1 344 153 ALTERED BDNF METHYLATION IN PATIENTS WITH CHRONIC MUSCULOSKELETAL PAIN AND HIGH BIOPSYCHOSOCIAL COMPLEXITY. PURPOSE: THE INTERMED INSTRUMENT, WHICH WAS DEVELOPED TO MEASURE PATIENT'S BIOPSYCHOSOCIAL (BPS) COMPLEXITY, REPRESENTS A POWERFUL DIAGNOSTIC AND THERAPEUTIC TOOL. EPIGENETIC CHANGES ARE THE INTERFACE BETWEEN SIGNALS FROM THE ENVIRONMENT AND GENETIC MODIFICATIONS, AFFECTING GENE EXPRESSION, IN PARTICULAR, BY DNA METHYLATION OF CPG DINUCLEOTIDES IN PROMOTOR REGIONS OF THE CORRESPONDING GENES. THE BRAIN-DERIVED NEUROTROPHIC FACTOR (BDNF) GENE PLAYS A CRUCIAL ROLE IN THE CENTRAL SENSITIZATION (CS) OF PAIN. IN THIS STUDY, WE HYPOTHESIZED THAT CHRONIC PAIN MODIFIES THE METHYLATION LEVELS OF THE BDNF GENE IN A MANNER THAT IS INTERCONNECTED WITH THE BPS STATUS. PATIENTS AND METHODS: FIFTY-EIGHT CHRONIC MUSCULOSKELETAL PAIN PATIENTS (CMSP) WERE ENROLLED IN THE STUDY. DNA WAS EXTRACTED FROM BLOOD SAMPLES, THE METHYLATION LEVELS OF 13 CPG SITES IN THE BDNF PROMOTER WERE MEASURED BY PYROSEQUENCING, AND ASSOCIATION STUDIES WITH VARIOUS PATIENT PARAMETERS AND THE INTERMED SCORES WERE PERFORMED. RESULTS: INTERESTINGLY, A NEGATIVE CORRELATION (-0.40) WAS FOUND BETWEEN THE TOTAL INTERMED SCORES AND THE AVERAGE CPG METHYLATION VALUES OF THE BDNF GENE, BUT NO CORRELATION WAS OBSERVED WITH THE SEVERITY OF PAIN, DEGREE OF ANXIETY, DEPRESSION, OR KINESIOPHOBIA AND CATASTROPHISM. MOREOVER, THE ASSOCIATION WAS INDEPENDENT OF AGE, SEX AND LEVEL OF COMORBIDITIES. CONCLUSION: THIS RESULT SHOWS THAT CMSP, IN ASSOCIATION WITH ITS BIOPSYCHOSOCIAL CONTEXT, EPIGENETICALLY DECREASES THE DEGREE OF METHYLATION OF THE BDNF PROMOTER AND SHOULD THEREFORE INCREASE THE LEVEL OF BDNF TRANSCRIPTION. IT ALSO SUGGESTS A ROLE OF THE INTERMED TOOL TO DETECT A RELATIONSHIP BETWEEN THE BPS COMPLEXITY AND THE EPIGENETIC CONTROL OF A TARGET GENE. THE POSSIBLE UPREGULATION OF BDNF EXPRESSION MIGHT BE, AT LEAST IN PART, THE SIGNAL FOR CHRONIC PAIN-INDUCED CENTRAL SENSITIZATION (CS). THIS COULD PARTLY EXPLAIN WHY PATIENTS WITH A HIGHER LEVEL OF COMPLEXITY FEEL MORE PAIN THAN THOSE WITH LOWER COMPLEXITY. 2020 2 990 38 CHRONIC SOCIAL STRESS INDUCES DNA METHYLATION CHANGES AT AN EVOLUTIONARY CONSERVED INTERGENIC REGION IN CHROMOSOME X. CHRONIC STRESS RESULTING FROM PROLONGED EXPOSURE TO NEGATIVE LIFE EVENTS INCREASES THE RISK OF MOOD AND ANXIETY DISORDERS. ALTHOUGH CHRONIC STRESS CAN CHANGE GENE EXPRESSION RELEVANT FOR BEHAVIOR, MOLECULAR REGULATORS OF THIS CHANGE HAVE NOT BEEN FULLY DETERMINED. ONE PROCESS THAT COULD PLAY A ROLE IS DNA METHYLATION, AN EPIGENETIC PROCESS WHEREBY A METHYL GROUP IS ADDED ONTO NUCLEOTIDES, PREDOMINANTLY CYTOSINE IN THE CPG CONTEXT, AND WHICH CAN BE INDUCED BY CHRONIC STRESS. IT IS UNKNOWN TO WHAT EXTENT CHRONIC SOCIAL DEFEAT, A MODEL OF HUMAN SOCIAL STRESS, INFLUENCES DNA METHYLATION PATTERNS ACROSS THE GENOME. OUR STUDY ADDRESSED THIS QUESTION BY USING A TARGETED-CAPTURE APPROACH CALLED METHYL-SEQ TO INVESTIGATE DNA METHYLATION PATTERNS OF THE DENTATE GYRUS AT PUTATIVE REGULATORY REGIONS ACROSS THE MOUSE GENOME FROM MICE EXPOSED TO 14 DAYS OF SOCIAL DEFEAT. FINDINGS WERE REPLICATED IN INDEPENDENT COHORTS BY BISULFITE-PYROSEQUENCING. TWO DIFFERENTIALLY METHYLATED REGIONS (DMRS) WERE IDENTIFIED. ONE DMR WAS LOCATED AT INTRON 9 OF DROSHA, AND IT SHOWED REDUCED METHYLATION IN STRESSED MICE. THIS OBSERVATION REPLICATED IN ONE OF TWO INDEPENDENT COHORTS. A SECOND DMR WAS IDENTIFIED AT AN INTERGENIC REGION OF CHROMOSOME X, AND METHYLATION IN THIS REGION WAS INCREASED IN STRESSED MICE. THIS METHYLATION DIFFERENCE REPLICATED IN TWO INDEPENDENT COHORTS AND IN MAJOR DEPRESSIVE DISORDER (MDD) POSTMORTEM BRAINS. THESE RESULTS HIGHLIGHT A REGION NOT PREVIOUSLY KNOWN TO BE DIFFERENTIALLY METHYLATED BY CHRONIC SOCIAL DEFEAT STRESS AND WHICH MAY BE INVOLVED IN MDD. 2018 3 1967 51 EPIGENETIC ALTERATION OF THE DOPAMINE TRANSPORTER GENE IN ALCOHOL-DEPENDENT PATIENTS IS ASSOCIATED WITH AGE. CHRONIC ALCOHOL ABUSE AND DEPENDENCE ARE ASSOCIATED WITH