1 3037 169 GENOME AND METHYLOME VARIATION IN HELICOBACTER PYLORI WITH A CAG PATHOGENICITY ISLAND DURING EARLY STAGES OF HUMAN INFECTION. BACKGROUND & AIMS: HELICOBACTER PYLORI IS REMARKABLE FOR ITS GENETIC VARIATION; YET, LITTLE IS KNOWN ABOUT ITS GENETIC CHANGES DURING EARLY STAGES OF HUMAN INFECTION, AS THE BACTERIA ADAPT TO THEIR NEW ENVIRONMENT. WE ANALYZED GENOME AND METHYLOME VARIATIONS IN A FULLY VIRULENT STRAIN OF H PYLORI DURING EXPERIMENTAL INFECTION. METHODS: WE PERFORMED A RANDOMIZED PHASE I/II, OBSERVER-BLIND, PLACEBO-CONTROLLED STUDY OF 12 HEALTHY, H PYLORI-NEGATIVE ADULTS IN GERMANY FROM OCTOBER 2008 THROUGH MARCH 2010. THE VOLUNTEERS WERE GIVEN A PROPHYLACTIC VACCINE CANDIDATE (N = 7) OR PLACEBO (N = 5) AND THEN CHALLENGED WITH H PYLORI STRAIN BCM-300. BIOPSY SAMPLES WERE COLLECTED AND H PYLORI WERE ISOLATED. GENOMES OF THE CHALLENGE STRAIN AND 12 REISOLATES, OBTAINED 12 WEEKS AFTER (OR IN 1 CASE, 62 WEEKS AFTER) INFECTION WERE SEQUENCED BY SINGLE-MOLECULE, REAL-TIME TECHNOLOGY, WHICH, IN PARALLEL, PERMITTED DETERMINATION OF GENOME-WIDE METHYLATION PATTERNS FOR ALL STRAINS. FUNCTIONAL EFFECTS OF GENETIC CHANGES OBSERVED IN H PYLORI STRAINS DURING HUMAN INFECTION WERE ASSESSED BY MEASURING RELEASE OF INTERLEUKIN 8 FROM AGS CELLS (TO DETECT CAG PATHOGENICITY ISLAND FUNCTION), NEUTRAL RED UPTAKE (TO DETECT VACUOLATING CYTOTOXIN ACTIVITY), AND ADHESION ASSAYS. RESULTS: THE OBSERVED MUTATION RATE WAS IN AGREEMENT WITH RATES PREVIOUSLY DETERMINED FROM PATIENTS WITH CHRONIC H PYLORI INFECTIONS, WITHOUT EVIDENCE OF A MUTATION BURST. A LOSS OF CAG PATHOGENICITY ISLAND FUNCTION WAS OBSERVED IN 3 REISOLATES. IN ADDITION, 3 REISOLATES FROM THE VACCINE GROUP ACQUIRED MUTATIONS IN THE VACUOLATING CYTOTOXIN GENE VACA, RESULTING IN LOSS OF VACUOLIZATION ACTIVITY. WE OBSERVED INTERSTRAIN VARIATION IN METHYLOMES DUE TO PHASE VARIATION IN GENES ENCODING METHYLTRANSFERASES. CONCLUSIONS: WE ANALYZED ADAPTATION OF A FULLY VIRULENT STRAIN OF H PYLORI TO 12 DIFFERENT VOLUNTEERS TO OBTAIN A ROBUST ESTIMATE OF THE FREQUENCY OF GENETIC AND EPIGENETIC CHANGES IN THE ABSENCE OF INTERSTRAIN RECOMBINATION. OUR FINDINGS INDICATE THAT THE LARGE AMOUNT OF GENETIC VARIATION IN H PYLORI POSES A CHALLENGE TO VACCINE DEVELOPMENT. CLINICALTRIALS.GOV NO: NCT00736476. 