Patent Description:
Chronic postsurgical pain (CPSP) is often defined as pain that lasts beyond three months post- surgery, in the absence of other preexisting problems or postoperative complications (<NPL>). In children, the median prevalence of CPSP is <NUM>% (<NPL>), however the incidence ranges from <NUM>-<NUM>% after spine fusion (<NPL>;<NPL>; <NPL>) a painful surgery that adolescents undergo. CPSP is a classic example of gene-environment interaction, and involves multiple peripheral and central signaling and modulatory pathways regulated by genes (<NPL>). It has a heritable risk of <NUM>%, (<NPL>) and genetic factors explain some of the individual differences in pain perception (<NPL>; <NPL>). However, a genetic basis for CPSP has been elusive (<NPL>) attributed partly to lack of replicability (<NPL>) and inconsistent findings (<NPL>) in genetic association studies (<NPL>;<NPL>) and lack of consideration of gene-environmental interactions. Especially in children, caregiving environment and psychological factors like anxiety, prime children's pain responses, influence the 'epigenetic landscape' and influence his or her response to further surgical stress (<NPL>; <NPL>). Twin studies have shown that environmental factors are involved in the inter-personal differences in pain sensitivity (<NPL>). Elucidating gene-environmental influences through epigenetics is expected to explain critical gaps in predisposition and mechanisms involved in CPSP (<NPL>; <NPL>).

DNA methylation via addition of a methyl group to the <NUM>' position of a cytosine - guanine residue (CpG dinucleotide) is a common epigenetic mechanism associated with decreased transcriptional activity, and altered expression of nociceptive genes. It affects pain processing and the transition from acute to chronic pain (<NPL>). We recently identified psychological, perioperative and µ-opioid receptor gene (OPRM1) DNA methylation markers as predictors of acute and chronic postsurgical pain in adolescents undergoing spine fusion surgery. The OPRM1 DNA methylation levels have been found to be elevated in opioid and heroin addicts (<NPL>). <CIT>) developed a DNA methylation-based test for detecting and monitoring methylation states of biomarker genes which differ in the diseased compared to the non-diseased state (e.g., multiple sclerosis and breast cancer). However, effect sizes of single CpG sites are small, and sometimes identify associations that cannot be replicated. Hence, in this study, we use epigenome-wide association studies (EWAS) and a global bioinformatics-based approach to identify pathways, histone marks, and protein-DNA binding events enriched in DNA methylation differences associated with CPSP and anxiety. This approach integrates epigenetic-level data with biologic processes, pathways, and networks, and overcomes pitfalls of hypothesis-driven candidate marker association studies (<NPL>). EWAS also allows novel candidate discovery, and have previously been used to study epigenetics of other conditions (e.g. panic disorder) (<NPL>) but not CPSP. We will test the hypothesis that shared biological processes enriched in DNA methylation will be associated with CPSP and anxiety, which will suggest new avenues for preventing and treating CPSP.

<NPL> teaches themethylation of the OPRMI gene as a predicator of acute and chronic postsurgical pain.

Therefore, there is a need to identify clinical markers for predicting a patient's susceptibility to CPSP in order to provide improved management of pain in the clinical setting.

The invention is defined by the appendant claims.

The present invention is based, in part, on the discovery that methylation status in genes of certain molecular signaling pathways, especially those of the GABA receptor and Dopamine-DARPP32 Feedback in cAMP (hereinafter referred to simply as "dopamine-DARPP-<NUM>") signaling pathways, can be used as biomarkers for susceptibility to perioperative pain, and particularly CPSP. In addition, the disclosure provides certain cytokines whose expression in peripheral blood can also be used as a biomarker for susceptibility to pain, and for risk of developing CPSP. Cytokines whose protein expression is associated with CPSP include tumor necrosis factor alpha (TNFα) and interleukin-1RA (IL1RA) as well as fractalkine, epidermal growth factor (EGF), FMS-like tyrosine kinase <NUM> ligand (FLT-<NUM>), macrophage derived chemokine (MDC), interleukin-<NUM> (IL-<NUM>), interleukin-<NUM> (IL-<NUM>), and interleukin-<NUM> (IL-<NUM>). Accordingly, the disclosure provides but does not claim methods for pain management in the perioperative context, particularly through methods comprising assaying the DNA methylation status of certain genes and/or the expression of certain cytokines in peripheral blood, in order to identify a patient as susceptible to perioperative pain. The disclosure also provides but does not claim methods for treating a patient identified as susceptible to perioperative pain or one who is at risk of developing CPSP, for example by administering demethylating agent or an inhibitor of the repressor element-<NUM> silencing transcription factor (REST), or by administering an anti-inflammatory agent where the patient presents with elevated levels of one or more of the cytokine biomarkers described herein.

In embodiments, the disclosure provides a method for the prophylaxis or treatment of perioperative pain in a patient in need thereof, the method comprising assaying, in vitro, a biological sample from the patient to determine the DNA methylation status of at least one CpG site in one or more of the human GABA-receptor and dopamine-DARPP-<NUM> signaling pathway genes. In embodiments, the disclosure provides a method for identifying a patient who is susceptible to perioperative pain, the method comprising assaying, in vitro, a biological sample from the patient to determine the DNA methylation status of at least one CpG site in one or more of the human GABA-receptor and dopamine-DARPP-<NUM> signaling pathway genes. In accordance with embodiments of the methods described here, the step of assaying a biological sample from the patient to determine the DNA methylation status of at least one CpG site in the GABA-receptor gene or in the dopamine-DARPP-<NUM> signaling pathway includes detecting one or more <NUM>-methylcytosine nucleotides in genomic DNA obtained from the sample. In embodiments, the step of assaying may further include one or more of isolating genomic DNA from the biological sample, treating the genomic DNA with bisulfite, and subjecting the genomic DNA to a polymerase chain reaction (DNA).

