one differently methylated CpG site, more preferably, at least two differently methylated CpG sites. It will be understood that the number of differently methylated CpG sites is independent of the total number of CpG sites present in the corresponding region.
  • the differentially methylated region preferably contains at least one differently methylated CpG site, more preferably, at least two differently methylated CpG sites.
  • a differentially methylated region contains at least one differently methylated CpG site, for example at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 differently methylated CpG sites, preferably at least 2 differently methylated CpG sites, in particular, a differentially methylated region (DMR) contains 1 to 20, 1 to 15, 2 to 15, 3 to 15, 4 to 15, 5 to 15 differently methylated CpG site, more particularly, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 differently methylated CpG site.
  • a differentially methylated region is one differently methylated CpG site.
  • the inventors demonstrated that the presence of at least one DMR located in a specific chromosomal region indicates that said subject is in transition to psychosis, the location of at least one DMR is therefore useful in context of the methods of the present invention.
  • the at least one differentially methylated region (DMR) is located in a chromosomal region selected from the group consisting of the chromosomal regions:
  • the at least one differentially methylated region is located in the chromosomal region starting at position 24348549 and ending at position 24348715 of chromosome 22, starting at position 55013946 and ending at position 55013954 of chromosome 19, starting at position 24384400 and ending at position 24384525 of chromosome 22, starting at position 146549909 and ending at position 146550467 of chromosome 1 or starting at position 1 10254662 and ending at position 1 10254835 of chromosome 1 , more preferably, starting at position 146549909 and ending at position 146550467 of chromosome 1 or starting at position 1 10254662 and ending at position 1 10254835 of chromosome 1 .
  • DMR differentially methylated region
  • the at least one differentially methylated region is located in the chromosomal region starting at position 24348549 and ending at position 24348715 of chromosome 22, starting at position 55013946 and ending at position 55013954 of chromosome 19 or starting at position 24384400 and ending at position 24384525 of chromosome 22.
  • the at least one differentially methylated region is located in the chromosomal regions selected from the group consisting of the chromosomal regions:
  • the at least one differentially methylated region is located in the chromosomal regions selected from the group consisting of the chromosomal regions:
  • the chromosomal region starting at position 146549909 and ending at position 146550467 of chromosome 1 corresponds to region 1 q21 .1
  • the chromosomal region starting at position 1 10254662 and ending at position 1 10254835 of chromosome 1 includes the GSTM5 gene, in particular the GSTM5 promoter.
  • said differentially methylated region is located in a gene, wherein said gene is selected from the group consisting of the genes as listed below: Probe CHR Physical Position UCSC gene name P cg23655769 7 56358512 LOC441228 2,69E-06 cg05588757 2 95825608 ZNF514 2,90E-06 cg06657560 12 121841372 RNF34 5,32E-06 cg24939380 4 84189574 COQ2 l,55E-05 cg27461213 4 3468649 DOK7 l,80E-05 cg26448137 5 962206 l,95E-05 cgl6807643 6 27862331 HIST1H2AM 2,34E-05 cg05498047 11 124714902 3,34E-05 cg21539600 14 81729570 UNGP3; STON2 3,45E-05 cgl6178622 10 72271510
  • the differentially methylated region is located in a gene, wherein said gene is selected from the group consisting of GSTT1 , GSTP1 , GSTM5, LOC441228, ZNF514, RNF34, COQ2, DOK7, HIST1 H2AM, UNGP3 (also referred to as STON2), KIAA1274, LRP2BP, CDH13, TRAF3IP2, PTPRN2, COL9A2, TRIM39, NRP1 , GGA3, CHL1 , SIM1 , TFAP2E, SCRN1 , RIPPLY3 (also referred to as MRPL20P1 ), UHRF1 BP1 L, KIA1026, t-RNA-Leu, MIR3683 (also referred to as LOC100121257), ATRN, EFNA3, CBS, ZNF282, BAH , NFIX, PPM1 E, CEP152,IL17RE, L
  • said gene is GSTT1 , GSTP1 or GSTM5, in particular GSTM5, more particularly the promoter of GSTM5.
  • the differentially methylated region is located in the gene GSTT1 , GSTP1 or GSTM5, preferably GSTM5, in particular, in the promoter region of the GSTM5 gene.
  • the positions of the nucleotides for the regions in the chromosome are indicated according to the genome sequence as indicated in the UCSC Genome Browser (according to the 18 April 2016).
  • the positions of the nucleotides for the genes are indicated according to the genome sequence as used in the UCSC Genome Browser (according to the 18 April 2016).
  • reference sequences are obtainable from the UCSC genome browser under the listed UCSC gene name. They are also typically accessible under the UCSC gene name in other databases known to the skilled in the art, such as the NCBI database "Gene” (according to the 18 April 2016).
  • the biological sample includes nucleic acids.
  • nucleic acid generally refers to at least one molecule or strand of DNA, RNA, miRNA or a derivative or mimic thereof, comprising at least one nucleobase, such as, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g., adenine "A,” guanine “G,” thymine “T,” and cytosine “C”) or RNA (e.g. A, G, uracil “U,” and C).
  • nucleic acid encompasses the terms “oligonucleotide” and “polynucleotide”.
  • biological sample means a substance of biological origin.
  • the biological sample comprises cells from the subject to be diagnosed.
  • the biological samples comprises in particular nucleic acids.
  • examples of biological samples include, but are not limited to, blood and components thereof such as plasma, platelets, subpopulations of blood cells and the like; bone marrow samples, organs such as kidney, liver, heart, lung, and the like, saliva and mouth epithelial cells.
  • the biological sample according to the invention may be obtained from the subject by any appropriate means of sampling known from the skilled person.
  • the biological sample is a blood sample.
  • the biological sample is a peripheral blood sample.
  • a peripheral blood sample typically comprises red blood cells, white blood cells and platelets.
  • the sample comprises peripheral blood mononuclear cells (PMBC).
  • PMBC peripheral blood mononuclear cells
  • PMBC peripheral blood mononuclear cells
  • monocytes and/or lymphocytes include monocytes and/or lymphocytes.
  • the sample comprises white blood cells.
  • White blood cells include monocytes, granulocytes and lymphocytes.
  • the sample comprises lymphocytes.
  • DNA may be extracted from a sample of the subject and treated with sodium bisulfite and then analyzed by a DNA array.
  • DNA is extracted from typically a blood sample and then treated with sodium bisulfite using for example EZ-96DNA Methylation Kit (Catalogue No D5004, Zymo Research, Irvine, CA, USA) typically according to manufacturer recommendations.
  • the extracted and bisulfite converted DNA may be analyzed by a DNA-array, in particular, by a DNA-array, as defined in the section "CpG methylation" herein above.
  • bisulfite converted DNA samples are analyzed for example on the using lllumina Infinium HumanMethylation450 BeadChip (lllumina, San Diego, CA, USA) typically according to the manufacturer's protocol. Primary data analysis might be done with lllumina Genome Studio Software (lllumina). Further Data processing and clean-up are typically performed as herein described in the experimental section.
  • the methods of the invention comprise a step b) of comparing said CpG methylations determined in step a) with reference CpG methylations.
  • the "reference CpG methylations" corresponds to the CpG methylations determined in the sample of a healthy subject.
  • the reference CpG methylations are determined in a sample from the same tissular origin as the biological sample of the subject of step a) of the methods of the invention. More preferably, the reference CpG methylations are determined in a blood sample.
  • the reference CpG methylation corresponds to the CpG methylations observed in a healthy population.
  • a "healthy population” means a population constituted of subjects who have not previously been diagnosed with psychosis, as defined herein above, or who is not at risk of psychosis, as defined in the section "Subject" herein above.
  • Subjects of a healthy population also preferably do not otherwise exhibit symptoms of disease. In other words, such subjects, if examined by a medical professional, would be characterized as healthy and free of symptoms of disease.
  • a “healthy subject” means a subject who has not previously been diagnosed with psychosis or who is not at risk of psychosis as defined in the section "Psychosis” and/or “Subject” herein above.
  • a healthy subject also preferably does not otherwise exhibit symptoms of disease. In other words, such healthy subject, if examined by a medical professional, would be characterized as healthy and free of symptoms of disease.
  • a healthy subject does not display any of the primary symptoms of psychosis or psychotic symptoms as defined herein above.
  • the healthy subject has the same age as the subject who is at risk of psychosis.
  • the healthy subject has the same gender as the subject who is at risk of psychosis.
  • diagnosis refers to the process of attempting to determine or identify a possible disease or disorder in a subject, as defined in the section "subject” herein above.
  • method of diagnosis refers herein to a process of determining in a subject if said subject is in transition to psychosis.
  • the inventors determined CpG methylations in a sample containing nucleic acids, wherein said sample is from a subject at risk of psychosis, and compared the CpG methylations with CpG methylations of a reference sample, thus determining differentially methylated regions (DMR) in the sample of the subject at risk of psychosis.
  • DMR differentially methylated regions
  • DMR differentially methylated region
  • the presence of at least one DMR located in a chromosomal region indicates said subject is in transition to psychosis.
  • the at least one DMR, the chromosomal region, the gene and the subject are as defined in the sections 'CpG methylation' and 'subject' herein above.
  • DMRs in regions as defined herein above, in particular chromosomal regions or genes as defined herein above, can be associated with psychotic transition.
  • a subject diagnosed to be in transition to psychosis has therefore, preferably, at least one DMR located in a chromosomal region or a gene as defined herein above.
  • the inventors discovered that the presence of differentially methylated regions, for example in the region 1 q21 .1 or in the gene GSTM5, and/or in about 100 identified genes that are listed for example in the Table of Figure 9, indicate that a subject is at high risk of a transition to psychosis, also called onset of psychosis.
  • the invention further refers to a method for predicting onset of psychosis in a subject at risk of psychosis comprising:
  • step c based on the comparison of step b), determining differentially methylated regions (DMR) in the sample of step a), and
  • DMR differentially methylated region
  • a "method for predicting” or “predicting method” refers to a method for determining whether an individual is likely to develop a disease or illness.
  • risk of onset of a disease refers to the probability that a disease will appear in a studied subject, in particular within a given period of time.
  • the method for predicting onset of psychosis comprises a step b) of comparing CpG methylations determined in step a) with reference CpG methylations, wherein said step is as defined in the section "CpG methylation" herein above.
  • Said method further comprises a step c) of determining, based on the comparison of step b) differentially methylated regions (DMRs) in the sample of step a).
  • the step of determining the DMRs is as further defined in the section 'CpG methylation' herein above.
  • the DMR refers to the presence or absence of the methylation of at least one CpG contained in said DMR compared to the reference CpG methylation.
  • the inventors further identified 14 genes and 2 gene networks, in particular the genes CPT1 A and NRP1 , which are differentially expressed during psychotic transition and which are thus genetic markers or so-called biomarkers for the transition into psychosis.
  • the present invention further refers to an in vitro method for diagnosing the transition to psychosis in a subject at risk of psychosis, said method comprising
  • step b) comparing said level of expression of the at least one gene determined in step a) to a reference value
  • step c) deducing from the comparison of step b) if the subject is in transition to psychosis.
  • the invention further refers to a method for predicting onset of psychosis in a subject at risk of psychosis comprising
  • step c) deducing from the comparison of step b) if the subject is at risk of onset of psychosis.
  • diagnosis means diagnosis based on CpG methylations
  • method for predicting means for predicting based on CpG methylations
  • the inventors of the present invention identified that the difference of the level of expression of the genes CPT1 A, NRP1 , CREB3L3, EPB49, EREG, GYPA, HBB, HBD, HSPB7, MUC16, OSBP2, PEG3, TBX1 , TMOD1 , TRIM58 and CPT1 A is significantly correlated with the transition into psychosis by RNAsequencing using the edgeR package or the DESeq package, wherein "significantly correlated" herein refers to genes having a difference in expression and having a p-value of ⁇ 0.05.
  • the gene NRP1 has been identified in the methylomic study of example 1 and has been confirmed by the inventors to be a gene that is differently expressed during the transition into psychosis with a p-value of ⁇ 0.05. Accordingly, in one embodiment in context of the methods of the invention, in step a) the level of expression of at least one gene selected from the group consisting of CPT1 A, NRP1 , CREB3L3, EPB49, EREG, GYPA, HBB, HBD, HSPB7, MUC16, OSBP2, PEG3, TBX1 , TMOD1 and TRIM58 is determined.
  • step a) the level of expression of at least one gene selected from the group consisting of CPT1 A, CREB3L3, EPB49, EREG, GYPA, HBB, HBD, HSPB7, MUC16, OSBP2, PEG3, TBX1 , TMOD1 and TRIM58 is determined.
  • the inventors further confirmed in a confirmation cohort that differences in the level of expression of the genes CPT1 A and/or NRP1 are significantly correlated with the transition to psychosis.
  • the at least one gene in step a) of the methods of the invention is CPT1 A and/or NRP1 .
  • the level of expression is determined in step a) of the methods of the invention of CPT1 A and/or NRP1 .
  • CPU A also known as “carnitine palmitoyltransferase 1 A” is encoded on chromosome 1 1 , for example, from position 68754620 until 68844410 (reference genome GRCh38.p7), and has two isoforms where the mRNA sequence is available under the NCBI Reference number NM_001031847.2 for transcript variant 2, and under NCBI Reference Number NM 001876 for transcript variant 1 , as accessible on 2 December 2016.
  • NRP1 also known as "Neuropilin 1” is encoded on chromosome 10, for example, from position 33177491 until 33334905 (reference genome GRCh38.p7), and has six isoforms where the mRNA sequence is available under the NCBI Reference numbers: NM_003873.5 (neuropilin-1 isoform a precursor) NM_001024628.2 (neuropilin-1 isoform b precursor), NM_001024629.2 (neuropilin-1 isoform c precursor), NM_001244972.1 (neuropilin-1 isoform d precursor), N M O 01244973.1 (neuropilin-1 isoform e precursor), NM 001330068.1 (neuropilin-1 isoform f precursor) as accessible on 2 December 2016.
  • At least one gene herein refers to at least one gene, at least two, three, four, five, six, seven, eight genes, more preferably two genes.
  • the level of expression of more than one gene such as two, three, four, five, six, seven, eight genes selected from the group of genes as defined herein above may be determined in step a) of the methods of the invention.
  • Level of expression or “Expression level” of a gene in context of the methods of the invention refers to the level of the gene product of said gene and thus refers to an amino acid sequence or protein encoded by said gene and/or a nucleic acid transcribed from said gene.
  • the nucleic acid transcribed from said gene refers in particular to a nucleic acid selected from the group consisting of mRNA, long and short non-coding RNA (such as tRNA, rRNA, catalytic RNA, miRNA), in particular mRNA.
  • level of expression refers to the level of expression of the protein of said at least one gene and/or the level of expression of the mRNA of said at least one gene.
  • the level of expression refers to the level of expression of all proteins encoded by said at least one gene or all mRNAs encoded by said at least one gene, thus including all protein isoforms and mRNA isoforms encoded by said at least one gene.
  • determining includes in particular qualitative and/or quantitative detection (i.e. detecting and/or measuring the level of expression) with or without reference to a control or a predetermined value.
  • detecting means determining if the gene product of the at least one gene (mRNA and/or protein), is present or not in a biological sample and “measuring” means determining the amount of the level of expression of said at least one gene in a biological sample.
  • Determining the level of expression of said at least one gene can be performed by methods which are well known to the person skilled in the art, including in particular immunologic methods, in case of determining the translation products (i.e. proteins) as further described herein below or quantitative methods to determine the quantity of nucleic acids, including reverse transcriptase PCR (RT-PCR), real-time quantitative RT-qPCR, and methods involving the use of DNA arrays (macroarrays or microarrays), RNA sequencing or in situ hybridizations, in particular reverse transcriptase PCR (RT-PCR), such as real-time quantitative RT-qPCR.
  • RT-PCR reverse transcriptase PCR
  • RT-PCR reverse transcriptase PCR
  • RT-PCR reverse transcriptase PCR
  • the level of expression of said at least one gene is measured by RT-qPCR or RNA sequencing, preferably RT-qPCR.
  • the level of expression is determined using sequencing.
  • total RNA can typically be extracted and purified from, typically, blood samples using a standard protocol with, for example, a QIAcube robot and, for example, a PAXgene Blood RNA kit (QIAGEN). Quality and quantity checks can typically be performed by spectrophotometer (BioAnalyzer & kit QUANT IT RNA).
  • Total RNA can be processed using, typically, the mRNA-Seq Sample Prep Kit
  • Poly(A) RNA can typically be isolated from the total RNA with a two-step magnetic bead protocol. The resulting mRNA can typically be fragmented in a buffer containing divalent cation at 94 °C for 5 min and purified by ethanol precipitation. The RNA can typically be resuspended in water and a reverse transcription reaction can be performed following manufacturer's instructions.
  • a paired-end 75-bp sequencing run can be performed, typically, on the lllumina HiSeq 2000.
  • each incorporated base can typically be color coded with a fluorophore.
  • the camera can record the color which base was incorporated.
  • the lllumina software can convert this fluorophore information to sequence data and provide fasta files.
  • the level of expression of said at least one gene in particular the level of expression of the protein encoded by said at least one gene, can be determined by detecting the translation product(s) using immunologic methods such as detection of the translation product(s) of the at least one gene using polyclonal or monoclonal antibodies.
  • Suitable immunologic methods include immuno-histochemistry (IHC), enzyme linked immunoassays (ELISA), sandwich, direct, indirect, or competitive ELISA assays, enzyme linked immunospotassays (ELIspot), radio immunoassays (RIA), flow-cytometry assays (FACS), Western Blot, fluorescence resonance energy transfer (FRET) assays, protein chip assays using for example antibodies, antibody fragments, receptor ligands or other agents binding the proteins encoded by said at least one gene.
  • IHC immuno-histochemistry
  • ELISA enzyme linked immunoassays
  • sandwich direct, indirect, or competitive ELISA assays
  • enzyme linked immunospotassays ELIspot
  • RIA radio immunoassays
  • FACS flow-cytometry assays
  • FRET fluorescence resonance energy transfer
  • protein chip assays using for example antibodies, antibody fragments, receptor ligands or other agents binding the proteins encoded by said at least one gene.
  • antibodies directed against the proteins encoded by a gene selected from the group consisting of CPT1 A, NRP1 , CREB3L3, EPB49, EREG, GYPA, HBB, HBD, HSPB7, MUC16, OSBP2, PEG3, TBX1 , TMOD1 , TRIM58, AKT1 , CHL1 , GSTM5 and IL17RE for this purpose are commercially available.
  • the level of expression of said at least one gene is determined using immunohistochemistry or Western Blot.
  • determining the level of expression of said at least one gene further contains normalizing said level of expression as described herein above. It will be understood by the skilled in the art, that the level of expression of said at least one gene as determined in a biological sample in step a) of the methods of the invention and depends on the amount, quality and the representativity of the biological sample used. It will therefore be understood by the skilled in the art that the level of expression of said at least one gene preferably refers to a normalized level of expression.
  • the level of expression as referred to in context of the present invention is a normalized level of expression.
  • Normalization refers to scaling data in such a way that different data sets obtained, for example, for different samples, can be compared. Normalization typically relies on genes whose expression does not change, so-called reference gene(s) or housekeeping gene(s) or on the total amount of gene expression products that were extracted.
  • the amount of the level of expression of at least one gene is normalized using the level of expression of at least one housekeeping genes.
  • Housekeeping genes are involved in basic cell maintenance and, therefore, are expected to maintain constant expression levels in all cells and conditions. It will however be understood by the skilled in the art, that the choice of a housekeeping gene to be used as the at least one housekeeping gene, typically depends on the nature of the biological sample to be normalized, i.e if the biological sample is saliva, blood, or tissue. Furthermore, as it will be understood by the skilled in the art, the housekeeping genes used for normalization of, for instance, mRNAs preferably encode mRNAs and not, for example, miRNAs.
  • the amount of the level of expression of at least one gene such as the amount of RNA of at least one gene, typically the amount of mRNA of at least one gene is determined and said amount may be normalized using the total amount of extracted RNA, in particular mRNA.
  • the amount of a protein encoded by at least one gene is determined as the level of expression of at least one gene, said amount may be normalized using the total amount of extracted proteins.
  • RNAs or mRNA Methods to determine the total amount of proteins, RNAs or mRNA are known to the skilled in the art.
  • the total amount of protein is determined using BCA protein assay kit (Thermo Fisher Scientific, IL, USA).
  • the total amount of RNA is determined using a spectrophotometer (BioAnalyzer & kit QUANT IT RNA).
  • At least one housekeeping genes herein refers to at least one, at least two or three, more preferably three housekeeping genes, most preferably one, two, or three housekeeping genes, in particular three housekeeping genes.
  • the methods in context of the invention further comprise a step b) of comparing said level of expression of the at least one gene determined in step a) to a reference value.
  • reference value refers to the reference value of said at least one gene selected from the group of genes as defined herein above.