2018 2 3625 72 IN VIVO GENOME AND METHYLOME ADAPTATION OF CAG-NEGATIVE HELICOBACTER PYLORI DURING EXPERIMENTAL HUMAN INFECTION. MULTIPLE STUDIES HAVE DEMONSTRATED RAPID BACTERIAL GENOME EVOLUTION DURING CHRONIC INFECTION WITH HELICOBACTER PYLORI IN CONTRAST, LITTLE WAS KNOWN ABOUT GENETIC CHANGES DURING THE FIRST STAGES OF INFECTION, WHEN SELECTIVE PRESSURE IS LIKELY TO BE HIGHEST. USING SINGLE-MOLECULE, REAL-TIME (SMRT) AND ILLUMINA SEQUENCING TECHNOLOGIES, WE ANALYZED GENOME AND METHYLOME EVOLUTION DURING THE FIRST 10 WEEKS OF INFECTION BY COMPARING THE CAG PATHOGENICITY ISLAND (CAGPAI)-NEGATIVE H. PYLORI CHALLENGE STRAIN BCS 100 WITH PAIRS OF H. PYLORI REISOLATES FROM GASTRIC ANTRUM AND CORPUS BIOPSY SPECIMENS OF 10 HUMAN VOLUNTEERS WHO HAD BEEN INFECTED WITH THIS STRAIN AS PART OF A VACCINE TRIAL. MOST GENETIC CHANGES DETECTED IN THE REISOLATES AFFECTED GENES WITH A SURFACE-RELATED ROLE OR A PREDICTED FUNCTION IN PEPTIDE UPTAKE. APART FROM PHENOTYPIC CHANGES OF THE BACTERIAL ENVELOPE, A DUPLICATION OF THE CATALASE GENE WAS OBSERVED IN ONE REISOLATE, WHICH RESULTED IN HIGHER CATALASE ACTIVITY AND IMPROVED SURVIVAL UNDER OXIDATIVE STRESS CONDITIONS. THE METHYLOMES ALSO VARIED IN SOME OF THE REISOLATES, MOSTLY BY ACTIVITY SWITCHING OF PHASE-VARIABLE METHYLTRANSFERASE (MTASE) GENES. THE OBSERVED IN VIVO MUTATION SPECTRUM WAS REMARKABLE FOR A VERY HIGH PROPORTION OF NONSYNONYMOUS MUTATIONS. ALTHOUGH THE DATA SHOWED SUBSTANTIAL WITHIN-STRAIN GENOME DIVERSITY IN THE CHALLENGE STRAIN, MOST ANTRUM AND CORPUS REISOLATES FROM THE SAME VOLUNTEERS WERE HIGHLY SIMILAR TO EACH OTHER, INDICATING THAT THE CHALLENGE INFECTION REPRESENTS A MAJOR SELECTIVE BOTTLENECK SHAPING THE TRANSMITTED POPULATION. OUR FINDINGS SUGGEST RAPID IN VIVO SELECTION OF H. PYLORI DURING EARLY-PHASE INFECTION PROVIDING ADAPTATION TO DIFFERENT INDIVIDUALS BY COMMON MECHANISMS OF GENETIC AND EPIGENETIC ALTERATIONS.IMPORTANCE EXCEPTIONAL GENETIC DIVERSITY AND VARIABILITY ARE HALLMARKS OF HELICOBACTER PYLORI, BUT THE BIOLOGICAL ROLE OF THIS PLASTICITY REMAINS INCOMPLETELY UNDERSTOOD. HERE, WE HAD THE RARE OPPORTUNITY TO INVESTIGATE THE MOLECULAR EVOLUTION DURING THE FIRST WEEKS OF H. PYLORI INFECTION BY COMPARING THE GENOMES AND EPIGENOMES OF H. PYLORI STRAIN BCS 100 USED TO CHALLENGE HUMAN VOLUNTEERS IN A VACCINE TRIAL WITH THOSE OF BACTERIA REISOLATED FROM THE VOLUNTEERS 10 WEEKS AFTER THE CHALLENGE. THE DATA PROVIDE MOLECULAR INSIGHTS INTO THE PROCESS OF ESTABLISHMENT OF THIS HIGHLY VERSATILE PATHOGEN IN 10 DIFFERENT HUMAN INDIVIDUAL HOSTS, SHOWING, FOR EXAMPLE, SELECTION FOR CHANGES IN HOST-INTERACTION MOLECULES AS WELL AS CHANGES IN EPIGENETIC METHYLATION PATTERNS. THE DATA PROVIDE IMPORTANT CLUES TO THE EARLY ADAPTATION OF H. PYLORI TO NEW HOST NICHES AFTER TRANSMISSION, WHICH WE BELIEVE IS VITAL TO UNDERSTAND ITS SUCCESS AS A CHRONIC PATHOGEN AND DEVELOP MORE EFFICIENT TREATMENTS AND VACCINES. 