DYSFUNCTIONAL DOPAMINERGIC NEUROTRANSMISSION IN MESOCORTICOLIMBIC CIRCUITS. GENETIC AND ENVIRONMENTAL FACTORS HAVE BEEN SHOWN TO MODULATE SUSCEPTIBILITY TO ALCOHOL DEPENDENCE, AND BOTH MAY ACT THROUGH EPIGENETIC MECHANISMS THAT CAN MODULATE GENE EXPRESSION, E.G. DNA METHYLATION AT CPG SITES. RECENT STUDIES HAVE SUGGESTED THAT DNA METHYLATION PATTERNS MAY CHANGE OVER TIME. HOWEVER, FEW DATA ARE AVAILABLE CONCERNING THE RATE OF THESE CHANGES IN SPECIFIC GENES. A RECENT STUDY FOUND THAT HYPERMETHYLATION OF THE PROMOTER OF THE DOPAMINE TRANSPORTER (DAT) GENE WAS POSITIVELY CORRELATED WITH ALCOHOL DEPENDENCE AND NEGATIVELY CORRELATED WITH ALCOHOL CRAVING. THE AIM OF THE PRESENT STUDY WAS TO REPLICATE THESE FINDINGS IN A LARGER SAMPLE OF ALCOHOL-DEPENDENT PATIENTS AND POPULATION-BASED CONTROLS MATCHED FOR AGE AND SEX. NO DIFFERENCE IN METHYLATION LEVEL WAS OBSERVED BETWEEN PATIENTS AND CONTROLS, AND NO DIFFERENCE IN METHYLATION LEVEL WAS OBSERVED BEFORE AND AFTER ALCOHOL WITHDRAWAL IN PATIENTS. HOWEVER, PATIENTS WITH MORE SEVERE CRAVING SHOWED A TREND TOWARDS LOWER DAT METHYLATION LEVELS (P = 0.07), WHICH IS CONSISTENT WITH PREVIOUS FINDINGS. FURTHERMORE, IN OUR OVERALL SAMPLE, DAT METHYLATION LEVELS INCREASED WITH AGE. INTERESTINGLY, A SEPARATE ANALYSIS OF PATIENTS SUGGESTED THAT THIS FINDING WAS MAINLY DRIVEN BY THE PATIENT GROUP. ALTHOUGH THE PRESENT DATA DO NOT CLARIFY WHETHER CHRONIC ALCOHOL ABUSE IS RESPONSIBLE FOR THIS PHENOMENON OR MERELY ENHANCES AN AGEING-SPECIFIC PROCESS, OUR FINDINGS SUGGEST THAT HYPERMETHYLATION IN ALCOHOL-DEPENDENT PATIENTS IS A CONSEQUENCE, RATHER THAN A CAUSE, OF THE DISORDER. 2014 4 1503 39 DNA METHYLATION AND GENE EXPRESSION DIFFERENCES IN CHILDREN CONCEIVED IN VITRO OR IN VIVO. EPIDEMIOLOGICAL DATA INDICATE THAT CHILDREN CONCEIVED IN VITRO HAVE A GREATER RELATIVE RISK OF LOW BIRTH-WEIGHT, MAJOR AND MINOR BIRTH DEFECTS, AND RARE DISORDERS INVOLVING IMPRINTED GENES, SUGGESTING THAT EPIGENETIC CHANGES MAY BE ASSOCIATED WITH ASSISTED REPRODUCTION. WE EXAMINED DNA METHYLATION AT MORE THAN 700 GENES (1536 CPG SITES) IN PLACENTA AND CORD BLOOD AND MEASURED GENE EXPRESSION LEVELS OF A SUBSET OF GENES THAT DIFFERED IN METHYLATION LEVELS BETWEEN CHILDREN CONCEIVED IN VITRO VERSUS IN VIVO. OUR RESULTS SUGGEST THAT IN VITRO CONCEPTION IS ASSOCIATED WITH LOWER MEAN METHYLATION AT CPG SITES IN PLACENTA AND HIGHER MEAN METHYLATION AT CPG SITES IN CORD BLOOD. WE ALSO FIND THAT IN VITRO CONCEPTION-ASSOCIATED DNA METHYLATION DIFFERENCES ARE ASSOCIATED WITH GENE EXPRESSION DIFFERENCES AT BOTH IMPRINTED AND NON-IMPRINTED GENES. THE RANGE OF INTER-INDIVIDUAL VARIATION IN GENE EXPRESSION OF THE IN VITRO AND IN VIVO GROUPS OVERLAPS SUBSTANTIALLY BUT SOME INDIVIDUALS FROM THE IN VITRO GROUP DIFFER FROM THE IN VIVO GROUP MEAN BY MORE THAN TWO STANDARD DEVIATIONS. SEVERAL OF THE GENES WHOSE EXPRESSION DIFFERS BETWEEN THE TWO GROUPS HAVE BEEN IMPLICATED IN CHRONIC METABOLIC DISORDERS, SUCH AS OBESITY AND TYPE II DIABETES. THESE FINDINGS SUGGEST THAT THERE MAY BE EPIGENETIC DIFFERENCES IN THE GAMETES OR EARLY EMBRYOS DERIVED FROM COUPLES UNDERGOING TREATMENT FOR INFERTILITY. ALTERNATIVELY, ASSISTED REPRODUCTION TECHNOLOGY MAY HAVE AN EFFECT ON GLOBAL PATTERNS OF DNA METHYLATION AND GENE EXPRESSION. IN EITHER CASE, THESE DIFFERENCES OR CHANGES MAY AFFECT LONG-TERM PATTERNS OF GENE EXPRESSION. 2009 5 4879 24 OVERLAPPING SIGNATURES OF CHRONIC PAIN IN THE DNA METHYLATION LANDSCAPE OF PREFRONTAL CORTEX AND PERIPHERAL T CELLS. WE TESTED THE HYPOTHESIS THAT EPIGENETIC MECHANISMS IN THE BRAIN AND THE IMMUNE SYSTEM ARE ASSOCIATED WITH CHRONIC PAIN. GENOME-WIDE DNA METHYLATION ASSESSED IN 9 MONTHS POST NERVE-INJURY (SNI) AND SHAM RATS, IN THE PREFRONTAL CORTEX (PFC) AS WELL AS IN T CELLS REVEALED A VAST DIFFERENCE IN THE DNA METHYLATION LANDSCAPE IN THE BRAIN BETWEEN THE GROUPS AND A REMARKABLE OVERLAP (72%) BETWEEN DIFFERENTIALLY METHYLATED PROBES IN T CELLS AND PREFRONTAL CORTEX. DNA METHYLATION STATES IN THE PFC SHOWED ROBUST CORRELATION WITH PAIN SCORE OF ANIMALS IN SEVERAL GENES INVOLVED IN PAIN. FINALLY, ONLY 11 DIFFERENTIALLY METHYLATED PROBES IN T CELLS WERE SUFFICIENT TO DISTINGUISH SNI OR SHAM INDIVIDUAL RATS. THIS STUDY SUPPORTS THE PLAUSIBILITY OF DNA METHYLATION INVOLVEMENT IN CHRONIC PAIN AND DEMONSTRATES THE POTENTIAL FEASIBILITY OF DNA METHYLATION MARKERS IN T CELLS AS NONINVASIVE BIOMARKERS OF CHRONIC PAIN SUSCEPTIBILITY. 