  • the reference value refers to the reference value of CPT1 A and to a reference value for NRP1 . Accordingly, for example, in step b), the measured level of expression of CPT1 A is compared with a reference level of CPT1 A and the measured level of expression of NRP1 is compared with a reference level of NRP1 .
  • the reference value is a predetermined reference value, a threshold value or a range of values.
  • the reference value can be any number of statistical measures to distinguish between a level indicative for a subject at risk of psychosis (also referred to as "in transition to psychosis” or "at risk of onset of psychosis”) i.e. a reference value obtained in subject(s) who converted to psychosis and a level indicative that a subject is not at risk of converting into psychosis i.e. a reference value obtained in subject(s) who did not convert into psychosis, while including median level of expression, and/or cut-off or threshold expression or fold change values as determined in an subject or a group of subjects.
  • the reference of said at least one gene herein refers to the median level of expression determined in a biological sample obtained from a subject or a population of subjects, said subject(s) being as defined herein above, who converted into psychosis. Accordingly, the reference of said at least one gene may be determined just after conversion into psychosis such as 1 day, 2 days, 3 days, 4 days, 5 days, 6, days, 1 week, 2 weeks after conversion into psychosis.
  • the reference of said at least one gene herein refers to the median level of expression determined in biological samples obtained from a subject or a population of subjects, said subject being as defined herein above, who, for example, did not convert into psychosis.
  • said reference value is the level of expression of the at least one gene determined in a sample of a healthy subject or a population of healthy subjects.
  • the healthy subject is as defined herein above in the section 'Reference value in context of the methods based on CpG methylations'.
  • the sample used for determining the reference value is a sample of the same type as the sample used in step a) in context of the methods of the invention, i.e. // the sample of the subject used in step a) is a blood sample, the sample of a healthy subject or a population of healthy subjects used for determining a reference value is preferably as well a blood sample.
  • the inventors have determined that over a certain time, for example, CPT1 A and/or NRP1 are downregulated in individuals that convert into psychosis.
  • the reference value might be a reference value from the same subject taken at an earlier time point, also referred to as a preceding reference value from the same subject.
  • Preceding thus refers in the chronologic order of events to a reference value that has been determined in a sample taken at an earlier time point.
  • a level of expression of said at least one gene being higher or lower than the reference value may be indicative for the subject being in transition to psychosis.
  • a level of expression of said at least one gene as determined in step a) higher or lower than the reference value of said at least one gene is indicative for said subject being in transition to psychosis.
  • the reference value refers to a reference value obtained in a healthy subject or population or a sample taken at an earlier time point of the same subject and the at least one gene is CPT1 A and/or NRP1 .
  • a level of expression of CPT1 A and/or NRP1 that is lower than the reference value of CPT1 A and/or NRP1 is indicative for said subject being in transition to psychosis.
  • a level of expression of CPT1 A that is lower than the reference value of CPT1 A and/or a level of expression of NRP1 that is lower than the reference value of NRP1 is indicative for said subject being in transition to psychosis.
  • the level of expression is lower than the reference value
  • the level of expression of the said at least one gene as determined in the biological sample is inferior to the reference value, the difference between said values being typically statistically significant.
  • the level of expression is higher than the reference value as used herein, means that the level of expression of the said at least one gene as determined in the biological sample is superior to the reference value, the difference between said values being typically statistically significant.
  • the invention also refers to a method of treatment of the transition to psychosis in a subject in need thereof which method comprises
  • a methylation inhibitor a gene expression modulator
  • said gene being selected from the group constituted of GSTT1 , GSTP1 , GSTM5, LOC441228, ZNF514, RNF34, COQ2, DOK7, HIST1 H2AM, UNGP3, KIAA1274, LRP2BP, CDH13, TRAF3IP2, PTPRN2, COL9A2, TRIM39, NRP1 , GGA3, CHL1 , SIM1 , TFAP2E, SCRN1 , RIPPLY3, UHRF1 BP1 L, KIA1026, t-RNA-Leu, MIR3683, ATRN, EFNA3, CBS, ZNF282, BAH , NFIX, PPM1 E, CEP152,IL17RE, LZTS2, FBRSL1 , EREG, CRELD1 , KCNIP4, AHSP, ASAM, INPP5A, LRWD1 , COX
  • the invention also refers to a method of treatment of psychosis or treatment of the transition in psychosis in a subject in need thereof which method comprises administering said subject with a therapeutically effective amount of a DNA methylation inhibitor and/or a gene expression modulator, said gene being selected from the group as defined herein above.
  • treating means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.
  • treatment of the transition to psychosis refers to preventing or delaying the onset of psychosis or the first-episode of psychosis.
  • the CAARMS score will thus, as a result of the treatment, stay stable.
  • such treatment can also lead to complete recovery.
  • the treatment will lead to the reduction of symptoms associated with psychosis.
  • the symptoms associated with psychosis are as define in the section 'psychosis' or 'subject' herein above.
  • Treatment of psychosis refers to reversing, alleviating, inhibiting psychosis.
  • the treatment of the transition to psychosis is chosen from a DNA methylation inhibitor and/or a gene expression modulator, said gene being selected from the group constituted of GSTT1 , GSTP1 , GSTM5, LOC441228, ZNF514, RNF34, COQ2, DOK7, HIST1 H2AM, UNGP3, KIAA1274, LRP2BP, CDH13, TRAF3IP2, PTPRN2, COL9A2, TRIM39, NRP1 , GGA3, CHL1 , SIM1 , TFAP2E, SCRN1 , RIPPLY3, UHRF1 BP1 L, KIA1026, t-RNA-Leu, MIR3683, ATRN, EFNA3, CBS, ZNF282, BAH , NFIX, PPM1 E, CEP152JL17RE, LZTS2, FBRSL1 , ER
  • a "DNA methylation inhibitor” has the ability to inhibit CpG methylation.
  • the methylation inhibitor is selected from the group constituted of 5- azacytidine, 5-aza-2'-deoxycytidine, 1 -3-D-arabinofuranosyl-5-azacytosine and dihydro-5- azacytidine.
  • a “gene expression modulator” refers to a substance that increase or decrease the expression of a specific gene.
  • the gene expression modulator is an enhancer of gene expression. In another embodiment, the gene expression modulator is an inhibitor of gene expression.
  • the inventors have in particular discovered that DMR in the promotor region of GSTM5 is associated with psychosis.
  • the DMR in the promotor region of GSTM5 refers to the absence or presence of CpG methylation in a subject at risk of psychosis.
  • the transition to psychosis may be treated with a GSTM5 expression inhibitor.
  • transition to psychosis may be treated with a GSTM5 expression enhancer.
  • the gene expression modulator is a GSTM5 expression modulator.
  • a antipsychotic treatment is selected from the group consisting of chlorpromazine, flupenthixol, fluphenazine, haloperidol, loxapine, perphenazine, pimozide, thioridazine, thiothixene, trifluoperazine, zuclopenthixol, clozapine (Clozaril), olanzapine (Zyprexa), quetiapine (Seroquel) and risperidone (Risperdal), preferably haloperidol, chlorpromazine and clozapine.
  • a "subject in need of” is intended for a subject likely to suffer from transition to psychosis. Said subject was preferably diagnosed as being at risk of psychosis as defined herein above Throughout the instant application, the term “comprising” is to be interpreted as encompassing all specifically mentioned features as well optional, additional, unspecified ones. As used herein, the use of the term “comprising” also discloses the embodiment wherein no features other than the specifically mentioned features are present (i.e. "consisting of").
  • Figure 1 depicts a table containing the clinical description of the population used for the study in context of the present invention, (a) is the p value as calculated by Fisher's Test and (b) is the p value as calculated by non parametric Mann-Whitney test.
  • the biological interval represents time between the two blood samples and the clinical follow-up represents the time between inclusion and final status assessments.
  • FIG. 2 is a table summarizing the significant pathways obtained in overrepresentation analysis of the multi-CpG pipeleine.
  • CpG refers to cytidine-phosphate-guanosine.
  • Figure 3 is a graph representing the mean of methylation in each CpG located in the GSTM5 promoter.
  • the full line represents the non- converter whereas the dashed line represents the converter.
  • a specific CpG might be methylated in one cell of an individual and not methylated in another cell, accordingly in one individual the methylation of a CpG position is an average value being between 0 and 100% methylation.
  • 100% methylation signifies that the specific CpG position is methylated in every cell, i.e. in all nucleic acids tested.
  • the mean of methylation herein refers to the percentage of methylation in the population tested, which in analogy to the above is between 0 and 100%.
  • Figure 4 is a table summarizing the main criteria for UHR and psychosis.
  • Figure 5 is a table summarizing the information about data clean-up procedure.
  • Figure 6 is a table summarizing the technical conditions of the PCR as further described in the section 1 .2.2. referring to the pyrosequencing of samples.
  • Figure 7 is a table summarizing the DMRs identified by Minfi.
  • the table lists the chromosomes and the locations of identified DMRs.
  • Figure 8 is a table summarizing the results of the exploratory analysis in context of the present invention.
  • the table lists the chromosomes and the locations of identified DMR.
  • Fwer refers to a family-wise error rate referring to the probability of making one or more false discoveries, or type I errors when performing multiple hypotheses tests.
  • the P value refers to the nominal significance value of the DMR difference test using a linear model without correction for multiple tests.
  • the Beta value refers to the amplitude of the methylation difference between the two groups for a given DMR.
  • the beta value varies from 0 to 1 and the sign + indicates hypermethylation of the cases (converters) vs controls (non converters) and the sign - hypomethylation of the (converters) vs controls (non converters).
  • Figure 9 is a table listing the Top 100 genes that were obtained for the test of difference between converters and non-converters in methylation change between baseline and final measure with adjustment for sex. P corresponds to the significance of this test of difference.
  • the physical position indicates the physical position of the detected CpG according to the annotations obtained from the lllumina's Infinium HumanMethylation450 on the 28th of November, 2013.
  • Figure 10 depicts a table containing the clinical description of the two sample groups used for the study in context of the present invention.
  • Sample 1 refers to samples used for RNAseq and sample 2 includes samples of sample 1 and samples of other subjects and was explored by Q-PCR analyses.
  • Clinical scales include BPRS (Brief Psychiatric Rating Scale) and CGI (Clinical Global Impressions Scale).
  • RIN RNA Integrity Number. In each sample, converters did not differ from non-converters excepted in clinical score at the end of follow-up (as expected).
  • Figure 11 depicts a table summarizing the findings from RNAseq and multiplex Q-PCR.
  • RNAseq RNA sequencing.
  • Q-PCR quantitative polymerase chain reaction, ⁇ -value referred to the level of methylation in specific CpGs.
  • Example 1 1.1. Patients and Methods: 1.1.1. Population
  • Exclusion criteria included manifest symptoms of psychosis (fulfilling DSM-IV criteria), pervasive developmental or bipolar disorders, and individuals with other established diagnoses such as obsessive-compulsive disorder.
  • Other exclusion criteria were: current antipsychotic treatment (> 100 mg Chlorpromazine equivalent) for more than 12 weeks; psychoactive substance dependence or abuse during the previous year and/or >5 years; serious or non-stabilized somatic and neurological disorders; head injury and IQ lower than 70. All subjects were examined with the Comprehensive Assessment for at risk mental state (CAARMS, in its translated version (see Yung AR et al., Aust N Z J Psychiatry 2005; 39: 964-971 and the corresponding French version Krebs M-0 et al.
  • genomic DNA 500ng was extracted from whole blood and treated with sodium bisulfite using the EZ-96DNA Methylation KIT (Catalog No D5004, Zymo Research, USA) following the manufacturer's standard protocol. Methylation was measured at baseline (M0), and after the longitudinal follow-up (MF) by the same technique in the same time for all samples. Genome-wide DNA methylation was assessed using lllumina Infinium HumanMethylation450 BeadChip (lllumina, San Diego, CA, USA), which interrogates the DNA methylation profile of more than 485 000 CpG loci across the genome at single-nucleotide resolution.
  • lllumina GenomeStudio software (lllumina, San Diego, CA, USA) was used to extract signal intensities for each probe. All computations and statistical analyses were performed within the R statistical analysis environment (http://www.r-project.org), and all scripts are available on request from the authors. R packages methylumi and wateRmelon were used for data quality check and normalization. Steps used for data clean-up procedure and normalization comprised gender check between phenotype file and methylation data set and evaluation of SNP genotypes concordance between the 2 samples from the same individuals.
  • Subsequent clean-up steps comprised flagging and removing individuals with no result or gender discrepancies or discordant genotypes, samples with ⁇ 1 % of sites with a detection p- value ⁇ 0.05, probes with beadcount ⁇ 3 in ⁇ 5 % of samples, probes with ⁇ 1 % of samples with a detection p-value ⁇ 0.05. Additionally, probes on chromosomes X and Y, SNP probes, probes with a SNP at the CpG site, and non-specific probes that map to more than one location in the genome were removed (Price ME et al. Epigenetics Chromatin, 2013; 6: 4.).
  • the initial methylation data file includes 485 577 probes and after normalization and data clean up, 41 1 947 probes were kept for the final analysis (Figure 5).
  • R Minfi package was used for supplementary quality control (data not shown) and no sample was removed. No batch effect was detected (according to Combat R Package using SVA function). Cellular populations were estimated by EstimateCellCounts function (Minfi package).
  • Association analysis Global methylation change was investigated by computing the difference between mean methylation changes for all probes in each individual and comparing converters to non-converters. Differentially methylated regions (DMR) were investigated using Minfi package in R (script available upon request). In summary, time and group were used as factors in a linear model adjusted by cellular populations with a paired design. DMR analysis was performed using bumphunter function (bootstrap with 1000 permutations and a methylation differential cut-off of 10). Significance was established for fiver correction ⁇ 0,1 .
  • Infinium HumanMethylation450 BeadChip is a current and reliable array to detect CpG methylation (Roessler J et al. Inc. BMC Res Notes 2012; 5: 210). However, some of the present findings were further confirmed using a technical reference based on pyrosequencing (Tost J and Gut IG, Nat Protoc 2007; 2: 2265-2275.). After bisulfite conversion by EpiTect Plus Bisulfite Kits (Qiagen) and DNA purification on column, non- methylation specific PCR were achieved using Platinium Taq DNA polymerase kit (invitrogen - Life Technologies).
  • MAEL promoter was used as positive control for the bisulfite treatment, in bisulfited and non-bisulfited samples (Data not shown).
  • Primers were designed by PyroMark Assay design Software 2.0 QIAGEN and technical conditions for PCR are shown in Figure 6. Biotinylated primers were used to keep the single DNA strand for pyrosequencing. Pyrosequencing was performed using PyroMark Q24 (Qiagen) according the manufacturer's instructions and data about methylation in each CpG were extracted and analysed using PyroMark Q24 2.0.6.20 software (Qiagen). 1.2. Results
  • GSTM5 is selectively expressed in the brain and is the most commonly expressed member of its gene family in this tissue. Its involvement in dopamine metabolism has also been suggested. Moreover, its expression has been shown to be decreased in the prefrontal cortex of patients with schizophrenia. Furthermore, GSTM5 levels displayed an inverse correlation with promoter DNA methylation in brain tissue, supporting the idea that GSTM5 CpG methylation status controls gene expression. Interestingly, the exploratory approach of the herein described study provided evidence that two other genes of GST family might be differentially methylated after conversion to psychosis: the GSTT1 and GSTP1 regions were hypomethylated and hypermethylated in converters, respectively, without differences between the groups during the initial sampling interval. These findings suggest the possibility that conversion to psychosis may depend on the specific control of oxidative metabolism and balance between these genes.
  • axon guidance pathway included the neural cell adhesion protein CHL1 gene (cell adhesion molecule L1 -like), which codes for the L1 CAM2 protein.
  • the L1 family encompasses immunoglobulin-class recognition proteins that promote axon growth and migration in developing neurons. In preclinical models, a deficit of CHL1 in adult mice impairs working memory, social behavior and synaptic transmission.
  • NRP1 neuropilinl
  • EFNA3 the third gene found in the analysis, is highly expressed in mature neurons suggesting that an imbalance in expression exists during cerebral maturation between CHL1 , NRP1 and EFNA3.
  • EFNA3 encodes ephrin-A3, which is a critical protein for the regulation of synaptic function and plasticity in astrocytes.
  • the second signaling pathway namely the IL-17 (interleukin-17) pathway
  • IL-17 interleukin-17 pathway
  • Immune response is a recurrent finding in association studies of schizophrenia.
  • a recent proteomic study identified interleukins as potential diagnostic biomarkers in the onset of psychosis. Differences in the level of several inflammatory cytokine were found in individuals with schizophrenia compared to healthy controls, with a positive correlation between levels of cytokines in the IL-17 pathway and scores on the Positive and Negative Symptoms Scale.
  • This pathway includes AKT1 , a serine-threonine kinase and a critical mediator of growth-factor-induced neuronal survival in the developing nervous system. Decreased AKT1 protein levels and phosphorylation activity were documented in the lymphocytes and brains of individuals with schizophrenia. In addition, it was reported that AKT1 genetic variants were associated with schizophrenia, in relation to cannabis use.
  • the present work was conducted in adolescents and young adults consecutively referred to a clinic specialized for early detection of psychosis and enrolled in a longitudinal follow-up program.
  • the inventors of the present invention did not find any differences in environmental exposure between those who converted to psychosis and those who did not, and the methylation changes associated with conversion to psychosis were not related to the initiation of medication. The observed modifications in methylation are thus more likely to be linked to psychosis conversion than to medication initiation or other environmental changes. Even if the sample size were sufficient to identify some significant DMRs, larger samples are needed to identify other DMRs. Another issue to identify DMRs is the molecular and clinical heterogeneity between individuals, a well- known issue in the genetics of psychosis. The sample size could not allow us to overpass this heterogeneity.
  • the present study has several strengths: A long-term prospective follow-up was conducted in both individuals at UHR for psychosis and non-UHR subjects. Rapidly frozen samples were used enabling the study of a larger number of methylation sites (even more labile ones).
  • the inventors of the present invention report longitudinal variations in methylation, which are more suitable for reflecting dynamic epigenetic processes compared to single time point analyses. A genome-wide strategy was herein used rather than limited candidate genes strategy. A newly-developed pathway and clustering analyses were used to investigate the functional relevance of top CpG methylation sites.
  • the inventors of the present invention found that the conversion to psychosis in young help seekers is accompanied by epigenetic changes in genes involved in relevant genes and pathways. Possible candidate mechanisms were also identified, including alterations in oxidative stress regulation, axon guidance and in inflammatory pathways. These candidate genes could represent multiple theaters for the disruption in homeostasis that accompanies the emergence of full-blown psychosis. At this point, it is unknown whether the observed methylation changes play a causal role in the processes leading to psychosis, or whether they are simply reflective of psychosis onset.
  • Subjects were selected from the French ICAAR cohort (PHRC AOM-07-1 18, promoted by Hopital Sainte-Anne). This cohort was approved by the institutional ethics committee "Comite de protection des did, lle-de-France III, Paris, France” and written informed consent was obtained from all participants in accordance with the Declaration of Helsinki (and their parents for individual under 18 years old). Help-seeking individuals (16 to 30 years old) consecutively referred to the Adolescent and Young Adults Assessment Centre (Service Hospitalo-Universitaire, Hopital Sainte-Anne, Paris, France) between 2009 and 2013 were enrolled in the ICAAR collaborative study (see description of this cohort in Chaumette et al).
  • Inclusion criteria were alterations in global functioning (Social and Occupational Functioning Assessment Scale score ⁇ 70) during the past year that were associated with psychiatric symptoms and/or subjective cognitive complaints. Exclusion criteria included manifest symptoms of psychosis (fulfilling DSM-IV criteria), pervasive developmental or bipolar disorders, and individuals with other established diagnoses such as obsessive-compulsive disorder. Other exclusion criteria were: current antipsychotic treatment (> 100 mg Chlorpromazine equivalent) for more than 12 weeks; psychoactive substance dependence or abuse during the previous year and/or >5 years; serious or non-stabilized somatic and neurological disorders; head injury and IQ lower than 70.
  • CAARMS (see Yung AR et al., Aust N Z J Psychiatry 2005; 39: 964-971 and the corresponding French version Krebs M-0 et al. L'Encephale)) by specifically trained psychiatrists followed by a consensus meeting for best estimate diagnoses.
  • Individuals underwent clinical assessment and blood samples at initial time (MO) and after the follow-up (MF). The duration of follow-up was to 1 year or less in case of psychotic transition.
  • the psychotic transition was characterized using the CAARMS-defined psychosis onset threshold (i.e. supra-threshold psychotic symptoms— thought content, perceptual abnormalities and/or disorganized speech— present for more than 1 week) was used (Data not shown).