2020 3 4725 31 NORMAL GASTRIC TISSUE HELICOBACTER PYLORI INFECTION IS ASSOCIATED WITH EPIGENETIC AGE ACCELERATION, INCREASED MITOTIC TICK RATE, TISSUE CELL COMPOSITION, AND NATURAL KILLER CELL METHYLATION ALTERATIONS. BACKGROUND: GASTRIC ADENOCARCINOMAS ARE A LEADING CAUSE OF GLOBAL MORTALITY, ASSOCIATED WITH CHRONIC INFECTION WITH HELICOBACTER PYLORI . THE MECHANISMS BY WHICH INFECTION WITH H. PYLORI CONTRIBUTES TO CARCINOGENESIS ARE NOT WELL UNDERSTOOD. RECENT STUDIES FROM SUBJECTS WITH AND WITHOUT GASTRIC CANCER HAVE IDENTIFIED SIGNIFICANT DNA METHYLATION ALTERATIONS IN NORMAL GASTRIC MUCOSA ASSOCIATED WITH H. PYLORI INFECTION AND GASTRIC CANCER RISK. HERE WE FURTHER INVESTIGATED DNA METHYLATION ALTERATIONS IN NORMAL GASTRIC MUCOSA IN GASTRIC CANCER CASES (N = 42) AND CONTROL SUBJECTS (N = 42) WITH H. PYLORI INFECTION DATA. WE ASSESSED TISSUE CELL TYPE COMPOSITION, DNA METHYLATION ALTERATIONS WITHIN CELL POPULATIONS, EPIGENETIC AGING, AND REPETITIVE ELEMENT METHYLATION. RESULTS: IN NORMAL GASTRIC MUCOSA OF BOTH GASTRIC CANCER CASES AND CONTROL SUBJECTS, WE OBSERVED INCREASED EPIGENETIC AGE ACCELERATION ASSOCIATED WITH H. PYLORI INFECTION. WE ALSO OBSERVED AN INCREASED MITOTIC TICK RATE ASSOCIATED WITH H. PYLORI INFECTION IN BOTH GASTRIC CANCER CASES AND CONTROLS. SIGNIFICANT DIFFERENCES IN IMMUNE CELL POPULATIONS ASSOCIATED WITH H. PYLORI INFECTION IN NORMAL TISSUE FROM CANCER CASES AND CONTROLS WERE IDENTIFIED USING DNA METHYLATION CELL TYPE DECONVOLUTION. WE ALSO FOUND NATURAL KILLER CELL-SPECIFIC METHYLATION ALTERATIONS IN NORMAL MUCOSA FROM GASTRIC CANCER PATIENTS WITH H. PYLORI INFECTION. CONCLUSIONS: OUR FINDINGS FROM NORMAL GASTRIC MUCOSA PROVIDE INSIGHT INTO UNDERLYING CELLULAR COMPOSITION AND EPIGENETIC ASPECTS OF H. PYLORI ASSOCIATED GASTRIC CANCER ETIOLOGY. 