2016 6 3652 38 INDIVIDUAL DNA METHYLATION PATTERN SHIFTS IN NANOPARTICLES-EXPOSED WORKERS ANALYZED IN FOUR CONSECUTIVE YEARS. A DNA METHYLATION PATTERN REPRESENTS AN ORIGINAL PLAN OF THE FUNCTION SETTINGS OF INDIVIDUAL CELLS AND TISSUES. THE BASIC STRATEGIES OF ITS DEVELOPMENT AND CHANGES DURING THE HUMAN LIFETIME ARE KNOWN, BUT THE DETAILS RELATED TO ITS MODIFICATION OVER THE YEARS ON AN INDIVIDUAL BASIS HAVE NOT YET BEEN STUDIED. MOREOVER, CURRENT EVIDENCE SHOWS THAT ENVIRONMENTAL EXPOSURE COULD GENERATE CHANGES IN DNA METHYLATION SETTINGS AND, SUBSEQUENTLY, THE FUNCTION OF GENES. IN THIS STUDY, WE ANALYZED THE EFFECT OF CHRONIC EXPOSURE TO NANOPARTICLES (NP) IN OCCUPATIONALLY EXPOSED WORKERS REPEATEDLY SAMPLED IN FOUR CONSECUTIVE YEARS (2016-2019). A DETAILED METHYLATION PATTERN ANALYSIS OF 14 PERSONS (10 EXPOSED AND 4 CONTROLS) WAS PERFORMED ON AN INDIVIDUAL BASIS. A MICROARRAY-BASED APPROACH USING CHIPS, ALLOWING THE ASSESSMENT OF MORE THAN 850 K CPG LOCI, WAS USED. INDIVIDUAL DNA METHYLATION PATTERNS WERE COMPARED BY PRINCIPAL COMPONENT ANALYSIS (PCA). THE RESULTS SHOW THE SHIFT IN DNA METHYLATION PATTERNS IN INDIVIDUAL YEARS IN ALL THE EXPOSED AND CONTROL SUBJECTS. THE OVERALL RANGE OF DIFFERENCES VARIED BETWEEN THE YEARS IN INDIVIDUAL PERSONS. THE DIFFERENCES BETWEEN THE FIRST AND LAST YEAR OF EXAMINATION (A THREE-YEAR TIME PERIOD) SEEM TO BE CONSISTENTLY GREATER IN THE NP-EXPOSED SUBJECTS IN COMPARISON WITH THE CONTROLS. THE SELECTED 14 MOST DIFFERENTLY METHYLATED CG LOCI WERE RELATIVELY STABLE IN THE CHRONICALLY EXPOSED SUBJECTS. IN SUMMARY, THE SPECIFIC TYPE OF LONG-TERM EXPOSURE CAN CONTRIBUTE TO THE FIXING OF RELEVANT EPIGENETIC CHANGES RELATED TO A SPECIFIC ENVIRONMENT AS, E.G., NP INHALATION. 2021 7 1599 40 DNA METHYLATION SIGNATURE OF CHILDHOOD CHRONIC PHYSICAL AGGRESSION IN T CELLS OF BOTH MEN AND WOMEN. BACKGROUND: HIGH FREQUENCY OF PHYSICAL AGGRESSION IS THE CENTRAL FEATURE OF SEVERE CONDUCT DISORDER AND IS ASSOCIATED WITH A WIDE RANGE OF SOCIAL, MENTAL AND PHYSICAL HEALTH PROBLEMS. WE HAVE PREVIOUSLY TESTED THE HYPOTHESIS THAT DIFFERENTIAL DNA METHYLATION SIGNATURES IN PERIPHERAL T CELLS ARE ASSOCIATED WITH A CHRONIC AGGRESSION TRAJECTORY IN MALES. DESPITE THE FACT THAT SEX DIFFERENCES APPEAR TO PLAY A PIVOTAL ROLE IN DETERMINING THE DEVELOPMENT, MAGNITUDE AND FREQUENCY OF AGGRESSION, MOST OF PREVIOUS STUDIES FOCUSED ON MALES, SO LITTLE IS KNOWN ABOUT FEMALE CHRONIC PHYSICAL AGGRESSION. WE THEREFORE TESTED HERE WHETHER OR NOT THERE IS A SIGNATURE OF PHYSICAL AGGRESSION IN FEMALE DNA METHYLATION AND, IF THERE IS, HOW IT RELATES TO THE SIGNATURE OBSERVED IN MALES. METHODOLOGY/PRINCIPAL FINDINGS: METHYLATION PROFILES WERE CREATED USING THE METHOD OF METHYLATED DNA IMMUNOPRECIPITATION (MEDIP) FOLLOWED BY MICROARRAY HYBRIDIZATION AND STATISTICAL AND BIOINFORMATIC ANALYSES ON T CELL DNA OBTAINED FROM ADULT WOMEN WHO WERE FOUND TO BE ON A CHRONIC PHYSICAL AGGRESSION TRAJECTORY (CPA) BETWEEN 6 AND 12 YEARS OF AGE COMPARED TO WOMEN WHO FOLLOWED A NORMAL PHYSICAL AGGRESSION TRAJECTORY. WE CONFIRMED THE EXISTENCE OF A WELL-DEFINED, GENOME-WIDE SIGNATURE OF DNA METHYLATION ASSOCIATED WITH CHRONIC PHYSICAL AGGRESSION IN THE PERIPHERAL T CELLS OF ADULT FEMALES THAT INCLUDES MANY OF THE GENES SIMILARLY ASSOCIATED WITH PHYSICAL AGGRESSION IN THE SAME CELL TYPES OF ADULT MALES. CONCLUSIONS: THIS STUDY IN A SMALL NUMBER OF WOMEN PRESENTS PRELIMINARY EVIDENCE FOR A GENOME-WIDE VARIATION IN PROMOTER DNA METHYLATION THAT ASSOCIATES WITH CPA IN WOMEN THAT WARRANT LARGER STUDIES FOR FURTHER VERIFICATION. A SIGNIFICANT PROPORTION OF THESE ASSOCIATIONS WERE PREVIOUSLY OBSERVED IN MEN WITH CPA SUPPORTING THE HYPOTHESIS THAT THE EPIGENETIC SIGNATURE OF EARLY LIFE AGGRESSION IN FEMALES IS COMPOSED OF A COMPONENT SPECIFIC TO FEMALES AND ANOTHER COMMON TO BOTH MALES AND FEMALES. 