  • RNA was extracted and purified from blood samples (PAXgene tubes) using a standard protocol with a QIAcube robot and PAXgene Blood RNA kit (QIAGEN). Quality and quantity checks were performed by spectrophotometer (BioAnalyzer & kit QUANT IT RNA - Data not shown). Mean concentration was 59.4 ng/mL and mean of RNA Integrity Number (RIN) was 8.4 without significant difference across groups.
  • a paired-end 75-bp sequencing run was performed on the lllumina HiSeq 2000 with more than 80 millions of reads per sample.
  • each incorporated base is color coded with a fluorophore.
  • the camera recorded the color which base was incorporated.
  • the lllumina software converts this fluorophore information to sequence data and provides fasta files.
  • the co-expression network analysis was conducted using the WGCNA package in R (Langfelder et al). After automatic, one-step network construction and module detection based on MO data, conservation of networks was tested after psychotic transition in MF data.
  • RNA was extracted from PAXgene using the same protocol as for RNA sequencing. Quality control was done using LabChip GX (Perkin Elmer, Waltham USA). Complementary DNA (cDNA) synthesis was performed using Reverse Transcription Master Mix from Fluidigm® according to the manufacturer's protocol with random primers in a final volume of 5 ⁇ _ containing 100 ng total RNA using a Nexus thermocycler (Eppendorf). cDNA samples were diluted by adding 20 ⁇ _ of low TE buffer [10 mM Tris ; 0.1 mM EDTA ; pH 8.0 (TEKNOVA)] and stored at -20°C.
  • TEKNOVA TEKNOVA
  • the pre-amplified cDNA were diluted 5X by adding 20 ⁇ _ of low TE buffer (TEKNOVA) and stored at -20°C before qPCR.
  • High-throughput real time PCR was performed on the qPCR-HD-Genomic Paris Centre platform and was supported by grants from Region lle-de-France, using the high- throughput platform BioMarkTM HD System and the 96.96 GE Dynamic Arrays (Fluidigm).
  • sample master mix consisted of 1 .8 ⁇ _ of 5X diluted pre-amplified cDNA, 0.3 ⁇ _ of 20X GE Sample Loading Reagent (Fluidigm) and 3 ⁇ of TaqMan® Gene Expression PCR Master Mix (Life Technologies, ThermoFisher).
  • AMM assay master mix
  • 5 ⁇ of each SMM and each AMM premixes were added to the dedicated wells. The samples and assays were mixed inside the chip using HX IFC controller (Fluidigm).
  • Thermal conditions for qPCR were: 25°C for 30 min and 70°C for 60 min for thermal mix ; 50°C for 2 min and 95°C for 10 min for hot start ; 40 cycles at 95°C for 15 s and 60°C for 1 min.
  • Data were processed by automatic threshold for each assay, with linear derivative baseline correction using BioMark Real-Time PCR Analysis Software 4.0.1 (Fluidigm). The quality threshold was set at the default setting of 0.65. Normalization was done using the GAPDH rate. Livak normalization provided the expression level in each sample with a transformation by the 2 ⁇ method (Schmittgen & Livak). Scripts are available in GitHub (https://github.com/jpouch/qPCR-Biomark). Genes expressed in less than 10 individuals were not further analyzed. Statistical analyses were done using R and SPSS Statistics 20 (IBM): non-parametric Wilcoxon test were performed.
  • example 1 of the present patent application the inventors reported longitudinal epigenetic changes in DNA methylation during psychotic transition, suggesting alterations in gene expression contemporary to psychosis onset. Accordingly, it was hypothesised that psychotic transition is accompanied with specific changes in gene (or gene network) expression.
  • the inventors were able to prospectively explore clinical evolution along with biological changes. One third of these individuals achieved the one-year follow-up, allowing intra-individual analyses in which each individual served as his/her own control, and among these, 41 % converted to psychosis.
  • the first network included genes from the Wnt pathway including Akt1 , CPT1 A and semaphorins. This biological network was recently shown to be decreased in subjects with schizophrenia compared to controls in the peripheral blood mononuclear cells transcriptome.
  • the second group corresponded to genes involved in the signaling cascade of Toll-like receptors. Toll-like receptors could account for innate immunity abnormalities in schizophrenia and mice deficient in Toll-like receptor show 'schizophrenic-like' features .
  • Post-hoc analyses comparing the level of expression for each gene analyzed in medicated and unmedicated subjects found no difference, suggesting that the results reported here are not related to antipsychotic medication.
  • methylomic and transcriptomic data were limited to 5% of overlap. This is not surprising given that the dysregulation of gene expression could be largely modulated by other epigenetic mechanisms like post-traductional histones modifications or miRNA regulation, not investigated in our study. Moreover, the methylome seemed less dynamic than the transcriptome: methylomic changes could occur several months before the transition whereas transcriptomic analysis reflects more rapid changes.

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Abstract

The present invention concerns an in vitro method for diagnosing the transition to psychosis in a subject at risk of psychosis and a method for predicting onset of psychosis. Identifying ultra-high-risk state for psychosis in subjects is challenging and predictive markers are needed. By studying two cohorts, one of 39 and one of 306 help-seekers individuals comprising young individuals at ultra-high risk (UHR) of conversion to psychosis, the inventors identified in subjects at ultra-high risk (UHR) of conversion to psychosis 139 differentially methylated Cp Gs and 14 genes being differentially expressed during psychotic transition. In particular, the invention refers to an in vitro method wherein in a biological sample the level of expression of at least one of the identified genes is determined and/or Cp G methylations are determined. Typically, the expression level is determined by RT-q PCR or RNA sequencing. Typically, Cp G methylations are determined using bisulfite conversion and/or microarrays.

Description

METHYLOMIC AND TRANSCRIPTOMIC CHANGES DURING CONVERSION TO
PSYCHOSIS
The present invention refers to an in vitro method for diagnosing the transition to psychosis in a subject at risk of psychosis and a method for predicting onset of psychosis.
Schizophrenia is a progressive illness, generally occurring in late adolescence, with several stages from early vulnerability, at-risk mental state (also called ultra-high risk abbreviated UHR), first-episode psychosis, to chronic disease. The molecular mechanisms triggering the progression of the illness are still largely unknown. The onset of psychosis is the consequence of complex interactions between genetic vulnerability to psychosis and response to environmental and/or maturational changes. Epigenetics is hypothesized to mediate the interplay between genes and environment leading to the onset of psychosis. The inventors of the present invention performed the first longitudinal prospective study of genomic DNA methylation during psychotic transition in help-seeking young individuals referred to a specialized outpatient unit for early detection of psychosis, and enrolled in a one-year follow-up. For methylation analysis the Infinium HumanMethylation450 BeadChip array was used after bisulfite conversion and longitudinal variations in methylation at 41 1 947 CpG sites were analyzed. The inventors surprisingly discovered that conversion to psychosis was associated with specific methylation changes. Changes in DNA methylation were significantly different between converters and non-converters in two regions: one located in 1 q21 .1 and a cluster of 6 CpG located in GSTM5 gene promoter. Methylation data were confirmed by pyrosequencing in the same population. The 100 top CpGs associated with conversion to psychosis were subjected to exploratory analyses regarding the related gene networks and their capacity to distinguish between converters and non-converters. Cluster analysis showed that the top CpG sites correctly distinguished between converters and non- converters. Methylation changes associated with conversion to psychosis preferentially occurred in gene promoters and pathways relevant for psychosis, including oxidative stress regulation, axon guidance and inflammatory pathways. Longitudinal variations in DNA methylation may reflect the biological mechanisms that precipitate some prodromal individuals into full-blown psychosis, under the influence of environmental factors and maturational processes at adolescence.
The identification of clinical ultra-high-risk state for psychosis (hereinafter UHR; also known as the "at-risk mental state") has been a relatively recent development in the field of psychiatry, and it has provided a means to capture the pre-psychotic phase and to describe individuals with prodromal symptoms that may transition into psychosis and schizophrenia (Yung, AR and McGorry, 1996, Aust. N. Z. J. Psychiatry 30:587-99). Operational criteria for UHR have been proposed based on specific comprehensive interviews. In individuals reaching these criteria, the conversion rate of individuals at UHR to full-blown psychosis is 30 to 40% in the following 24 to 36 months. Nevertheless, the populations reaching UHR criteria remain heterogeneous with the possibility of several outcomes, including symptomatic regression or development of non-psychotic disorders, rather than psychosis, which underscores the need for more predictive markers. Deciphering the biological mechanisms underlying the onset of psychosis requires longitudinal measures in help-seekers patients which includes characterization of their outcomes.
Understanding the different pathophysiological pathways leading to conversion to psychosis is a major issue of the field. The literature about conversion to psychosis, however, is still maturing and molecular findings remain limited. From the molecular point of view, conversion to psychosis is viewed as the complex interaction between biological vulnerability and exposure to many potentially harmful environmental risk factors. This is in line with the overall gene χ environment interaction hypothesis in schizophrenia, whereby the influence of the environment is thought to induce epigenetic changes. Until now, however, the epigenetic signature of conversion to psychosis has not been studied. Epigenetic regulation involves dynamic processes that have a role in controlling gene expression levels, among which histone post-transcriptional modifications and methylation of genes on CpG dinucleotides have been the main focus of the research. New methylomic technologies enable investigation of CpG methylation sites at the genomic scale. A large methylome-wide-association study recently compared patients with established schizophrenia to controls and found 139 differentially methylated CpGs, including FAM63B and RELN.
To the inventors' knowledge, no previous study has considered pangenomic methylation longitudinal changes accompanying conversion to psychosis. In the present study, blood methylation biomarkers were explored and associated with conversion to psychosis in a methylomic association study involving young help-seeking individuals who were enrolled in a longitudinal follow-up program. Significant differentially methylated regions (DMRs) were detected by the inventors. Furthermore, an original exploration of multiple CpG sites was conducted and followed by pathway and cluster analyses of the top methylation changes. The top findings were further confirmed by the inventors using pyrosequencing. The longitudinal epigenetic changes in DNA methylation during psychotic transition which were surprisingly discovered by the inventors suggest alterations in gene expression contemporary to psychosis onset.
Different peripheral whole-transcriptome studies were published in chronic schizophrenia but only one study, limited to a candidate gene approach, has analyzed gene expression in ultra-high risk (UHR) individuals.
Accordingly, the inventors further studied longitudinal variations in gene expression during the onset of psychosis. In a cohort of 306 help-seekers individuals comprising young individuals at ultra-high risk (UHR) of conversion to psychosis, among which 41 % converted to psychosis, a longitudinal transcriptomic analysis was performed in blood samples. 14 genes and 2 gene networks, in particular the genes CPT1 A and NRP1 , were identified by the inventors as being differentially expressed during psychotic transition.
Summary of the invention
The present invention thus refers to an in vitro method for diagnosing the transition to psychosis in a subject at risk of psychosis, comprising
a) determining, in a biological sample from the subject which includes nucleic acids, CpG methylations;
b) comparing said CpG methylations determined in step a) with reference CpG methylations;
c) based on the comparison of step b), determining differentially methylated regions (DMR) in the sample of step a), and
d) deducing if the subject is in transition to psychosis;
wherein the presence of at least one differentially methylated region (DMR) located in a chromosomal region selected from the group consisting of the chromosomal regions:
- starting at position 146549909 and ending at position 146550467 of chromosome 1 ,
- starting at position 1 10254662 and ending at position 1 10254835 of chromosome 1 ,
- starting at position 1594282 and ending at position 1594733 of chromosome 5,
- starting at position 195489306 and ending at position 195489782 of chromosome 3,
- starting at position 1856713 and ending at position 1857477 of chromosome 5,
- starting at position 325915 and ending at position 325964 of chromosome 1 1 ,
- starting at position 17599784 and ending at position 17600122 of chromosome 19,
- starting at position 121496875 and ending at position 121497334 of chromosome 2,
- starting at position 101093834 and ending at position 101093900 of chromosome 15,
- starting at position 176797999 and ending at position 176798049 of chromosome 5, - starting at position 13875014 and ending at position 138751 1 1 of chromosome 19,
- starting at position 724273 and ending at position 724374 of chromosome 17,
- starting at position 143751796 and ending at position 143751801 of chromosome 8,
- starting at position 24348549 and ending at position 24348715 of chromosome 22,
- starting at position 1 10254919 and ending at position 1 10255096 of chromosome 1 ,
- starting at position 128776861 and ending at position 128777132 of chromosome 9,
- starting at position 55013946 and ending at position 55013954 of chromosome 19,
- starting at position 50981 121 and ending at position 50981406 of chromosome 22,
- starting at position 4081325 and ending at position 4081428 of chromosome 17,
- starting at position 12876846 and ending at position 12877000 of chromosome 19,
- starting at position 1 14429965 and ending at position 1 14430031 of chromosome 1 ,
- starting at position 20036684 and ending at position 20036779 of chromosome 20,
- starting at position 52492209 and ending at position 52492248 of chromosome 20, and
- starting at position 24384400 and ending at position 24384525 of chromosome 22, is indicative for said subject being in transition to psychosis.
In one embodiment, said differentially methylated region (DMR) is located in a gene, wherein said gene is selected from the group consisting of GSTT1 , GSTP1 , GSTM5, LOC441228, ZNF514, RNF34, COQ2, DOK7, HIST1 H2AM, UNGP3, KIAA1274, LRP2BP, CDH13, TRAF3IP2, PTPRN2, COL9A2, TRIM39, NRP1 , GGA3, CHL1 , SIM1 , TFAP2E, SCRN1 , RIPPLY3, UHRF1 BP1 L, KIA1026, t-RNA-Leu, MIR3683, ATRN, EFNA3, CBS, ZNF282, BAH , NFIX, PPM1 E, CEP152,IL17RE, LZTS2, FBRSL1 , EREG, CRELD1 , KCNIP4, AHSP, ASAM, INPP5A, LRWD1 , COX10, MRC2, CYP2R1 , PAFAH2, FAM187B, FAM123C, HHCA2, COL22A1 , MAL2, PROCA1 , CNIH3, C1 orf106, C22orf45, PDE2A, NUDCD3, ZNF212, CC2D2A, PRPF39, UNCX, LOC100422263, C4orf42, SLC7A5, MNX1 , AN09, FBX018, TCERG1 L, PTCHD3P3, NDUFV3, HTA, OR1 F12, C17orf82, FKBP10, TAP1 , AP2A2, RTF1 , AKT1 , FOXF1 , DENND1 B, KIAA1274, LIN28, PLD3, LOC285205, TOB2, KREMEN2, C16orf78, SDK1 , CYFIP1 , LOC150381 and M6PR.
The invention further refers to a method for predicting onset of psychosis in a subject at risk of psychosis comprising
a) determining, in a biological sample from the subject which includes nucleic acids, CpG methylations;
b) comparing said CpG methylations determined in step a) with reference CpG methylations;
c) based on the comparison of step b), determining differentially methylated regions (DMR) in the sample of step a), and
d) deducing if the subject is at risk of developing psychosis; wherein the presence of at least one differentially methylated region (DMR) located in a chromosomal as defined herein above in the 'in vitro method for diagnosing the transition to psychosis' indicates that said subject is at risk of developing psychosis.
In one aspect the invention further refers to the use of at least one differentially methylated region (DMR) located in a chromosomal region in a chromosomal as defined herein above in the 'in vitro method for diagnosing the transition to psychosis'.
The invention further refers to a DNA methylation inhibitor and/or a gene expression modulator, said gene being selected from the group consisting of GSTT1 , GSTP1 , GSTM5, LOC441228, ZNF514, RNF34, COQ2, DOK7, HIST1 H2AM, UNGP3 (also called STON2), KIAA1274, LRP2BP, CDH13, TRAF3IP2, PTPRN2, COL9A2, TRIM39, NRP1 , GGA3, CHL1 , SIM1 , TFAP2E, SCRN1 , RIPPLY3 (also called MRPL20P1 ), UHRF1 BP1 L, KIA1026, T-RNA-Leu, MIR3683 (also called LOC100121257), ATRN, EFNA3, CBS, ZNF282, BAH , NFIX, PPM1 E, CEP152,IL17RE, LZTS2, FBRSL1 , EREG, CRELD1 , KCNIP4, AHSP, ASAM, INPP5A, LRWD1 , COX10, MRC2, CYP2R1 , PAFAH2, FAM187B (also called LOC100421621 ), FAM123C, HHCA2 (also called CTSD), COL22A1 , MAL2, PROCA1 , CNIH3, C1 orf 106, C22orf45 (also called UPB1 ), PDE2A, NUDCD3, ZNF212, CC2D2A, PRPF39, UNCX, LOC100422263, C4orf42, SLC7A5, MNX1 , AN09, FBX018, TCERG1 L, PTCHD3P3 (also called CCDC162P), NDUFV3, HTA, OR1 F12 (also called ZNF165), C17orf82, FKBP10, TAP1 , AP2A2, RTF1 (also called ITPKA), AKT1 , FOXF1 , DENND1 B, KIAA1274, LIN28, PLD3 (also called C19orf47), LOC285205, TOB2, KREMEN2, C16orf78, SDK1 , CYFIP1 , LOC150381 (also called LOC642648) and M6PR, for use in the treatment of psychosis or transition to psychosis.
In a further aspect, the invention refers to a method of treatment of psychosis or transition in psychosis in a subject in need thereof which method comprises administering said subject with a therapeutically effective amount of a DNA methylation inhibitor and/or a gene expression modulator, said gene being as defined herein above.
In another aspect, the invention refers to a method of treatment of psychosis or transition in psychosis in a subject in need thereof which method comprises
i) determining if a subject is in transition to psychosis using one of the in vitro methods of diagnosis of the invention, and
(ii) administering to said subject being in transition to psychosis a DNA methylation inhibitor, a gene expression modulator, said gene being defined as herein above, and/or an antipsychotic treatment.
The present invention further refers to an in vitro method for diagnosing the transition to psychosis in a subject at risk of psychosis, said method comprising a) determining the level of expression of at least one gene selected from the group consisting of CPT1 A, NRP1 , CREB3L3, EPB49, EREG, GYPA, HBB, HBD, HSPB7, MUC16, OSBP2, PEG3, TBX1 , TMOD1 , TRIM58, AKT1 , CHL1 , GSTM5 and IL17RE in a biological sample from said subject,
b) comparing said level of expression of the at least one gene determined in step a) to a reference value; and
c) deducing from the comparison of step b) if the subject is in transition to psychosis.
The present invention further refers to a method for predicting onset of psychosis in a subject at risk of psychosis comprising
a) determining the level of expression of at least one gene selected from the group consisting of CPT1 A, NRP1 , CREB3L3, EPB49, EREG, GYPA, HBB, HBD, HSPB7, MUC16, OSBP2, PEG3, TBX1 , TMOD1 , TRIM58, AKT1 , CHL1 , GSTM5 and IL17RE in a biological sample from said subject,
b) comparing said measured level of expression of the at least one gene to a reference value; and
c) deducing from the comparison of step b) if the subject is at risk of onset of psychosis. In some embodiments the methods based on CpG methylations and the methods based on the level of expression of at least one gene may be combined.
Accordingly in one embodiment, the present invention further refers to an in vitro method for diagnosing the transition to psychosis in a subject at risk of psychosis, said method comprising
a) the steps of:
i) determining, in a biological sample from the subject which includes nucleic acids, CpG methylations;
ii) comparing said CpG methylations determined in step ai) with reference CpG methylations;
iii) based on the comparison of step aii), determining differentially methylated regions (DMR) in the sample of step ai), and
b) the steps of
i) determining the level of expression of at least one gene selected from the group consisting of CPT1 A, NRP1 , CREB3L3, EPB49, EREG, GYPA, HBB, HBD, HSPB7, MUC16, OSBP2, PEG3, TBX1 , TMOD1 , TRIM58, AKT1 , CHL1 , GSTM5 and IL17RE in a biological sample from said subject, ii) comparing said level of expression of step bi) with the at least one gene determined in step bii) to a reference value; and
c) deducing from the comparison of steps aiii) and bii) if the subject is in transition to psychosis.
As described herein above, the presence of at least one differentially methylated region (DMR) located in a chromosomal region as defined herein above in the 'in vitro method for diagnosing the transition to psychosis indicates that said subject is at risk of developing psychosis.
It will be understood that in one embodiment, steps a) including ai), aii) and aiii) and b) including bi) and bii) may be switched, i.e. the steps as indicated under b) may be performed first, followed by the steps as indicated under a).
The present invention further refers to a method for predicting onset of psychosis in a subject at risk of psychosis comprising
a) the steps of
i) determining, in a biological sample from the subject which includes nucleic acids, CpG methylations;
ii) comparing said CpG methylations determined in step ai) with reference CpG methylations;
iii) based on the comparison of step aii), determining differentially methylated regions (DMR) in the sample of step ai), and
b) the steps of
i) determining the level of expression of at least one gene selected from the group consisting of CPT1 A, NRP1 , CREB3L3, EPB49, EREG, GYPA, HBB, HBD, HSPB7, MUC16, OSBP2, PEG3, TBX1 , TMOD1 , TRIM58, AKT1 , CHL1 , GSTM5 and IL17RE in a biological sample from said subject,
ii) comparing said measured level of expression of the at least one gene to a reference value; and
c) deducing if the subject is at risk of developing psychosis.