In embodiments, the perioperative pain is selected from preoperative pain, acute postoperative The invention concerns perioperative pain is chronic postoperative pain.

In embodiments, the at least one CpG site in the GABA-receptor gene or in the dopamine-DARPP-<NUM> signaling pathway is identified in Tables 6A-6C.

In embodiments, the biological sample is a blood sample. For assaying DNA methylation, the blood sample is preferably a sample of whole blood, or one containing blood cells such as leukocytes and erythrocytes. In embodiments where the assay is for the expression of one or more cytokines in peripheral blood, the biological sample is preferably a serum sample.

In embodiments, a patient having a DNA methylation status of 'methylated' at the at least one CpG site is identified as a patient susceptible to perioperative pain or CPSP. In embodiments, a patient having a DNA methylation status of 'methylated' at the at least one CpG site is identified as a patient susceptible to CPSP or anxiety (as measured by the Childhood Anxiety Sensitivity Index (CASI). In embodiments, the patient identified as susceptible is administered a therapeutic agent selected from a demethylating agent and an inhibitor of the repressor element-<NUM> silencing transcription factor (REST). In embodiments the agent is administered before or after a surgical procedure is performed on the patient. In embodiments, the demethylating agent is selected from procaine, zebularine and decitabine, or a combination of two or more of the foregoing. In embodiments, the demethylating agent is zebularine, decitabine, or a combination of two or more of the foregoing.

In embodiments, the biological sample is assayed by a method comprising isolation of genomic DNA from the biological sample, for example a sample of whole blood or serum. In embodiments, the biological sample is assayed by a method comprising, or further comprising, pyrosequencing. In embodiments, the pyrosequencing comprises two or more rounds of a polymerase chain reaction.

In embodiments, the patient is a female patient.

In embodiments, the patient is self-reported Caucasian or white.

In embodiments, the disclosure provides but does not claim a kit comprising a set of recombinant enzymes including one or more of DNA polymerase, ATP sulfurylase, luciferase, and apyrase, two substrates selected from one or both of adenosine <NUM>' phosphosulfate (APS) and luciferin, at least one detectably labeled oligonucleotide primer designed to amplify in a polymerase chain reaction a DNA segment corresponding to at least one of the CpG sites defined in Table <NUM> and a methylated DNA polynucleotide of known sequence, as a positive control.

The present disclosure is based, in part, on associations between epigenetic modifications in the genomic DNA of certain genes and preoperative pain, acute postoperative pain, and chronic postoperative pain following surgery. These findings allow for the identification of patients who are likely to be particularly susceptible to perioperative pain, especially acute and chronic postoperative pain. The ability to identify such patients allows for the development of targeted prevention and treatment regimens for acute and chronic postoperative pain.

In the context of the present disclosure, the term "CpG site" refers to a site in genomic DNA where a cytosine nucleotide is followed by a guanine nucleotide when the linear sequence of bases is read in its <NUM> prime (<NUM>') to <NUM> prime (<NUM>') direction. The 'p' in "CpG" refers to a phosphate moiety and indicates that the cytosine and guanine are separated by only one phosphate group. A status of "methylated" in reference to a CpG site refers to methylation of the cytosine of the CpG dinucleotide to form a <NUM>-methylcytosine.

In the context of the present disclosure, the terms "acute postoperative pain" and "chronic postoperative pain" are synonymous, respectively, with the terms "acute postsurgical pain" and "chronic postsurgical pain". The term "chronic postsurgical pain" may be abbreviated "CPSP". In this context, the term "chronic" refers to pain that persists for more than two or three months after surgery. Likewise, the term "acute" refers to pain occurring within the first two months after surgery.

In the context of the present disclosure, the term 'patient' refers to a human subject and a patient who is "susceptible" is one who is predisposed to suffering from perioperative pain, especially acute and chronic postsurgical pain. The identification of such patients according to the methods described herein is intended to provide for more effective personalized pain management and, in embodiments, for the targeted prevention and/or treatment of acute and/or chronic postsurgical pain. The patient identified as susceptible to perioperative pain or as susceptible to having an atypical perioperative anxiety response may be administered an agent to mitigate that susceptibility, such as a demethylating agent or an inhibitor of the repressor element-<NUM> silencing transcription factor (REST). In embodiments, the demethylating agent may be selected from procaine, zebularine and decitabine, or a combination of two or more of the foregoing. In embodiments, the demethylating agent is zebularine, decitabine, or a combination of two or more of the foregoing.

In accordance with embodiments of the methods described here, the biological sample from the patient which is used to isolate genomic DNA and determine methylation status is a blood sample. In these embodiments, blood is used as a proxy for the target tissue, brain, because brain tissue is generally inaccessible in the clinical context in which the present methods are performed. The use of blood as a substitute for various target tissues has been validated for example, by ChlP assay findings showing similar transcription factors at the identified CpG sites across tissues and regulatory regions in brain tissue relevant to pain, which may be indicative of methylation at these sites having an effect on expression. Last but not least, methylation profiles derived from <NUM> tissues were compared in a previous study and found to be highly correlated between somatic tissues (<NPL>). Davies et al. also reported that inter-individual variation in DNA methylation was reflected across brain and blood, indicating that peripheral tissues may have utility in studies of complex neurobiological phenotypes (<NPL>). For example, a comparison of methylation profiles of human chromosome <NUM> derived from different twelve tissues showed that CpG island methylation profiles were highly correlated (<NPL>). More recently, some inter-individual variation in DNA methylation was found to be conserved across brain and blood, indicating that peripheral tissues such as blood can have utility in studies of complex neurobiological phenotypes (<NPL>).