2014 8 2078 38 EPIGENETIC DIVERGENCE IN THE TRPA1 PROMOTER CORRELATES WITH PRESSURE PAIN THRESHOLDS IN HEALTHY INDIVIDUALS. THE EXPRESSION PATTERN OF IMPORTANT TRANSDUCTION MOLECULES IN NOCICEPTIVE SENSORY NEURONS IS LIKELY TO DICTATE PAIN SENSITIVITY. WHILE THIS NOTION IS WELL ESTABLISHED FOR INCREASED PAIN SENSITIVITIES UNDER CONDITIONS LIKE INFLAMMATION AND NEUROPATHY, LESS IS KNOWN AS TO WHICH MOLECULES ARE DEFINING INTERINDIVIDUAL DIFFERENCES IN PAIN SENSITIVITY IN HEALTHY SUBJECTS. A GENOME-WIDE METHYLATION ANALYSIS ON MONOZYGOTIC TWINS FOUND THAT METHYLATION OF A CPG DINUCLEOTIDE IN THE PROMOTER OF TRANSIENT RECEPTOR POTENTIAL ANKYRIN 1 (TRPA1) IS INVERSELY ASSOCIATED WITH THE THRESHOLD FOR HEAT-INDUCED PAIN. SEVERAL IN VITRO STUDIES ALSO SUGGEST THAT TRPA1 MEDIATES MECHANICAL SENSITIVITY OF SENSORY AFFERENTS, THUS POTENTIALLY MEDIATING PRESSURE-EVOKED PAIN. IN THE PRESENT STUDY, WE THEREFORE INVESTIGATED THE EPIGENETIC PREDISPOSITION FOR PRESSURE PAIN BY ANALYZING THE METHYLATION STATUS OF 47 CPG SITES IN THE PROMOTER REGION OF TRPA1. USING DNA FROM WHOLE-BLOOD SAMPLES OF 75 HEALTHY VOLUNTEERS, WE FOUND THAT THE SAME CPG SITE PREVIOUSLY FOUND TO AFFECT THE THRESHOLD FOR HEAT-EVOKED PAIN IS HYPERMETHYLATED IN SUBJECTS WITH A LOW THRESHOLD FOR PRESSURE PAIN. WE ALSO FOUND GENDER DIFFERENCES, WITH FEMALES DISPLAYING HIGHER METHYLATION RATES COMBINED WITH HIGHER PRESSURE PAIN SENSITIVITIES AS COMPARED WITH MALES. IN CONCLUSION, OUR FINDINGS SUPPORT THE NOTION THAT EPIGENETIC REGULATION OF TRPA1 SEEMS TO REGULATE THERMAL AND MECHANICAL PAIN SENSITIVITIES. 2017 9 287 33 AGING AND CHRONIC SUN EXPOSURE CAUSE DISTINCT EPIGENETIC CHANGES IN HUMAN SKIN. EPIGENETIC CHANGES ARE WIDELY CONSIDERED TO PLAY AN IMPORTANT ROLE IN AGING, BUT EXPERIMENTAL EVIDENCE TO SUPPORT THIS HYPOTHESIS HAS BEEN SCARCE. WE HAVE USED ARRAY-BASED ANALYSIS TO DETERMINE GENOME-SCALE DNA METHYLATION PATTERNS FROM HUMAN SKIN SAMPLES AND TO INVESTIGATE THE EFFECTS OF AGING, CHRONIC SUN EXPOSURE, AND TISSUE VARIATION. OUR RESULTS REVEAL A HIGH DEGREE OF TISSUE SPECIFICITY IN THE METHYLATION PATTERNS AND ALSO SHOWED VERY LITTLE INTERINDIVIDUAL VARIATION WITHIN TISSUES. DATA STRATIFICATION BY AGE REVEALED THAT DNA FROM OLDER INDIVIDUALS WAS CHARACTERIZED BY A SPECIFIC HYPERMETHYLATION PATTERN AFFECTING LESS THAN 1% OF THE MARKERS ANALYZED. INTERESTINGLY, STRATIFICATION BY SUN EXPOSURE PRODUCED A FUNDAMENTALLY DIFFERENT PATTERN WITH A SIGNIFICANT TREND TOWARDS HYPOMETHYLATION. OUR RESULTS THUS IDENTIFY DEFINED AGE-RELATED DNA METHYLATION CHANGES AND SUGGEST THAT THESE ALTERATIONS MIGHT CONTRIBUTE TO THE PHENOTYPIC CHANGES ASSOCIATED WITH SKIN AGING. 2010 10 2297 33 EPIGENETIC REGULATION OF ACUTE INFLAMMATORY PAIN. ACUTE PAIN IS ASSOCIATED WITH TISSUE DAMAGE, WHICH RESULTS IN THE RELEASE OF INFLAMMATORY MEDIATORS. RECENT STUDIES POINT TO THE INVOLVEMENT OF EPIGENETIC MECHANISMS (DNA METHYLATION) IN THE DEVELOPMENT OF PAIN. WE HAVE FOUND THAT DURING ACUTE INFLAMMATORY PAIN INDUCED BY THE APPLICATION OF 10% MUSTARD OIL ON THE TONGUES OF RATS, LEVELS OF DNMT3A AND 3B WERE ELEVATED MARKEDLY (36 AND 42 % RESPECTIVELY), WHEREAS THE LEVEL OF DNMT1 WAS NOT CHANGED SIGNIFICANTLY. PREVIOUS INJECTION OF XEFOCAM WITH 0,4 MG/KG DOSE DECREASED LEVELS OF DNMT3A AND 3B (25 AND 24% RESPECTIVELY). THE LEVEL OF DNMT1 WAS NOT CHANGED SIGNIFICANTLY COMPARED TO THE CONTROL GROUP. THE FINDINGS SUPPORT THE IDEA THAT INHIBITORS OF DNA-METHYLTRANSFERASES COULD BE USEFUL FOR PAIN MANAGEMENT. OUR DATA SUGGEST THAT NSAIDS (ALONE OR IN COMBINATION WITH DNMT INHIBITORS) MAY BE PROPOSED AS POSSIBLE EPIGENETIC REGULATORY AGENTS, WHICH MAY PLAY A ROLE IN EPIGENETIC MECHANISMS INDIRECTLY THROUGH ALTERING THE ACTIVITY OF INFLAMMATORY MEDIATORS INVOLVED IN PAIN DEVELOPMENT. 