As described herein above, the presence of at least one differentially methylated region (DMR) located in a chromosomal region as defined herein above in the 'in vitro method for diagnosing the transition to psychosis' indicates that said subject is at risk of developing psychosis.
It will be understood that in one embodiment, steps a) including ai), aii) and aiii) and b) including bi) and bii) may be switched, i.e. the steps as indicated under b) may be performed first, followed by the steps as indicated under a). In these embodiments, concerning the combined methods, it will be further understood by the skilled in the art, that the sample used in steps a) and b) in particular step ai) and bi) is a sample from the same subject and the same origin. For example, one blood sample is obtained from said subject and then divided into two, one part of said sample being used for the steps a) of the combined methods of the invention and the other part of said sample being used for the steps b) of the combined methods of the invention.
Detailed description of the invention
As used herein, the term "determining" includes qualitative and/or quantitative detection (i.e. detecting and/or measuring the level of expression of at least one gene as defined herein above or determining CpG methylations as defined herein above) with or without reference to a control.
Psychosis
"Psychosis" is a clinical term and herein refers to a family of serious mental illnesses, referred to as psychotic disorders. Psychosis, in general, is characterized by radical changes in personality, impaired functioning, and a distorted or nonexistent sense of objective reality. Accordingly, subjects suffering from psychosis have impaired reality testing; that is, they are unable to distinguish personal subjective experience from the reality of the external world.
"Primary symptoms of psychosis" include hallucinations, delusions, disorganized speech (except for substance/medication-induced psychotic disorder and psychotic disorder due to another medical condition), abnormal psychomotor behavior, as well as dimensional assessments of depression and mania.
Symptoms of psychosis may be relatively constant or come and go, and is therefore also referred to as "psychotic symptoms", "psychotic episode" or "psychotic experience". Psychosis refers to psychotic disorders, as mentioned above, and as further defined herein below, but psychosis can also occur for many other reasons, including substance abuse, brain injury, seizure disorders, or conditions of extreme sleep deprivation or isolation.
"Psychotic disorders" include, but are not limited to, schizotypal (Personality) disorders, delusional disorder, brief psychotic disorder, schizophreniform disorder, schizophrenia, schizoaffective disorder, substance/medication-induced psychotic disorder, psychotic disorder due to another medical condition, catatonia, depression and bipolar disorders. Accordingly, in one embodiment, psychosis herein refers to psychotic disorders such as schizotypal (Personality) disorders, delusional disorder, brief psychotic disorder, schizophreniform disorder, schizophrenia, schizoaffective disorder, substance/medication- induced psychotic disorder, psychotic disorder due to another medical condition, catatonia, depression and bipolar disorders.
In one embodiment, psychosis is not schizophrenia.
In one embodiment, psychosis is not chronic schizophrenia
The diagnostic criteria of the psychotic disorders listed above are generally known to the skilled in the art and are, for example, further described in "Diagnostic and Statistical Manual of Mental disorders », DSM-5, American Psychiatic Association, 2013 or in the 4th edition, DSM-4, American Psychiatic Association, 1994.
"Transition to psychosis" herein refers to a "pre-psychotic" or "prodromal stage" of psychosis, which has been defined as the period marked by changes from a person's premorbid mental state and level of functioning up to the appearance of psychotic features (Yung, AR and McGorry, 1996, Aust. N. Z. J. Psychiatry 30:587-99 and Yung AR, McGorry PD. 1996, Schizophr. Bull. 22(2):353-70).
Those changes may be, for example, changes in drive, perception, beliefs, attention, concentration, mood, affect, and behavior (Yung, AR and McGorry, 1996, Aust. N. Z. J. Psychiatry 30:587-99 and Yung AR, McGorry PD. 1996, Schizophr. Bull. 22(2):353-70). It will be understood by the skilled in the art, that a precondition for early intervention is the accurate detection of prodromal states, i.e., knowing who may be at high risk of developing a psychotic disease.
One attempt to determine such a transition state is a diagnostic interview and rating system called Comprehensive Assessment of At-Risk Mental States (CAARMS) developed by the Australian researchers Yung AR and McGorry PD. The researchers defined putative risk criteria for psychosis. These criteria reflect an ultra-high-risk state for developing a psychotic disorder in the near future and include attenuated positive symptoms, brief intermittent psychotic states, and genetic/familial risk for psychotic illness coupled with recent and dramatic decline in functioning.
"Ultra-high-risk state for psychosis (UHR)" herein refers to a state of a subject with prodromal symptoms that is thus at risk of transition into psychosis. In one example, such state is determined according to CAARMS.
"Comprehensive Assessment of At-Risk Mental States (CAARMS)" herein refers to a semistructured interview schedule designed for use by mental health professionals who are already able to assess and evaluate patients' information. It is designed for repeated use over time, for example, monthly to 6 times monthly. The CAARMS includes the following subscales: disorders of thought content (e.g. delusional mood, overvalued ideas and delusions), perceptual abnormalities (e.g. distortions, illusions and hallucinations), conceptual disorganization (e.g. subjectively experienced difficulties with forming thoughts and objective assessment of formal thought disorder), motor changes (e.g. subjectively experienced difficulties with movement and objective signs of catatonia), concentration and attention (assessing both the subjective experience and objective rating), emotion and affect (assessing subjective sense of change in emotions and objective rating of blunting of affect), subjectively impaired energy (a basic symptom) and impaired tolerance to normal stress (a basic symptom). Scores for each subscale range from 0 to 6. Accordingly, a subject obtains a CAARMS score depending on his assessment results in each of the above mentioned subscales. Typically, the assessment is done by a trained psychiatrist.
So far the populations reaching the UHR criteria remain heterogeneous with the possibility of several outcomes, including symptomatic regression or development of non- psychotic disorders, rather than psychosis, which underscores the need for more predictive markers.
Subject
In the context of the invention, "subject" refers to an animal, preferably a non- human or human mammal. Examples of non-human mammals include rodents and primates. Most preferably, the subject is a human.
More preferably, the "subject" denotes herein an individual that is under medical care, surveillance or treatment.
The present invention refers to the diagnosis of the transition to psychosis in a subject at risk of psychosis, or, in other words, diagnosing in a subject at risk of psychosis a high-risk state for psychosis or a Ultra-high-risk state for psychosis (UHR) using the methods of the invention.
Accordingly, in one embodiment, the subject does not have
- manifest symptoms of psychosis,
- pervasive developmental disorder,
- bipolar disorder, and/or
- obsessive compulsive disorder.
"Manifest symptoms of psychosis" herein preferably refers to fulfilling typically the DSM-IV criteria for psychosis.
In one embodiment, in context of the present invention, the subject is at risk of psychosis. A subject "at risk of psychosis" herein refers to a subject having alterations in global functioning.
"Alterations in global functioning" herein refers to a social and occupational functioning assessment scale score of typically less than 70, preferably, obtained during the during the year preceding the time point of the application of the methods of the invention.
In one embodiment, the subject at risk of psychosis does not have a CAARMS score above the psychosis threshold as defined by CAARMS. The CAARMS score, threshold and methods to determine the CAARMS score of an individual at risk of psychosis are known to the skilled in the art and further described in "Diagnostic and Statistical Manual of Mental disorders », DSM-5, American Psychiatic Association, 2013 or DSM-4, American Psychiatic Association, 1994.
Furthermore, in one embodiment, the subject is not under antipsychotic treatment for more than 12 weeks, wherein antipsychotic treatment preferably refers to more than 10Omg of a Chlorpromazine equivalent.
In a further embodiment, the subject has no psychoactive substance dependence or abuse during the year preceding the time point of the application of the methods of the invention and/or during more than 5 years.
Furthermore, in one embodiment, the subject does not suffer from serious or non- stabilized somatic and neurological disorders.
In a further aspect of the invention, the subject does not suffer from a head injury.
In another aspect, the subject has an intelligent quotient of more than 70.
In context of the present invention, the subject has any age; preferably, the age of the subject is 16 to 30.
Symptoms that might indicate a risk of psychosis are generally known to the skilled in the art and are, for example, further described in "Diagnostic and Statistical Manual of Mental disorders », DSM-5, American Psychiatic Association, 2013 or in the articles of, for example, Yung, AR and McGorry (see Yung AR, McGorry PD. 1996a. Aust. N. Z. J. Psychiatry 30:587-99, Yung AR, McGorry PD. 1996b. Schizophr. Bull. 22(2):353-70, Yung AR et al. 1996 Schizophr. Bull. 22(2):283-303, and Yung AR, et al. 2005. Aust. N. Z. J. Psychiatry 39(1 1 -12):964-71 ).
In one embodiment, a "subject that is at risk of psychosis", for example, shows at least one symptom selected from the group constituted of subtle changes in drive, subtle changes in affect, subtle changes in thought, subtle changes in speech, subtle changes in perception, subtle changes in motor activity, first vegetative symptoms, social difficulties, academic problems, vocational problems, mood symptoms (anxiety, depression); social withdrawal, impaired social cognition (affect recognition; understanding others' emotions, actions, intentions), attenuated psychotic symptoms such as unusual thoughts, suspiciousness, paranoia, bizarre, thinking, and primary symptoms of psychosis such as hallucinations, delusions, disorganized speech, abnormal psychomotor behavior, as well as dimensional assessments of depression and mania.
In one particular embodiment, a subject that is at risk of psychosis, for example, shows at least one symptom selected from the group constituted of disorders of thought content (e.g. delusional mood, overvalued ideas and delusions), perceptual abnormalities (e.g. distortions, illusions and hallucinations), conceptual disorganization (e.g. subjectively experienced difficulties with forming thoughts and objective assessment of formal thought disorder), motor changes (e.g. subjectively experienced difficulties with movement and objective signs of catatonia), concentration and attention (assessing both the subjective experience and objective rating), emotion and affect (assessing subjective sense of change in emotions and objective rating of blunting of affect), subjectively impaired energy (a basic symptom) and impaired tolerance to normal stress (a basic symptom).
"At least one symptom" herein refers to at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more symptoms as defined above. The subject that is at risk of psychosis might suffer from, for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more symptoms selected from the list of symptoms mentioned above, preferably, the subject that is at risk of psychosis suffers from 3, 4, 5 or more symptoms selected from the group of symptoms indicated above.
In one embodiment, the subject at risk of psychosis is at ultra-high-risk for psychosis, wherein ultra-high-risk for psychosis is defined as herein above, preferably,
In several embodiments, the subject at risk of psychosis has beforehand been identified by a physician, in particular by a psychiatrist, as being at risk of psychosis, in particular, using standard criteria for best-estimate diagnosis, more particularly using comprehensive assessment for at risk mental state.
In one embodiment, ultra-high-risk for psychosis is determined in a subject using the comprehensive assessment for at risk mental state (CAARMS).
"Comprehensive assessment for at risk mental state (CAARMS)" is the diagnostic interview and rating system developed by Australian researchers to assess psychosis risk criteria prospectively and is further described in the following articles (Yung AR, McGorry PD. 1996a. Aust. N. Z. J. Psychiatry 30:587-99, Yung AR, McGorry PD. 1996b. Schizophr. Bull. 22(2):353-70, Yung AR et al. 1996 Schizophr. Bull. 22(2):283-303). Furthermore, the CAARMS manual provides detailed definitions, questions, and anchor points for eliciting and rating 27 symptoms across seven dimensions of psychopathology, including positive symptoms, negative symptoms, deterioration of role functioning, sleep disturbance, and impaired tolerance to normal stress (see Yung AR, et al. 2005. Aust. N. Z. J. Psychiatry 39(1 1 -12):964-71 ).
In one embodiment, the subject at risk of psychosis has been beforehand diagnosed by a trained psychiatrist using the comprehensive assessment for at risk mental state (CAARMS).
CpG methylation
The word "methylome" refers to the DNA methylations in an organism's genome or in a particular cell.
"Methylomic changes" refer accordingly to changes of the DNA methylation in an organism's genome or in a particular cell.
"DNA methylation" is a process by which methyl groups are added to DNA. Methylation modifies the function of the DNA. When located in a gene promoter, DNA methylation typically acts to repress gene transcription. DNA methylation is essential for normal development and is associated with a number of key processes including genomic imprinting, X-chromosome inactivation, repression of repetitive elements, aging and carcinogenesis. Two of DNA's four nucleotides, cytosine and adenine, can be methylated. Adenine methylation is restricted to prokaryotes.
"Cytosine methylation" involves the addition of a methyl group at the 5-carbon position of cytosine thus forming 5-methylcytosine. In humans, cytosine methylation is the most common epigenetic modification of DNA, which occurs typically at symmetric CG sites on the DNA double helix and is referred to as CpG dinucleotides. The CpG sites or CG sites refer to DNA nucleotide sequence wherein a cytosine nucleotide is followed by a guanine nucleotide in the linear sequence of bases along its 5' → 3' direction. CpG is shorthand for 5'-C-phosphate-G-3', that is, cytosine and guanine separated by only one phosphate; phosphate links any two nucleosides together in DNA. The CpG notation is used to distinguish this single-stranded linear sequence from the CG base-pairing of cytosine and guanine for double-stranded sequences.
Accordingly, CpG methylation herein refers to cytosine methylation of cytosine within CpG dinucleotides. CpG methylation occurs typically in humans in adult somatic cells.
In the context of the invention, CpG methylation can be determined in a biological sample with techniques that are well known from the one skilled in the art. These methods include, without being limited, chemical modification of the DNA with bisulfite (represented by bisulfite genomic sequencing), restriction enzyme digestion of the DNA (represented by methylation-sensitive restriction endonucleases) and affinity-based isolation of methylated DNA (represented by methylated DNA immunoprecipitation). In a preferred embodiment, CpG methylation can be determined in a biological sample containing DNA using chemical modification of said DNA with bisulfite also referred to as "bisulfite conversion" of said DNA and further using standard methods, known to the skilled in the art, such as methylation specific restriction enzymes, Sanger sequencing or pyrosequencing, microarrays, and/or PCR Techniques such as bisulfite specific PCR (BSP) or COBRA (determination of methylation at specific restriction enzyme sites within PCR amplicon).
In a preferred embodiment, the presence of CpG methylation can be determined using bisulfite conversion and microarrays, in particular by high-throughput micro arrays.
In one example, bisulfite conversion and a Human Methylation450 Bead Chip
(from lllumina) are used to determine CpG methylations.
In another example, bisulfite conversion and pyosequencing (from lllumina) are used to determine CpG methylations.
"Determining CpG methylation" herein refers to determining the presence of a CpG methylation and at the same time determining the region or the position of said methylated CpG site.
"Region" herein refers to a stretch of DNA, preferably to a region of a chromosome, particularly to a gene, more preferably to region of a gene.
The term "chromosome" herein refers to a packaged and organized structure containing most of the DNA of a living organism. In case of eukaryotes the chromosomes are located in the nucleus.
The term "gene" means a DNA sequence that codes for, or corresponds to, a particular sequence of amino acids which comprises all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. Some genes, which are not structural genes, may be transcribed from DNA to RNA, but are not translated into an amino acid sequence. Other genes may function as regulators of structural genes or as regulators of DNA transcription. In particular, the term gene may be intended for the genomic sequence encoding a protein, i.e. a sequence comprising regulator, promoter, intron and exon sequences.
Accordingly, in one embodiment the gene is the promoter, intron or exon sequence of said gene.
As it will be understood by the skilled in the art that the comparison of CpG methylation determined in one sample with the CpG methylation determined in a reference sample leads to the determination of differentially methylated regions (DMR) in said sample, in particular in the sample which is not the reference sample. Accordingly, "differentially methylated region (DMR)" herein refers to a region of DNA in the genome of a subject containing at least one CpG site, wherein said at least one CpG site is differently methylated compared to the same at least one CpG site of a reference sample.
A "region" or "region of DNA" as defined herein above, such as a chromosomal region or a gene, contains more than one CpG site, preferably at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 CpG sites, more preferably at least 1 , 2, 3, 4,
5, 6, 7, 8, 9, 10, CpG sites, in particular, a region as herein defined contains 1 , 2, 3, 4, 5,
6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 CpG sites. Accordingly, in one embodiment, the DMR contains at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 CpG sites, more preferably at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, CpG sites, in particular, a region as herein defined contains 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 CpG sites.
It will be understood by the skilled in the art that if a differentially methylated region (DMR) refers to region containing, for example, 5 CpG sites, not all of those 5 CpG sites must be differently methylated.
Accordingly, in one embodiment, the differentially methylated region (DMR) preferably contains at least one differently methylated CpG site, more preferably, at least two differently methylated CpG sites. It will be understood that the number of differently methylated CpG sites is independent of the total number of CpG sites present in the corresponding region.
In one embodiment, the differentially methylated region (DMR) preferably contains at least one differently methylated CpG site, more preferably, at least two differently methylated CpG sites.
Accordingly, a differentially methylated region (DMR) contains at least one differently methylated CpG site, for example at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 differently methylated CpG sites, preferably at least 2 differently methylated CpG sites, in particular, a differentially methylated region (DMR) contains 1 to 20, 1 to 15, 2 to 15, 3 to 15, 4 to 15, 5 to 15 differently methylated CpG site, more particularly, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 differently methylated CpG site.
In a particular embodiment, a differentially methylated region (DMR) is one differently methylated CpG site.
The inventors demonstrated that the presence of at least one DMR located in a specific chromosomal region indicates that said subject is in transition to psychosis, the location of at least one DMR is therefore useful in context of the methods of the present invention. In one embodiment, the at least one differentially methylated region (DMR) is located in a chromosomal region selected from the group consisting of the chromosomal regions:
- starting at position 146549909 and ending at position 146550467 of chromosome 1 , - starting at position 1 10254662 and ending at position 1 10254835 of chromosome 1 ,
- starting at position 1594282 and ending at position 1594733 of chromosome 5,
- starting at position 195489306 and ending at position 195489782 of chromosome 3,
- starting at position 1856713 and ending at position 1857477 of chromosome 5,
- starting at position 325915 and ending at position 325964 of chromosome 1 1 ,
- starting at position 17599784 and ending at position 17600122 of chromosome 19,
- starting at position 121496875 and ending at position 121497334 of chromosome 2,
- starting at position 101093834 and ending at position 101093900 of chromosome 15,
- starting at position 176797999 and ending at position 176798049 of chromosome 5,
- starting at position 13875014 and ending at position 138751 1 1 of chromosome 19, - starting at position 724273 and ending at position 724374 of chromosome 17,
- starting at position 143751796 and ending at position 143751801 of chromosome 8,
- starting at position 24348549 and ending at position 24348715 of chromosome 22,
- starting at position 1 10254919 and ending at position 1 10255096 of chromosome 1 ,
- starting at position 128776861 and ending at position 128777132 of chromosome 9, - starting at position 55013946 and ending at position 55013954 of chromosome 19,
- starting at position 50981 121 and ending at position 50981406 of chromosome 22,
- starting at position 4081325 and ending at position 4081428 of chromosome 17,
- starting at position 12876846 and ending at position 12877000 of chromosome 19,
- starting at position 1 14429965 and ending at position 1 14430031 of chromosome 1 , - starting at position 20036684 and ending at position 20036779 of chromosome 20,
- starting at position 52492209 and ending at position 52492248 of chromosome 20, and starting at position 24384400 and ending at position 24384525 of chromosome 22, preferably, the at least one differentially methylated region (DMR) is located in the chromosomal region starting at position 24348549 and ending at position 24348715 of chromosome 22, starting at position 55013946 and ending at position 55013954 of chromosome 19, starting at position 24384400 and ending at position 24384525 of chromosome 22, starting at position 146549909 and ending at position 146550467 of chromosome 1 or starting at position 1 10254662 and ending at position 1 10254835 of chromosome 1 , more preferably, starting at position 146549909 and ending at position 146550467 of chromosome 1 or starting at position 1 10254662 and ending at position 1 10254835 of chromosome 1 . In a particular embodiment, the at least one differentially methylated region (DMR) is located in the chromosomal region starting at position 24348549 and ending at position 24348715 of chromosome 22, starting at position 55013946 and ending at position 55013954 of chromosome 19 or starting at position 24384400 and ending at position 24384525 of chromosome 22.
In one preferred embodiment, the at least one differentially methylated region (DMR) is located in the chromosomal regions selected from the group consisting of the chromosomal regions:
- starting at position 146549909 and ending at position 146550467 of chromosome 1 , - starting at position 1 10254662 and ending at position 1 10254835 of chromosome 1 ,
- starting at position 1594282 and ending at position 1594733 of chromosome 5,
- starting at position 195489306 and ending at position 195489782 of chromosome 3,
- starting at position 1856713 and ending at position 1857477 of chromosome 5,
- starting at position 325915 and ending at position 325964 of chromosome 1 1 ,
- starting at position 17599784 and ending at position 17600122 of chromosome 19,
- starting at position 121496875 and ending at position 121497334 of chromosome 2,
- starting at position 176797999 and ending at position 176798049 of chromosome 5, and
- starting at position 724273 and ending at position 724374 of chromosome 17.