2023 4 6311 33 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 5 3652 35 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 6 1345 37 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 7 287 32 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 8 70 30 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 9 2920 37 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 10 5276 35 PROMOTER METHYLATION OF TUMOR-RELATED GENES IN GASTRIC CARCINOGENESIS. ABERRANT PROMOTER METHYLATION AND SUBSEQUENT SILENCING OF CANCER-RELATED GENES HAS BEEN RECOGNIZED AS AN IMPORTANT PATHWAY INVOLVED IN GASTRIC CARCINOGENESIS. IN FACT, SEVERAL FACTORS ARE BELIEVED TO CONTRIBUTE TO ITS INDUCTION IN GASTRIC EPITHELIA, INCLUDING AGING, DIET, CHRONIC INFLAMMATION AND INFECTION OF HELICOBACTER PYLORI (H. PYLORI) AND EPSTEIN-BARR VIRUS (EBV). HOWEVER, THE UNDERLING MECHANISMS ARE NOT COMPLETELY IDENTIFIED, DESPITE THE BELIEF THAT INCREASED EXPRESSION OR ACTIVITY OF DNA METHYLTRANSFERASES (DNMTS), OR DECREASED DEMETHYLATION ACTIVITY MAY CONTRIBUTE TO THE EXCESSIVE METHYLATION. A GREAT NUMBER OF GENES WITH PROMOTER METHYLATION HAVE BEEN OBSERVED IN GASTRIC CANCER (GC), AMONG WHICH P16INK4A (P16), MUT L HOMOLOGUE 1 (MLH1), EPITHELIAL-CADHERIN (E-CADHERIN), RUNT-RELATED TRANSCRIPTION FACTOR 3 (RUNX3), ADENOMATOUS POLYPOSIS COLI (APC), O(6)-METHYLGUANINE-DNA METHYLTRANSFERASE (MGMT), RAS ASSOCIATION DOMAIN FAMILY 1A (RASSF1A) AND DEATH-ASSOCIATED PROTEIN KINASE (DAPK) HAVE BEEN EXTENSIVELY STUDIED. UNLIKE THE DISTINCT METHYLATION CHARACTERIZATION IN SINGLE GENES, METHYLATION ANALYSIS OF MULTIPLE GENES MAY PROVIDE MORE INFORMATION IN RISK PREDICTION, EARLY DETECTION, PROGNOSIS ASSESSMENT AND CHEMOTHERAPY CHOICE FOR GC. SPECIFICALLY, PARTICULAR MONITORING AND SCREENING SHOULD BE PERFORMED ON THOSE OVER 45 YEARS OLD, WITH PRECANCEROUS GASTRIC DISEASE OR INFECTION OF H. PYLORI OR EBV. AS AN ALTERNATIVE TO TUMOR TISSUES, METHYLATION DETECTION IN PATIENT SERA OR GASTRIC WASHES MAY ALSO BE USED IN RISK PREDICTION AND EARLY DETECTION. HOWEVER, WHAT STILL POSES A GREAT CHALLENGE AS WELL AS A PUZZLE IS THE DETERMINATION OF THE VERY GENES THAT SHOULD BE USED IN METHYLATION ANALYSIS. BECAUSE EPIGENETIC ALTERATIONS ARE NORMALLY REVERSIBLE, DRUGS OR CHEMICAL COMPOUNDS WITH DEMETHYLATING ACTIVITY, SUCH AS 5-AZA-2'-DEOXYCYTIDINE (5-AZA-DC) COULD BE USED IN THE TREATMENT OF PATIENTS WITH MULTIPLE GENE METHYLATION. IN VIEW OF THE ADVERSE EFFECTS OF 5-AZA-DC, DNMT-TARGETED STRATEGY HAS BEEN PROPOSED AND MAY PROVE TO BE MORE EFFECTIVE THAN DEMETHYLATING AGENTS. 