2014 11 1345 48 DETECTION OF DIFFERENTIALLY METHYLATED REGIONS USING BAYES FACTOR FOR ORDINAL GROUP RESPONSES. RESEARCHERS IN GENOMICS ARE INCREASINGLY INTERESTED IN EPIGENETIC FACTORS SUCH AS DNA METHYLATION, BECAUSE THEY PLAY AN IMPORTANT ROLE IN REGULATING GENE EXPRESSION WITHOUT CHANGES IN THE DNA SEQUENCE. THERE HAVE BEEN SIGNIFICANT ADVANCES IN DEVELOPING STATISTICAL METHODS TO DETECT DIFFERENTIALLY METHYLATED REGIONS (DMRS) ASSOCIATED WITH BINARY DISEASE STATUS. MOST OF THESE METHODS ARE BEING DEVELOPED FOR DETECTING DIFFERENTIAL METHYLATION RATES BETWEEN CASES AND CONTROLS. WE CONSIDER MULTIPLE SEVERITY LEVELS OF DISEASE, AND DEVELOP A BAYESIAN STATISTICAL METHOD TO DETECT THE REGION WITH INCREASING (OR DECREASING) METHYLATION RATES AS THE DISEASE SEVERITY INCREASES. PATIENTS ARE CLASSIFIED INTO MORE THAN TWO GROUPS, BASED ON THE DISEASE SEVERITY (E.G., STAGES OF CANCER), AND DMRS ARE DETECTED BY USING MOVING WINDOWS ALONG THE GENOME. WITHIN EACH WINDOW, THE BAYES FACTOR IS CALCULATED TO TEST THE HYPOTHESIS OF MONOTONIC INCREASE IN METHYLATION RATES CORRESPONDING TO SEVERITY OF THE DISEASE VERSUS NO DIFFERENCE. A MIXED-EFFECT MODEL IS USED TO INCORPORATE THE CORRELATION OF METHYLATION RATES OF NEARBY CPG SITES IN THE REGION. RESULTS FROM EXTENSIVE SIMULATION INDICATE THAT OUR PROPOSED METHOD IS STATISTICALLY VALID AND REASONABLY POWERFUL. WE DEMONSTRATE OUR APPROACH ON A BISULFITE SEQUENCING DATASET FROM A CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) STUDY. 2019 12 1537 38 DNA METHYLATION IN ADOLESCENTS WITH ANXIETY DISORDER: A LONGITUDINAL STUDY. ANXIETY DISORDERS (AD) TYPICALLY MANIFEST IN CHILDREN AND ADOLESCENTS AND MIGHT PERSIST INTO ADULTHOOD. HOWEVER, THERE ARE STILL FEW DATA CONCERNING EPIGENETIC MECHANISMS ASSOCIATED WITH ONSET, PERSISTENCE OR REMISSION OF AD OVER TIME. WE INVESTIGATED A COHORT OF ADOLESCENTS AND YOUNG ADULTS AT BASELINE (AGE; 13.19 +/- 2.38) AND AFTER 5 YEARS AND CLASSIFIED THEM ACCORDING TO THE AD DIAGNOSIS AND THEIR LONGITUDINAL TRAJECTORIES INTO 4 GROUPS: (1) TYPICALLY DEVELOPING COMPARISONS (TDC; CONTROL GROUP, N = 14); (2) INCIDENT (AD IN THE SECOND EVALUATION ONLY, N = 11); (3) PERSISTENT (AD IN BOTH EVALUATIONS, N = 14) AND (4) REMITTENT (AD IN THE FIRST EVALUATION ONLY, N = 8). DNA METHYLATION WAS EVALUATED WITH THE INFINIUM HUMANMETHYLATION450 BEADCHIP FROM SALIVA SAMPLES COLLECTED AT BOTH EVALUATIONS. GENE SET ENRICHMENT ANALYSIS WAS APPLIED TO CONSIDER BIOLOGICAL PATHWAYS. WE FOUND DECREASED DNA METHYLATION IN TDC GROUP WHILE THE CHRONIC CASES OF AD PRESENTED HYPERMETHYLATION IN CENTRAL NERVOUS SYSTEM DEVELOPMENT PATHWAYS. MOREOVER, WE SHOWED THAT THIS PERSISTENT GROUP ALSO PRESENTED HYPERMETHYLATION WHILE THE OTHER THREE GROUPS WERE ASSOCIATED WITH HYPOMETHYLATION IN NERVOUS SYSTEM DEVELOPMENT PATHWAY. INCIDENCE AND REMISSION GROUPS WERE ASSOCIATED WITH INCREASED AND DECREASED METHYLATION IN NEURON DEVELOPMENT PATHWAYS, RESPECTIVELY. LARGER STUDIES ARE LIKELY TO DETECT SPECIFIC GENES RELEVANT TO AD. 2018 13 3077 33 GENOME-WIDE METHYL-SEQ ANALYSIS OF BLOOD-BRAIN TARGETS OF GLUCOCORTICOID EXPOSURE. CHRONIC EXPOSURE TO GLUCOCORTICOIDS (GCS) CAN LEAD TO PSYCHIATRIC COMPLICATIONS THROUGH EPIGENETIC MECHANISMS SUCH AS DNA METHYLATION (DNAM). WE SOUGHT TO DETERMINE WHETHER EPIGENETIC CHANGES IN A PERIPHERAL TISSUE CAN SERVE AS A SURROGATE FOR THOSE IN A RELATIVELY INACCESSIBLE TISSUE SUCH AS THE BRAIN. DNA EXTRACTED FROM THE HIPPOCAMPUS AND BLOOD OF MICE TREATED WITH GCS OR VEHICLE SOLUTION WAS ASSAYED USING A GENOME-WIDE DNAM PLATFORM (METHYL-SEQ) TO IDENTIFY DIFFERENTIALLY METHYLATED REGIONS (DMRS) INDUCED BY GC TREATMENT. WE OBSERVED THAT APPROXIMATELY 70% OF THE DMRS IN BOTH TISSUES LOST METHYLATION FOLLOWING GC TREATMENT. OF THE 3,095 DMRS THAT MAPPED TO THE SAME GENES IN BOTH TISSUES, 1,853 DMRS UNDERWENT DNAM CHANGES IN THE SAME DIRECTION. INTERESTINGLY, ONLY 209 DMRS (<7%) OVERLAPPED IN GENOMIC COORDINATES BETWEEN THE 2 