In one further preferred embodiment, the at least one differentially methylated region (DMR) is located in the chromosomal regions selected from the group consisting of the chromosomal regions:
- starting at position 146549909 and ending at position 146550467 of chromosome 1 , and
- starting at position 1 10254662 and ending at position 1 10254835 of chromosome 1 .
In one embodiment, the chromosomal region starting at position 146549909 and ending at position 146550467 of chromosome 1 , corresponds to region 1 q21 .1 , the chromosomal region starting at position 1 10254662 and ending at position 1 10254835 of chromosome 1 , includes the GSTM5 gene, in particular the GSTM5 promoter.
In another embodiment, said differentially methylated region (DMR) is located in a gene, wherein said gene is selected from the group consisting of the genes as listed below: Probe CHR Physical Position UCSC gene name P cg23655769 7 56358512 LOC441228 2,69E-06 cg05588757 2 95825608 ZNF514 2,90E-06 cg06657560 12 121841372 RNF34 5,32E-06 cg24939380 4 84189574 COQ2 l,55E-05 cg27461213 4 3468649 DOK7 l,80E-05 cg26448137 5 962206 l,95E-05 cgl6807643 6 27862331 HIST1H2AM 2,34E-05 cg05498047 11 124714902 3,34E-05 cg21539600 14 81729570 UNGP3; STON2 3,45E-05 cgl6178622 10 72271510 KIAA1274 3,64E-05 cg24069286 4 186300477 LRP2BP 3,68E-05 cg01750200 16 83273795 CDH13 3,93E-05 cgl6704530 6 111889939 TRAF3IP2 4,94E-05 cgl7255108 7 157338433 PTPRN2 4,99E-05 cgl3970437 11 24275293 5,22E-05 cg06636289 1 40781617 COL9A2 5,34E-05 cg21686797 6 30303645 TRIM39 5,42E-05 cgl2870705 10 33624623 NRP1 5,49E-05 cgl8531405 17 73234288 GGA3 5,81E-05 cglll63933 3 240139 CHL1 6,23E-05 cg20232986 6 100915767 SIM1 6,63E-05 cg26372517 1 36039159 TFAP2E 7,38E-05 cgl6594999 7 29974575 SCRN1 7,69E-05 cg06592890 21 38376101 RIPPLY3; MRPL20P1 7,78E-05 cg07744832 12 100537267 UHRF1BP1L 7,85E-05 cgl9520372 1 15256127 KIAA1026 7,98E-05 cg04736453 16 22205090 tRNA-Leu 9,10E-05 cgl3229969 7 7105916 MIR3683; LOC100131257 9,61E-05 cg20868728 17 30072661 9,95E-05 cgl2339292 20 3452280 ATRN 0,00010197 cg25745642 1 155050609 EFNA3 0,00010289 cgl5108590 21 44494906 CBS 0,00010942 cgl4925158 7 148892553 ZNF282 0,00011167 cgl4250251 8 143599611 BAI1 0,00011487 cgl2522311 19 13113893 NFIX 0,00011946 cg21131343 22 42686019 0,00012145 cgl9401340 17 56833197 PPM1E 0,00012662 cgl7834717 15 49102800 CEP152 0,00013367 cgl8738581 3 9944183 IL17RE 0,0001365 cg02613435 10 102756977 LZTS2 0,00013921 cgl6088107 12 133066646 FBRSL1 0,000143 cgll646192 4 75230615 EREG 0,0001434 cg01087880 3 9975336 CRELD1 0,00014552 cg00688962 4 21950567 KCNIP4 0,00014816 cg00148355 16 31538871 AHSP 0,00014825 cg09019035 11 1357820 0,00014835 cg01816768 11 123063169 ASAM 0,00015403 cg09270366 10 134587114 INPP5A 0,00016397 cg07112456 7 102109247 LRWD1 0,00016709 cgl3663116 9 116372034 0,00016842 cg20170777 17 13972099 COX10 0,00017421 cg05651778 17 60729731 MRC2 0,00017474 cg00455178 11 14913981 CYP2R1 0,00017907 cg04108612 1 26324550 PAFAH2 0,00018129 cg23495820 19 35715624 -■AM187B ; LOC100421621 0,00018157 cgl8194734 2 131513724 FAM123C 0,00018283 cg06300658 11 1774907 HCCA2; CTSD 0,00018564 cg09853792 8 139735754 COL22A1 0,00018612 cg02225716 8 120220856 MAL2 0,00018659 cg02685896 17 27038861 PROCA1 0,00018755 cgl0105262 1 224730478 CNIH3 0,0001925 cgl8279842 1 200879897 ciorfioe 0,00019414 cg08036764 22 24891220 C22orf45; UPB1 0,00019534 cg09612929 11 72353049 PDE2A 0,00020879 cg07383268 7 44530730 NUDCD3 0,00021232 cg05476998 7 148941307 ZNF212 0,00021326 cgl6509355 4 15471240 CC2D2A 0,00021555 cg03014427 14 45552678 PRPF39 0,00022388 cg07296447 7 1286023 UNCX 0,0002318 cg27665188 6 168168453 LOC100422263 0,00023233 cgl0874251 4 1245021 C4orf42 0,00023378 cg02614661 16 87878292 SLC7A5 0,0002352 cg07691705 7 156802325 MNX1 0,00023753 cg27420135 11 425289 AN 09 0,00023758 cgll222219 2 5584614 0,00024706 cg09952564 10 5979174 FBX018 0,00024791 cgl8006379 10 132907652 TCERG1L 0,00024992 cg01475377 6 109611718 PTCHD3P3 ; CCDC162P 0,00025218 cg00940809 21 44312947 NDUFV3 0,00025759 cg08855708 16 73127100 HTA 0,00026193 cg20656154 6 28041663 0R1F12; ZNF165 0,00026324 cg04406071 17 59489784 C17orf82 0,0002656 cg26750927 17 39977959 FKBP10 0,0002671 cg01979171 6 32818189 TAP1 0,00027383 cg01261206 11 1011281 AP2A2 0,00027618 cg21237133 15 41778987 RTF1 ; ITPKA 0,00028372 cg06934468 14 105248634 AKT1 0,00028422 cg00551679 16 86547530 F0XF1 0,00028655 cg02736602 1 197744226 DENND1B 0,0002919 cg04221624 10 72274527 KIAA1274 0,00029304 cg05768558 1 26737005 UN28 0,00029539 cg00043080 19 40853155 PLD3 ; C19orf47 0,00029582 cg26023908 3 107601974 LOC285205 0,00029649 cglll73202 22 41843125 T0B2 0,0002985 cg09786098 16 3016378 KREMEN2 0,00030075 cg08989564 16 49411456 C16orf78 0,00030476 cg04358834 7 4030413 SDK1 0,00030534 cg25124211 15 22981162 CYFIP1 0,00031061 cg27648216 22 46460338 LOC150381 ; LOC642648 0,00031091 cgl9642100 12 9102345 M6PR 0,00031629
GSTM5, GSTT1 and GSTP1 . In one particular embodiment, the differentially methylated region (DMR) is located in a gene, wherein said gene is selected from the group consisting of GSTT1 , GSTP1 , GSTM5, LOC441228, ZNF514, RNF34, COQ2, DOK7, HIST1 H2AM, UNGP3 (also referred to as STON2), KIAA1274, LRP2BP, CDH13, TRAF3IP2, PTPRN2, COL9A2, TRIM39, NRP1 , GGA3, CHL1 , SIM1 , TFAP2E, SCRN1 , RIPPLY3 (also referred to as MRPL20P1 ), UHRF1 BP1 L, KIA1026, t-RNA-Leu, MIR3683 (also referred to as LOC100121257), ATRN, EFNA3, CBS, ZNF282, BAH , NFIX, PPM1 E, CEP152,IL17RE, LZTS2, FBRSL1 , EREG, CRELD1 , KCNIP4, AHSP, ASAM, INPP5A, LRWD1 , COX10, MRC2, CYP2R1 , PAFAH2, FAM187B (also referred to as LOC100421621 ), FAM123C, HHCA2 (also referred to as CTSD), COL22A1 , MAL2, PROCA1 , CNIH3, C1 orf106, C22orf45 (also referred to as UPB1 ), PDE2A, NUDCD3, ZNF212, CC2D2A, PRPF39, UNCX, LOC100422263, C4orf42, SLC7A5, MNX1 , AN09, FBX018, TCERG1 L, PTCHD3P3 (also referred to as CCDC162P), NDUFV3, HTA, OR1 F12 (also called ZNF165), C17orf82, FKBP10, TAP1 , AP2A2, RTF1 (also referred to as ITPKA), AKT1 , FOXF1 , DENND1 B, KIAA1274, LIN28, PLD3 (also referred to as C19orf47), LOC285205, TOB2, KREMEN2, C16orf78, SDK1 , CYFIP1 , LOC150381 (also referred to as LOC642648) and M6PR.
In a preferred embodiment, said gene is GSTT1 , GSTP1 or GSTM5, in particular GSTM5, more particularly the promoter of GSTM5.
Accordingly, in one preferred embodiment the differentially methylated region (DMR) is located in the gene GSTT1 , GSTP1 or GSTM5, preferably GSTM5, in particular, in the promoter region of the GSTM5 gene.
In the context of the present invention, the positions of the nucleotides for the regions in the chromosome are indicated according to the genome sequence as indicated in the UCSC Genome Browser (according to the 18 April 2016). The positions of the nucleotides for the genes are indicated according to the genome sequence as used in the UCSC Genome Browser (according to the 18 April 2016).
Furthermore, in the context of the invention reference sequences are obtainable from the UCSC genome browser under the listed UCSC gene name. They are also typically accessible under the UCSC gene name in other databases known to the skilled in the art, such as the NCBI database "Gene" (according to the 18 April 2016).
It is known to the one skilled in the art, that a genome sequence is variable from an individual to another. Therefore, the positions defined herein may slightly change according to the human genome sequence used. However, methods to compare genomic sequences and nucleotide positions are well known to the one skilled in the art. Accordingly, DMR of sequences situated on different nucleotide positions on another human genome than the genome sequence as indicated in the UCSC reference sequence, but which sequence matches with the sequence delimited by the nucleotide positions defined above are within the scope of the invention. Biological sample
In one embodiment, the biological sample includes nucleic acids.
The term "nucleic acid" generally refers to at least one molecule or strand of DNA, RNA, miRNA or a derivative or mimic thereof, comprising at least one nucleobase, such as, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g., adenine "A," guanine "G," thymine "T," and cytosine "C") or RNA (e.g. A, G, uracil "U," and C). The term "nucleic acid" encompasses the terms "oligonucleotide" and "polynucleotide".
As used herein, the term "biological sample" means a substance of biological origin. In particular the biological sample comprises cells from the subject to be diagnosed. The biological samples comprises in particular nucleic acids. Examples of biological samples include, but are not limited to, blood and components thereof such as plasma, platelets, subpopulations of blood cells and the like; bone marrow samples, organs such as kidney, liver, heart, lung, and the like, saliva and mouth epithelial cells. The biological sample according to the invention may be obtained from the subject by any appropriate means of sampling known from the skilled person.
In a preferred embodiment of the invention, the biological sample is a blood sample.
In a further preferred embodiment of the invention, the biological sample is a peripheral blood sample.
A peripheral blood sample typically comprises red blood cells, white blood cells and platelets.
In one embodiment the sample comprises peripheral blood mononuclear cells (PMBC).
"Peripheral blood mononuclear cells" (PMBC) include monocytes and/or lymphocytes.
In one embodiment the sample comprises white blood cells.
"White blood cells" include monocytes, granulocytes and lymphocytes.
In one embodiment the sample comprises lymphocytes.
For genome-wide analysis of DNA methylation, DNA may be extracted from a sample of the subject and treated with sodium bisulfite and then analyzed by a DNA array. In one example, DNA is extracted from typically a blood sample and then treated with sodium bisulfite using for example EZ-96DNA Methylation Kit (Catalogue No D5004, Zymo Research, Irvine, CA, USA) typically according to manufacturer recommendations.
In the following step the extracted and bisulfite converted DNA may be analyzed by a DNA-array, in particular, by a DNA-array, as defined in the section "CpG methylation" herein above.
In one example, bisulfite converted DNA samples are analyzed for example on the using lllumina Infinium HumanMethylation450 BeadChip (lllumina, San Diego, CA, USA) typically according to the manufacturer's protocol. Primary data analysis might be done with lllumina Genome Studio Software (lllumina). Further Data processing and clean-up are typically performed as herein described in the experimental section.
Reference Value in context of the methods based on CpG methylations
The methods of the invention comprise a step b) of comparing said CpG methylations determined in step a) with reference CpG methylations.
Preferably, the "reference CpG methylations" corresponds to the CpG methylations determined in the sample of a healthy subject.
Preferably, the reference CpG methylations are determined in a sample from the same tissular origin as the biological sample of the subject of step a) of the methods of the invention. More preferably, the reference CpG methylations are determined in a blood sample.
In one embodiment, the reference CpG methylation corresponds to the CpG methylations observed in a healthy population.
As used herein, a "healthy population" means a population constituted of subjects who have not previously been diagnosed with psychosis, as defined herein above, or who is not at risk of psychosis, as defined in the section "Subject" herein above. Subjects of a healthy population also preferably do not otherwise exhibit symptoms of disease. In other words, such subjects, if examined by a medical professional, would be characterized as healthy and free of symptoms of disease.
Accordingly, a "healthy subject" means a subject who has not previously been diagnosed with psychosis or who is not at risk of psychosis as defined in the section "Psychosis" and/or "Subject" herein above. A healthy subject also preferably does not otherwise exhibit symptoms of disease. In other words, such healthy subject, if examined by a medical professional, would be characterized as healthy and free of symptoms of disease. Preferably, a healthy subject does not display any of the primary symptoms of psychosis or psychotic symptoms as defined herein above.
In one particular embodiment, the healthy subject has the same age as the subject who is at risk of psychosis.
In a further particular embodiment, the healthy subject has the same gender as the subject who is at risk of psychosis.
Method of diagnosis based on CpG methylations
As used herein the term "diagnosis" refers to the process of attempting to determine or identify a possible disease or disorder in a subject, as defined in the section "subject" herein above. In particular, "method of diagnosis" refers herein to a process of determining in a subject if said subject is in transition to psychosis.
In context of the present invention, the inventors determined CpG methylations in a sample containing nucleic acids, wherein said sample is from a subject at risk of psychosis, and compared the CpG methylations with CpG methylations of a reference sample, thus determining differentially methylated regions (DMR) in the sample of the subject at risk of psychosis.
The inventors discovered surprisingly that the presence of at least one differentially methylated region (DMR) located in a chromosomal region or a gene as defined herein above in the section "CpG methylation" is indicative for said subject being in transition to psychosis.
Accordingly, in one embodiment the presence of at least one DMR located in a chromosomal region, indicates said subject is in transition to psychosis. The at least one DMR, the chromosomal region, the gene and the subject are as defined in the sections 'CpG methylation' and 'subject' herein above.
The inventors showed that the presence of DMRs in regions as defined herein above, in particular chromosomal regions or genes as defined herein above, can be associated with psychotic transition.
More particularly, they demonstrated that detecting the presence of at least one DMR at specific chromosomal locations allows diagnosing that a subject is in transition to psychosis.
According to the invention, a subject diagnosed to be in transition to psychosis has therefore, preferably, at least one DMR located in a chromosomal region or a gene as defined herein above. In vitro method for predicting based on CpG methylations
The inventors discovered that the presence of differentially methylated regions, for example in the region 1 q21 .1 or in the gene GSTM5, and/or in about 100 identified genes that are listed for example in the Table of Figure 9, indicate that a subject is at high risk of a transition to psychosis, also called onset of psychosis.
Accordingly, the invention further refers to a method for predicting onset of psychosis in a subject at risk of psychosis comprising:
a) determining, in a biological sample from the subject which includes nucleic acids, CpG methylations;
b) comparing said CpG methylations determined in step a) with reference CpG methylations;
c) based on the comparison of step b), determining differentially methylated regions (DMR) in the sample of step a), and
d) deducing if the subject is at risk of developing psychosis;
wherein the presence of at least one differentially methylated region (DMR) located in a chromosomal region as defined in the section 'CpG methylations' herein above, indicates that said subject is at risk of developing psychosis.
As used herein, a "method for predicting" or "predicting method" refers to a method for determining whether an individual is likely to develop a disease or illness.
As used herein, the expression "risk of onset" of a disease refers to the probability that a disease will appear in a studied subject, in particular within a given period of time.
The method for predicting onset of psychosis according to the invention comprises a step b) of comparing CpG methylations determined in step a) with reference CpG methylations, wherein said step is as defined in the section "CpG methylation" herein above.
Said method further comprises a step c) of determining, based on the comparison of step b) differentially methylated regions (DMRs) in the sample of step a). The step of determining the DMRs is as further defined in the section 'CpG methylation' herein above.
Preferably, in the methods of the invention, it is further determined whether the DMR refers to the presence or absence of the methylation of at least one CpG contained in said DMR compared to the reference CpG methylation.
Methods of diagnosis and prediction based on gene expression
As mentioned herein above, the inventors further identified 14 genes and 2 gene networks, in particular the genes CPT1 A and NRP1 , which are differentially expressed during psychotic transition and which are thus genetic markers or so-called biomarkers for the transition into psychosis.
Accordingly, the present invention further refers to an in vitro method for diagnosing the transition to psychosis in a subject at risk of psychosis, said method comprising
a) determining the level of expression of at least one gene selected from the group consisting of CPT1 A, NRP1 , CREB3L3, EPB49, EREG, GYPA, HBB, HBD, HSPB7, MUC16, OSBP2, PEG3, TBX1 , TMOD1 , TRIM58, AKT1 , CHL1 , GSTM5 and IL17RE in a biological sample from said subject,
b) comparing said level of expression of the at least one gene determined in step a) to a reference value; and
c) deducing from the comparison of step b) if the subject is in transition to psychosis.
Accordingly the invention further refers to a method for predicting onset of psychosis in a subject at risk of psychosis comprising
a) determining the level of expression of at least one gene selected from the group consisting of CPT1 A, NRP1 , CREB3L3, EPB49, EREG, GYPA, HBB, HBD, HSPB7, MUC16, OSBP2, PEG3, TBX1 , TMOD1 , TRIM58, AKT1 , CHL1 , GSTM5 and IL17RE in a biological sample from said subject,
b) comparing said measured level of expression of the at least one gene to a reference value; and
c) deducing from the comparison of step b) if the subject is at risk of onset of psychosis.
The terms "diagnosis", "method of diagnosis", "method for predicting" and "risk of onset" are the same as defined herein above under the section "Method of diagnosis based on CpG methylations" and "In vitro method for predicting based on CpG methylations".
In particular, the inventors of the present invention identified that the difference of the level of expression of the genes CPT1 A, NRP1 , CREB3L3, EPB49, EREG, GYPA, HBB, HBD, HSPB7, MUC16, OSBP2, PEG3, TBX1 , TMOD1 , TRIM58 and CPT1 A is significantly correlated with the transition into psychosis by RNAsequencing using the edgeR package or the DESeq package, wherein "significantly correlated" herein refers to genes having a difference in expression and having a p-value of <0.05. Furthermore, the gene NRP1 has been identified in the methylomic study of example 1 and has been confirmed by the inventors to be a gene that is differently expressed during the transition into psychosis with a p-value of <0.05. Accordingly, in one embodiment in context of the methods of the invention, in step a) the level of expression of at least one gene selected from the group consisting of CPT1 A, NRP1 , CREB3L3, EPB49, EREG, GYPA, HBB, HBD, HSPB7, MUC16, OSBP2, PEG3, TBX1 , TMOD1 and TRIM58 is determined.
In one further embodiment in context of the methods of the invention, in step a) the level of expression of at least one gene selected from the group consisting of CPT1 A, CREB3L3, EPB49, EREG, GYPA, HBB, HBD, HSPB7, MUC16, OSBP2, PEG3, TBX1 , TMOD1 and TRIM58 is determined.
Furthermore, the inventors further confirmed in a confirmation cohort that differences in the level of expression of the genes CPT1 A and/or NRP1 are significantly correlated with the transition to psychosis.
Therefore, according to a further preferred embodiment, the at least one gene in step a) of the methods of the invention is CPT1 A and/or NRP1 .
Accordingly, in the same preferred embodiment, the level of expression is determined in step a) of the methods of the invention of CPT1 A and/or NRP1 .
"CPU A" also known as "carnitine palmitoyltransferase 1 A" is encoded on chromosome 1 1 , for example, from position 68754620 until 68844410 (reference genome GRCh38.p7), and has two isoforms where the mRNA sequence is available under the NCBI Reference number NM_001031847.2 for transcript variant 2, and under NCBI Reference Number NM 001876 for transcript variant 1 , as accessible on 2 December 2016.