2012 11 1599 39 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 12 344 38 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 13 4994 33 PERFORMANCE OF DNA METHYLATION ON THE MOLECULAR PATHOGENESIS OF HELICOBACTER PYLORI IN GASTRIC CANCER; TARGETED THERAPY APPROACH. GASTRIC CANCER (GC) IS A SIGNIFICANT CAUSE OF CANCER MORTALITY WHICH HAS LED TO FOCUSED EXPLORATION OF THE PATHOLOGY OF GC. THE ADVENT OF GENOME-WIDE ANALYSIS METHODS HAS MADE IT POSSIBLE TO UNCOVER GENETIC AND EPIGENETIC FLUCTUATION SUCH AS ABNORMAL DNA METHYLATION IN GENE PROMOTER REGIONS THAT IS EXPECTED TO PLAY A KEY ROLE IN GC. THE STUDY OF GASTRIC MALIGNANCIES REQUIRES AN ETIOLOGICAL PERSPECTIVE, AND HELICOBACTER PYLORI (H. PYLORI) WAS IDENTIFIED TO PLAY A ROLE IN GC. H. PYLORI INFECTION CAUSES CHRONIC INFLAMMATION OF THE GASTRIC EPITHELIUM CAUSING ABNORMAL POLYCLONAL METHYLATION, WHICH MIGHT RAISE THE RISK OF GC. IN THE LAST TWO DECADES, VARIOUS PATHOGENIC FACTORS BY WHICH H. PYLORI INFECTION CAUSES GC HAVE BEEN DISCOVERED. ABNORMAL DNA METHYLATION IS TRIGGERED IN SEVERAL GENES, RENDERING THEM INACTIVE. IN GC, METHYLATION PATTERNS ARE LINKED TO CERTAIN SUBTYPES INCLUDING MICROSATELLITE INSTABILITY. MULTIPLE CANCER-RELATED PROCESSES ARE MORE USUALLY CHANGED BY ABNORMAL DNA METHYLATION THAN THROUGH MUTATIONS, ACCORDING TO CURRENT GENERAL AND COMBINED INVESTIGATIONS. FURTHERMORE, THE AMOUNT OF ACQUIRED ABNORMAL DNA METHYLATION IS HEAVILY LINKED TO THE CHANCES OF DEVELOPING GC. THEREFORE, WE INVESTIGATED ABNORMAL DNA METHYLATION IN GC AND THE LINK BETWEEN METHYLATION AND H. PYLORI INFECTION. 2022 14 3219 36 HELICOBACTER PYLORI AND GASTRIC CANCER. INFECTION WITH HELICOBACTER PYLORI IS ESTABLISHED AS THE MAJOR RISK FACTOR FOR GASTRIC CANCER DEVELOPMENT. DAMAGE OF THE MUCOSAL BARRIER DUE TO H. PYLORI-INDUCED INFLAMMATION ENHANCES THE CARCINOGENIC EFFECT OF OTHER RISK FACTORS SUCH AS SALT INTAKE OR TOBACCO SMOKING. THE GENETIC DISPOSITION OF BOTH THE BACTERIAL STRAIN AND THE HOST CAN INCREASE THE POTENTIAL TOWARDS GASTRIC CANCER FORMATION. GENETIC VARIANCE OF THE BACTERIAL PROTEINS CAGA AND VACA IS ASSOCIATED WITH A HIGHER GASTRIC CANCER RISK, AS ARE POLYMORPHISMS AND EPIGENETIC CHANGES IN HOST GENE CODING FOR INTERLEUKINS (IL1BETA, IL8), TRANSCRIPTION FACTORS (CDX2, RUNX3) AND DNA REPAIR ENZYMES. APPLICATION OF HIGH-THROUGHPUT ASSAYS FOR GENOME-WIDE ASSESSMENT OF EITHER GENETIC STRUCTURAL VARIANCE OR GENE EXPRESSION PATTERNS MAY LEAD TO A BETTER UNDERSTANDING OF THE PATHOBIOLOGICAL BACKGROUND OF THESE PROCESSES, INCLUDING THE UNDERLYING SIGNALING PATHWAYS. UNDERSTANDING OF THE STEPWISE ALTERATIONS THAT TAKE PLACE IN THE TRANSITION