In accordance with embodiments of the methods described here, the methylation status at a genomic site, for example, at a CpG site as described herein, is binary, i.e., it is either methylated or unmethylated. In some embodiments where multiple CpG sites are assays, if at least one CpG site is methylated the region may be designated as methylated according to the claimed methods. This is because even if only one of several possible sites is methylated, if that site is a critical one for gene expression, its methylation may be sufficient. In other embodiments, where more than one of several possible CpG sites in a genomic region is methylated, the region may be designated as methylated or hypermethylated.

Embodiments of the methods described here include assaying a patient's genomic DNA to determine the DNA methylation status at one or more CpG sites in a plurality of genes described infra.

As noted above, a status of "methylated" in reference to a CpG site refers to methylation of the cytosine of the CpG dinucleotide to form a <NUM>-methylcytosine. Accordingly, methods of determining the DNA methylation status at one or more CpG sites in a genomic region of DNA generally involve detecting the presence of a <NUM>-methylcytosine at the site, or multiple <NUM>-methylcytosine in the region of interest. The determination of DNA methylation status can be performed by methods known to the skilled person. Typically such methods involve a determination of whether one or more particular sites are methylated or unmethylated, or a determination of whether a particular region of the genome is methylated, unmethylated, or hypermethylated, through direct or indirect detection of <NUM>-methylcytosine at a particular CpG site, or in the genomic region of interest.

Whole-genome methylation can be detected by methods including whole-genome bisulfite sequencing (WGBS), high-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (LC-MS/MS), enzyme-linked immunosorbent assay (ELISA)-based methods, as well as amplification fragment length polymorphism (AFLP), restriction fragment length polymorphism (FRLP) and luminometric methylation assay (LUMA).

Generally, in the context of the methods described herein, the methylation status of one or more specific CpG sites is determined. Suitable methods may include bead array, DNA amplification utilizing a polymerase chain reaction (PCR) followed by sequencing, pyrosequencing, methylation-specific PCR, PCR with high resolution melting, cold-PCR for the detection of unmethylated islands, and digestion-based assays. Bisulfite conversion is typically an initial step in these methods. Accordingly, in embodiments, the method for assaying DNA methylation status in accordance with the present disclosure may include a step of bisulfite conversion, for example, a step of treating a sample of genomic DNA with bisulfite thereby converting cytosine nucleotides to uracil nucleotides except where the cytosine is methylated.

In embodiments, the step of assaying DNA methylation status comprises pyrosequencing. The analysis of DNA methylation by pyrosequencing is known in the art and can be performed in accordance with published protocols, such as described in Delaney et al. This technique detects single-nucleotide polymorphisms which are artificially created at CpG sites through bisulfite modification of genomic DNA, which selectively converts cytosine to uracil except where the cytosine is methylated, in which case the <NUM>-methylcytosine is protected from deamination and the CG sequence is preserved in downstream reactions. Generally, the method comprises treating extracted genomic DNA with bisulfite and amplifying the DNA segment of interest with suitable primers, i.e., using a PCR-based amplification.

DNA demethylating agents inhibit DNA methyltransferases (DNMTs) such as DNMT1, which is responsible for the maintenance of methylation patterns after DNA replication, DNMT3A, and DNMT3B, each of which carries out de novo methylation.

In accordance with certain embodiments of the methods described here, a patient identified as susceptible to perioperative pain and anxiety based on the patient's methylation status as described herein may be administered one or a combination of two or more demethylating agents, for example, as part of a personalized pain management regimen.

In embodiments, a demethylating agent administered in accordance with embodiments of the methods described here may be a nucleoside-like DNMT inhibitor or a non-nucleoside DNMT inhibitor.

In an embodiment, the agent is a nucleoside-like DNMT inhibitor. In embodiments, the nucleoside-like DNMT inhibitor is selected from azacytidine (VIDAZA™), and analogs thereof, including <NUM>-aza-<NUM>'-deoxycytidine (decitabine, <NUM>-AZA-CdR), <NUM>-fluoro-<NUM>'-deoxycytidine, and <NUM>,<NUM>-dihydro-<NUM>-azacytidine. In embodiments, the nucleoside-like DNMT inhibitor is selected from pyrimidine-<NUM>-one ribonucleoside (zebularine).

In an embodiment, the agent is a non-nucleoside-like DNMT inhibitor. In embodiments, the agent is an antisense oligonucleotide. In embodiments, the antisense oligonucleotide is MG98, a <NUM>-base pair antisense oligonucleotide that binds to the <NUM>' untranslated region of DMNT1, preventing transcription of the DNMT1 gene. In embodiments, the non-nucleoside-like DNMT inhibitor is RG108, a small molecule DNA methylation inhibitor (<NPL>).

In accordance with embodiments of the methods described here, a patient identified as susceptible to perioperative pain based on the patient's methylation status as described herein, including a patient identified as susceptible to perioperative pain or hyperalgesia, may be administered an inhibitor of the repressor elements-<NUM> silencing transcription factor (REST). In embodiments, the REST inhibitor is denzoimidazole-<NUM>-carboxamide derivative (X5050) (<NPL>).