TISSUES, SUGGESTING TISSUE-SPECIFIC DIFFERENCES IN GC-TARGETED LOCI. PATHWAY ANALYSIS SHOWED THAT THE DMR-ASSOCIATED GENES WERE MEMBERS OF PATHWAYS INVOLVED IN METABOLISM, IMMUNE FUNCTION, AND NEURODEVELOPMENT. ALSO, CHANGES IN CELL TYPE COMPOSITION OF BLOOD AND BRAIN WERE EXAMINED BY FLUORESCENCE-ACTIVATED CELL SORTING. SEPARATION OF THE CORTEX INTO NEURONAL AND NON-NEURONAL FRACTIONS AND THE LEUKOCYTES INTO T-CELLS, B-CELLS, AND NEUTROPHILS SHOWED THAT GC-INDUCED METHYLATION CHANGES PRIMARILY OCCURRED IN NEURONS AND T-CELLS, WITH THE BLOOD TISSUE ALSO UNDERGOING A SHIFT IN THE PROPORTION OF CONSTITUENT CELL TYPES WHILE THE PROPORTION OF NEURONS AND GLIA IN THE BRAIN REMAINED STABLE. FROM THE CURRENT PILOT STUDY, WE FOUND THAT DESPITE TISSUE-SPECIFIC EPIGENETIC CHANGES AND CELLULAR HETEROGENEITY, BLOOD CAN SERVE AS A SURROGATE FOR GC-INDUCED CHANGES IN THE BRAIN. 2017 14 4093 35 MATERNAL SEPARATION FOLLOWED BY CHRONIC MILD STRESS IN ADULTHOOD IS ASSOCIATED WITH CONCERTED EPIGENETIC REGULATION OF AP-1 COMPLEX GENES. DEPRESSION IS ONE OF THE MOST PREVALENT MENTAL DISEASES WORLDWIDE. PATIENTS WITH PSYCHIATRIC DISEASES OFTEN HAVE A HISTORY OF CHILDHOOD NEGLECT, INDICATING THAT EARLY-LIFE EXPERIENCES PREDISPOSE TO PSYCHIATRIC DISEASES IN ADULTHOOD. TWO STRONG MODELS WERE USED IN THE PRESENT STUDY: THE MATERNAL SEPARATION/EARLY DEPRIVATION MODEL (MS) AND THE CHRONIC MILD STRESS MODEL (CMS). IN BOTH MODELS, WE FOUND CHANGES IN THE EXPRESSION OF A NUMBER OF GENES SUCH AS CREB AND NPY. STRIKINGLY, THERE WAS A CLEAR REGULATION OF EXPRESSION OF FOUR GENES INVOLVED IN THE AP-1 COMPLEX: C-FOS, C-JUN, FOSB, AND JUN-B. INTERESTINGLY, DIFFERENT EXPRESSION LEVELS WERE OBSERVED DEPENDING ON THE MODEL, WHEREAS THE COMBINATION OF THE MODELS RESULTED IN A NORMAL LEVEL OF GENE EXPRESSION. THE EFFECTS OF MS AND CMS ON GENE EXPRESSION WERE ASSOCIATED WITH DISTINCT HISTONE METHYLATION/ACETYLATION PATTERNS OF ALL FOUR GENES. THE EPIGENETIC CHANGES, LIKE GENE EXPRESSION, WERE ALSO DEPENDENT ON THE SPECIFIC STRESSOR OR THEIR COMBINATION. THE OBTAINED RESULTS SUGGEST THAT SINGLE LIFE EVENTS LEAVE A MARK ON GENE EXPRESSION AND THE EPIGENETIC SIGNATURE OF GENE PROMOTERS, BUT A COMBINATION OF DIFFERENT STRESSORS AT DIFFERENT LIFE STAGES CAN FURTHER CHANGE GENE EXPRESSION THROUGH EPIGENETIC FACTORS, POSSIBLY CAUSING THE LONG-LASTING ADVERSE EFFECTS OF STRESS. 2021 15 6311 42 THE RELATION BETWEEN DNA METHYLATION PATTERNS AND SERUM CYTOKINE LEVELS IN COMMUNITY-DWELLING ADULTS: A PRELIMINARY STUDY. BACKGROUND: THE LEVELS OF CIRCULATING CYTOKINES FLUCTUATE WITH AGE, ACUTE ILLNESS, AND CHRONIC DISEASE, AND ARE PREDICTIVE OF MORTALITY; THIS IS ALSO TRUE FOR PATTERNS OF DNA (CPG) METHYLATION. GIVEN THAT IMMUNE CELLS ARE PARTICULARLY SENSITIVE TO CHANGES IN THE CONCENTRATION OF CYTOKINES IN THEIR MICROENVIRONMENT, WE HYPOTHESIZED THAT SERUM LEVELS OF TNF, IL-6, IL-8 AND IL-10 WOULD CORRELATE WITH GENOME-WIDE ALTERATIONS IN THE DNA METHYLATION LEVELS OF BLOOD LEUKOCYTES. TO TEST THIS, WE EVALUATED COMMUNITY-DWELLING ADULTS (N = 14; 48-78 YEARS OLD) RECRUITED TO A PILOT STUDY FOR THE CANADIAN LONGITUDINAL STUDY ON AGING (CLSA), EXAMINING DNA METHYLATION PATTERNS IN PERIPHERAL BLOOD MONONUCLEAR CELLS USING THE ILLUMINA HUMANMETHYLATION 450 K BEADCHIP. RESULTS: WE SHOW THAT, APART FROM AGE, SERUM IL-10 LEVELS EXHIBITED THE MOST SUBSTANTIAL ASSOCIATION TO DNA METHYLATION PATTERNS, FOLLOWED BY TNF, IL-6 AND IL-8. FURTHERMORE, WHILE THE LEVELS OF THESE CYTOKINES WERE HIGHER IN ELDERLY ADULTS, NO ASSOCIATIONS WITH EPIGENETIC ACCELERATED AGING, DERIVED USING THE EPIGENETIC CLOCK, WERE OBSERVED. CONCLUSIONS: AS A PRELIMINARY STUDY WITH A SMALL SAMPLE SIZE, THE CONCLUSIONS DRAWN FROM THIS WORK MUST BE VIEWED WITH CAUTION; HOWEVER, OUR OBSERVATIONS ARE ENCOURAGING AND CERTAINLY WARRANT MORE SUITABLY POWERED STUDIES OF THIS RELATIONSHIP. 