FROM CHRONIC ATROPHIC GASTRITIS, VIA METAPLASTIC CHANGES, TO INVASIVE NEOPLASIA IS VITAL TO DEFINE THE 'POINT OF NO RETURN' BEFORE WHICH ERADICATION OF H. PYLORI HAS THE POTENTIAL TO PREVENT GASTRIC CANCER. CURRENTLY, ERADICATION AS PREVENTIVE STRATEGY IS ONLY RECOMMENDED FOR HIGH-INCIDENCE REGIONS IN ASIA; LARGE POPULATION STUDIES WITH AN ADEQUATE FOLLOW-UP ARE REQUIRED TO DEMONSTRATE THE EFFECTIVENESS OF SUCH AN APPROACH IN WESTERN POPULATIONS. 2014 15 2766 36 EXPRESSION, POLYMORPHISM AND METHYLATION PATTERN OF INTERLEUKIN-6 IN PERIODONTAL TISSUES. PERIODONTITIS IS CONSIDERED AN INFLAMMATORY DISORDER OF BACTERIAL ETIOLOGY THAT RESULTS IN PERIODONTAL TISSUE DESTRUCTION, AS A RESULT OF COMPLEX INTERACTIONS BETWEEN PERIODONTAL PATHOGENS, HOST AND IMMUNE RESPONSE. GENETIC AND EPIGENETIC MECHANISMS MAY MODULATE THE INDIVIDUAL RESPONSE SINCE IT IS ABLE TO INFLUENCE THE GENE EXPRESSION. THE AIM OF THIS STUDY WAS TO EVALUATE THE IMPACT OF -174 G/C POLYMORPHISM AND THE METHYLATION STATUS OF THE PROMOTER REGION OF IL-6 GENE ON THE EXPRESSION OF IL-6 IN GINGIVAL SAMPLES FROM INDIVIDUALS WITH CHRONIC PERIODONTITIS. GINGIVAL BIOPSIES WERE COLLECTED FROM 21 PATIENTS WITH CHRONIC PERIODONTITIS AND 21 CONTROLS. HISTOLOGIC SECTIONS STAINED BY HEMATOXYLIN-EOSIN WERE USED FOR HISTOPATHOLOGICAL EVALUATION. THE IL-6 GENE EXPRESSION WAS ASSESSED BY QUANTITATIVE REAL-TIME PCR. THE POLYMORPHISM IL-6 -174 C/G WAS STUDIED BY POLYMERASE CHAIN REACTION (PCR) AMPLIFICATION AND RESTRICTION ENDONUCLEASE DIGESTION (HSPII). METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION WAS USED TO VERIFY THE DNA METHYLATION PATTERN. THE NUMBER OF INFLAMMATORY CELLS IN TISSUE FRAGMENTS FROM INDIVIDUALS WITH CHRONIC PERIODONTITIS WAS HIGHER THAN IN THE CONTROL GROUP AND THE INFLAMMATORY INFILTRATE WAS PREDOMINANTLY MONONUCLEAR. THE EXPRESSION OF IL-6 WAS HIGHER IN THE GROUP WITH PERIODONTITIS. IN POLYMORPHISM ASSAY, NO STATISTICAL DIFFERENCE IN THE DISTRIBUTION OF GENOTYPES AND ALLELES IN BOTH GROUPS WERE OBSERVED. THE MOST OF SAMPLES WERE PARTIALLY METHYLATED. NO DIFFERENCE WAS OBSERVED IN METHYLATION PATTERN FROM TWO DIFFERENT REGIONS OF THE IL-6 GENE AMONG GROUPS. THE HIGH EXPRESSION OF IL-6 IS AN IMPORTANT FACTOR RELATED TO CHRONIC PERIODONTITIS, BUT WAS NOT ASSOCIATED WITH METHYLATION STATUS OR THE -174 (G/C) GENETIC POLYMORPHISM, SUGGESTING THAT OTHER MECHANISMS ARE INVOLVED IN THIS GENE TRANSCRIPTION REGULATION. 