In embodiments of the methods described here, the methods are directed to a target population of patients in need of prophylaxis or treatment of perioperative pain. In embodiments, the target patient population may be further defined as discussed below. In the context of the methods described here, the term "patient" refers to a human subject. In embodiments, the term may more particularly refer to a human subject under the care of a medical professional.

In embodiments, the target patient population may be further defined by sex, age, or self-reported human population or ethnic group. For example, in embodiments the patient is a female. In embodiments, the patient is an adolescent, as that term is understood by the skilled medical practitioner. In embodiments, the patient's race or ethnicity is self-reported as white or Caucasian.

Kits useful in the methods disclosed here comprise components such as primers for nucleic acid amplification, hybridization probes, means for analyzing the methylation state of a deoxyribonucleic acid sequence, and the like. The kits can, for example, include necessary buffers, nucleic acid primers, and reagents for detection of methylation, as well as suitable controls, including for example bisulfite conversion controls, such as a bisulfite-treated DNA oligonucleotide of known sequence, and template free negative controls for pyrosequencing, as well as necessary enzymes (e.g. DNA polymerase), and suitable buffers.

In some embodiments, the kit comprises one or more nucleic acids, including for example PCR primers and bisulfite-treated DNA for use as a control, for use in the detection of the methylation status of one or more of the specific CpG sites identified herein, as well as suitable reagents, e.g., for bisulfite conversion, for amplification by PCR and/or for detection and/or sequencing of the amplified products.

In embodiments, the kit comprises a set of PCR primers for detecting the methylation status of one or more of the CpG sites identified herein. In embodiments, the kit comprises at least two sets of primers, long and nested.

In certain embodiments, the kit further comprises a set of instructions for using the reagents comprising the kit.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. <NPL>); <NPL>); and <NPL>) provide one skilled in the art with a general guide to many of the terms used in the present application.

A prospective observational cohort study was conducted in <NUM> adolescents with idiopathic scoliosis undergoing posterior spine fusion under standard anesthesia (Propofol-remifentanil total intravenous anesthesia) and postoperative analgesia (Patient Controlled Analgesia (PCA) plus scheduled intravenous acetaminophen, ketorolac, diazepam as needed and methocarbamol) protocols. The study was approved by the Cincinnati's Children's Hospital Medical Center (CCHMC) institutional review board. This study was registered with Clinicaltrials. gov identifiers NCT01839461 and NCT01731873. Written informed consent was obtained from parents, and assent was obtained from children before enrollment.

Healthy non-obese subjects with an American Society of Anesthesiologists (ASA) physical status less than or equal to two (mild systemic disease), aged ten to <NUM> years, with a diagnosis of idiopathic scoliosis and/or kyphosis, undergoing elective spinal fusion were recruited. Exclusion criteria included pregnant or breastfeeding females, presence of chronic pain defined as use of opioids in the past six months, liver or renal diseases and developmental delays.

Preoperatively, data regarding demographics (sex, age, race), weight, pain scores (numerical rating scale/<NUM>-<NUM> NRS) and home medication use were obtained. Anxiety scores for both child and a parent were assessed using the <NUM>-<NUM> visual analog scale (VAS), a simple validated scale which has been used previously in children. Questionnaires were administered as described in Table <NUM>. Intraoperative data collected included propofol and remifentanil doses, duration of surgery, and number of vertebral levels fused. In the immediate postoperative period (postoperative days (POD) one and two), pain scores (every four hours), morphine and diazepam doses administered were noted. After hospital discharge, questionnaires were administered over phone/email in a standard fashion, per schedule presented in Table <NUM> to obtain psychosocial and pain measures.

Outcomes evaluated were: a) CPSP, defined as NRS><NUM>/<NUM> at <NUM>-<NUM> months post-surgery (Macrae and Davies, Seattle: IASP Press; <NUM>). These cut-offs were used because NRS pain scores ><NUM> (moderate/severe pain) have been described as a predictor for persistence of pain and associated with functional disability (<NPL>); and b) Child Anxiety Sensitivity Index (CASI), an <NUM>-item self-report tool designed to measure symptoms of anxiety in children and adolescents, with total scores ranging from <NUM>-<NUM>, was chosen as the anxiety measure, because CASI is strongly correlated with state and trait anxiety (<NPL>). It is a measure of the degree to which one interprets anxiety-related symptoms as being associated with potentially harmful somatic, psychological, or social consequences (<NPL>). The CASI has demonstrated high internal consistency in both clinical and nonclinical samples (aged <NUM>-<NUM> years), good test-retest reliability and good construct validity (<NPL>). Our own studies have shown that the odds of pain persistence at <NUM> year after spine surgery was <NUM> higher for every unit increase in CASI score (<NUM>% CI <NUM>-<NUM>, p=<NUM>) (<NPL>) and is supported by studies in other pediatric cohorts (<NPL>).

Blood was drawn upon intravenous line placement before surgery, from which DNA was isolated on the same day and frozen at -<NUM>. To study DNA methylation, <NUM> ng of genomic DNA of acceptable quality (measured by Thermo Scientific NanoDrop spectrophotometer and with a <NUM>/<NUM> ratio ranging from <NUM> to <NUM>) was extracted, treated with bisulfite using Zymo EZ DNA Methylation Gold kit (Zymo Research, Orange, CA, USA), according to the manufacturer's instructions. DNA methylation was analyzed using the Infinium MethylationEPIC kit (©Illumina Inc. , San Diego, CA) which provides unparalleled coverage of CpG islands, genes, and enhancers.