"NRP1 " also known as "Neuropilin 1 " is encoded on chromosome 10, for example, from position 33177491 until 33334905 (reference genome GRCh38.p7), and has six isoforms where the mRNA sequence is available under the NCBI Reference numbers: NM_003873.5 (neuropilin-1 isoform a precursor) NM_001024628.2 (neuropilin-1 isoform b precursor), NM_001024629.2 (neuropilin-1 isoform c precursor), NM_001244972.1 (neuropilin-1 isoform d precursor), N M O 01244973.1 (neuropilin-1 isoform e precursor), NM 001330068.1 (neuropilin-1 isoform f precursor) as accessible on 2 December 2016.
"At least one gene" herein refers to at least one gene, at least two, three, four, five, six, seven, eight genes, more preferably two genes.
It will be therefore understood by the skilled in the art, that according to some embodiments, the level of expression of more than one gene, such as two, three, four, five, six, seven, eight genes selected from the group of genes as defined herein above may be determined in step a) of the methods of the invention.
"Level of expression" or "Expression level" of a gene in context of the methods of the invention refers to the level of the gene product of said gene and thus refers to an amino acid sequence or protein encoded by said gene and/or a nucleic acid transcribed from said gene. The nucleic acid transcribed from said gene refers in particular to a nucleic acid selected from the group consisting of mRNA, long and short non-coding RNA (such as tRNA, rRNA, catalytic RNA, miRNA), in particular mRNA.
In particular, in context of the invention "level of expression" refers to the level of expression of the protein of said at least one gene and/or the level of expression of the mRNA of said at least one gene.
The level of expression refers to the level of expression of all proteins encoded by said at least one gene or all mRNAs encoded by said at least one gene, thus including all protein isoforms and mRNA isoforms encoded by said at least one gene.
As used in context of these methods, the term "determining" includes in particular qualitative and/or quantitative detection (i.e. detecting and/or measuring the level of expression) with or without reference to a control or a predetermined value.
As used herein, "detecting" means determining if the gene product of the at least one gene (mRNA and/or protein), is present or not in a biological sample and "measuring" means determining the amount of the level of expression of said at least one gene in a biological sample.
Determining the level of expression of said at least one gene can be performed by methods which are well known to the person skilled in the art, including in particular immunologic methods, in case of determining the translation products (i.e. proteins) as further described herein below or quantitative methods to determine the quantity of nucleic acids, including reverse transcriptase PCR (RT-PCR), real-time quantitative RT-qPCR, and methods involving the use of DNA arrays (macroarrays or microarrays), RNA sequencing or in situ hybridizations, in particular reverse transcriptase PCR (RT-PCR), such as real-time quantitative RT-qPCR.
In one particular embodiment, the level of expression of said at least one gene is measured by RT-qPCR or RNA sequencing, preferably RT-qPCR.
Methods such as RT-qPCR and/or RNA sequencing are generally known to the skilled in the art.
In one example the level of expression is determined using sequencing.
Accordingly, total RNA can typically be extracted and purified from, typically, blood samples using a standard protocol with, for example, a QIAcube robot and, for example, a PAXgene Blood RNA kit (QIAGEN). Quality and quantity checks can typically be performed by spectrophotometer (BioAnalyzer & kit QUANT IT RNA).
Total RNA can be processed using, typically, the mRNA-Seq Sample Prep Kit
(lllumina). Poly(A) RNA can typically be isolated from the total RNA with a two-step magnetic bead protocol. The resulting mRNA can typically be fragmented in a buffer containing divalent cation at 94 °C for 5 min and purified by ethanol precipitation. The RNA can typically be resuspended in water and a reverse transcription reaction can be performed following manufacturer's instructions.
In one example, then, a paired-end 75-bp sequencing run can be performed, typically, on the lllumina HiSeq 2000. During sequencing, each incorporated base can typically be color coded with a fluorophore. The camera can record the color which base was incorporated. The lllumina software can convert this fluorophore information to sequence data and provide fasta files.
In a further embodiment, the level of expression of said at least one gene, in particular the level of expression of the protein encoded by said at least one gene, can be determined by detecting the translation product(s) using immunologic methods such as detection of the translation product(s) of the at least one gene using polyclonal or monoclonal antibodies. Suitable immunologic methods include immuno-histochemistry (IHC), enzyme linked immunoassays (ELISA), sandwich, direct, indirect, or competitive ELISA assays, enzyme linked immunospotassays (ELIspot), radio immunoassays (RIA), flow-cytometry assays (FACS), Western Blot, fluorescence resonance energy transfer (FRET) assays, protein chip assays using for example antibodies, antibody fragments, receptor ligands or other agents binding the proteins encoded by said at least one gene.
As known by the skilled in the art antibodies directed against the proteins encoded by a gene selected from the group consisting of CPT1 A, NRP1 , CREB3L3, EPB49, EREG, GYPA, HBB, HBD, HSPB7, MUC16, OSBP2, PEG3, TBX1 , TMOD1 , TRIM58, AKT1 , CHL1 , GSTM5 and IL17RE for this purpose are commercially available.
Accordingly, in a one embodiment, the level of expression of said at least one gene is determined using immunohistochemistry or Western Blot.
As mentioned above, in one embodiment, determining the level of expression of said at least one gene further contains normalizing said level of expression as described herein above. It will be understood by the skilled in the art, that the level of expression of said at least one gene as determined in a biological sample in step a) of the methods of the invention and depends on the amount, quality and the representativity of the biological sample used. It will therefore be understood by the skilled in the art that the level of expression of said at least one gene preferably refers to a normalized level of expression.
Accordingly, in one embodiment, the level of expression as referred to in context of the present invention is a normalized level of expression. "Normalization" herein refers to scaling data in such a way that different data sets obtained, for example, for different samples, can be compared. Normalization typically relies on genes whose expression does not change, so-called reference gene(s) or housekeeping gene(s) or on the total amount of gene expression products that were extracted.
Accordingly, in one embodiment, the amount of the level of expression of at least one gene is normalized using the level of expression of at least one housekeeping genes.
"Housekeeping genes" are involved in basic cell maintenance and, therefore, are expected to maintain constant expression levels in all cells and conditions. It will however be understood by the skilled in the art, that the choice of a housekeeping gene to be used as the at least one housekeeping gene, typically depends on the nature of the biological sample to be normalized, i.e if the biological sample is saliva, blood, or tissue. Furthermore, as it will be understood by the skilled in the art, the housekeeping genes used for normalization of, for instance, mRNAs preferably encode mRNAs and not, for example, miRNAs.
In another embodiment the amount of the level of expression of at least one gene, such as the amount of RNA of at least one gene, typically the amount of mRNA of at least one gene is determined and said amount may be normalized using the total amount of extracted RNA, in particular mRNA.
Alternatively, if for example the amount of a protein encoded by at least one gene is determined as the level of expression of at least one gene, said amount may be normalized using the total amount of extracted proteins.
Methods to determine the total amount of proteins, RNAs or mRNA are known to the skilled in the art. In one example, the total amount of protein is determined using BCA protein assay kit (Thermo Fisher Scientific, IL, USA). In another example the total amount of RNA is determined using a spectrophotometer (BioAnalyzer & kit QUANT IT RNA).
In context of the normalization, "at least one housekeeping genes" herein refers to at least one, at least two or three, more preferably three housekeeping genes, most preferably one, two, or three housekeeping genes, in particular three housekeeping genes.
As it will be understood by the skilled in the art when three housekeeping genes are used for normalization, typically the geometrical mean of said three genes is used for normalization. The methods in context of the invention further comprise a step b) of comparing said level of expression of the at least one gene determined in step a) to a reference value.
In context of these methods, the term "reference value" refers to the reference value of said at least one gene selected from the group of genes as defined herein above.
Accordingly, It will be understood by the skilled in the art that if the at least one gene refers to two genes, for example CPT1 A and NRP1 , the reference value refers to the reference value of CPT1 A and to a reference value for NRP1 . Accordingly, for example, in step b), the measured level of expression of CPT1 A is compared with a reference level of CPT1 A and the measured level of expression of NRP1 is compared with a reference level of NRP1 .
In one embodiment, the reference value is a predetermined reference value, a threshold value or a range of values.
The reference value can be any number of statistical measures to distinguish between a level indicative for a subject at risk of psychosis (also referred to as "in transition to psychosis" or "at risk of onset of psychosis") i.e. a reference value obtained in subject(s) who converted to psychosis and a level indicative that a subject is not at risk of converting into psychosis i.e. a reference value obtained in subject(s) who did not convert into psychosis, while including median level of expression, and/or cut-off or threshold expression or fold change values as determined in an subject or a group of subjects.
In a further embodiment, the reference of said at least one gene herein refers to the median level of expression determined in a biological sample obtained from a subject or a population of subjects, said subject(s) being as defined herein above, who converted into psychosis. Accordingly, the reference of said at least one gene may be determined just after conversion into psychosis such as 1 day, 2 days, 3 days, 4 days, 5 days, 6, days, 1 week, 2 weeks after conversion into psychosis.
In a further embodiment, the reference of said at least one gene herein refers to the median level of expression determined in biological samples obtained from a subject or a population of subjects, said subject being as defined herein above, who, for example, did not convert into psychosis.
In one particular embodiment, said reference value is the level of expression of the at least one gene determined in a sample of a healthy subject or a population of healthy subjects. The healthy subject is as defined herein above in the section 'Reference value in context of the methods based on CpG methylations'.
As it will be understood by the skilled in the art, the sample used for determining the reference value is a sample of the same type as the sample used in step a) in context of the methods of the invention, i.e. // the sample of the subject used in step a) is a blood sample, the sample of a healthy subject or a population of healthy subjects used for determining a reference value is preferably as well a blood sample. In context of the present invention, the inventors have determined that over a certain time, for example, CPT1 A and/or NRP1 are downregulated in individuals that convert into psychosis.
Accordingly, in one embodiment the reference value might be a reference value from the same subject taken at an earlier time point, also referred to as a preceding reference value from the same subject.
"Preceding" thus refers in the chronologic order of events to a reference value that has been determined in a sample taken at an earlier time point.
It will be understood by the skilled in the art, that, depending on the reference value used, a level of expression of said at least one gene being higher or lower than the reference value may be indicative for the subject being in transition to psychosis.
Accordingly, in one embodiment a level of expression of said at least one gene as determined in step a) higher or lower than the reference value of said at least one gene is indicative for said subject being in transition to psychosis.
For example, in one particular embodiment, the reference value refers to a reference value obtained in a healthy subject or population or a sample taken at an earlier time point of the same subject and the at least one gene is CPT1 A and/or NRP1 . In this example, a level of expression of CPT1 A and/or NRP1 that is lower than the reference value of CPT1 A and/or NRP1 is indicative for said subject being in transition to psychosis.
Accordingly, in one embodiment, a level of expression of CPT1 A that is lower than the reference value of CPT1 A and/or a level of expression of NRP1 that is lower than the reference value of NRP1 is indicative for said subject being in transition to psychosis.
The term "the level of expression is lower than the reference value" as used herein, means that the level of expression of the said at least one gene as determined in the biological sample is inferior to the reference value, the difference between said values being typically statistically significant.
The term "the level of expression is higher than the reference value" as used herein, means that the level of expression of the said at least one gene as determined in the biological sample is superior to the reference value, the difference between said values being typically statistically significant.
"Statistically significant" typically refers to a p value that is smaller than 0.05. Treatment
The invention also refers to a method of treatment of the transition to psychosis in a subject in need thereof which method comprises
i) determining if a subject is in transition to psychosis according to the in vitro method of diagnosis of the invention, and
ii) administering to said subject being in transition to psychosis a methylation inhibitor, a gene expression modulator, said gene being selected from the group constituted of GSTT1 , GSTP1 , GSTM5, LOC441228, ZNF514, RNF34, COQ2, DOK7, HIST1 H2AM, UNGP3, KIAA1274, LRP2BP, CDH13, TRAF3IP2, PTPRN2, COL9A2, TRIM39, NRP1 , GGA3, CHL1 , SIM1 , TFAP2E, SCRN1 , RIPPLY3, UHRF1 BP1 L, KIA1026, t-RNA-Leu, MIR3683, ATRN, EFNA3, CBS, ZNF282, BAH , NFIX, PPM1 E, CEP152,IL17RE, LZTS2, FBRSL1 , EREG, CRELD1 , KCNIP4, AHSP, ASAM, INPP5A, LRWD1 , COX10, MRC2, CYP2R1 , PAFAH2, FAM187B, FAM123C, HHCA2, COL22A1 , MAL2, PROCA1 , CNIH3, C1 orf106, C22orf45, PDE2A, NUDCD3, ZNF212, CC2D2A, PRPF39, UNCX, LOC100422263, C4orf42, SLC7A5, MNX1 , AN09, FBX018, TCERG1 L, PTCHD3P3, NDUFV3, HTA, OR1 F12, C17orf82, FKBP10, TAP1 , AP2A2, RTF1 , AKT1 , FOXF1 , DENND1 B, KIAA1274, LIN28, PLD3, LOC285205, TOB2, KREMEN2, C16orf78, SDK1 , CYFIP1 , LOC150381 and M6PR, and/or an antipsychotic treatment.
The invention also refers to a method of treatment of psychosis or treatment of the transition in psychosis in a subject in need thereof which method comprises administering said subject with a therapeutically effective amount of a DNA methylation inhibitor and/or a gene expression modulator, said gene being selected from the group as defined herein above.
In the context of the invention, the term "treating" or "treatment", as used herein, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.
The term "treatment of the transition to psychosis" as used herein refers to preventing or delaying the onset of psychosis or the first-episode of psychosis. In one embodiment, the CAARMS score will thus, as a result of the treatment, stay stable. In one embodiment, such treatment can also lead to complete recovery. In one embodiment, the treatment will lead to the reduction of symptoms associated with psychosis. The symptoms associated with psychosis are as define in the section 'psychosis' or 'subject' herein above.
"Treatment of psychosis" herein refers to reversing, alleviating, inhibiting psychosis. In one embodiment, the treatment of the transition to psychosis is chosen from a DNA methylation inhibitor and/or a gene expression modulator, said gene being selected from the group constituted of GSTT1 , GSTP1 , GSTM5, LOC441228, ZNF514, RNF34, COQ2, DOK7, HIST1 H2AM, UNGP3, KIAA1274, LRP2BP, CDH13, TRAF3IP2, PTPRN2, COL9A2, TRIM39, NRP1 , GGA3, CHL1 , SIM1 , TFAP2E, SCRN1 , RIPPLY3, UHRF1 BP1 L, KIA1026, t-RNA-Leu, MIR3683, ATRN, EFNA3, CBS, ZNF282, BAH , NFIX, PPM1 E, CEP152JL17RE, LZTS2, FBRSL1 , EREG, CRELD1 , KCNIP4, AHSP, ASAM, INPP5A, LRWD1 , COX10, MRC2, CYP2R1 , PAFAH2, FAM187B, FAM123C, HHCA2, COL22A1 , MAL2, PROCA1 , CNIH3, C1 orf106, C22orf45, PDE2A, NUDCD3, ZNF212, CC2D2A, PRPF39, UNCX, LOC100422263, C4orf42, SLC7A5, MNX1 , AN09, FBX018, TCERG1 L, PTCHD3P3, NDUFV3, HTA, OR1 F12, C17orf82, FKBP10, TAP1 , AP2A2, RTF1 , AKT1 , FOXF1 , DENND1 B, KIAA1274, LIN28, PLD3, LOC285205, TOB2, KREMEN2, C16orf78, SDK1 , CYFIP1 , LOC150381 and M6PR, and/or an antipsychotic treatment.
A "DNA methylation inhibitor" has the ability to inhibit CpG methylation. In one embodiment, the methylation inhibitor is selected from the group constituted of 5- azacytidine, 5-aza-2'-deoxycytidine, 1 -3-D-arabinofuranosyl-5-azacytosine and dihydro-5- azacytidine.
A "gene expression modulator" refers to a substance that increase or decrease the expression of a specific gene.
In one embodiment, the gene expression modulator is an enhancer of gene expression. In another embodiment, the gene expression modulator is an inhibitor of gene expression.
The inventors have in particular discovered that DMR in the promotor region of GSTM5 is associated with psychosis. The DMR in the promotor region of GSTM5 refers to the absence or presence of CpG methylation in a subject at risk of psychosis.
Therefore in one example, the transition to psychosis may be treated with a GSTM5 expression inhibitor.
In another example, the transition to psychosis may be treated with a GSTM5 expression enhancer.
Accordingly, in one embodiment the gene expression modulator is a GSTM5 expression modulator. In one embodiment a antipsychotic treatment is selected from the group consisting of chlorpromazine, flupenthixol, fluphenazine, haloperidol, loxapine, perphenazine, pimozide, thioridazine, thiothixene, trifluoperazine, zuclopenthixol, clozapine (Clozaril), olanzapine (Zyprexa), quetiapine (Seroquel) and risperidone (Risperdal), preferably haloperidol, chlorpromazine and clozapine.
According to the invention, a "subject in need of" is intended for a subject likely to suffer from transition to psychosis. Said subject was preferably diagnosed as being at risk of psychosis as defined herein above Throughout the instant application, the term "comprising" is to be interpreted as encompassing all specifically mentioned features as well optional, additional, unspecified ones. As used herein, the use of the term "comprising" also discloses the embodiment wherein no features other than the specifically mentioned features are present (i.e. "consisting of").
The invention will now be described in more detail with reference to the following examples. All literature and patent documents cited herein are hereby incorporated by reference. While the invention has been illustrated and described in detail in the the foregoing description, the examples are to be considered illustrative or exemplary and not restrictive.
Figures
Figure 1 depicts a table containing the clinical description of the population used for the study in context of the present invention, (a) is the p value as calculated by Fisher's Test and (b) is the p value as calculated by non parametric Mann-Whitney test. The biological interval represents time between the two blood samples and the clinical follow-up represents the time between inclusion and final status assessments.
Figure 2 is a table summarizing the significant pathways obtained in overrepresentation analysis of the multi-CpG pipeleine. CpG refers to cytidine-phosphate-guanosine.
Figure 3 is a graph representing the mean of methylation in each CpG located in the GSTM5 promoter. The full line represents the non- converter whereas the dashed line represents the converter. A specific CpG might be methylated in one cell of an individual and not methylated in another cell, accordingly in one individual the methylation of a CpG position is an average value being between 0 and 100% methylation. 100% methylation signifies that the specific CpG position is methylated in every cell, i.e. in all nucleic acids tested. The mean of methylation herein refers to the percentage of methylation in the population tested, which in analogy to the above is between 0 and 100%. Figure 4 is a table summarizing the main criteria for UHR and psychosis.
Figure 5 is a table summarizing the information about data clean-up procedure.
Figure 6 is a table summarizing the technical conditions of the PCR as further described in the section 1 .2.2. referring to the pyrosequencing of samples.
Figure 7 is a table summarizing the DMRs identified by Minfi. The table lists the chromosomes and the locations of identified DMRs. Figure 8 is a table summarizing the results of the exploratory analysis in context of the present invention. The table lists the chromosomes and the locations of identified DMR. Fwer refers to a family-wise error rate referring to the probability of making one or more false discoveries, or type I errors when performing multiple hypotheses tests. The P value refers to the nominal significance value of the DMR difference test using a linear model without correction for multiple tests. The Beta value refers to the amplitude of the methylation difference between the two groups for a given DMR. The beta value varies from 0 to 1 and the sign + indicates hypermethylation of the cases (converters) vs controls (non converters) and the sign - hypomethylation of the (converters) vs controls (non converters).
Figure 9 is a table listing the Top 100 genes that were obtained for the test of difference between converters and non-converters in methylation change between baseline and final measure with adjustment for sex. P corresponds to the significance of this test of difference. The physical position indicates the physical position of the detected CpG according to the annotations obtained from the lllumina's Infinium HumanMethylation450 on the 28th of November, 2013.
Figure 10 depicts a table containing the clinical description of the two sample groups used for the study in context of the present invention. Sample 1 refers to samples used for RNAseq and sample 2 includes samples of sample 1 and samples of other subjects and was explored by Q-PCR analyses. Clinical scales include BPRS (Brief Psychiatric Rating Scale) and CGI (Clinical Global Impressions Scale). RIN: RNA Integrity Number. In each sample, converters did not differ from non-converters excepted in clinical score at the end of follow-up (as expected).
Figure 11 depicts a table summarizing the findings from RNAseq and multiplex Q-PCR. RNAseq: RNA sequencing. Q-PCR: quantitative polymerase chain reaction, β-value referred to the level of methylation in specific CpGs.