2013 16 1503 31 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 17 1537 32 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 18 5706 33 SINGLE-MOLECULE QUANTIFICATION OF 5-HYDROXYMETHYLCYTOSINE FOR DIAGNOSIS OF BLOOD AND COLON CANCERS. BACKGROUND: THE DNA MODIFICATION 5-HYDROXYMETHYLCYTOSINE (5HMC) IS NOW REFERRED TO AS THE SIXTH BASE OF DNA WITH EVIDENCE OF TISSUE-SPECIFIC PATTERNS AND CORRELATION WITH GENE REGULATION AND EXPRESSION. THIS EPIGENETIC MARK WAS RECENTLY REPORTED AS A POTENTIAL BIOMARKER FOR MULTIPLE TYPES OF CANCER, BUT ITS APPLICATION IN THE CLINIC IS LIMITED BY THE UTILITY OF RECENT 5HMC QUANTIFICATION ASSAYS. WE USE A RECENTLY DEVELOPED, ULTRA-SENSITIVE, FLUORESCENCE-BASED SINGLE-MOLECULE METHOD FOR GLOBAL QUANTIFICATION OF 5HMC IN GENOMIC DNA. THE HIGH SENSITIVITY OF THE METHOD GIVES ACCESS TO PRECISE QUANTIFICATION OF EXTREMELY LOW 5HMC LEVELS COMMON IN MANY CANCERS. METHODS: WE ASSESSED 5HMC LEVELS IN DNA EXTRACTED FROM A SET OF COLON AND BLOOD CANCER SAMPLES AND COMPARED 5HMC LEVELS WITH HEALTHY CONTROLS, IN A SINGLE-MOLECULE APPROACH. RESULTS: USING OUR METHOD, WE OBSERVED A SIGNIFICANTLY REDUCED LEVEL OF 5HMC IN BLOOD AND COLON CANCERS AND COULD DISTINGUISH BETWEEN COLON TUMOR AND COLON TISSUE ADJACENT TO THE TUMOR BASED ON THE GLOBAL LEVELS OF THIS MOLECULAR BIOMARKER. CONCLUSIONS: SINGLE-MOLECULE DETECTION OF 5HMC ALLOWS DISTINGUISHING BETWEEN MALIGNANT AND HEALTHY TISSUE IN CLINICALLY RELEVANT AND ACCESSIBLE TISSUE SUCH AS BLOOD AND COLON. THE PRESENTED METHOD OUTPERFORMS CURRENT COMMERCIALLY AVAILABLE QUANTIFICATION KITS AND MAY POTENTIALLY BE DEVELOPED INTO A WIDELY USED, 5HMC QUANTIFICATION ASSAY FOR RESEARCH AND CLINICAL DIAGNOSTICS. FURTHERMORE, USING THIS METHOD, WE CONFIRM THAT 5HMC IS A GOOD MOLECULAR BIOMARKER FOR DIAGNOSING COLON AND VARIOUS TYPES OF BLOOD CANCER. 2017 19 1805 29 EFFECT OF SMOKING ON THE DNA METHYLATION PATTERN OF THE SOCS1 PROMOTER IN EPITHELIAL CELLS FROM THE SALIVA OF PATIENTS WITH CHRONIC PERIODONTITIS. BACKGROUND: THE AIM OF THE PRESENT STUDY WAS TO EVALUATE THE METHYLATION PATTERN IN THE SUPPRESSOR OF CYTOKINE SIGNALING 1 (SOCS1) GENE IN SMOKERS AND NON-SMOKERS WITH CHRONIC PERIODONTITIS (CP). METHODS: METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION (PCR) WAS PERFORMED TO DETERMINE THE METHYLATION STATUS OF THE SOCS1 PROMOTER IN 45 SALIVA SAMPLES FROM SMOKERS AND NON-SMOKERS WITH CP. RESULTS: CELLS FROM THE SALIVA OF CP PATIENTS WHO SMOKED