Demographics and patients' clinical characteristics were summarized as mean (with standard deviation), median (IQR) and frequency (percentage) according to the distribution of the data. Prior to the DNA methylation analysis, the quality of the methylation arrays was assessed using sample-independent and -dependent internal control probes included on the array for staining, extension, hybridization, specificity and bisulfite conversion. The number of probes with detection P value ≤<NUM> was examined for each sample. Only samples that passed the quality control with ><NUM>% probes detected were included in the analysis. CpG sites that were not detected in all samples at p=<NUM> level or located on the X and Y chromosomes were excluded. The signal intensities were background-adjusted using out-of-band probes (noob), and normalized using subset-quantile within array normalization (swan in R package 'minfi'). Beta values, calculated as beta = <MAT>, and M values , the logit transformation of the beta values (<NPL>) were used. Surrogate variable analysis (SVA) was used to control batch effect and unknown confounders such as cell composition. For each of the CpG sites, the association of DNAm with CPSP and CASI was tested with linear regression. Age, sex, race and significant surrogate variables were adjusted. CpG sites whose DNAm (both beta and M values) were associated with CPSP or CASI at p≤<NUM> level were selected for further evaluation. The selected sites should also have differences ≥ <NUM> in beta between CPSP yes and no groups. As impact of non-genetic covariates were previously found on CPSP and CASI (<NPL>) to ensure the robustness of the association identified from the above analyses in which the DNAm was used as the dependent variable, we conducted logistic and linear regression for CPSP and CASI, respectively, in which CPSP and CASI were used as dependent variables, beta value as primary independent variable, and significant non-genetic co-variables were adjusted. Significant non-genetic co-variables were identified by univariate analysis for CPSP (factors tested: age, sex, race, morphine dose in mg/kg POD1 and POD2, preoperative anxiety score (VAS) for child and parent, duration of surgery, vertebral levels fused, PCS-P and CASI) and CASI (factors tested: age, sex, race, PCS-P, diazepam doses and parent anxiety score), and selection of co-variables associated at p < <NUM>. Analyses were performed using Statistical Analysis System (SAS), version <NUM> (SAS Institute Inc. , Cary, NC) and R. Only CpG sites showing significance association with beta values in these models were extracted from MethylationEPIC array annotation files and imported into Ingenuity Pathway Analysis software (IPA, Ingenuity Systems, Redwood City, CA) for pathway mapping, gene network detection, and upstream regulator identification.

To identify potential regulatory mechanisms altered by CpG methylation differences, we evaluated CpG sites that were significant in the previous step (p<<NUM>) against a control set of CpG sites (p><NUM>) using a compiled large collection of functional genomics datasets from various sources, including ENCODE (<NPL>), Roadmap Epigenomics (<NPL>), Cistrome (<NPL>), and ReMap-ChIP (<NPL>). In total, this database contains <NUM>,<NUM> datasets performed in <NUM>,<NUM> different cell types and conditions. Monitoring of protein binding interactions of <NUM>,<NUM> data sets including transcription factors with the human genome was performed using ChIP-seq. A particular histone mark was measured in <NUM>,<NUM> data sets using ChIP-seq. ; <NUM> data sets measured open chromatin through DNase-seq; <NUM> data sets measured expression-quantitative loci (eQTLs); and <NUM> data sets predicted "ActiveChromatin" states using combinations of histone marks (<NPL>). Collectively, <NUM> of these experiments were performed in brain-related cell lines and cell types.

We next used the RELI algorithm to estimate the statistical enrichment of histone marks and protein-binding events at the genomic loci displaying altered DNA methylation (<NPL>). As input, the method took a set of genomic loci (in this case, regions with differential methylation marks). The coordinates of each locus were padded by <NUM> bases in either direction to account for experimental resolution. The resulting loci were then systematically intersected with the ChIP-seq and the epigenetic data set libraries described above, and the number of input regions overlapping each dataset by at least one base was counted. Next, a P-value describing the significance of this overlap was estimated using a simulation-based procedure. To this end, the control set of CpG sites that do not change (p><NUM>) was used as a negative, background set. A distribution of expected overlap values was then created from <NUM>,<NUM> iterations of randomly sampling from the negative set, each time choosing a set of negative examples that match the input set in terms of the total number of genomic loci and the length of each locus. The distribution of the expected overlap values from the randomized data resembles a normal distribution, and can thus be used to generate a Z-score and corresponding P-value estimating the significance of the observed number of input regions that overlap each data set. Collectively, this procedure controlled for the count and sizes of the input loci, and the count and sizes of each individual dataset in the library. The final output of the method is a p-value based ranking of all of the functional genomics datasets, in terms of their overlap with the input set.

With the goal of further elucidating pathways and potential regulatory mechanisms underlying the observed epigenetic changes, we performed enrichment analysis using a comprehensive, curated library of transcription factor targets that combines results from ENCODE and literature based CHEA ChIP-seq experiments, available through Enrichr (http://amp. edu/Enrichr/). Next, we used the Library of Integrated Network-based Cellular Signatures (LINCS) of genetic perturbations (gene knockdowns of the <NUM> genes common to both outcomes with DNA methylation changes) and connectivity analysis, with the focus on kinase signaling pathways, available through Pinet (http://pinet-server. org) and Enrichr (<NPL>). One of the goals of LINCS library is to enable analysis of connectivity between genetic (<NPL>) and chemical perturbations by measuring correlations between their transcriptional echo (correlation between landmark gene expression vectors). Here, we use signatures of genetic knock-downs of gene encoding protein kinases, which consist of genes whose mRNA expression is downregulated in response to the loss of function for each kinase.