Example 1 1.1. Patients and Methods: 1.1.1. Population
The present study was approved by the institutional ethics committee "Comite de protection des personnes, lle-de-France III, Paris, France" and written informed consent was obtained from all participants in accordance with the Declaration of Helsinki. Help- seeking individuals (16 to 30 years old) consecutively referred to the Adolescent and Young Adults Assessment Centre (Service Hospitalo-Universitaire, Hopital Sainte-Anne, Paris, France) between 2009 and 2013 were enrolled in the ICAAR collaborative study promoted by Sainte-Anne Hospital as already described (Chaumette B et al. Psychoneuroendocrinology, 2015). Inclusion criteria were alterations in global functioning (Social and Occupational Functioning Assessment Scale score <70) during the past year that were associated with psychiatric symptoms and/or subjective cognitive complaints. Exclusion criteria included manifest symptoms of psychosis (fulfilling DSM-IV criteria), pervasive developmental or bipolar disorders, and individuals with other established diagnoses such as obsessive-compulsive disorder. Other exclusion criteria were: current antipsychotic treatment (> 100 mg Chlorpromazine equivalent) for more than 12 weeks; psychoactive substance dependence or abuse during the previous year and/or >5 years; serious or non-stabilized somatic and neurological disorders; head injury and IQ lower than 70. All subjects were examined with the Comprehensive Assessment for at risk mental state (CAARMS, in its translated version (see Yung AR et al., Aust N Z J Psychiatry 2005; 39: 964-971 and the corresponding French version Krebs M-0 et al. L'Encephale) by specifically trained psychiatrists followed by a consensus meeting for best estimate diagnoses. Individuals fulfilling the criteria for at risk mental state were characterized as UHR; conversion to psychosis was characterized using the CAARMS- defined psychosis onset threshold (i.e. supra-threshold psychotic symptoms— thought content, perceptual abnormalities and/or disorganized speech— present for more than 1 week) was used (see Table in Figure 4). All subjects excepted those above the psychosis threshold at M0 were included in the longitudinal follow-up, whether or not they were UHR. Subjects who reach the psychosis threshold during follow-up were classified as converters. The clinical assessment and blood sample collection were repeated after 6 and 12 months or after psychosis onset. In this study, 39 individuals were included and enrolled in the longitudinal follow-up, among whom 14 subsequently developed full-blown psychosis (converters) while 25 did not (non-converters). There were no significant differences between these two groups at baseline in sex ratio, age, follow-up duration, body mass index, substance abuse or psychotropic treatment introduction (Figure 1 ). Of the 25 non-converters individuals: 13 were UHR and 12 were non-UHR at baseline. Non- UHR individuals had variable sub-threshold symptoms (anxiety and depressive symptoms) without reaching criteria of a fully characterized disorder.
1.1.2 Genome-wide analysis of DNA methylation
1.1.2.1 Using Infinium HumanMethylation450 BeadChip
Preparation: For each individual, genomic DNA (500ng) was extracted from whole blood and treated with sodium bisulfite using the EZ-96DNA Methylation KIT (Catalog No D5004, Zymo Research, USA) following the manufacturer's standard protocol. Methylation was measured at baseline (M0), and after the longitudinal follow-up (MF) by the same technique in the same time for all samples. Genome-wide DNA methylation was assessed using lllumina Infinium HumanMethylation450 BeadChip (lllumina, San Diego, CA, USA), which interrogates the DNA methylation profile of more than 485 000 CpG loci across the genome at single-nucleotide resolution.
Data preprocessing and clean-up: lllumina GenomeStudio software (lllumina, San Diego, CA, USA) was used to extract signal intensities for each probe. All computations and statistical analyses were performed within the R statistical analysis environment (http://www.r-project.org), and all scripts are available on request from the authors. R packages methylumi and wateRmelon were used for data quality check and normalization. Steps used for data clean-up procedure and normalization comprised gender check between phenotype file and methylation data set and evaluation of SNP genotypes concordance between the 2 samples from the same individuals. Subsequent clean-up steps comprised flagging and removing individuals with no result or gender discrepancies or discordant genotypes, samples with≥ 1 % of sites with a detection p- value≥ 0.05, probes with beadcount < 3 in ≥ 5 % of samples, probes with ≥ 1 % of samples with a detection p-value≥ 0.05. Additionally, probes on chromosomes X and Y, SNP probes, probes with a SNP at the CpG site, and non-specific probes that map to more than one location in the genome were removed (Price ME et al. Epigenetics Chromatin, 2013; 6: 4.). The initial methylation data file includes 485 577 probes and after normalization and data clean up, 41 1 947 probes were kept for the final analysis (Figure 5). R Minfi package was used for supplementary quality control (data not shown) and no sample was removed. No batch effect was detected (according to Combat R Package using SVA function). Cellular populations were estimated by EstimateCellCounts function (Minfi package). Association analysis: Global methylation change was investigated by computing the difference between mean methylation changes for all probes in each individual and comparing converters to non-converters. Differentially methylated regions (DMR) were investigated using Minfi package in R (script available upon request). In summary, time and group were used as factors in a linear model adjusted by cellular populations with a paired design. DMR analysis was performed using bumphunter function (bootstrap with 1000 permutations and a methylation differential cut-off of 10). Significance was established for fiver correction < 0,1 .
Multi-CpG analysis: Because methylation changes could occur in different CpG from various genes converging to the same pathways, the inventors of the present invention developed a new pipeline to examine methylation longitudinal changes at each CpG sites. Linear model with moderated t-statistic was used (Diboun I et al. BMC Genomics 2006; 7: 252). Statistical analysis was performed with R script (using R Limma Package) testing the following model: Difference in methylation (MF minus MO) = psychosis (converters vs. non-converters) + gender. The multiple-testing-adjusted significance threshold for probe- wise analysis was established at p=1 .2x10"7 (0.05/41 1 947 analyzed probes) (Data not shown). Top results of this analysis were pasted in ConsensusPathDB (Kamburov A. et al., Nucleic Acids Res 2013; 41 : D793-800.) and Enrichr (Chen EY et al. BMC Bioinformatics 2013; 14: 128) and an over-representation analysis based on Reactome, wikipathways and KEGG database was used with a hypergeometric correction to explore whether the top findings are linked to specific biological pathways more than expected by chance. Clustering analysis of the same top results was performed using the MultiExperimentViewer software (version 4.9.0) (Saeed Al et al. BioTechniques 2003; 34: 374-378.). Methylation differential rates were inputted and normalized by genes. For hierarchical clustering, 'K-Nearest Neighbors imputation engine' (number of neighbors = 10) was used and 'Average linkage clustering' using Pearson correlation and to construct a gene/sample tree.
1.1.2.2. Using pyrosequencinq
Infinium HumanMethylation450 BeadChip is a current and reliable array to detect CpG methylation (Roessler J et al. Inc. BMC Res Notes 2012; 5: 210). However, some of the present findings were further confirmed using a technical reference based on pyrosequencing (Tost J and Gut IG, Nat Protoc 2007; 2: 2265-2275.). After bisulfite conversion by EpiTect Plus Bisulfite Kits (Qiagen) and DNA purification on column, non- methylation specific PCR were achieved using Platinium Taq DNA polymerase kit (invitrogen - Life Technologies). MAEL promoter was used as positive control for the bisulfite treatment, in bisulfited and non-bisulfited samples (Data not shown). Two findings were assessed: One specific CpG selected from the top results of multi-CpGs analysis (CpG located in CHL1 gene) and one significant DMR in GSTM5 (the only one significant DMR including a promoter) identified by minfi package were chosen to be further confirm by pyrosequencing. Primers were designed by PyroMark Assay design Software 2.0 QIAGEN and technical conditions for PCR are shown in Figure 6. Biotinylated primers were used to keep the single DNA strand for pyrosequencing. Pyrosequencing was performed using PyroMark Q24 (Qiagen) according the manufacturer's instructions and data about methylation in each CpG were extracted and analysed using PyroMark Q24 2.0.6.20 software (Qiagen). 1.2. Results
1.2.1 Longitudinal global methylation change in converters vs. non converters
No significant changes in global methylation were associated with the occurrence of conversion to psychosis (p=0.41 ). 1.2.2. Longitudinal methylation changes at specific regions in converters vs. non converters
After paired-analysis, two significant Differentially Methylated Regions (DMRs) (fwer < 0,1 ) including at least 2 CpGs were identified (Figure 7). The region including HLA- DQ and HLA-DRB (chromosome 6 [32523136; 32633163]) was excluded because of frequent recurrence of this finding by minfi package suggesting spurious results due to the algorithm (according to its authors). The two DMRs were identified in chromosome 1 : first region located in [146549909;146550467] corresponding to 1 q21 .1 and second region in [1 10254662; 1 10254835] including the GSTM5 gene promoter. Significant and suggestive results are shown in Figure 7 and 8. These DMRs are quite stable across time, which could suggest that differences in methylation pattern in these regions could predate conversion to psychosis. A transversal exploratory analysis was conducted comparing subjects at M0 and subjects at MF (Figure 8). Fifteen DMRs were concordant before and after conversion but three appeared different across groups after transition only. Interestingly, two of these three DMRs were in 22q1 1 region and are located near GSTT1 and GSTP1 , two genes from the same family as GSTM5.
1.2.3 Longitudinal methylation changes in CpG sites between converters and non converters
It was tested if changes of methylation in different CpG sites located in distinct genes were associated with psychotic transition. Longitudinal methylation changes at specific CpGs associated with conversion to psychosis are shown in a Manhattan plot (Data not shown, see also the top 100 CpGs in Figure 9). None of the individual CpG changes alone reached significance at a genome-wide level. The best associated CpG sites with conversion to psychosis (top 100 CpGs) were kept for biological pathways analysis and revealed two networks implicating eight genes: an axon guidance pathway and the IL-17 signaling pathway (Figure 2).
A cluster analysis of individual data from the top 100 CpGs was performed. Hierarchical clustering of the methylation changes of these top 100 CpGs successfully discriminated between the converters and non-converters (Data not shown). It was examined whether the prescription of psychotropic treatment in 10 individuals (6 converters and 4 non-converters) during follow-up could account for the observed methylomic changes. It was tested whether the same top CpGs sites display significant methylation modifications in patients in whom medication (antipsychotics or valproate) is initiated compared to those who have no treatment changes. Only two DMPs showed a significant difference in methylation change in relation to medication initiation (eg 09270366 located in the inositol-polyphosphate 5-phosphatase gene (nominal p=0.0019) and eg 05768558 located in the Lin-28 homolog A gene (nominal p=0.03)).
1.2.4 Confirmation of significant results by pyrosequencing
Pyrosequencing was performed of CHL1 gene (ch3: 240139) in the 78 samples. Pyrosequencing results were significantly correlated with Meth450K beadchip results (p=0.005; Spearman's rho = 0.32).
Pyrosequencing was also performed of GSTM5. It shows large and significant differences between converters and non-converters regardless of the time of assessment (Figure 3), with converters showing hypermethylation of GSTM5 promoter. Whereas bio- informatical analyses identified a cluster of 6 differentially methylated CpG, pyrosequencing further revealed that 4 additional CpGs located in the promoter, not targeted by the Meth450 beadchip, showed significant methylation change.
Discussion
This study represents the first genome-wide analysis of DNA methylation profiles in individuals during conversion to psychosis and one of its strength is the longitudinal design. The inventors of the present invention observed that conversion to psychosis was not associated with a global change in methylation and there was no individual CpGs significantly associated with psychotic transition, in line with previous findings showing that one individual CpG is rarely associated with one disease. By contrast, it was found that conversion to psychosis was associated with specific methylation changes in genes involved in axon guidance, as well as genes of the Interleukine 17 pathway and the Glutathion-S-transferase family.
Both genome wide and confirmatory experiments suggested that methylation changes, especially in the 1 q21 .1 region and in the promoter of the GSTM5 gene, were associated with psychosis onset. Deletion of 1 q21 .1 region has previously been associated with schizophrenia. This deletion classically encompasses several genes including HYDIN2 associated with macrocephaly and autism, suggesting an alteration of neurodevelopment. GSTM5 is a member of Glutathion-S-transferase family and is implicated in the synthesis of glutathione and protection against oxidative stress, which seems to be part of the pathophysiology of schizophrenia. Oxidative stress has recurrently been suggested to be related to different stages of schizophrenic illness. GSTM5 is selectively expressed in the brain and is the most commonly expressed member of its gene family in this tissue. Its involvement in dopamine metabolism has also been suggested. Moreover, its expression has been shown to be decreased in the prefrontal cortex of patients with schizophrenia. Furthermore, GSTM5 levels displayed an inverse correlation with promoter DNA methylation in brain tissue, supporting the idea that GSTM5 CpG methylation status controls gene expression. Interestingly, the exploratory approach of the herein described study provided evidence that two other genes of GST family might be differentially methylated after conversion to psychosis: the GSTT1 and GSTP1 regions were hypomethylated and hypermethylated in converters, respectively, without differences between the groups during the initial sampling interval. These findings suggest the possibility that conversion to psychosis may depend on the specific control of oxidative metabolism and balance between these genes.
Cluster analysis showed that a subset of top CpGs with the most significant changes in methylation during psychotic conversion correctly classified converters and non-converters, with no influence of medication initiation. Pathway analysis revealed that these top epigenetic changes were over-represented in certain biological pathways, including an axon guidance pathway and the IL-17 pathway. The axon guidance pathway included the neural cell adhesion protein CHL1 gene (cell adhesion molecule L1 -like), which codes for the L1 CAM2 protein. The L1 family encompasses immunoglobulin-class recognition proteins that promote axon growth and migration in developing neurons. In preclinical models, a deficit of CHL1 in adult mice impairs working memory, social behavior and synaptic transmission. Genetic variants in the CHL1 gene have been found to be associated with schizophrenia. NRP1 (neuropilinl ), also included in this pathway, acts as a receptor that mediates axonal inhibition or repulsion. Neuropilinl colocalizes with L1 CAM2 in thalamic axons and in immature neurons; they interact together in growth cone collapse, a process important for developing axons. EFNA3, the third gene found in the analysis, is highly expressed in mature neurons suggesting that an imbalance in expression exists during cerebral maturation between CHL1 , NRP1 and EFNA3. EFNA3 encodes ephrin-A3, which is a critical protein for the regulation of synaptic function and plasticity in astrocytes. The second signaling pathway, namely the IL-17 (interleukin-17) pathway, is involved in the regulation of inflammatory factors and in the immune response to bacterial pathogens. Immune response is a recurrent finding in association studies of schizophrenia. A recent proteomic study identified interleukins as potential diagnostic biomarkers in the onset of psychosis. Differences in the level of several inflammatory cytokine were found in individuals with schizophrenia compared to healthy controls, with a positive correlation between levels of cytokines in the IL-17 pathway and scores on the Positive and Negative Symptoms Scale. This pathway includes AKT1 , a serine-threonine kinase and a critical mediator of growth-factor-induced neuronal survival in the developing nervous system. Decreased AKT1 protein levels and phosphorylation activity were documented in the lymphocytes and brains of individuals with schizophrenia. In addition, it was reported that AKT1 genetic variants were associated with schizophrenia, in relation to cannabis use.
The genome-wide approach, without pre-definite candidate regions, was crucial for identifying new relevant regions that undergo differential methylation or demethylation changes in converters and non-converters across the baseline and follow-up intervals. lllumina Infinium HumanMethylation450 BeadChip interrogates about 485 000 CpG sites after bisulfite conversion of unmethylated cytosines. This design is valuable since it does not require the selection of a small number of 'candidate' genes or methylation sites. Further, these results were compared with a reference method based on pyrosequencing; the correlation was significant between the beadchip and pyrosequencing as reported in the literature (Roessler J. et al. BMC Res Notes 2012; 5: 210). Pyrosequencing identified additional differentially methylated neighboring CpGs in GSTM5 promoter (4 additional CpGs not initially interrogated by the Meth450K array) further strengthening the methylomic results.
The genes identified in this study had not been previously reported in methylomic studies of schizophrenia. These discoveries may be due to the fact that, in addition to methodological differences (notably differences in methylome coverage), an original methodology based on longitudinal variation in methylation levels was employed, which cannot easily be compared to methylation measurements from single time point studies in subjects with established schizophrenia. Moreover, in two of the three published studies, patients were thirty years older on average than the participants of the present study and methylation changes with age.
The present work was conducted in adolescents and young adults consecutively referred to a clinic specialized for early detection of psychosis and enrolled in a longitudinal follow-up program. The inventors of the present invention did not find any differences in environmental exposure between those who converted to psychosis and those who did not, and the methylation changes associated with conversion to psychosis were not related to the initiation of medication. The observed modifications in methylation are thus more likely to be linked to psychosis conversion than to medication initiation or other environmental changes. Even if the sample size were sufficient to identify some significant DMRs, larger samples are needed to identify other DMRs. Another issue to identify DMRs is the molecular and clinical heterogeneity between individuals, a well- known issue in the genetics of psychosis. The sample size could not allow us to overpass this heterogeneity.
The amplitude of methylation changes in DMR was similar to that found in previous studies in peripheral tissues comparing individuals with psychiatric disorders and healthy controls and seems to be biologically relevant (more than 10%). However, the observations in context of the present study suggest that individual methylation levels are relatively stable. The extent to which these findings (which were based on peripheral markers) reflect methylation processes in the brain cannot be definitively concluded. Mounting evidence favors a relative concordance between methylation profiles in brain and blood peripheral cells, although the amplitude of peripheral methylation levels might be lower for the equivalent loci in central tissues. Blood and brain convergence has been investigated by the beadchip suggesting that subset of peripheral data may proxy methylation status of brain tissue. Within-subjects design, as it was performed here, are recommended.
The present study has several strengths: A long-term prospective follow-up was conducted in both individuals at UHR for psychosis and non-UHR subjects. Rapidly frozen samples were used enabling the study of a larger number of methylation sites (even more labile ones). The inventors of the present invention report longitudinal variations in methylation, which are more suitable for reflecting dynamic epigenetic processes compared to single time point analyses. A genome-wide strategy was herein used rather than limited candidate genes strategy. A newly-developed pathway and clustering analyses were used to investigate the functional relevance of top CpG methylation sites. Several issues need to be addressed in future studies, however, including the problem of clinical heterogeneity and the possible influence of a larger number of environmental factors (e.g. early stressful events). It will also be important to make direct measures of maturational changes (e.g. using brain imaging) and to examine interactions between CpGs methylation and other mechanisms of epigenetic regulation. Finally, inter-individual heterogeneity raises a yet-to-be-investigated hypothesis that private epimutations might be involved in the conversion to psychosis.
In conclusion, the inventors of the present invention found that the conversion to psychosis in young help seekers is accompanied by epigenetic changes in genes involved in relevant genes and pathways. Possible candidate mechanisms were also identified, including alterations in oxidative stress regulation, axon guidance and in inflammatory pathways. These candidate genes could represent multiple theaters for the disruption in homeostasis that accompanies the emergence of full-blown psychosis. At this point, it is unknown whether the observed methylation changes play a causal role in the processes leading to psychosis, or whether they are simply reflective of psychosis onset. These new observations shed light on the biological processes underlying the interaction between early vulnerability and late environmental response and maturational processes at adolescence that precipitate some UHR individuals into full-blown psychosis, and they give fresh perspectives for early intervention and prevention in psychosis. Future developments could investigate the functional impact of these methylation changes. Altogether, this study is a first step towards the identification of the epigenetic signature of conversion to psychosis.
Example 2 2.1 Population
Subjects were selected from the French ICAAR cohort (PHRC AOM-07-1 18, promoted by Hopital Sainte-Anne). This cohort was approved by the institutional ethics committee "Comite de protection des personnes, lle-de-France III, Paris, France" and written informed consent was obtained from all participants in accordance with the Declaration of Helsinki (and their parents for individual under 18 years old). Help-seeking individuals (16 to 30 years old) consecutively referred to the Adolescent and Young Adults Assessment Centre (Service Hospitalo-Universitaire, Hopital Sainte-Anne, Paris, France) between 2009 and 2013 were enrolled in the ICAAR collaborative study (see description of this cohort in Chaumette et al). Inclusion criteria were alterations in global functioning (Social and Occupational Functioning Assessment Scale score <70) during the past year that were associated with psychiatric symptoms and/or subjective cognitive complaints. Exclusion criteria included manifest symptoms of psychosis (fulfilling DSM-IV criteria), pervasive developmental or bipolar disorders, and individuals with other established diagnoses such as obsessive-compulsive disorder. Other exclusion criteria were: current antipsychotic treatment (> 100 mg Chlorpromazine equivalent) for more than 12 weeks; psychoactive substance dependence or abuse during the previous year and/or >5 years; serious or non-stabilized somatic and neurological disorders; head injury and IQ lower than 70. All subjects were examined with the Comprehensive Assessment for at risk mental state, CAARMS ((see Yung AR et al., Aust N Z J Psychiatry 2005; 39: 964-971 and the corresponding French version Krebs M-0 et al. L'Encephale)) by specifically trained psychiatrists followed by a consensus meeting for best estimate diagnoses. Help- seeking individuals were distinguished between those not reaching the criteria for UHR (non-at-risk individuals- N-UHR, corresponding to clinical controls usually experiencing variable sub-threshold anxiety or depressive symptoms without reaching criteria of any characterized disorder, n=92), those reaching the CAARMS' criteria for at risk mental state (ultra-high risk individuals, UHR, n=170) and individuals reaching the threshold for psychosis (P, n=44). Individuals underwent clinical assessment and blood samples at initial time (MO) and after the follow-up (MF). The duration of follow-up was to 1 year or less in case of psychotic transition. The psychotic transition was characterized using the CAARMS-defined psychosis onset threshold (i.e. supra-threshold psychotic symptoms— thought content, perceptual abnormalities and/or disorganized speech— present for more than 1 week) was used (Data not shown).