2017 16 5034 37 PHARMACOEPIGENETICS OF THE ROLE OF DNA METHYLATION IN MU-OPIOID RECEPTOR EXPRESSION IN DIFFERENT HUMAN BRAIN REGIONS. AIM: EXPOSURE TO OPIOIDS HAS BEEN ASSOCIATED WITH EPIGENETIC EFFECTS. STUDIES IN RODENTS SUGGESTED A ROLE OF VARYING DEGREES OF DNA METHYLATION IN THE DIFFERENTIAL REGULATION OF MU-OPIOID RECEPTOR EXPRESSION ACROSS THE BRAIN. METHODS: IN A TRANSLATIONAL INVESTIGATION, USING TISSUE ACQUIRED POSTMORTEM FROM 21 BRAIN REGIONS OF FORMER OPIATE ADDICTS, REPRESENTING A HUMAN COHORT WITH CHRONIC OPIOID EXPOSURE, MU-OPIOID RECEPTOR EXPRESSION WAS ANALYZED AT THE LEVEL OF DNA METHYLATION, MRNA AND PROTEIN. RESULTS & CONCLUSION: WHILE HIGH OR LOW MU-OPIOID RECEPTOR EXPRESSION SIGNIFICANTLY CORRELATED WITH LOCAL OPRM1 MRNA LEVELS, THERE WAS NO CORRESPONDING ASSOCIATION WITH OPRM1 METHYLATION STATUS. ADDITIONAL EXPERIMENTS IN HUMAN CELL LINES SHOWED THAT CHANGES IN DNA METHYLATION ASSOCIATED WITH CHANGES IN MU-OPIOID EXPRESSION WERE AN ORDER OF MAGNITUDE GREATER THAN DIFFERENCES IN BRAIN. HENCE, DIFFERENT DEGREES OF DNA METHYLATION ASSOCIATED WITH CHRONIC OPIOID EXPOSURE ARE UNLIKELY TO EXERT A MAJOR ROLE IN THE REGION-SPECIFICITY OF MU-OPIOID RECEPTOR EXPRESSION IN THE HUMAN BRAIN. 2016 17 1583 40 DNA METHYLATION PROFILES OF BLOOD CELLS ARE DISTINCT BETWEEN EARLY-ONSET OBESE AND CONTROL INDIVIDUALS. OBESITY IS A HIGHLY PREVALENT, CHRONIC DISORDER THAT HAS BEEN INCREASING IN INCIDENCE IN YOUNG PATIENTS. BOTH EPIGENETIC AND GENETIC ABERRATIONS MAY PLAY A ROLE IN THE PATHOGENESIS OF OBESITY. THEREFORE, IN-DEPTH EPIGENOMIC AND GENOMIC ANALYSES WILL ADVANCE OUR UNDERSTANDING OF THE DETAILED MOLECULAR MECHANISMS UNDERLYING OBESITY AND AID IN THE SELECTION OF POTENTIAL BIOMARKERS FOR OBESITY IN YOUTH. HERE, WE PERFORMED MICROARRAY-BASED DNA METHYLATION AND GENE EXPRESSION PROFILING OF PERIPHERAL WHITE BLOOD CELLS OBTAINED FROM SIX YOUNG, OBESE INDIVIDUALS AND SIX HEALTHY CONTROLS. WE OBSERVED THAT THE HIERARCHICAL CLUSTERING OF DNA METHYLATION, BUT NOT GENE EXPRESSION, CLEARLY SEGREGATES THE OBESE INDIVIDUALS FROM THE CONTROLS, SUGGESTING THAT THE METABOLIC DISTURBANCE THAT OCCURS AS A RESULT OF OBESITY AT A YOUNG AGE MAY AFFECT THE DNA METHYLATION OF PERIPHERAL BLOOD CELLS WITHOUT ACCOMPANYING TRANSCRIPTIONAL CHANGES. TO EXAMINE THE GENOME-WIDE DIFFERENCES IN THE DNA METHYLATION PROFILES OF YOUNG OBESE AND CONTROL INDIVIDUALS, WE IDENTIFIED DIFFERENTIALLY METHYLATED CPG SITES AND INVESTIGATED THEIR GENOMIC AND EPIGENOMIC CONTEXTS. THE ABERRANT DNA METHYLATION PATTERNS IN OBESE INDIVIDUALS CAN BE SUMMARIZED AS RELATIVE GAINS AND LOSSES OF DNA METHYLATION IN GENE PROMOTERS AND GENE BODIES, RESPECTIVELY. WE ALSO OBSERVED THAT THE CPG ISLANDS OF OBESE INDIVIDUALS ARE MORE SUSCEPTIBLE TO DNA METHYLATION COMPARED TO CONTROLS. OUR PILOT STUDY SUGGESTS THAT THE GENOME-WIDE ABERRANT DNA METHYLATION PATTERNS OF OBESE INDIVIDUALS MAY ADVANCE NOT ONLY OUR UNDERSTANDING OF THE EPIGENOMIC PATHOGENESIS BUT ALSO EARLY SCREENING OF OBESITY IN YOUTH. 2017 18 2920 42 GENE-SET ANALYSIS IS SEVERELY BIASED WHEN APPLIED TO GENOME-WIDE METHYLATION DATA. MOTIVATION: DNA METHYLATION IS AN EPIGENETIC MARK THAT CAN STABLY REPRESS GENE EXPRESSION. BECAUSE OF ITS BIOLOGICAL AND CLINICAL SIGNIFICANCE, SEVERAL METHODS HAVE BEEN DEVELOPED TO COMPARE GENOME-WIDE PATTERNS OF METHYLATION BETWEEN GROUPS OF SAMPLES. THE APPLICATION OF GENE SET ANALYSIS TO IDENTIFY RELEVANT GROUPS OF GENES THAT ARE ENRICHED FOR DIFFERENTIALLY METHYLATED GENES IS OFTEN A MAJOR COMPONENT OF THE ANALYSIS OF THESE DATA. THIS CAN BE USED, FOR EXAMPLE, TO IDENTIFY PROCESSES OR PATHWAYS THAT ARE PERTURBED IN DISEASE DEVELOPMENT. WE SHOW THAT GENE-SET ANALYSIS, AS IT IS TYPICALLY APPLIED TO GENOME-WIDE METHYLATION ASSAYS, IS SEVERELY BIASED AS A RESULT OF DIFFERENCES IN THE NUMBERS OF CPG SITES ASSOCIATED WITH DIFFERENT CLASSES OF GENES AND GENE PROMOTERS. RESULTS: WE DEMONSTRATE THIS BIAS USING PUBLISHED DATA FROM A STUDY OF DIFFERENTIAL CPG ISLAND METHYLATION IN LUNG CANCER AND A DATASET WE GENERATED TO STUDY METHYLATION CHANGES IN PATIENTS WITH LONG-STANDING ULCERATIVE COLITIS. WE SHOW THAT SEVERAL OF THE GENE