The mean age of participants was <NUM> years (SD <NUM>); they were mostly white (<NUM>%) and female (<NUM>%). Demographics and description of variables evaluated, are presented in Table <NUM>. Median preoperative pain score was <NUM> (IQR <NUM>-<NUM>) and mean (SD) for AUC on POD1 and POD2 was <NUM> (<NUM>). As expected, there was a significant difference in NRS pain scores at <NUM>-<NUM> months between the non-CPSP (<NUM> (<NUM>-<NUM>)) and CPSP (<NUM> (<NUM>-<NUM>)) groups (p<<NUM>). Of <NUM> subjects recruited, follow-up for CPSP outcomes was successful for <NUM> subjects. Incidence of CPSP in this cohort was <NUM>/<NUM> (<NUM>%).

At a significance threshold of p<<NUM>, univariate analyses identified age and CASI were significant determinants of CPSP (p= <NUM> and <NUM> respectively) (Table <NUM>); and PCS-P (p=<NUM>) and diazepam dose (p=<NUM>) for CASI. Preoperative pain, AUC and pain at <NUM>-<NUM> months were all significantly higher in the CPSP group compared to the non-CPSP group (p=<NUM>, <NUM> and <<NUM> respectively). Since preoperative pain, AUC and CPSP are correlated pain outcomes, with possible overlap of DNA methylation associations, we did not include them as co-variables in the multivariate model for CPSP.

Of the <NUM> samples, one was excluded from analysis due to bad array quality. The remaining samples all had more than <NUM>% of the probes detected. For CPSP, a final set of <NUM> CpG sites were selected; for CASI, <NUM>,<NUM> CpG sites were selected for IPA analyses (<FIG>). The distribution of differentially methylated regions in association with CPSP and the CASI (P<. <NUM> and delta-beta > <NUM>) with regard to genomic location, are presented in Table <NUM>.

To assess the possible overall influence of the significant differences in DNA methylation enrichment for CPSP and CASI, two pathway analyses were performed. Annotation information on the significantly associated CpG sites was used for the analysis. In total, <NUM> genes (CPSP) and <NUM>,<NUM> genes (CASI) were annotated to the CpG sites. The top canonical pathways mapped to significantly methylated CpG sites for CPSP and CASI and overlap with gene sets defining the pathways at a p-value<<NUM> are shown in Table <NUM>.

Significant CpG sites associated with CPSP were annotated to <NUM> genes, and those for CASI were located on <NUM> genes. At the gene level, <NUM> genes had CpG sites with significant DNA methylation associations with both outcomes, and they mapped to <NUM> pathways. Shared overlapping pathways identified by gene overlap for CpG sites associated with both chronic post-surgical pain and child anxiety sensitivity index are detailed in Table <NUM>. The top pathways were GABA receptor signaling and Dopamine-DARPP32 Feedback in cAMP Signaling (<FIG>).

Significantly methylated CpG sites associated with CPSP are located in active regulatory regions with open chromatin marked by H3K27ac, H3K4me1 and H3K4me3 in brain cells from the hippocampus, frontal lobe, temporal lobe, anterior cingulate cortex, etc. (Table 6A). Also depicted in Table 6A are the significant (p<<NUM>, after correction for multiple testing) protein (e.g., transcription factor) binding events identified to overlap significantly at the CpG sites, significant for CASI. Of note, many involve the RNA polymerase subunit POLR2A, suggesting that many differential methylation events might result in altered gene expression. Table 6B shows the genomic locations of the specific histone markers of the CpG sites associated with Chronic postsurgical pain (CPSP) and Table 6B-<NUM> shows similar data for Childhood Anxiety Sensitivity Index (CASI). Table 6C shows the location of certain histone markers found in the regulatory regions of the indicated genes in multiple cell lines. Although we do not have expression data from the brain, the CpG sites depicted in Table 6C are located within brain sites associated with several active chromatin markers. For example, H3K27me3 ChIP-seq peaks in brain cells is a repressive (polycomb) signal. Finding these sites overlapping with similar markers from several brain sites important for nociception indicates that they are functional in those areas, and hence alter gene expression. In each of Tables 6B and 6C, the CpG sites are designated by their Illumina Identification number, "cg" followed by a number, and their position is relative to the human reference genome released February <NUM>, <NUM> by the Genome Reference Consortium (GRC) referred to as GRCh37 or HG19.

We have previously shown that psychological variables (CASI), (<NPL>) (clinical variables and OPRM1 promoter DNA methylation (<NPL>) associated with CPSP. In this study, we performed an EW AS of CPSP (acute or chronic perioperative pain) and CASI (anxiety sensitivity) in children undergoing spinal surgery and followed up on our findings with an integrative computational analysis to identify common, targetable pathways and transcription factors associated with significantly methylated CpG sites associated with CPSP and anxiety. Our findings open new avenues for personalized interventions based on epigenetics, and add to the emerging evidence linking epigenetic mechanisms to the development of chronic pain and psychological states. (<NPL>;<NPL>).

Epigenetic research into acute to chronic pain transitions (<NPL>) is still in its infancy. To our knowledge, there are only a handful of clinical epigenetic studies in postsurgical patients. DNA methylation of the Secreted Protein, Acidic, Rich in Cysteine (SPARC) promoter was shown to play a role in chronic low-back pain related to degenerated intervertebral discs (<NPL>). CpG methylation within the Tumor Necrosis Factor (TNF) gene promoter has also been identified as an additional mechanism through which TNF alters the risk for mild persistent breast pain after breast cancer surgery (<NPL>). We previously reported on two CpG sites in an active regulatory region of the OPRM1 gene that binds multiple transcription factors to be predictive of CPSP in another subset of the spine surgery cohort. The present EWAS study provides further evidence for the role of epigenetics in CPSP.