2.2 RNA sequencing
2.2.1 RNA extraction, library and sequencing
Total RNA was extracted and purified from blood samples (PAXgene tubes) using a standard protocol with a QIAcube robot and PAXgene Blood RNA kit (QIAGEN). Quality and quantity checks were performed by spectrophotometer (BioAnalyzer & kit QUANT IT RNA - Data not shown). Mean concentration was 59.4 ng/mL and mean of RNA Integrity Number (RIN) was 8.4 without significant difference across groups.
Total RNA was processed using the mRNA-Seq Sample Prep Kit (lllumina). Poly(A) RNA was isolated from the total RNA with a two-step magnetic bead protocol. The resulting mRNA was fragmented in a buffer containing divalent cation at 94 °C for 5 min and purified by ethanol precipitation. The RNA was resuspended in water and a reverse transcription reaction was performed following manufacturer's instructions.
A paired-end 75-bp sequencing run was performed on the lllumina HiSeq 2000 with more than 80 millions of reads per sample. During sequencing, each incorporated base is color coded with a fluorophore. The camera recorded the color which base was incorporated. The lllumina software converts this fluorophore information to sequence data and provides fasta files.
2.2.2. RNAseq analysis
Quality control was performed using FastQC software (Data not shown). As bio-informatic softwares are various, in parallel two pipelines using reads counts approaches were conducted. Using reads counts is the only available method allowing complex design like interaction between time and groups. The data were analyzed in the R statistical language using edgeR and DESeq packages according to the recommendations (Anders et al.) with a sum of number of counts >=3. Results were taken into consideration if the two pipelines ranged them among the top results (p<0.05).
2.2.3 Co-Expression Network Analysis
The co-expression network analysis was conducted using the WGCNA package in R (Langfelder et al). After automatic, one-step network construction and module detection based on MO data, conservation of networks was tested after psychotic transition in MF data.
2. 3 Q-PCR validation
For confirmation in the whole cohort, total RNA was extracted from PAXgene using the same protocol as for RNA sequencing. Quality control was done using LabChip GX (Perkin Elmer, Waltham USA). Complementary DNA (cDNA) synthesis was performed using Reverse Transcription Master Mix from Fluidigm® according to the manufacturer's protocol with random primers in a final volume of 5 μΙ_ containing 100 ng total RNA using a Nexus thermocycler (Eppendorf). cDNA samples were diluted by adding 20 μΙ_ of low TE buffer [10 mM Tris ; 0.1 mM EDTA ; pH = 8.0 (TEKNOVA)] and stored at -20°C. For specific target pre-amplification 1 .25 μΙ_ of each diluted cDNA was used for multiplex pre- amplification with Fluidigm® PreAmp Master Mix at 12 cycles. In a total volume of 5 μΙ_ the reaction contained 1 μΙ_ of pre-amplification mastermix, 1 .25 μΙ_ of cDNA, 1 .25 μΙ_ of pooled TaqMan® Gene Expression assays (Life Technologies, ThermoFisher) with a final concentration of each assay of 180 nM (0.2X) and 1 μί of PCR water. The cDNA samples were subjected to pre-amplification following the temperature protocol: 95°C for 2 min, followed by 12 cycles at 95°C for 15 s and 60°C for 4 min. The pre-amplified cDNA were diluted 5X by adding 20 μΙ_ of low TE buffer (TEKNOVA) and stored at -20°C before qPCR. High-throughput real time PCR was performed on the qPCR-HD-Genomic Paris Centre platform and was supported by grants from Region lle-de-France, using the high- throughput platform BioMark™ HD System and the 96.96 GE Dynamic Arrays (Fluidigm). 6 μΙ_ of sample master mix (SMM) consisted of 1 .8 μΙ_ of 5X diluted pre-amplified cDNA, 0.3 μΙ_ of 20X GE Sample Loading Reagent (Fluidigm) and 3 μί of TaqMan® Gene Expression PCR Master Mix (Life Technologies, ThermoFisher). Each 6 μί assay master mix (AMM) consisted of 3 μί of TaqMan® Gene Expression assay 20X (Life Technologies) and 3 μί of 2X Assay Loading Reagent (Fluidigm). 5 μί of each SMM and each AMM premixes were added to the dedicated wells. The samples and assays were mixed inside the chip using HX IFC controller (Fluidigm). Thermal conditions for qPCR were: 25°C for 30 min and 70°C for 60 min for thermal mix ; 50°C for 2 min and 95°C for 10 min for hot start ; 40 cycles at 95°C for 15 s and 60°C for 1 min. Data were processed by automatic threshold for each assay, with linear derivative baseline correction using BioMark Real-Time PCR Analysis Software 4.0.1 (Fluidigm). The quality threshold was set at the default setting of 0.65. Normalization was done using the GAPDH rate. Livak normalization provided the expression level in each sample with a transformation by the 2ΔΔΟΊ method (Schmittgen & Livak). Scripts are available in GitHub (https://github.com/jpouch/qPCR-Biomark). Genes expressed in less than 10 individuals were not further analyzed. Statistical analyses were done using R and SPSS Statistics 20 (IBM): non-parametric Wilcoxon test were performed.
2.4. Results
In example 1 of the present patent application the inventors reported longitudinal epigenetic changes in DNA methylation during psychotic transition, suggesting alterations in gene expression contemporary to psychosis onset. Accordingly, it was hypothesised that psychotic transition is accompanied with specific changes in gene (or gene network) expression. In a cohort of 306 help-seekers individuals, the inventors were able to prospectively explore clinical evolution along with biological changes. One third of these individuals achieved the one-year follow-up, allowing intra-individual analyses in which each individual served as his/her own control, and among these, 41 % converted to psychosis.
First, an exploratory data-driven analysis was conducted using RNA sequencing of blood samples from 15 individuals: 12 converters vs. 3 clinical controls (comparable help- seeking individuals not reaching criteria for UHR). Description of the samples is provided in Figure 10. Identification of longitudinal changes in gene expression was performed with DESeq6 and edgeR7 packages (Data not shown). Only significant and concordant results were retained (p-value in each package < 0.05 or genes expressed in only one condition) resulting in 14 genes with differential longitudinal changes in their expression between the two groups (Table of Figure 1 1 ). No differences in expressed gene isoforms or alternative gene promoters were detected. Co-expression network analysis was performed using WGCNA package: correlations between gene expressions were used to construct networks that were then compared between baseline and follow-up assessments. During psychotic transition, two co-expression networks were less preserved than others indicating that they could be more specifically altered during psychotic conversion (Data not shown). The first network included genes from the Wnt pathway including Akt1 , CPT1 A and semaphorins. This biological network was recently shown to be decreased in subjects with schizophrenia compared to controls in the peripheral blood mononuclear cells transcriptome. The second group corresponded to genes involved in the signaling cascade of Toll-like receptors. Toll-like receptors could account for innate immunity abnormalities in schizophrenia and mice deficient in Toll-like receptor show 'schizophrenic-like' features .
In the following confirmatory study using quantitative-PCR (Q-PCR), converters were compared to 63 non-converters. Description of the sample is shown in the table of Figure 10 (Sample 2). Duration of follow-up was statistically significant because non- converters were followed longer than converters to assess the absence of transition. Converters were sampled just after the transition. Fourteen genes were selected from our RNA-seq analysis and five from our methylomic data obtained previously in the same cohort (n=39) 2. Q-PCR identified CPT1 A (carnitine palmitoyltransferase 1 A) and NRP1 two differentially expressed genes (Wilcoxon p-value < 0.05; Table of Figure 1 1 ). CPT1 A is a key gene allowing an essential step in the beta-oxidation of long chain fatty acids in the mitochondria. This gene family has been implicated in schizophrenia. In our study, CPT1 A expression significantly increased during follow-up in controls (n=63), whereas its expression decreased during psychotic transition in converters (n=31 ). Interestingly, the expression of CPT1 A could be related to (ω-3) polyunsaturated fatty acids intake, which have been proposed to prevent psychotic transition 14, 15. NRP1 is part of the Semaphorin Receptor Complex implicated in neuronal development and axon guidance. Its peripheral expression is decreased during the psychotic transition as predicted by hypermethylation of CpG located in its promoter. NRP1 interacts with CHL1 , whose expression was modified during psychotic transition but did not reach significance (p=0.187; table of Figure 1 1 ). Post-hoc analyses comparing the level of expression for each gene analyzed in medicated and unmedicated subjects found no difference, suggesting that the results reported here are not related to antipsychotic medication.
Our study is the first to explore the dynamic of expression changes in peripheral blood samples during onset of psychosis. In peripheral sample from patients with first- episode of psychosis (FEP), just after the psychotic transition, CPT1 A was reported hypoexpressed compared to controls 16, (p=7.86E-03; fold change=-1 .1 1 ), in the same way as in our present cohort.
Because of the tissue specificity of gene expression, modifications seen in peripheral samples only imperfectly reflect changes in brain structures. Nevertheless, a direct comparison of gene expressions in different tissues showed that a large overlap exists between blood and many part of the brain in particular with prefrontal cortex17. This correlation was more significant in network in relation with immune response, metabolism of fatty-acid and axonogenesis, reinforcing our corresponding findings. On the contrary, Wnt pathway was not significantly correlated between blood and brain.
Concordance between methylomic and transcriptomic data was limited to 5% of overlap. This is not surprising given that the dysregulation of gene expression could be largely modulated by other epigenetic mechanisms like post-traductional histones modifications or miRNA regulation, not investigated in our study. Moreover, the methylome seemed less dynamic than the transcriptome: methylomic changes could occur several months before the transition whereas transcriptomic analysis reflects more rapid changes.
In summary, this first transcriptomic study suggested that the psychotic transition is associated with differential expression of the CPT1 A and NRP1 genes, and in two gene networks relevant for psychosis and involving the Wnt and Toll-like receptors pathways. These results open new perspectives towards the identification of peripheral staging biomarkers, new phase-specific therapeutic drugs and the prevention of psychosis.

Claims

1 . An in vitro method for diagnosing the transition to psychosis in a subject at risk of psychosis, said method comprising
a) determining the level of expression of at least one gene selected from the group consisting of CPT1 A, NRP1 , CREB3L3, EPB49, EREG, GYPA, HBB, HBD, HSPB7, MUC16, OSBP2, PEG3, TBX1 , TMOD1 , TRIM58, AKT1 , CHL1 , GSTM5 and IL17RE in a biological sample from said subject,
b) comparing said level of expression of the at least one gene determined in step a) to a reference value; and
c) deducing from the comparison of step b) if the subject is in transition to psychosis.
2. The method according to claim 1 , wherein the at least one gene in step a) is CPT1 A or NRP1 .
3. The method according to claim 1 , wherein the level of expression of at least one gene selected from the group consisting of CPT1 A, CREB3L3, EPB49, EREG, GYPA, HBB, HBD, HSPB7, MUC16, OSBP2, PEG3, TBX1 , TMOD1 and TRIM58 is determined.
4. The in vitro method according to any one of claims 1 to 3, wherein said reference value is the expression of the at least one gene determined in the sample of a healthy subject or a population of healthy subjects.
5. An in vitro method for diagnosing the transition to psychosis in a subject at risk of psychosis, comprising
a) determining, in a biological sample from the subject which includes nucleic acids, CpG methylations;
b) comparing said CpG methylations determined in step a) with reference CpG methylations;
c) based on the comparison of step b), determining differentially methylated regions (DMR) in the sample of step a), and
d) deducing if the subject is in transition to psychosis;
wherein the presence of at least one differentially methylated region (DMR) located in a chromosomal region selected from the group consisting of the chromosomal regions:
- starting at position 146549909 and ending at position 146550467 of chromosome 1 ,
- starting at position 1 10254662 and ending at position 1 10254835 of chromosome 1 , - starting at position 1594282 and ending at position 1594733 of chromosome 5,
- starting at position 195489306 and ending at position 195489782 of chromosome 3,
- starting at position 1856713 and ending at position 1857477 of chromosome 5,
- starting at position 325915 and ending at position 325964 of chromosome 1 1 ,
- starting at position 17599784 and ending at position 17600122 of chromosome 19,
- starting at position 121496875 and ending at position 121497334 of chromosome 2,
- starting at position 176797999 and ending at position 176798049 of chromosome 5, and
- starting at position 724273 and ending at position 724374 of chromosome 17,
is indicative for said subject being in transition to psychosis.
6. The in vitro method according to claim 5, wherein the at least one differentially methylated region (DMR) is located in the chromosomal regions selected from the group consisting of the chromosomal regions:
- starting at position 146549909 and ending at position 146550467 of chromosome 1 , and - starting at position 1 10254662 and ending at position 1 10254835 of chromosome 1 .
7. The in vitro method according to claim 5 or 6, wherein said differentially methylated region (DMR) is located in a gene, wherein said gene is selected from the group consisting of GSTT1 , GSTP1 , GSTM5, LOC441228, ZNF514, RNF34, COQ2, DOK7, HIST1 H2AM, UNGP3, KIAA1274, LRP2BP, CDH13, TRAF3IP2, PTPRN2, COL9A2, TRIM39, NRP1 , GGA3, CHL1 , SIM1 , TFAP2E, SCRN1 , RIPPLY3, UHRF1 BP1 L, KIA1026, t-RNA-Leu, MIR3683, ATRN, EFNA3, CBS, ZNF282, BAH , NFIX, PPM1 E, CEP152JL17RE, LZTS2, FBRSL1 , EREG, CRELD1 , KCNIP4, AHSP, ASAM, INPP5A, LRWD1 , COX10, MRC2, CYP2R1 , PAFAH2, FAM187B, FAM123C, HHCA2, COL22A1 , MAL2, PROCA1 , CNIH3, C1 orf106, C22orf45, PDE2A, NUDCD3, ZNF212, CC2D2A, PRPF39, UNCX, LOC100422263, C4orf42, SLC7A5, MNX1 , AN09, FBX018, TCERG1 L, PTCHD3P3, NDUFV3, HTA, OR1 F12, C17orf82, FKBP10, TAP1 , AP2A2, RTF1 , AKT1 , FOXF1 , DENND1 B, KIAA1274, LIN28, PLD3, LOC285205, TOB2, KREMEN2, C16orf78, SDK1 , CYFIP1 , LOC150381 and M6PR, preferably GSTM5.
8. The in vitro method according to any one of claims 5 to 7, wherein said reference CpG methylations are the CpG methylations determined in the sample of a healthy subject or a population of healthy subjects.
9. The in vitro method according to any one of claims 1 to 8, wherein the subject does not display any of the following - manifest symptoms of psychosis,
- pervasive developmental disorder,
- bipolar disorder,
- obsessive compulsive disorder.
10. The in vitro method according to any one of claims 1 to 9, wherein the subject is not under antipsychotic treatment for more than 12 weeks.
1 1 . The in vitro method according to any one of claims 1 to 10, wherein the age of the subject is 16 to 30.
12. The method according to any one of claims 1 to 4, said method further comprising the steps of:
i) determining, in a biological sample from the subject which includes nucleic acids, CpG methylations;
ii) comparing said CpG methylations determined in step i) with reference CpG methylations;
iii) based on the comparison of step ii), determining differentially methylated regions (DMR) in the sample of step i), and
deducing in step c) from the comparison of step b) and the comparison of step iii) if the subject is in transition to psychosis or at risk of onset of psychosis,
wherein the presence of at least one differentially methylated region (DMR) located in a chromosomal region selected from the group consisting of the chromosomal regions:
- starting at position 146549909 and ending at position 146550467 of chromosome 1 , - starting at position 1 10254662 and ending at position 1 10254835 of chromosome 1 ,
- starting at position 1594282 and ending at position 1594733 of chromosome 5,
- starting at position 195489306 and ending at position 195489782 of chromosome 3,
- starting at position 1856713 and ending at position 1857477 of chromosome 5,
- starting at position 325915 and ending at position 325964 of chromosome 1 1 ,
- starting at position 17599784 and ending at position 17600122 of chromosome 19,
- starting at position 121496875 and ending at position 121497334 of chromosome 2,
- starting at position 176797999 and ending at position 176798049 of chromosome 5, and
- starting at position 724273 and ending at position 724374 of chromosome 17, is indicative for said subject being in transition to psychosis or at risk of onset of psychosis.
13. A method for predicting onset of psychosis in a subject at risk of psychosis comprising a) determining, in a biological sample from the subject which includes nucleic acids, CpG methylations;
b) comparing said CpG methylations determined in step a) with reference CpG methylations;
c) based on the comparison of step b), determining differentially methylated regions (DMR) in the sample of step a), and
d) deducing if the subject is at risk of developing psychosis;
wherein the presence of at least one differentially methylated region (DMR) located in a chromosomal region selected from the group consisting of the chromosomal regions:
- starting at position 146549909 and ending at position 146550467 of chromosome 1 ,
- starting at position 1 10254662 and ending at position 1 10254835 of chromosome 1 ,
- starting at position 1594282 and ending at position 1594733 of chromosome 5,
- starting at position 195489306 and ending at position 195489782 of chromosome 3, - starting at position 1856713 and ending at position 1857477 of chromosome 5,
- starting at position 325915 and ending at position 325964 of chromosome 1 1 ,
- starting at position 17599784 and ending at position 17600122 of chromosome 19,
- starting at position 121496875 and ending at position 121497334 of chromosome 2,
- starting at position 176797999 and ending at position 176798049 of chromosome 5, and - starting at position 724273 and ending at position 724374 of chromosome 17,
indicates that said subject is at risk of onset of psychosis.
14. Use of at least one differentially methylated region (DMR) located in a chromosomal region selected from the group consisting of the chromosomal regions:
- starting at position 146549909 and ending at position 146550467 of chromosome 1 ,
- starting at position 1 10254662 and ending at position 1 10254835 of chromosome 1 ,
- starting at position 1594282 and ending at position 1594733 of chromosome 5,
- starting at position 195489306 and ending at position 195489782 of chromosome 3,
- starting at position 1856713 and ending at position 1857477 of chromosome 5,
- starting at position 325915 and ending at position 325964 of chromosome 1 1 ,
- starting at position 17599784 and ending at position 17600122 of chromosome 19,
- starting at position 121496875 and ending at position 121497334 of chromosome 2,
- starting at position 176797999 and ending at position 176798049 of chromosome 5, and
- starting at position 724273 and ending at position 724374 of chromosome 17, for diagnosing transition to psychosis in a subject at risk of psychosis.
15. A DNA methylation inhibitor, an enhancer of gene expression and/or an inhibitor of gene expression, said gene being selected from the group consisting of GSTT1, GSTP1, GSTM5, LOC441228, ZNF514, RNF34, COQ2, DOK7, HIST1 H2AM, UNGP3 (also called STON2), KIAA1274, LRP2BP, CDH13, TRAF3IP2, PTPRN2, COL9A2, TRIM39, NRP1, GGA3, CHL1, SIM1, TFAP2E, SCRN1, RIPPLY3 (also called MRPL20P1), UHRF1BP1L, KIA1026, T-RNA-Leu, MIR3683 (also called LOC100121257), ATRN, EFNA3, CBS, ZNF282, BAH , NFIX, PPM1E, CEP152,IL17RE, LZTS2, FBRSL1, EREG, CRELD1, KCNIP4, AHSP, ASAM, INPP5A, LRWD1, COX10, MRC2, CYP2R1, PAFAH2, FAM187B (also called LOC100421621), FAM123C, HHCA2 (also called CTSD), COL22A1, MAL2, PROCA1, CNIH3, C1 orf 106, C22orf45 (also called UPB1), PDE2A, NUDCD3, ZNF212, CC2D2A, PRPF39, UNCX, LOC100422263, C4orf42, SLC7A5, MNX1, AN09, FBX018, TCERG1L, PTCHD3P3 (alos called CCDC162P), NDUFV3, HTA, OR1F12 (also called ZNF165), C17orf82, FKBP10, TAP1, AP2A2, RTF1 (also called ITPKA), AKT1, FOXF1, DENND1B, KIAA1274, LIN28, PLD3 (also called C19orf47), LOC285205, TOB2, KREMEN2, C16orf78, SDK1, CYFIP1, LOC150381 (also called LOC642648) and M6PR, for use in the treatment of psychosis or transition to psychosis.
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