SETS THAT SEEM ENRICHED WOULD ALSO BE IDENTIFIED WITH RANDOMIZED DATA. WE SUGGEST TWO EXISTING APPROACHES THAT CAN BE ADAPTED TO CORRECT THE BIAS. ACCOUNTING FOR THE BIAS IN THE LUNG CANCER AND ULCERATIVE COLITIS DATASETS PROVIDES NOVEL BIOLOGICAL INSIGHTS INTO THE ROLE OF METHYLATION IN CANCER DEVELOPMENT AND CHRONIC INFLAMMATION, RESPECTIVELY. OUR RESULTS HAVE SIGNIFICANT IMPLICATIONS FOR MANY PREVIOUS GENOME-WIDE METHYLATION STUDIES THAT HAVE DRAWN CONCLUSIONS ON THE BASIS OF SUCH STRONGLY BIASED ANALYSIS. CONTACT: CATHAL.SEOIGHE@NUIGALWAY.IE SUPPLEMENTARY INFORMATION: SUPPLEMENTARY DATA ARE AVAILABLE AT BIOINFORMATICS ONLINE. 2013 19 2281 45 EPIGENETIC REGULATION IN EARLY CHILDHOOD: A MINIATURIZED AND VALIDATED METHOD TO ASSESS HISTONE ACETYLATION. INTRODUCTION: CHRONIC INFLAMMATORY DISEASES INCLUDING ALLERGIES AND ASTHMA ARE THE RESULT OF COMPLEX INTERACTIONS BETWEEN GENES AND ENVIRONMENTAL FACTORS. EPIGENETIC MECHANISMS COMPRISE A SET OF BIOCHEMICAL REACTIONS THAT REGULATE GENE EXPRESSION. IN ORDER TO UNDERSTAND THE CAUSE-EFFECT RELATIONSHIP BETWEEN ENVIRONMENTAL EXPOSURES AND DISEASE DEVELOPMENT, METHODS CAPABLE OF ASSESSING EPIGENETIC REGULATION (ALSO) IN LARGE COHORTS ARE NEEDED. METHODS: FOR THIS PURPOSE, WE DEVELOPED AND EVALUATED A MINIATURIZED CHROMATIN IMMUNOPRECIPITATION (CHIP) ASSAY ALLOWING FOR A COST-EFFECTIVE ASSESSMENT OF HISTONE ACETYLATION OF CANDIDATE GENES IN A QUANTITATIVE FASHION. THIS METHOD WAS THEN APPLIED TO ASSESS H3 AND H4 HISTONE ACETYLATION CHANGES IN CORD BLOOD (CB) SAMPLES FROM AN ESTABLISHED COHORT OF AUSTRALIAN CHILDREN EXPOSED IN THE FETAL PERIOD TO EITHER VERY LOW OR VERY HIGH LEVELS OF MATERNAL FOLATE. RESULTS: OUR CHIP ASSAY WAS VALIDATED FOR A MINIMUM REQUIREMENT OF 1 X 105 TARGET CELLS (E.G. CD4+ T CELLS). VERY HIGH LEVELS OF MATERNAL FOLATE WERE SIGNIFICANTLY ASSOCIATED WITH INCREASED H3/H4 ACETYLATION AT GATA3 AND/OR IL9 PROMOTER REGIONS IN CD4+ T CELLS IN CB. CONCLUSION: WE DEVELOPED A CHIP METHOD ALLOWING RELIABLE ASSESSMENT OF H3/H4 ACETYLATION USING 1 X 105 CELLS ONLY. PRACTICAL APPLICATION OF THIS ASSAY DEMONSTRATED AN ASSOCIATION BETWEEN HIGH MATERNAL FOLATE EXPOSURE AND INCREASED HISTONE ACETYLATION, CORRESPONDING TO A MORE TRANSCRIPTIONALLY PERMISSIVE CHROMATIN STATUS IN THE PROMOTER REGIONS OF SOME TH2-RELATED GENES. 2015 20 70 36 A METHOD TO DETECT DIFFERENTIALLY METHYLATED LOCI WITH NEXT-GENERATION SEQUENCING. EPIGENETIC CHANGES, ESPECIALLY DNA METHYLATION AT CPG LOCI HAVE IMPORTANT IMPLICATIONS IN CANCER AND OTHER COMPLEX DISEASES. WITH THE DEVELOPMENT OF NEXT-GENERATION SEQUENCING (NGS), IT IS FEASIBLE TO GENERATE DATA TO INTERROGATE THE DIFFERENCE IN METHYLATION STATUS FOR GENOME-WIDE LOCI USING CASE-CONTROL DESIGN. HOWEVER, A PROPER AND EFFICIENT STATISTICAL TEST IS LACKING. THERE ARE SEVERAL CHALLENGES. FIRST, UNLIKE METHYLATION EXPERIMENTS USING MICROARRAYS, WHERE THERE IS ONE MEASURE OF METHYLATION FOR ONE INDIVIDUAL AT A PARTICULAR CPG SITE, HERE WE HAVE THE COUNTS OF METHYLATION ALLELE AND UNMETHYLATION ALLELE FOR EACH INDIVIDUAL. SECOND, DUE TO THE NATURE OF SAMPLE PREPARATION, THE MEASURED METHYLATION REFLECTS THE METHYLATION STATUS OF A MIXTURE OF CELLS INVOLVED IN SAMPLE PREPARATION. THEREFORE, THE UNDERLYING DISTRIBUTION OF THE MEASURED METHYLATION LEVEL IS UNKNOWN, AND A ROBUST TEST IS MORE DESIRABLE THAN PARAMETRIC APPROACH. THIRD, CURRENTLY NGS MEASURES METHYLATION AT OVER 2 MILLION CPG SITES. ANY STATISTICAL TESTS HAVE TO BE COMPUTATIONALLY EFFICIENT IN ORDER TO BE APPLIED TO THE NGS DATA. TAKING THESE CHALLENGES INTO ACCOUNT, WE PROPOSE A TEST FOR DIFFERENTIAL METHYLATION BASED ON CLUSTERED DATA ANALYSIS BY MODELING THE METHYLATION COUNTS. WE PERFORMED SIMULATIONS TO SHOW THAT IT IS ROBUST UNDER SEVERAL DISTRIBUTIONS FOR THE MEASURED METHYLATION LEVELS. IT HAS GOOD POWER AND IS COMPUTATIONALLY EFFICIENT. FINALLY, WE APPLY THE TEST TO OUR NGS DATA ON CHRONIC LYMPHOCYTIC LEUKEMIA. THE RESULTS INDICATE THAT IT IS A PROMISING AND PRACTICAL TEST. 2013