Epigenome-based pathway analyses have been previously described using whole blood DNA in a large cohort of adults, with chronic widespread musculoskeletal pain (<NPL>). They found that <NUM>% of variance for the pain phenotype was explained by epigenetic factors, and showed enrichment for neurological pathways, including synaptic long-term depression, axonal guidance signaling, CREB, neuropathic pain signaling and melatonin signaling (<NPL>). While some of the pathways are similar to what we have identified for CPSP, the differences may be reflective of differences in the nature of the pain and cohorts evaluated.

Of great interest is that the top canonical pathways common to both CPSP and CASI were identified to be the GABA receptor signaling and Dopamine-DARPP32 pathways. This is aligned with previous literature citing hypofunction of GABAergic inhibitory tone in the dorsal horn of the spinal cord as a key factor in central neuropathic pain after spinal cord injury (<NPL>). Mechanisms proposed for GABAergic hypofunction include decreased number of GABA receptors (through apoptosis), downregulation of GABA synthesizing enzyme (GAD) and decreased GABA concentrations. Multiple in vitro and in vivo studies suggest the role of DNA methyltransferases in the epigenetic regulation of GABAergic gene expression in the cortex, striatum and hippocampus (<NPL>). DNA epigenetic modifications of GABAergic interneurons in the basolateral amygdala have also been shown to be involved in the etiology of anxiety-like phenotype in prenatal stress mice, which could be reversed by demethylating agent, <NUM>-Aza deoxycytidine (<NPL>). Our functional genomics analyses show that many of the CpG sites identified are located in regions of the brain marked by lysine <NUM> tri-methylation (H3K27me3), which is known to negatively regulate gene expression. Our study thus provides new evidence for DNA methylation as a mechanism for possibly reduced function of the GABA receptor pathway genes and its role in CPSP and anxiety pathogenesis.

Our findings are also aligned with postulated roles for the DARPP-<NUM> dopamine pathway in the actions of drugs of abuse inflammatory states and psychiatric conditions like schizophrenia and bipolar disorder. DARPP-<NUM> is a substrate of cAMP-dependent protein kinase (PKA) highly concentrated in dopamine-innervated brain areas, which functions as a PKA-regulated inhibitors of protein phosphatase-<NUM> (PP1). The identification of epigenetic enrichment of this pathway is exciting because animal studies suggest a role for this phosphoprotein as an intracellular detector of convergent dopamine-<NUM> receptor and N-methyl-D-aspartate (NMDA) receptor activation (<NPL>). These are target receptors for pain medications (opioids) and antipsychotic medications (for example haloperidol). Thus our studies indicate therapeutic interventions for CPSP based on epigenetic profile. A cyclin-dependent kinase <NUM> (Cdk5) inhibitor, roscovitine, was shown to decrease DARPP-<NUM> phosphorylation (<NPL>) and its intrathecal use decreased the formalin-induced nociceptive response in rats (<NPL>) and remifentanil-induced hyperalgesia (<NPL>). Dopamine is involved in reward mechanisms (<NPL>) and motivation to engage in pain self-management behaviors is an important predictor of adaptation/coping with acute pain. Accordingly, anxiety induced avoidance or lack of motivation (<NPL>) is a plausible mechanism by which dopamine signaling might be a player in development of CPSP in the presence of anxiety.

Among the other top shared pathways, nitric oxide signaling (NOS) also deserves mention. This appears to be essential for neural plasticity and modulation of opioid action (<NPL>). Nitric oxide formed by N-methyl-d-aspartate (NMDA)-receptor activation, may act at several levels of the nervous system to develop hyperexcitability, resulting in hyperalgesia or allodynia (<NPL>). While it has been shown to be an analgesic (<NPL>) and algesic (<NPL>) mediator at spinal, supraspinal and systemic sites in experimental animals, Pu et. postulated a dual-control mechanism composed of the excitatory NMDA and the inhibitory µ-opioid receptors in modulating cyclic GMP/nitric oxide release (<NPL>). Moreover, NOS also plays a role in morphine dependence and tolerance, which has been shown to be prevented using NOS inhibitors (<NPL>).

There is evidence from prior studies for the role of some of the other pathways we have identified to play a role in anxiety. Bioinformatics analysis of differently expressed microRNAs in anxiety disorder used for predicting target genes and functions using gene ontology and KEGG pathway analysis showed significant enrichment in several pathways related to neuronal brain functions such as the GnRH signaling pathway (<NPL>). The Protein Kinase A (PKA) signaling pathway, closely related to the DARPP-<NUM> pathway described above, is involved in neuronal plasticity in the amygdala, is responsible for amplification of anxiety behaviors in response to stressful stimuli. Results of clinical studies support the finding that alterations in PKA are associated with various anxiety, depression, and other psychiatric disorders (<NPL>.

We have found interesting evidence based on Enrichr and Pinet enrichment analyses, including overlaps with LINCS knockdowns signatures for the overlaps with TF targets. They reveal several TFs with neuronal phenotypes as regulators of significant subsets of overlapping (common to both outcomes) genes. These include, but are not limited to, REST, TRIM28, POUSF1, NFE2L2, GATA2 and NANOG (Table <NUM>, <FIG>).