WERE 7.08 TIMES MORE LIKELY TO HAVE A METHYLATED SOCS1 PROMOTER THAN CELLS FROM THE SALIVA OF NON-SMOKING PATIENTS. CONCLUSIONS: SOCS1 GENE PROMOTER METHYLATION, WITH ITS POTENTIAL EFFECTS ON THE EXPRESSION OF THIS GENE, SEEMS TO BE A CONSEQUENCE OF EXPOSURE TO TOBACCO AND NOT TO PERIODONTAL DISEASE. FURTHER STUDIES ARE NEEDED TO ELUCIDATE THE RELATIONSHIP BETWEEN THE EPIGENETIC CONTROL OF IMMUNE RESPONSE GENE EXPRESSION, EXPOSURE TO ENVIRONMENTAL FACTORS, AND THE DEVELOPMENT, PROGRESSION, AND PROGNOSIS OF CP. 2019 20 2400 36 EPIGENETIC REPROGRAMMING OF IMMUNE CELLS IN WOMEN WITH PCOS IMPACT GENES CONTROLLING REPRODUCTIVE FUNCTION. CONTEXT: POLYCYSTIC OVARY SYNDROME (PCOS) IS A CHRONIC DISEASE AFFECTING REPRODUCTIVE FUNCTION AND WHOLE-BODY METABOLISM. ALTHOUGH THE ETIOLOGY IS UNCLEAR, EMERGING EVIDENCE INDICATES THAT THE EPIGENETICS MAY BE A CONTRIBUTING FACTOR. OBJECTIVE: TO DETERMINE THE ROLE OF GLOBAL AND GENOME-WIDE EPIGENETIC MODIFICATIONS IN SPECIFIC IMMUNE CELLS IN PCOS COMPARED WITH CONTROLS AND WHETHER THESE COULD BE RELATED TO CLINICAL FEATURES OF PCOS. DESIGN: CROSS-SECTIONAL STUDY. PARTICIPANTS: WOMEN WITH (N = 17) OR WITHOUT PCOS (N = 17). SETTING: RECRUITED FROM THE GENERAL COMMUNITY. MAIN OUTCOME MEASURES: ISOLATED PERIPHERAL BLOOD MONONUCLEAR CELLS WERE ANALYZED USING MULTICOLOR FLOW CYTOMETRY METHODS TO DETERMINE GLOBAL DNA METHYLATION LEVELS IN A CELL-SPECIFIC FASHION. TRANSCRIPTOMIC AND GENOME-WIDE DNA METHYLATION ANALYSES WERE PERFORMED ON T HELPER CELLS USING RNA SEQUENCING AND REDUCED REPRESENTATION BISULFITE SEQUENCING. RESULTS: WOMEN WITH PCOS HAD LOWER GLOBAL DNA METHYLATION IN MONOCYTES (P = 0.006) AND IN T HELPER (P = 0.004), T CYTOTOXIC (P = 0.004), AND B CELLS (P = 0.03). SPECIFIC GENOME-WIDE DNA METHYLATION ANALYSIS OF T HELPER CELLS FROM WOMEN WITH PCOS IDENTIFIED 5581 DIFFERENTIALLY METHYLATED CPG SITES. FUNCTIONAL GENE ONTOLOGY ENRICHMENT ANALYSIS SHOWED THAT GENES LOCATED AT THE PROXIMITY OF DIFFERENTIALLY METHYLATED CPG SITES BELONG TO PATHWAYS RELATED TO REPRODUCTIVE FUNCTION AND IMMUNE CELL FUNCTION. HOWEVER, THESE GENES WERE NOT ALTERED AT THE TRANSCRIPTOMIC LEVEL. CONCLUSIONS: IT WAS SHOWN THAT PCOS IS ASSOCIATED WITH GLOBAL AND GENE-SPECIFIC DNA METHYLATION REMODELING IN A CELL TYPE-SPECIFIC MANNER. FURTHER INVESTIGATION IS WARRANTED TO DETERMINE WHETHER EPIGENETIC REPROGRAMMING OF IMMUNE CELLS IS IMPORTANT IN DETERMINING THE DIFFERENT PHENOTYPES OF PCOS. 2019