NANOG is also associated with POU5F1, KLF4, etc. and its LINCS knockdowns are strongly positively correlated (in multiple cell lines) with SMAD1/<NUM>/<NUM>, POLR2A (and other units of PolIIa), EP300 and other putative TFs targeting the overlap genes, which adds supporting evidence for them working together. Analysis of overlaps with LINCS knockdowns reveals GABA receptor subunits and GPRC5C, among other potential positive upstream regulators of overlap genes (Table <NUM>, <FIG>). The latter (GPRC5C) is associated with activation of NANOG (<NPL>), which seems to be consistent with its and related TFs targets being among (in this case predicted to be positively regulated) the overlap genes.

Although this study utilized blood samples for measurement of DNA methylation status, instead of a more relevant target tissue such as brain tissue, the translational relevance of findings in easily available tissues such as blood cannot be overemphasized. Also, the ChIP assay findings show similar transcription factors at the identified CpG sites across tissues and regulatory regions in brain tissue areas relevant to pain, which may be indicative of methylation at these sites having an effect on expression. Moreover, methylation profiles derived from <NUM> tissues were compared in a previous study and found to be highly correlated between somatic tissues (<NPL>). Davies et al. also reported that inter-individual variation in DNA methylation was reflected across brain and blood, indicating that peripheral tissues may have utility in studies of complex neurobiological phenotypes (<NPL>). Our study is comprehensive in evaluating several relevant covariates with known influence on CPSP and anxiety sensitivity. Further work may involve evaluation in larger prospective cohorts and longitudinal evaluation of methylation changes after surgery.

DNA methylation is influenced by multiple modifiable factors such as diet, exercise, stress, and meditation. Therefore, understanding the shared role of epigenetic regulation of CPSP and anxiety opens new avenues of pain research. Our findings provide a basis for biopsychosocial profiles involved in CPSP and suggest consideration of behavioral and other pathway- targeted strategies, based on the individual's methylation profile at one or more of the CpG sites described here as determined from a blood sample (<NPL>). There is also promise from animal models for epigenetic modification to prevent the progression to chronic postsurgical pain (<NPL>); <NPL>) and use of demethylating drugs in other diseases (<NPL>; <NPL>) for such therapies to be useful for the treatment of chronic pain. Recent advent of targeted epigenetic modification (<NPL>) also provides hope for decreasing non-specific effects and poor delivery of epigenetic modulation to target cells and tissues, a major impediment to the development and clinical application of such analgesics.

The following additional experiments provide a basis for the use of cytokine expression in blood obtained from patients as a proxy for surgery related inflammation leading to CPSP. The data supports our hypothesis that pain is characterized by a heightened inflammatory responsiveness in susceptible patients and suggests that the immune system is "primed" in pain patients. Patients presenting with such a 'primed' immune system can be identified, for example, by assaying for perioperative pro-inflammatory cytokine levels, e.g., TNFα and IL1RA, and/or for lower levels of anti-inflammatory cytokines such as IL-<NUM> in order to identify patients at risk of developing CPSP. At risk patients could be treated with one or more interventions aimed at decreasing risk of CPSP, for example treatment with an anti-inflammatory agent.

Perioperative pro-inflammatory cytokine levels are higher in those who develop CPSP. We also found that perioperative pro-inflammatory cytokine levels are higher in those who develop CPSP. Cytokine levels were measured in serum samples obtained at baseline and <NUM> time points within <NUM> hours of surgery (<NUM> samples from <NUM> patients) using Human Cytokine/Chemokine Panel I (Milliplex® Immunology Panel). Average levels in patients who developed CPSP (N=<NUM>) and those who did not are plotted in <FIG>. We found significantly increased pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα) and interleukin-1RA (IL1RA), as well as decreased levels of anti-inflammatory cytokines such as IL-<NUM> in patients with CPSP, compared to controls. Additional differentially expressed cytokines were fractalkine, epidermal growth factor (EGF), FMS-like tyrosine kinase <NUM> ligand (FLT-<NUM>), macrophage derived chemokine (MDC), interleukin-<NUM> (IL-<NUM>), interleukin-<NUM> (IL-<NUM>), and interleukin-<NUM> (IL-<NUM>).

Immune pathways are among the top canonical pathways enriched with DNAm associated with CPSP. Our pilot EWAS in spine patients showed DNAm enrichment of PKCθ Signaling in T Lymphocytes (p<<NUM>) (overlap of HLA-A, PLCG2, CACNA1H, CACNA1C, CACNA1A, HLA-DRB5, LCP2, CAMK2B) and FcγRIIB Signaling in B Lymphocytes (PLCG2, CACNA1H, CACNA1C, CACNA1A) (p=<NUM>). Gene-gene interaction network enrichment analysis revealed participation of pathways in cell signaling, molecular transport, immune responses, metabolism and neurological diseases (p-value < <NUM>-<NUM>) (<FIG>) in CPSP, with several target molecules. This is shown graphically in <FIG>.

Claim 1:
A therapeutic agent which is a demethylating agent for use in a method for the prophylaxis or treatment of perioperative pain in a human patient identified as susceptible to perioperative pain,
wherein the patient has been identified as susceptible to perioperative pain by a method comprising assaying, in vitro, a biological sample from the patient to determine the DNA methylation status of a plurality of CpG sites in a plurality of genes of a molecular signaling pathway selected from a GABA receptor signaling pathway and a dopamine-DARPP32 feedback in cAMP signaling pathway, or both, wherein the plurality of genes includes at least two genes selected from the group consisting of either ABAT, ADCY5, CACNA1H, CACNA1C, GABBR1, KCNH2, CACNA1A or PPP1R1B, ADCY5, PLCG2, CAMKK1, CACNA1C, DRD4, CACNA1A and wherein having a DNA methylation status of 'methylated' is identified as a patient susceptible to perioperative pain; and wherein the perioperative pain is chronic postsurgical pain.